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Review of the Study of Autosomal Aneuploidy

Part 1: Review of the Study of Aneuploidy

00:00:01;09 Hello, my name is Angelika Amon. I'm a Professor of
00:00:04;23 Biology in the Department of Biology at the
00:00:07;29 Massachusetts Institute of Technology. I'm also
00:00:11;06 Howard Hughes Medical Investigator. And today I will
00:00:14;19 tell you about our research that concerns the question
00:00:17;29 of what the consequences are for cells and for
00:00:21;24 organisms when their chromosome content is
00:00:25;02 unbalanced when these cells and these organisms are
00:00:28;03 aneuploid. So the title of my talk is, "The consequences
00:00:32;06 of aneuploidy." And what I'm going to do today is I'm
00:00:35;08 going to tell you three little, separate stories. In the
00:00:40;01 first part of my story, I will give you a historical
00:00:43;13 overview about the studies of aneuploidy, how people
00:00:46;26 over the centuries have tried to understand what it
00:00:50;07 means for a cell and an organism to have an
00:00:54;28 unbalanced karyotype, to have an unbalanced
00:00:57;16 genome. In the second part of the talk, I'll tell you
00:01:00;21 about work in my lab that addresses a very basic
00:01:04;07 question, that is, what is the impact on the physiology
00:01:07;13 of aneuploidy, an unbalanced karyotype, on cells and
00:01:13;04 on organisms? And as you will see, these studies and
00:01:16;18 questions were motivated by conundrums that have
00:01:19;28 plagued that field for as long as the field exists. And I
00:01:24;02 will try and put our work and our science in the context
00:01:28;05 of these particular questions. In the third part of my
00:01:31;16 talk, I will try and sort of branch out and ask, well, how
00:01:37;27 is our science important for medicine? Can we, with our
00:01:42;17 basic research in understanding basic biological
00:01:45;11 processes, shed light on diseases such as cancer and
00:01:51;03 neurodegenerative diseases? And it's my hope that at
00:01:54;11 the end, we'll all appreciate how important it is to do
00:01:59;04 basic science and to actually use model organisms such
00:02:02;20 as budding yeast (which my lab is using) to address
00:02:06;23 basic questions in biology, and that they do impact the
00:02:11;16 medicine of the future. So let me start out with a
00:02:17;04 historical review of aneuploidy. And again, I've defined
00:02:22;06 or subdivided this first part into a bunch of subparts, so
00:02:26;06 that we sort of move along smoothly. I will first define
00:02:30;16 the term aneuploidy for you. I think it's really important
00:02:33;24 that we have all the definitions in place so that we do
00:02:36;29 understand everything that I'm going to tell you about
00:02:39;23 today, we're all on the same page. I will discuss with
00:02:42;21 you very briefly the mechanisms and pathways that go
00:02:48;09 wrong and that do lead to this condition of aneuploidy.
00:02:52;12 I will tell you how it arises. I will then basically give you
00:02:56;25 a historical overview of the long tradition of the study
00:03:00;16 of aneuploidy. And then finally, in the first part of my
00:03:03;24 talk, I will try and put our research into context, and I
00:03:07;05 will sort of try to illustrate what the big questions are,
00:03:11;06 that arose from these earlier studies, and how we are
00:03:14;14 hoping to sort of address them. So let me define the
00:03:18;23 terms for you. If you go into Wikipedia and type in
00:03:22;26 "aneuploidy," this is what comes up. The answer that
00:03:26;06 you get is, "Chromosome composition that is not a
00:03:29;12 multiple of the haploid, normal chromosome
00:03:32;28 composition." And what's a really key aspect of
00:03:36;12 aneuploidy is that the genome is unbalanced. That is in
00:03:41;00 contrast to another condition, the name you've
00:03:44;02 probably heard frequently, that's called "polyploidy."
00:03:47;21 What happens in polyploidy is that the entire genome
00:03:50;10 gets duplicated. And as you will see from my talk, the
00:03:55;00 phenotypes and traits that are associated with
00:03:58;03 polyploidy are going to be very different from the ones
00:04:01;05 associated with aneuploidy. And as you will see, and as
00:04:05;11 we already know, polyploidy is actually well tolerated in
00:04:08;11 nature. It happens very frequently in plants, and
00:04:11;05 sometimes it's actually part of the normal development
00:04:14;19 of a particular tissue. In contrast, in all organisms
00:04:19;15 where we have studied aneuploidy so far, aneuploidy's
00:04:23;01 highly detrimental. And so, as you know, organisms
00:04:27;13 come in haploid -- some, like budding yeast, the model
00:04:30;21 organism that I study, is haploid. Most eukaryotes are
00:04:34;11 diploid, that means they have two complements. And
00:04:36;29 then sometimes, in rare cases, there are organisms,
00:04:39;29 and sometimes cells, that are tetraploid and polyploid.
00:04:43;15 And as you can see from this picture, the entire
00:04:47;14 chromosomes, like the blue, pink, and yellow sausages,
00:04:51;03 are all duplicated. So even though there's many more
00:04:53;24 chromosomes, the relative ratios of these
00:04:56;07 chromosomes is the same. Okay? In aneuploidy, single
00:05:00;29 chromosomes sort of get unbalanced. So for example,
00:05:04;24 here we have a haploid yeast strain, and as you can
00:05:07;21 see, this particular yeast strain lacks the yellow
00:05:10;24 chromosome. And so therefore, this is called a
00:05:14;10 "nullisome." It's lacking an entire chromosome,
00:05:16;26 obviously that's sort of lethal most of the time. This
00:05:20;04 particular cell has an extra copy of the blue
00:05:23;12 chromosome. We call this chromosome to be "disomic,"
00:05:27;24 instead of its normal, haploid complement, now you
00:05:30;18 have two copies of that blue chromosome. Then in the
00:05:33;24 case of diploid organisms, we can again distinguish
00:05:36;21 between chromosome gains and chromosome losses.
00:05:39;20 When you lose a chromosome, we call that a
00:05:41;22 "monosomy," and as I will tell you later, there are
00:05:45;14 actually diseases where entire chromosomes are lost,
00:05:48;05 for example, XO Turner syndrome, you have only one
00:05:51;14 copy of the X chromosome, rather than two. Then
00:05:54;16 there are conditions where we gain an extra
00:05:56;14 chromosome, and that well a "trisomy." And what
00:06:00;25 happens in a trisomy is you have three, instead of the
00:06:03;10 normal two, copies, and again, from human diseases, in
00:06:08;00 human conditions, we know a very popular example is
00:06:12;18 Down syndrome. Down syndrome individuals have
00:06:16;02 three copies of chromosome 21, instead of just two.
00:06:20;06 And then finally, there is a condition we call "high-grade
00:06:23;28 aneuploidy," and organisms basically do not exist that
00:06:28;18 have... basically, we call them in the lab "crazy
00:06:31;26 karyotypes," you know, they have lots of different
00:06:34;06 chromosomes gained, many chromosomes lost. These
00:06:37;11 high-grade aneuploidies are not observed in whole
00:06:40;00 organisms, but they're sometimes observed in
00:06:43;17 particular cells, and these cells are invariably cancer
00:06:47;06 cells, okay? So cancer is a disease that's characterized
00:06:51;21 by high, high grades of aneuploidy. And as you will
00:06:55;02 see, this was actually an observation that initially
00:06:58;27 motivated our studies of the effects of aneuploidy on
00:07:03;25 cell physiology. And then finally, I want to point out
00:07:07;14 one additional subtlety, which is sort of down here,
00:07:11;12 categorized as "partial aneuploidies." So sometimes,
00:07:15;22 you don't have whole chromosomes lost or gained, but
00:07:18;22 you've parts of chromosomes gained and lost. And
00:07:21;08 these partial aneuploidies are called frequently
00:07:24;25 "segmental aneuploidies," where you have either
00:07:27;16 deletions of a chromosome or duplications of a
00:07:30;16 chromosome or sometimes also nonreciprocal
00:07:33;21 translocations. And so, the last definition that I want to
00:07:39;21 bring up, which is sort of important again for thinking
00:07:42;24 about aneuploidy and its impact on physiology, is the
00:07:46;22 idea that basically aneuploidy can come in two flavors.
00:07:51;15 The entire organism can be aneuploid, for example,
00:07:55;10 individuals with Down syndrome. Most of them, the
00:07:58;21 vast majority of their cells have an extra copy of
00:08:02;06 chromosome 21. But then there are also "mosaic
00:08:04;22 aneuploidies," and only a particular tissue or a
00:08:09;15 particular cancer, for example, is aneuploid. And so
00:08:13;13 basically in fact we now know that there are some
00:08:16;18 tissues in humans, the liver for example, that is
00:08:20;05 basically aneuploid, so we call that a mosaic
00:08:22;18 aneuploidy. And so basically these types of
00:08:26;11 aneuploidies occur in nature. The more penetrant, the
00:08:31;11 more high degree, the aneuploidy is, the usually more
00:08:34;08 severe it is, and so a lot of times, mosaic aneuploidies
00:08:37;10 go unnoticed, but constitutional aneuploids, where the
00:08:41;19 entire organism is aneuploid, usually have very, very
00:08:45;12 severe impact on the fitness of this organism. So now
00:08:49;24 that we know what the terms now, now that we
00:08:52;09 understand what it's called basically, what I want to do
00:08:57;26 now is I want to sort of briefly summarize for you how
00:09:02;07 it happens that whole chromosomes get lost or gained.
00:09:07;17 This is not a lecture of the gymnastics of chromosome
00:09:10;25 segregation. There are a number of other wonderful
00:09:14;17 other lectures in the iBio series that illustrate the
00:09:18;11 detailed ins and outs of chromosome segregation, and I
00:09:22;08 refer you to those. What I will do is sort of very briefly
00:09:26;03 recap in a few minutes how chromosomes get
00:09:29;17 segregated, and in which ways that chromosome
00:09:33;01 segregation can go wrong. So basically what is
00:09:37;10 happens during chromosome segregation is you
00:09:40;13 duplicate your genetic material to form these very
00:09:44;02 characteristic pairs of sister chromatids. These pairs of
00:09:48;08 sister chromatids are then condensed into these X-
00:09:51;11 shaped structures that we call chromosomes, and they
00:09:54;25 then, through the process of the mitotic microtubules
00:09:58;20 and the mitotic spindle, get aligned in the middle of the
00:10:02;04 cell. And as you can see here, this chromosome here is
00:10:06;15 attached to one microtubule that comes from this
00:10:10;22 centrosome (sorry, it's this one here), and to another
00:10:14;19 microtubule that's coming from this centrosome here,
00:10:17;02 okay? And we call this chromosome to be "biorient," to
00:10:21;03 be properly attached, and it will get split in half during
00:10:25;20 the chromosome segregation process, okay?
00:10:28;20 Sometimes, this attaching to chromosomes to the
00:10:32;18 mitotic spindle doesn't work very well, and what
00:10:35;29 happens is that one of chromosomes is unattached,
00:10:42;20 and we say that its kinetochore (which is the
00:10:44;25 proteinaceous structure that hooks the chromosome
00:10:47;19 onto the spindle) is an unattached. There are
00:10:51;11 checkpoints, or we call them "surveillance" mechanisms,
00:10:54;20 that make sure that such a situation is recognized, and
00:10:59;04 the cell cycle (mitosis, in this particular case) halts until
00:11:03;21 the chromosome attaches. When these checkpoints are
00:11:06;23 faulty and they don't work, and the cells can't sense
00:11:10;07 that its chromosomes aren't attached right to the
00:11:13;07 spindle, you will get a faulty chromosome segregation,
00:11:17;02 and you can see here this cell is undergoing anaphase,
00:11:20;05 and look at that particular chromosome. It sort of
00:11:23;02 wasn't attached properly to the spindle and sort of
00:11:25;14 floats off, okay? And that will then lead to a cell, a
00:11:29;12 diploid cell, that has gained a chromosome, and a
00:11:32;24 diploid cell that's missing a particular chromosome. So
00:11:36;10 that's one way, you're not attaching these
00:11:38;15 chromosomes right, and you don't notice. Another
00:11:42;07 possibility by which you can missegregate chromosomes
00:11:45;07 has something to do with the fact how duplicated DNA
00:11:50;14 strands are linked together. So as you're replicating
00:11:53;20 your DNA and you make these two sister chromatids,
00:11:58;01 they're not sort of floating off into space, right?
00:12:02;04 They're actually being linked during that DNA replication
00:12:04;19 process by sort of a generic protein complex that's
00:12:09;29 called a "cohesion complex." So you can think about the
00:12:13;10 cohesion complex as a little clip that holds the
00:12:17;08 replicated chromosomes together, and that, as the
00:12:21;06 chromosomes segregate, this cohesion is resolved, and
00:12:24;03 the chromosomes segregate. So if you either have
00:12:28;04 mutations in this cohesion protein, or in the process
00:12:31;22 that puts that sort of proteinaceous glue onto
00:12:35;08 chromosomes, what can happen is that these sister
00:12:38;02 chromatids are floating away, and then they can't
00:12:41;05 sense each other anymore, they don't know where
00:12:43;08 they are. And they will start randomly attaching to the
00:12:47;14 microtubules. See this particular chromosome here? The
00:12:50;20 two sisters fell apart, they're floating away, and
00:12:54;06 instead of one attaching to a microtubule that's from
00:12:58;13 this pole, and the other one attaching to a microtubule
00:13:01;26 from that pole, what you realize is that both of them
00:13:04;28 are attached to microtubules from the same pole, this
00:13:07;00 pole. And so that will also lead to chromosome
00:13:09;21 missegregation in the subsequent anaphase, and again
00:13:14;12 it will result in a trisomy and a monosomy. And in the
00:13:22;27 final way in which you can missegregate chromosome
00:13:26;00 during mitosis is again a funny way in which the
00:13:31;21 chromosomes attach to the mitotic spindle, attach to
00:13:34;15 the microtubules. So to segregate a chromosome,
00:13:40;03 particularly in higher eukaryotes, you have to attach
00:13:43;06 multiple microtubules to one particular chromosome,
00:13:46;14 okay? And sometimes what happens, as is sort of
00:13:49;22 shown here, is that in this one kinetochore, instead of
00:13:54;29 attaching from the microtubule from only one pole right
00:13:58;16 here, it actually attaches to microtubules from both
00:14:02;16 poles, okay? And that configuration is called a
00:14:05;19 "merotelic attachment." And so now you can envision
00:14:10;01 they want to segregate their chromosomes, but this
00:14:13;05 one chromosome is being pulled on from both
00:14:15;04 directions, and obviously then you can't segregate it,
00:14:18;04 it's sort of, as you can see here, it gets stuck in the
00:14:20;17 middle of the cell, and as the spindle disassembles and
00:14:23;19 the cells are undergoing cytokinesis and the next G1
00:14:26;19 period, what you will see, sort of, this chromosome will
00:14:29;20 sort of float off into space and then randomly join one
00:14:33;01 of two nuclei. Okay? And so again that will lead to
00:14:37;19 aneuploidy. So these are various chromosome
00:14:40;06 missegregation events that lead to aneuploidy in
00:14:43;04 mitotically dividing cells. There's one last situation that I
00:14:48;25 would like to consider, and that is when chromosome
00:14:51;25 missegregation happens in the germline, because of
00:14:55;20 course, if chromosome missegregation happens during
00:14:59;27 the process of making sperm and egg, when this egg or
00:15:04;05 this sperm gets fertilized, right, it will lead to the entire
00:15:08;06 organism being aneuploid. And so again, what you can
00:15:13;07 see here is you see cells progressing through meiosis.
00:15:18;06 Here's... as you probably already know, meiosis is a
00:15:22;03 specialized cell division, and it basically reduces the
00:15:25;26 complement from a diploid to a haploid. And basically,
00:15:30;17 this is accomplished by having two chromosome
00:15:33;17 segregation phases following one replication phase.
00:15:37;04 There's a meiosis I chromosome segregation phase,
00:15:39;28 and there's a meiosis II chromosome segregation
00:15:42;08 phase. And without going through the details, all these
00:15:45;13 problems that I just explained to you during mitosis,
00:15:49;06 they can also occur during meiosis. So you can have
00:15:53;13 chromosome segregation errors during the first meiotic
00:15:55;28 division, and you have chromosome segregation errors
00:15:58;19 in the second meiotic division. And needless to say that
00:16:03;04 this will then of course lead to the formation of
00:16:05;23 aneuploid germ cells, aneuploid sperm and egg, and
00:16:09;15 upon fertilization, they will basically result in an
00:16:13;10 organism which, in its entirety, is in fact aneuploid. So
00:16:18;20 this is how you can get whole chromosomal
00:16:22;01 aneuploidies. The mechanisms that lead to the
00:16:24;26 formation of partial aneuploidies, or segmental
00:16:28;02 aneuploidies, are very different. They are due mostly
00:16:31;03 to defects in DNA repair or in DNA replication, and I will
00:16:36;14 not consider them here today. What we're going to
00:16:39;02 focus on for the entire rest of the talk is sort of these
00:16:41;26 whole chromosomal aneuploidies, and what their impact
00:16:44;26 is on cells. So let me now then move on and tell you a
00:16:49;03 little bit about the long tradition of the study of
00:16:53;06 aneuploidy. And really the first people to study
00:16:57;18 aneuploidy in some systematic fashion were a husband
00:17:04;01 and wife scientist team, Marcella O'Grady and Theodor
00:17:09;03 Boveri. And they were very interested in is trying to
00:17:13;07 understand what happens to a sea urchin embryo that
00:17:18;23 instead of being fertilized by one sperm, is fertilized by
00:17:22;21 two sperm. Okay? And what happens when you
00:17:26;16 instead of fertilizing an egg with one sperm with two
00:17:29;13 sperm is, first of all, you start out being triploid, right?
00:17:33;16 Instead of being your normal diploid, you have three
00:17:36;00 copies of the complement. That actually wouldn't be so
00:17:38;27 bad, I told you as long you're balanced, polyploidy is
00:17:42;17 actually not so bad. But what happens when you put in
00:17:47;01 two sperm, you will not only two copies of the genome,
00:17:51;19 you will actually also get two centrosomes, okay?
00:17:54;22 These are the organelles that organize the first mitotic
00:17:58;20 spindle of this embryo. So now instead of having one
00:18:03;01 centrosome from the mom (from the egg) and one
00:18:06;26 centrosome from the sperm, that form this proper, nice
00:18:10;01 spindle, all of a sudden you have three centrosomes,
00:18:13;04 and they're all trying to form a spindle. And so what
00:18:15;25 they do is they start making a tripolar mitosis. So
00:18:19;23 basically Marcella O'Grady here illustrates what this first
00:18:23;24 embryonic division looks like, right? So there are three
00:18:26;16 microtubule-organizing centers, three centrosomes,
00:18:30;00 and they start engaging the chromosomes. And instead
00:18:33;07 of making two cells in that first embryonic division, that
00:18:36;27 will actually result in three cells, right? And these cells,
00:18:41;17 these first three cells, will of course be highly
00:18:44;03 aneuploid, the chromosome segregation is going to be
00:18:47;06 completely random and messy, okay? And in this slide
00:18:50;29 here now shows you what Marcella O'Grady observed,
00:18:55;25 what was the effect of this first crazy, random
00:18:59;13 chromosome segregation on the development of these
00:19:02;12 sea urchins? So this here is a more-or-less normal
00:19:05;24 gastrulating sea urchin embryo, and these are all the
00:19:09;10 things that happen when you undergo a tripolar first
00:19:15;15 embryoic division, versus a dipolar one. So these
00:19:18;18 embryos don't develop, they're a huge mess, they're
00:19:21;23 these pile of cells, and invariably, all these sea urchins
00:19:26;02 will die. So that really was the first realization that
00:19:30;20 having an incorrect genetic complement, having an
00:19:34;04 incorrect karyotype, is highly detrimental for an
00:19:38;04 organism. In most cases, it's lethal. And throughout
00:19:42;11 history, in every single organism where people have
00:19:46;01 studied this, this turns out to be true. So here's a less
00:19:49;15 well-known example that I nevertheless like to point
00:19:52;09 out, because it was by the director of a botanical
00:19:56;19 garden in Connecticut, Albert Blakeslee. He was very
00:20:00;13 much interested in jimson weeds, and so he created a
00:20:04;25 series of jimson weeds that had incorrect karyotypes.
00:20:09;29 And so here on the top you see the fruit of a normal,
00:20:13;16 diploid jimson weed plant, it's very large. And then
00:20:17;16 down here you see the various abnormalities that occur
00:20:21;11 if these plants have one extra chromosome of any kind
00:20:27;22 of a flavor. There's sort of clearly specific effects, they
00:20:32;01 all look like they're different, they have different
00:20:33;22 phenotypes, but they're also some general things we
00:20:36;19 observe. Most of these plants are smaller, they're
00:20:39;22 runted, and they have a number of developmental
00:20:43;08 defects. And indeed in all organisms where this has
00:20:47;03 been looked at, that's sort of a very general feature.
00:20:50;12 These plants, animals, you name it, that are aneuploid
00:20:54;19 are growth retarded, and I'm actually going to come to
00:20:58;03 that point back in when I'll tell you about the work
00:21:04;18 that's ongoing on our lab. So here, Barbara McClintock,
00:21:09;08 very famous maize geneticist. She too studied the
00:21:13;12 effects of a wrong karyotype on her maize plants, and
00:21:17;04 you can see here there's a diploid plant that is normal.
00:21:22;15 And then here, I'm standing among all these little,
00:21:26;05 runted, aneuploid maize plants that have all sorts of
00:21:29;21 problems: they're sterile and can't grow very well. And
00:21:33;08 of course, the last example that I want to bring up is
00:21:36;25 from humans. I mentioned it before. In 1959, Jérôme
00:21:41;27 Lejeune actually showed that a very characteristic
00:21:45;19 syndrome known as Down syndrome is actually due to
00:21:49;06 the fact that these individuals, instead of having two
00:21:52;14 copies of chromosome 21, having three copies of
00:21:55;17 chromosome 21. And I also want to take a minute now
00:21:59;09 to sort of actually tell you about the impact that
00:22:02;23 aneuploidy has on humans. As I said before, they're
00:22:07;02 severe, but here are the stats. All autosomal
00:22:10;11 monosomies, and most autosomal trisomies, are lethal
00:22:13;19 in humans. Aneuploidy is the leading cause of mental
00:22:17;12 retardation in humans, and it's also the leading cause
00:22:20;15 of miscarriages in humans. Thirty-five percent of all
00:22:24;10 clinically recognized spontaneous abortions are due to
00:22:27;27 the fact that the fetus has the wrong chromosome
00:22:30;16 number, is aneuploid. And of course another point that
00:22:33;19 is very interesting to us in the lab is sort of this
00:22:37;00 observation that aneuploidy is associated with cancer.
00:22:41;01 More than 90% of all human cancers are in fact
00:22:45;04 aneuploid. So to summarize this sort of first review part
00:22:51;26 of the talk, and the main points that I wanted to drive
00:22:55;02 home here before I move on and sort of discuss what
00:22:58;04 the big questions are in the field, is I want to
00:23:02;04 summarize for you very clearly: aneuploidy, having the
00:23:06;16 wrong karyotype, is detrimental at the organismal
00:23:09;16 level. Bad news for any organism that has ever been
00:23:13;19 looked at. What's also very clear from these very
00:23:17;06 correlative studies is that chromosome losses are more
00:23:20;14 detrimental than chromosome gains. Now that we
00:23:23;14 understand the gene dosage is frequently very
00:23:26;20 important, that's probably very easy to understand.
00:23:29;24 Losing a copy of a chromosome is probably much more
00:23:33;05 detrimental than gaining a chromosome. And then what
00:23:38;01 is also generally true is that the severity of the
00:23:40;23 phenotypes usually correlates with the degree of
00:23:44;05 aneuploidy. What do I mean by this? Usually, the more
00:23:49;25 chromosomes are aneuploid, the larger the
00:23:52;13 chromosome that is aneuploid, the more detrimental
00:23:55;00 the phenotype. Let me give you an example. For
00:23:58;04 example, chromosome 21 is the most gene-poorest
00:24:04;22 chromosome in humans. Trisomy 21 is in fact viable. I
00:24:08;26 mean, these people have various symptoms, and they
00:24:11;22 have a reduced lifespan and so forth, but they actually
00:24:14;27 are very high-functioning individuals. On the other
00:24:18;15 hand, a trisomy of chromosome 1, the largest
00:24:21;07 chromosome in humans, has not even ever been
00:24:24;14 detected in spontaneous abortions, indicating that that
00:24:27;28 trisomy is very early embryonic lethal. Okay? So these
00:24:32;04 are the general points that, you know, 120 years of
00:24:41;09 studying aneuploids has led us to conclude. So what
00:24:45;16 are the big questions? What is it that we need to
00:24:49;04 understand to understand the impact of aneuploidy on
00:24:53;22 cellular and organismal physiology? A big question in
00:24:58;16 the field of aneuploidy is a very simple question. When
00:25:02;27 you are becoming aneuploid for an entire chromosome,
00:25:06;28 you're changing the gene dosage of not a few genes
00:25:10;06 but hundreds, if not thousands, of genes at the same
00:25:12;25 time. So a very basic question that we need to
00:25:16;07 understand is: The various phenotypes and traits that
00:25:20;13 we see associated with the particular chromosomal
00:25:23;05 aneuploidy, are they due to a single gene, or a small
00:25:29;03 group of genes, that have, when you change their
00:25:32;08 gene dosage, a very severe impact on a particular
00:25:36;00 pathway on the fitness of the cell of the organism? The
00:25:40;10 alternative hypothesis that, remind you, not mutually
00:25:43;12 exclusive, and I will actually argue at the end of the
00:25:46;07 second part of my talk that both are true, is that a
00:25:50;15 large change in the gene copy number of a large
00:25:52;28 number of genes, which one their own do not have a
00:25:57;00 particular strong effect on fitness or on the health of
00:26:01;27 organism, when you add them all up, they will actually
00:26:05;01 have a significant impact on physiology. So that is sort
00:26:09;15 of the cumulative gene dosage hypothesis. And as you
00:26:14;04 will see, there are examples for both of this. So again
00:26:17;29 I'm using human aneuploidies as a case study here. So
00:26:22;17 an example of a single gene effect is the APP
00:26:26;13 duplication. APP encodes the precursor of a protein
00:26:32;23 that forms plaques during Alzheimer's disease, makes
00:26:36;15 amyloid beta plaques. So it turns out that individuals
00:26:41;09 with Down syndrome all show early onset Alzheimer's
00:26:45;23 disease. By 40 years of age, virtually every single
00:26:49;22 individual with Down syndrome will have Alzheimer's
00:26:53;03 characteristics. And so what researchers believe is that
00:26:58;18 it's the duplication of the APP gene that will lead to this
00:27:03;29 early onset Alzheimer's disease. Are there potentially
00:27:08;16 indications that there is also evidence for this
00:27:12;01 cumulative gene dosage hypothesis? Yes there are.
00:27:16;07 Another example from Down syndrome that I'd like to
00:27:19;12 give here is the Down syndrome critical region. So ever
00:27:23;13 since the realization in 1959 that Down syndrome is due
00:27:28;13 to the duplication of an entire chromosome 21,
00:27:31;24 researchers have tirelessly tried to find the genes that
00:27:36;28 cause Down syndrome, the phenotypes associated
00:27:39;25 with this disease, very characteristic craniofacial
00:27:43;03 features, heart diseases, mental retardation, learning
00:27:46;21 disabilities, and so forth. And over the years they've
00:27:49;27 tried to map it down to smaller and smaller regions, and
00:27:53;17 they are, what they call, in the hunt for the Down
00:27:57;26 syndrome "critical region," the piece of chromosome 21
00:28:00;22 that gives rise to all these phenotypes. And it's quite
00:28:03;24 clear that, while they have found a particular region,
00:28:07;14 "Down syndrome critical region 1," that is responsible
00:28:11;10 for some of the phenotypes, by no means can all
00:28:20;06 phenotypes be ascribed to that particular region. And
00:28:24;07 another example I want to give here that is evidence
00:28:27;21 for a multi-gene defect, as I mentioned it earlier, it is
00:28:31;08 quite clear that embryonic lethality of trisomies in
00:28:35;07 humans correlates with the gene density of that
00:28:38;29 particular chromosome. So I'm showing you here very
00:28:42;06 nice correlation between chromosome size and survival.
00:28:53;04 And as you know, in humans chromosomes are
00:28:58;00 numbered by size, so chromosome 1 is the largest
00:29:00;08 chromosome. And as you can very clearly see, there's
00:29:02;24 actually three (humans are kind of unusual in that)
00:29:06;00 trisomies that survive to birth in humans. This is trisomy
00:29:09;26 13, Patau syndrome; trisomy 18, Edwards syndrome;
00:29:14;15 and trisomy 21, Down syndrome. And as you can
00:29:16;26 clearly see from this correlation here, those are the
00:29:20;24 three, excuse me, chromosomes that have the fewest
00:29:27;07 number of genes located on them. And so these types
00:29:30;26 of correlations that we not only see them in humans,
00:29:33;12 we actually see them in all organisms, also raise this
00:29:37;14 very interesting possibility that not only are there
00:29:42;04 additive, cumulative effects, but that there perhaps is
00:29:44;28 actually sort of a general response to the aneuploid
00:29:47;20 state. And in the second part of my talk, I will actually
00:29:50;26 argue this is probably the case, okay? And I will explain
00:29:55;00 to why we believe there is sort of a generic response
00:29:59;04 to the aneuploid state and what its origins are. So the
00:30:04;09 last, and to us most motivating, question in the
00:30:08;12 aneuploidy field is the cancer conundrum, I call it the
00:30:12;13 cancer conundrum. So I've just spent the last 25
00:30:16;28 minutes or so telling you how really bad it is to be
00:30:22;28 aneuploid, okay? And a single extra chromosome kills
00:30:26;26 an entire organism, it's lethal, okay? But now here, I'm
00:30:31;19 showing you an example of a karyotype, an aneuploid
00:30:34;18 karyotype, that is seen in cancer cells. In this particular
00:30:37;27 case, many chromosomes are duplicated, many
00:30:42;03 chromosomes are lost. These are highly, highly
00:30:45;25 aneuploid cells. So this observation now raises this very
00:30:50;10 interesting question to us. How is this possible? A single
00:30:54;13 extra chromosome kills an organism, but at the cellular
00:30:58;03 level, apparently it doesn't seem to matter how many
00:31:01;12 chromosomes you have. It actually even looks the
00:31:04;15 more the better, right? Clearly, cancer is a disease
00:31:07;21 that's characterized by unrestricted growth. These cells
00:31:10;27 grow terribly well, okay? So one argument you can
00:31:16;04 make is, well, it's difficult to make a heart or a brain,
00:31:23;05 and so if you have imbalances in the gene dosage of a
00:31:26;29 small number of genes, right, that leads to
00:31:30;05 malformation of the brain and malformation of the
00:31:32;21 heart, and that leads to the death of the organism. But
00:31:36;09 maybe at the cellular level, in the context of cancer,
00:31:41;03 having many extra chromosomes doesn't matter, or
00:31:44;02 perhaps at the cellular level, having many more
00:31:46;15 chromosomes is actually a good thing. It allows you to
00:31:48;22 proliferate better. We as yeast geneticists, who study
00:31:52;21 single-cell organisms for a living, didn't think so, but of
00:31:56;06 course it was a specific possibility. And so because of
00:32:00;24 this significant discrepancy of what's observed at the
00:32:03;27 organismal level and at the cellular level in context of
00:32:06;23 cancer, what we wanted to do was we wanted to ask a
00:32:10;09 very simple question. What we wanted to know was
00:32:13;02 sort of, what are the consequences of aneuploidy on
00:32:16;27 normal cell physiology? What happens to a normal cell
00:32:21;23 when you change its karyotype. And in the second part
00:32:24;27 of my talk, I will tell you about the research in our lab
00:32:28;13 that is aimed at addressing this question. Thank you
00:32:31;21 very much for listening, I'll see you.
00:32:35;16

Part 2: Effects of Aneuploidy on Cell Physiology

00:00:00;23 Hello, my name is Angelika Amon, I'm a professor in the
00:00:04;13 Biology Department at MIT, and I'm also an
00:00:08;19 Investigator of the Howard Hughes Medical Institute.
00:00:11;24 In the second part of my talk, I will tell you about our
00:00:14;24 work in trying to understand what the consequences of
00:00:18;22 aneuploidy are on organismal and cell physiology. In
00:00:23;12 the first part of that talk, I defined the terms for you, I
00:00:30;02 gave you a historical overview of the study of
00:00:33;07 aneuploidy, and I ended with what the big questions
00:00:38;00 are in the field, especially how aneuploidy relates to
00:00:43;02 cancer. And so this part now, having set up this
00:00:47;16 question how can it be that aneuploidy at the
00:00:51;20 organismal level is highly detrimental, but at the cellular
00:00:55;10 level it is also associated with a disease that's
00:00:58;26 characterized by unrestricted growth, cancer, how we
00:01:03;08 can merge these two questions. I will start with the
00:01:07;00 research question that we started about 10 years ago,
00:01:11;17 asking, well, what are the consequences of aneuploidy
00:01:15;06 on cell physiology. So this is basically the second part
00:01:20;25 of this three-part series. How can we understand how
00:01:25;21 aneuploidy impacts cellular fitness? And again, like the
00:01:30;11 first part of my talk, I subdivided this research
00:01:34;11 presentation into three parts. I will first explain to you
00:01:40;29 what kind of model systems and cellular systems we've
00:01:43;23 established and developed over the years to study the
00:01:46;26 effect of aneuploidy on cell physiology. I will then tell
00:01:51;04 you that basically aneuploidy causes two sets of broad
00:01:56;06 phenotypes. One which I will call a gene-specific
00:01:59;28 phenotype, where changing the dosage of one
00:02:03;19 particular gene will cause a particular phenotype, and I
00:02:07;03 will give you examples for this. And then the main part
00:02:10;28 of my talk will deal with this more surprising discovery
00:02:14;26 that we made in the lab, that sort of suggests that in
00:02:18;04 addition to these gene-specific effects, there are
00:02:21;01 general effects of aneuploidy, where changing the
00:02:24;12 dosage of many genes simultaneously will actually
00:02:28;07 impose a very significant burden on various cellular
00:02:34;12 housekeeping functions, and therefore will lead to what
00:02:37;28 we call an aneuploidy stress response. So let me start
00:02:42;13 out by telling you about the model systems that we
00:02:46;17 have developed over the years to study the effects of
00:02:50;11 aneuploidy. So we have generally used two broad
00:02:55;10 systems, one we define as the low-complexity
00:02:59;00 aneuploidies, and what we mean by that is these are
00:03:02;15 usually cells, yeast cells or mammalian cells, that carry
00:03:06;19 one or two additional chromosomes. In our study of
00:03:11;01 aneuploidy, we initially started with budding yeast, and
00:03:16;08 we created yeast strains that carry one particular
00:03:21;15 chromosome in two copies instead of one copy, and we
00:03:25;07 created a series of 20 different strains that carry 20
00:03:28;27 different additional chromosomes. So this slide here
00:03:32;19 shows you how we know that this particular yeast
00:03:37;20 strain has an additional copy of a particular
00:03:40;00 chromosome. So what you see here, each box
00:03:43;23 presents a particular karyotype of a particular yeast
00:03:48;24 strain. And what we're showing here is we're showing
00:03:51;10 the DNA content of each of the chromosomes, and
00:03:54;24 we're ordering the chromosomes according to their
00:03:58;05 numbers. The left arm of chromosome 1 is at the left
00:04:01;12 end of the box, the right arm of the chromosome 16
00:04:05;07 (budding yeast has 16 chromosomes) is the right end of
00:04:08;07 the box. And you see there's the same amount of DNA
00:04:11;23 for all of them, except for this particular stretch here
00:04:15;13 shown in red. This, turns out, is present in two copies
00:04:20;03 instead of one. And as you can see here, this
00:04:22;16 corresponds to the entire chromosome 8. And so we
00:04:27;26 now know from this DNA content analysis that this
00:04:31;21 particular yeast strain has two copies of chromosome
00:04:35;13 8. And as you can see here, we generated many
00:04:39;02 different strains with all sorts of different aneuploidies,
00:04:44;08 and we began to study them. After we began to study
00:04:49;07 the aneuploidies in budding yeast, and we actually
00:04:54;22 identified a number of phenotypes that were shared
00:04:58;09 among all these different budding yeast strains, we
00:05:01;14 then were also curious to ask, these shared
00:05:03;12 phenotypes that we see in yeast, do they exist also in
00:05:07;13 other systems? And we were especially interested in
00:05:09;27 whether they exist in mammalian cells. So we used
00:05:12;29 some old genetic tricks to generate mouse embryos
00:05:18;09 that instead of being euploid, they had one particular
00:05:21;26 chromosome extra. So in the mouse, unlike in humans,
00:05:25;18 all trisomies are lethal, but we can isolate embryos from
00:05:31;01 pregnant mothers that have a particular trisomy. So
00:05:34;09 initially we made four of them: trisomy 1, trisomy 13,
00:05:39;17 trisomy 19, and trisomy 16. So we isolate these
00:05:44;17 embryos, we then dissociate these embryos, make cell
00:05:47;28 lines from them, we're making mouse embryonic
00:05:50;05 fibroblasts from them, and then we can study them. So
00:05:53;27 these are the two systems that we use to study what
00:05:57;08 we call "constitutional, low-grade aneuploidies." We
00:06:01;19 also have a way of generating more complex and
00:06:05;14 heterogeneous aneuploidies. And here we take
00:06:08;16 advantage of mutations that increase the frequency
00:06:14;05 with which chromosomes are missegregated. For
00:06:17;21 example, in budding yeast, we can use mutations that
00:06:23;13 induce chromosome nondisjunction, or chromosome
00:06:27;02 missegregation, both during mitosis and meiosis. We
00:06:30;24 can also make triploid yeast strains by various tricks,
00:06:35;17 sporulate them, and the progeny of these triplet
00:06:39;10 meioses are highly aneuploid yeast strains. And as I
00:06:43;16 said in both the mouse and humans, we just use
00:06:46;07 mutation. Down below here you see an example of
00:06:49;07 what we, for example, do in mammalian cells. So this is
00:06:52;12 a mammalian cell that's about to undergo chromosome
00:06:54;28 segregation, and we actually treat it with a drug that
00:06:59;04 inhibits a protein that's critically important for faithful
00:07:03;10 chromosome segregation. So we give the cells the
00:07:06;01 drug, and as a result, this protein is inactive. And now,
00:07:10;18 the cells will missegregate its chromosomes and will
00:07:13;12 result in aneuploid cells. So, the advantage of these
00:07:21;03 sort of random, high-complexity aneuploidies that we
00:07:25;17 induce with various mutations or chemicals, is that
00:07:29;21 these chromosomal aneuploidies are very similar to the
00:07:33;18 kinds of aneuploidies that we see in cancer. And from
00:07:36;27 that perspective, they're very interesting. However,
00:07:40;08 these types of aneuploidies are very unstable, they're
00:07:42;23 a moving target, so they're very difficult to study. So
00:07:46;27 what we usually do is we usually conduct our initial
00:07:51;06 studies and the discovery process in the low-complexity
00:07:55;29 aneuploidies that I told you about before, these
00:07:59;01 disomic yeast strains and these trisomic mouse
00:08:02;06 embryonic fibroblasts. And when we discover a
00:08:05;03 phenotype that we're particularly interested in, where
00:08:07;22 we want to know whether there are general
00:08:08;17 phenotypes, we then ask, do these phenotypes that
00:08:14;05 we see in these low-grade aneuploidies also occur in
00:08:18;14 these various aneuploidies when we induce massive
00:08:23;04 chromosome missegregation? And to our great delight
00:08:26;10 and satisfaction, so far this has always been the case.
00:08:30;13 If we discover a general phenotype among the various
00:08:34;14 low-grade, constitutional aneuploids (the disomic yeast
00:08:37;13 strains, the trisomic MEFs), we also usually see them in
00:08:41;22 these high-grade, random aneuploidies. So, having
00:08:46;15 introduced to you the model system that we use to
00:08:50;08 study the effects of aneuploidy on cells, let me now
00:08:54;22 move on and sort of very briefly highlight for you the
00:08:58;06 sort of gene-specific effects. The fact that aneuploidy
00:09:03;10 duplication of particular genes can lead to a particular
00:09:06;12 phenotype has been known for a very long time. And
00:09:09;29 I'm showing you here one example from the work of
00:09:14;14 Judith Berman's lab, where she was actually studying a
00:09:19;09 pathogenic fungus called Candida albicans. Candida
00:09:23;13 albicans is a fungus that can become pathogenic to
00:09:27;05 humans, especially immunocompromised humans. And
00:09:31;08 so if you are immunocompromised and you become
00:09:35;12 afflicted with this yeast infection, what doctors usually
00:09:40;22 do is they start giving you an antifungal drug called
00:09:44;00 fluconazole. Unfortunately, when you are treated for
00:09:47;19 long periods of time with that drug, the fungus
00:09:51;09 eventually develops resistance, so it will no longer be
00:09:55;29 responsive to the drug. And Judith Berman's lab
00:10:00;08 showed here is that when you duplicate a part of
00:10:03;24 chromosome 5, that can lead to fluconazole resistance.
00:10:10;20 And she actually then figured out which gene in that
00:10:13;11 particular region was responsible, so a specific gene on
00:10:16;14 this part of chromosome 5 can provide a new trait, it
00:10:21;04 can provide resistance to a particular drug. Down here
00:10:25;10 you see an example from our lab, where we asked,
00:10:29;14 being disomic for a particular chromosome, can that
00:10:34;18 confer a new trait? What we're looking at here is we're
00:10:38;24 looking at benomyl resistance. Benomyl is actually a
00:10:42;11 microtubule depolymerizing drug, it sort of
00:10:45;11 depolymerizes microtubules, it kills all cells basically.
00:10:50;01 And what Eduardo Torres, a postdoctoral fellow in the
00:10:53;06 lab, observed: that this particular strain here, you can
00:10:55;13 see this is a yeast strain that has an extra copy of
00:10:59;13 chromosome 16, it is actually able to grow much better
00:11:03;24 in the presence of the drug, than the wild-type euploid
00:11:08;06 cells. So it acquired the ability to grow better in the
00:11:12;13 presence of high concentrations of benomyl. So it is
00:11:17;00 quite clear that gene-specific effects exist, that
00:11:21;26 duplicating particular genes on a particular chromosome
00:11:25;24 can confer either a drug resistance or some other new
00:11:30;17 trait. So, there's many more examples for this, I
00:11:34;04 actually mentioned one in the first part of my talk: APP
00:11:37;14 duplication in early onset Alzheimer's and Down
00:11:40;09 syndrome; and this list goes on and on and on. What to
00:11:43;25 us was much more interesting is that our basic studies
00:11:47;26 of these aneuploid yeast strains revealed that, in
00:11:50;26 addition to these gene-specific effects, there were
00:11:53;20 very general effects. And those are sort of listed here.
00:11:57;15 What we find is that aneuploidy causes proteotoxic
00:12:00;05 stress. Now I'm going to tell you in a minute what this is
00:12:03;08 all about, and what proteotoxic stress means. We
00:12:06;02 found that aneuploidy causes a transcriptional
00:12:08;12 response. And then aneuploidy also causes a delay in
00:12:12;16 G1. And so we basically, since these are all signs of a
00:12:16;25 stress response -- you will see very quickly where
00:12:19;22 these stresses are coming from, I'm going to explain
00:12:21;24 this to you -- we sort of generally refer to this as the
00:12:25;05 "aneuploidy stress response." Okay? So what I'm going
00:12:29;06 go do next is I'm actually going to show you the data
00:12:32;25 that led us to the conclusion that aneuploidy causes
00:12:35;24 proteotoxic stress, and I'm going to show you the data
00:12:38;13 that led us to the conclusion that there's a stress
00:12:40;18 response. I'm not going to show you the data for that,
00:12:44;01 I'm just going to mention to you that in addition to all
00:12:46;20 these other phenotypes, cell proliferation is impaired in
00:12:50;04 these aneuploid cells, it causes a G1 delay. So, let me
00:12:55;05 tell you how we initially developed the hypothesis that
00:13:00;28 budding yeast strains are under proteotoxic stress. So
00:13:04;12 what is proteotoxic stress? Proteotoxic stress is a
00:13:09;05 condition where, when the proteins in the cell are
00:13:12;16 misfolded or they're not working right, that's obviously
00:13:16;03 bad news for the cells. And the cells try to fix it. And
00:13:19;25 usually what it does is it sort of activates chaperones
00:13:23;09 and ubiquitin-dependent protein degradation to sort of
00:13:27;09 eliminate these unfolded and malfunctioning proteins,
00:13:31;07 and sort of making new, better ones, okay? And so we
00:13:35;05 initially realized that there perhaps was proteotoxic
00:13:38;05 stress in these aneuploid cells because compromising
00:13:41;24 proteasome function, either by chemical or genetic
00:13:45;14 means, was more detrimental in the aneuploid cells
00:13:49;12 than in the euploid cells, so lowering the proteasome
00:13:52;23 function really affected these aneuploid cells in a
00:13:56;00 substantial manner. And the other thing that we
00:13:58;26 realized early on was that the aneuploid yeast strains,
00:14:02;00 all of them, were sensitive to a protein synthesis
00:14:05;23 inhibitor called cycloheximide, and they were also
00:14:08;20 sensitive to temperature, high temperature. High
00:14:10;17 temperature is known to unfold proteins, causes a lot
00:14:15;13 of stress for the cells, and so raising the temperature in
00:14:18;18 these particular disomic strains had actually quite
00:14:21;24 detrimental effects, okay? And so these types of
00:14:26;19 results led us to hypothesize that the protein quality
00:14:30;26 control systems of the cells are either not functioning
00:14:34;07 right, or they are overburdened, okay, and therefore
00:14:37;26 can't catch up with proteotoxic stress. So to address
00:14:42;13 this question in more detail, we actually wanted to look
00:14:46;14 at markers for proteotoxicity, and I'm showing you one
00:14:49;05 marker here. We're looking here at a chaperone, a
00:14:53;17 disaggregase known as Hsp104, and it's fused to GFP,
00:14:59;25 and normally, this protein is sort of diffusely present
00:15:03;28 throughout the yeast cells. But when aggregates are
00:15:06;28 forming in these yeast cells, the disaggregase, the
00:15:09;19 chaperone, will go to these places and you will start
00:15:13;17 seeing these foci accumulating in yeast strains. And
00:15:17;06 here's an extreme case where the cell is actually heat-
00:15:19;27 shocked at 37°, which causes a lot the proteins to
00:15:24;18 unfold and aggregate, and see, now these cells have a
00:15:27;23 large number of these Hsp104 foci. And what we found
00:15:33;09 that was very exciting is that every single disomic
00:15:36;14 yeast strain (so here's a wild type, this is the
00:15:39;03 percentage of normal wild-type cells that harbor
00:15:42;22 Hsp104 aggregates... and here are all the disomic
00:15:45;16 yeast strains), and every single one of them has a
00:15:48;24 higher percentage of cells that harbor these Hsp104
00:15:53;09 aggregates, indicating that endogenous proteins in
00:15:57;12 these disomic yeast strains tend to aggregate. We also
00:16:01;29 looked at another marker for proteotoxic stress, this is
00:16:06;17 a reference protein called VHL. VHL is actually a human
00:16:13;09 protein that, when you express it in yeast, it doesn't
00:16:16;22 fold right, and it's actually very quickly degraded by the
00:16:20;18 ubiquitin-proteasome system. But if the ubiquitin-
00:16:23;17 proteasome system and some other chaperones are
00:16:26;28 either not working right or overburdened, what will
00:16:30;01 happen is that VHL will start accumulating in the cells,
00:16:33;09 and again it will start forming aggregates. And so again
00:16:36;23 you can see, whereas wild type only a small
00:16:39;25 percentage of cells has VHL aggregates, you can see
00:16:43;15 that all the disomes have much higher levels of
00:16:48;00 aggregates, indicating that indeed the protein quality
00:16:50;19 control systems in the cells are compromised. This is not
00:16:55;12 just a unique feature of these constitutional, low-grade
00:16:59;26 aneuploidies, these disomic yeast strains that we
00:17:03;01 constructed. We also see protein aggregates in these
00:17:07;13 random, high-grade aneuploids that we create when
00:17:12;09 we make progeny of a triploid strain, that will create
00:17:17;10 highly aneuploid progeny. So we're looking at Hsp104
00:17:21;14 here. This is sort of the distribution that you get in
00:17:24;22 euploid cells. Upon sporulation, this is the distribution
00:17:28;25 that you get in these highly aneuploid cells, and the
00:17:32;23 same is true for VHL. And then finally we're very
00:17:36;08 interested in addressing the question of how quickly do
00:17:40;03 these protein aggregates start accumulating upon
00:17:43;25 chromosome missegregation, upon generating
00:17:46;08 aneuploidy. And so to address this question, we took
00:17:49;28 advantage of two mutants that missegregate
00:17:53;12 chromosomes at a high frequency. For the purpose of
00:17:56;25 my talk, it doesn't really matter what ndc10 is and what
00:18:00;14 ipl1 is. The only thing you need to know is that these
00:18:04;00 are temperature-sensitive mutants, and when these
00:18:06;20 strains are grown at the intermediate temperature,
00:18:09;20 they will missegregate chromosomes at a very high
00:18:12;11 frequency. So we take these cells, we arrest them in
00:18:16;00 G1, and then we'll send them through the cell cycle,
00:18:20;07 and then ask two hours later, after they've
00:18:23;27 missegregated their chromosomes, have they started
00:18:26;24 to accumulate these aggregates? And the answer here
00:18:30;16 is very clearly yes, it's elevated in ndc10 mutants, it's
00:18:34;11 elevated in ipl1 mutants, compared to the euploid
00:18:37;16 control. The gray bars here are an important control.
00:18:42;01 What we did here is we actually prevented these cells
00:18:46;02 from undergoing mitosis, so we prevented these cells
00:18:48;23 from missegregating their chromosomes by basically
00:18:51;25 depolymerizing their microtubule cytoskeleton. So even
00:18:55;03 though they want to segregate, they can't because
00:18:57;00 they don't have any microtubules. And you can see
00:18:59;18 under those conditions, we do not see an increase in
00:19:02;25 aggregate formation. So from these experiments we
00:19:06;09 concluded that aneuploidy causes proteotoxic stress, it
00:19:14;20 causes protein aggregation, and that occurs very
00:19:18;06 quickly, very soon after chromosome missegregation.
00:19:23;00 We then also asked: Hsp104, this disaggregase that's
00:19:28;01 very important for cells, does the amount of
00:19:30;29 disaggregase that you make, is that actually correlated
00:19:34;19 with the degree of aneuploidy that occurs in the cells?
00:19:39;06 And so what I'm showing you here is I'm showing you
00:19:41;16 the amount of expression of Hsp104 on the y-axis,
00:19:45;03 with respect to what the percentage of aneuploidy in a
00:19:49;20 particular strain. And as you can see, this actually
00:19:53;05 tracks very nicely, again indicating that there is a very
00:19:56;19 general response to the aneuploid state that's
00:20:00;23 proportional to the amount of disaggregating protein.
00:20:06;12 So in yeast cells this suggests very clearly that there's
00:20:10;01 proteotoxic stress in these strains. Do we have
00:20:12;22 evidence that this proteotoxic stress also exists in
00:20:15;25 mammalian cells? The answer is yes here. I'm not going
00:20:19;17 to show you the data, but instead I'm just going to
00:20:22;00 summarize the data for you. Here, I'm showing you
00:20:25;25 that aneuploid mouse cells are sensitive to drugs that
00:20:30;26 cause proteotoxic stress, they're sensitive to this drug
00:20:34;19 called autophagy inhibitor chloroquine. We actually find
00:20:38;19 that the basal levels of autophagy (that's a protein
00:20:42;03 quality control pathway that clears protein aggregates
00:20:44;17 from cells) are increased in trisomic mouse embryonic
00:20:48;12 fibroblasts. When we inhibit chaperones by drugs, in
00:20:52;13 this case 17-AAG inhibits the chaperone Hsp90, again
00:20:56;28 then trisomic mouse embryonic fibroblasts are more
00:21:00;28 sensitive to this than euploid cells. And then very
00:21:05;04 interestingly, the downregulation of a subset of
00:21:08;18 protein-folding factors is actually delayed upon
00:21:12;10 transient heat shock, indicating that the protein quality
00:21:15;15 control pathways in these cells are either not working
00:21:19;27 right or they're again overloaded. There's one piece of
00:21:22;27 data that I want to show you that, like in budding
00:21:25;27 yeast, this proteotoxic stress is seen very quickly upon
00:21:32;00 the creation of aneuploid cells. This is the system that I
00:21:36;06 showed you before, where we take a dividing
00:21:39;03 mammalian cell, we treat it with a drug that induces
00:21:43;27 chromosome missegregation, that leads to the
00:21:47;28 formation of aneuploid cells. And here on the bottom,
00:21:51;29 we're looking at a marker for autophagy. I told you
00:21:55;02 autophagy is this protein elimination system that gets
00:21:58;13 rid of aggregated proteins and misfolded proteins. And
00:22:01;21 as you can see, in the drug-treated cells shown in red,
00:22:05;14 these autophagy markers are dramatically increased,
00:22:09;03 compared to in green, the control cells. So a key
00:22:14;08 question that arises from these findings is, where is this
00:22:18;12 proteotoxic stress coming from? Obviously, a very
00:22:22;12 simple hypothesis is there are additional chromosomes
00:22:26;25 in these cells, these chromosomes make RNAs and
00:22:31;12 proteins, and perhaps these proteins then change the
00:22:34;26 proteome in the cell, and therefore lead to changes in
00:22:38;06 protein homeostasis and increased aggregation. That's
00:22:42;07 clearly the simplest hypothesis, and the hypothesis
00:22:44;20 that we favored. However, before we test this
00:22:48;10 hypothesis, we actually needed to ask a much more
00:22:51;04 basic question, which is: Are these additional
00:22:56;14 chromosomes actually active? Do they actually make
00:22:59;11 RNAs and proteins? And this experiment here shows
00:23:02;29 you that this is indeed the case. I'm showing you here
00:23:05;20 a strain again, I'm showing you the DNA content, the
00:23:08;12 RNA content, and the protein content, of a yeast
00:23:12;23 strain that carries an extra copy of chromosome 5.
00:23:16;02 Remember, this is sort of the stretch of the DNA that's
00:23:18;21 present in two copies. And you can see very clearly,
00:23:21;16 there's a transcriptional response. You see there's
00:23:24;29 much more biological noise here that we actually
00:23:27;01 understand where this is coming from, but what I want
00:23:30;01 you to draw from this picture here is that, generally
00:23:32;22 speaking, the amount of RNA is made according to
00:23:36;10 gene copy number. And about 80% of the proteins are
00:23:40;17 also produced according to gene copy number. So we
00:23:44;15 now understand that these additional chromosomes are
00:23:47;29 indeed active. So now can we acquire evidence to
00:23:54;07 indicate that it is actually the fact that these additional
00:23:58;00 chromosomes are active that caused the problems?
00:24:00;26 And I'm not showing you the data here, I'm just
00:24:03;00 summarizing the pieces of data here that are actually
00:24:05;27 both the most telling (we've done many more
00:24:07;29 experiments, but these I think are the most critical
00:24:10;06 data). First of all, in yeast, we have the ability of
00:24:14;21 introducing human DNA or mouse DNA into these cells,
00:24:19;20 and because the splicing machinery is so fundamentally
00:24:22;03 different between yeast and mouse (and humans),
00:24:25;19 these pieces of DNA, they're called "yeast artificial
00:24:28;08 chromosomes," will make few if any gene products. And
00:24:32;06 so one can now ask... we can stick a chromosome's
00:24:35;05 worth of human or mouse DNA into these yeast cells,
00:24:38;14 and ask, well, what's the phenotype of that? And the
00:24:41;11 fact is that all these phenotypes that we see in the
00:24:44;14 aneuploid strains that have extra yeast DNA are not
00:24:48;01 seen in the strains that have extra mouse and extra
00:24:52;05 human DNA. The perhaps most telling experiment that
00:24:56;08 we did that it's actually the fact that these additional
00:25:01;15 chromosomes are active is what's critical here, is the
00:25:04;24 observation that ploidy "buffers." What do I mean by
00:25:10;05 that? What we routinely observe is that a haploid strain
00:25:14;28 that carries an extra copy of a chromosome has much
00:25:18;07 more severe phenotypes than a diploid strain that
00:25:21;29 carries an extra copy of a particular chromosome.
00:25:25;13 What that result actually tells you is that what the cells
00:25:28;27 care about here is relative ratios. That is, if you
00:25:34;10 double, in the case of a haploid, the gene products by
00:25:38;00 adding an extra copy, that's significantly more worse
00:25:41;03 than if you just add an extra third, in the case of a
00:25:44;04 diploid, where you add an extra copy. Okay? So from
00:25:49;11 these, and many more, experiments that I don't have
00:25:51;24 time to tell you about is, this is our current working
00:25:55;03 hypothesis. We believe that aneuploidy leads to excess
00:25:59;17 protein production. And what we propose is that this
00:26:03;11 causes, of course among other deleterious outcomes,
00:26:07;10 proteotoxic stress, because overproduction of certain
00:26:10;28 proteins saturates the protein quality control pathways
00:26:14;26 of the cell. And so the question is, how can you
00:26:18;26 saturate the protein quality control pathway of the
00:26:22;04 cells? There's several possibilities. Of course, there's
00:26:25;18 some proteins that are now made in excess in these
00:26:29;14 aneuploid cells because you doubled their gene copy
00:26:32;08 number, that are obligatory chaperone clients, or that
00:26:35;29 obligatorily require some protein quality control for their
00:26:39;15 function: protein kinases, WD40-repeat proteins,
00:26:43;01 tubulin, actin, and so forth, many proteins. So that
00:26:46;17 could cause an additional burden on the protein quality
00:26:49;06 control pathways of the cell. And secondly, what we
00:26:53;04 really believe is at the heart of the aneuploidy problem
00:26:57;13 is protein stoichiometry imbalances. And I want to
00:27:01;00 illustrate this here with this particular slide. I want you
00:27:04;09 to visualize a protein complex that's made out of
00:27:08;01 protein A and made out of protein B, and they need to
00:27:12;05 function together. So generally speaking, it's very
00:27:15;28 difficult for a cell make exactly the same amount of
00:27:19;12 protein A and protein B. And so the way the cell solves
00:27:23;04 this problem is, they make approximately the same
00:27:26;06 amount of each, and then they neutralize excess
00:27:30;10 subunits, for example "A" here, by using protein quality
00:27:34;22 control pathways and sometimes feedback
00:27:37;13 mechanisms. And there's wonderful examples for this in
00:27:40;10 the literature: α- and β-tubulin, ribosomal subunits,
00:27:43;25 histones, and so forth. Now imagine that you're
00:27:47;14 doubling the gene dosage of hundreds if not thousands
00:27:52;00 of proteins at the same time, by introducing an extra
00:27:55;21 copy of a chromosome. All of a sudden, you do not
00:27:59;21 have 5% excess of protein A, you have a 110%
00:28:05;11 excess of protein A. And what we propose is that that
00:28:09;17 causes an increased burden on the protein quality
00:28:12;17 control pathways of the cells, and it also leads to other
00:28:16;02 phenotypes that I don't have time to tell you about. It
00:28:18;24 is there's clearly energy stress, and there's also cell
00:28:21;24 proliferation defects. And we speculate that these
00:28:24;29 phenotypes are also caused by protein stoichiometry
00:28:28;00 imbalances. Where we're going with this project right
00:28:31;25 now, clearly we are very much interested in
00:28:34;19 understanding how the protein quality control systems
00:28:39;01 are impacted, so we're now probing the activities of
00:28:43;00 individual protein quality control systems in these
00:28:46;11 various aneuploid strains. So I'm showing you one
00:28:49;23 example here. We're looking at one chaperone, it's
00:28:53;13 called Hsp90, and what I'm showing you here's a
00:28:56;19 biological assay for Hsp90 activity. Doesn't really
00:29:00;19 matter how the assay works, but if there's a lot of
00:29:03;15 bands here on this gel, that means that the chaperone
00:29:07;14 is working well, okay? And I hope you can appreciate
00:29:10;27 that there's many disomic yeast strains in which the
00:29:15;06 Hsp90 chaperone is actually not working very well, so
00:29:19;05 indicating that indeed in some of these aneuploid cells,
00:29:23;02 there's a profound impact on at least one chaperone
00:29:28;16 system. We're now going about trying to understand
00:29:32;14 what the other ones are. So this is what I wanted to
00:29:36;03 tell you, protein quality control. The second general
00:29:39;21 phenotype that I wanted to briefly discuss is the
00:29:43;20 realization that there is a transcriptional response to
00:29:47;01 the aneuploid state. And we initially discovered this by
00:29:51;24 simply querying the RNA gene expression profile of all
00:29:57;13 the disomic yeast strains that we initially created. So
00:30:03;13 what I'm showing you here is I'm showing you here all
00:30:06;28 the disomic yeast strains that are sort of listed here.
00:30:09;25 Each column represents a yeast strain. Each row
00:30:14;02 represents a gene. And you can see here red- and
00:30:18;08 green-colored images here. Red means the gene is
00:30:24;21 downregulated, green means it's upregulated. And you
00:30:28;24 can see here very clearly all these disomic yeast
00:30:32;05 strains, there's a cluster of genes that's
00:30:35;00 downregulated, these are genes associated with growth
00:30:38;14 (ribosome biogenesis and RNA metabolism). And that
00:30:42;06 there are some genes that are upregulated, and they
00:30:45;12 all fall into the category of stress. This signature has
00:30:49;21 been observed before by David Botstein and
00:30:52;02 colleagues, and they call it the "environmental stress
00:30:55;02 response" in yeast, the ESR. And so, a graduate
00:30:59;14 student in the lab, Jason Sheltzer, was very much
00:31:02;08 interested in asking whether this environmental stress
00:31:06;00 response in also seen not just in these disomic yeast
00:31:10;03 strains that we created, but also seen in all sorts of
00:31:13;28 other aneuploid organisms. And the first thing he did is
00:31:18;27 he asked... There's a collection of yeast strains called
00:31:21;24 the Yeast Knockout Collection, and for reasons that are
00:31:24;29 not important here, there's among these actually a
00:31:28;05 large number of strains that are aneuploid, naturally
00:31:30;13 occurring aneuploidies. And he asked, is this ESR also
00:31:36;05 seen in this collection of aneuploid yeast strains made
00:31:40;13 while the Yeast Knockout Collection was made? And
00:31:42;26 more importantly, does the strength of this
00:31:46;10 transcriptional response correlate with the amount or
00:31:50;01 the degree of aneuploidy? And that's shown here.
00:31:52;18 We're showing here the percent of aneuploid, meaning
00:31:55;13 the percent of excess gene products present in the
00:31:58;16 cells, we can calculate this in yeast. And here we're at
00:32:01;26 the intensity of the stress response, and you can very
00:32:05;04 clearly see that there's a striking correlation. What was
00:32:08;17 very exciting to us, he not only saw this in budding
00:32:11;18 yeast, he saw also say that in fission yeast, another
00:32:15;09 related yeast. And he also saw that in plants, indicating
00:32:20;08 that at least among fungi and plants, there's a general
00:32:23;16 transcriptional response. He was also curious to see
00:32:27;05 whether the transcriptional signature for aneuploidy
00:32:32;13 was also observed in mammalian cells. And what he did
00:32:35;25 here is he used data collected by others that were
00:32:40;04 basically gene expression arrays of various trisomies in
00:32:43;12 both mouse and humans, and the question that he
00:32:46;20 asked here is, if we look at the percent of genes that
00:32:52;21 are upregulated for example here in trisomy 21, what's
00:32:56;28 the likelihood that these same genes are upregulated in
00:33:01;02 other trisomies, too, in this case trisomy 13, 16, and
00:33:04;29 19, for example? And you can see very clearly that
00:33:08;21 again there's a correlation. If something is upregulated
00:33:11;20 in one trisomy, the likelihood of this gene being
00:33:15;01 upregulated in other trisomies is very high. And these
00:33:18;24 are all the various data here for trisomies in the mouse,
00:33:23;00 and down below here for trisomies in humans. And the
00:33:29;01 question is what kind of genes are upregulated and
00:33:31;28 downregulated in these mammalian cells? And to our
00:33:35;23 great satisfaction, we saw that the same types of
00:33:40;06 categories of genes are upregulated and
00:33:42;18 downregulated as in fungi and in plants. So for example,
00:33:50;12 you see that upregulated are genes that are involved
00:33:54;15 in stress response and inflammatory response. And
00:33:57;10 genes that are downregulated are genes involved in
00:34:00;19 cell proliferation and cell growth, DNA replication, and
00:34:04;06 cell division. So these results to us suggested that
00:34:10;03 aneuploidy is also detrimental at the cellular level. I
00:34:15;24 gave you examples for gene-specific effects that
00:34:19;01 undoubtedly existed, and then I summarized for you
00:34:22;02 the evidence that led us to conclude that, in addition to
00:34:26;18 these gene-specific effects, aneuploidy causes a set of
00:34:30;06 phenotypes that's independent of the identity of the
00:34:33;29 aneuploidy, and is indicative of cellular stress. And we
00:34:38;09 generically call this of phenotypes that are shared
00:34:41;29 among many different aneuploid cells, the "aneuploidy
00:34:44;26 stress response." And with that, I would like to end this
00:34:48;13 part of my presentation, giving you some insights into
00:34:52;05 the kind of questions that we're addressing, to get at
00:34:56;05 how does aneuploidy affect cells. And now in the third
00:35:00;25 part of my talk, I will address the questions of how
00:35:05;11 aneuploidy impacts human diseases. And I will also
00:35:10;07 address this, to us, very exciting possibility that
00:35:13;10 aneuploidy could actually be developed as a new
00:35:17;10 therapeutic. So let me end by acknowledging the
00:35:20;16 people who've done all this work. On the top row are
00:35:24;23 the yeast researchers in the lab, Eduardo Torres, a
00:35:28;21 postdoctoral fellow in the lab, now has his independent
00:35:31;10 faculty position at UMass Worcester. He actually
00:35:34;12 developed the aneuploidy system in yeast in the lab
00:35:38;10 many years ago. The work on the transcriptional
00:35:41;19 response that I showed you was work by a graduate
00:35:45;08 student in the lab, Jason Sheltzer. The work on
00:35:48;28 proteotoxic stress in aneuploids was work done by
00:35:52;26 another graduate student in the lab, Ana Oromendia. I
00:35:56;27 want to especially acknowledge Bret Williams. He was a
00:36:00;07 postdoctoral fellow in the lab who actually was brave
00:36:03;10 enough to start studying mouse cells in an entirely
00:36:06;14 yeast lab, and so he really set up the mouse
00:36:10;11 aneuploidy system in the lab. Stefano Santaguida,
00:36:15;09 another postdoc in the lab, characterized the effects of
00:36:20;14 aneuploidy on autophagy. And Yun-Chi Tang did some
00:36:24;20 of the work of proteotoxicity in mammalian cells. And of
00:36:27;23 course, I would like to acknowledge the funding
00:36:29;15 sources. Without, none of this would have been
00:36:32;10 possible. We've been generously supported over many
00:36:35;24 years by NIGMS and also by the Howard Hughes
00:36:40;00 Medical Institute. And I thank you very much for
00:36:42;24 listening.
00:36:45;26

Part 3: Disease Implications

00:00:01;10 Hello, my name is Angelika Amon. I'm a professor in the
00:00:04;18 Biology Department at MIT, and I'm also an
00:00:07;29 Investigator of the Howard Hughes Medical Institute.
00:00:11;10 In Part 1, I introduced aneuploidy and its impact on
00:00:16;03 human health to you. In Part 2 of this series, I told you
00:00:20;04 a little bit about the work we're doing in the lab. And in
00:00:23;15 this part, the third part, I would put aneuploidy in the
00:00:27;24 context of human diseases, and again, I would like to,
00:00:31;16 with work from my lab, illustrate to you how we think
00:00:35;28 about the role of aneuploidy in human diseases,
00:00:38;22 especially cancer. And I want to raise some possibilities
00:00:42;23 by which we can use or exploit aneuploidy, perhaps,
00:00:46;26 even in developing new therapies for this disease. Like
00:00:51;08 all my other talks, I have basically subdivided this part
00:00:57;10 of the talk into a few subsections. I will first discuss
00:01:02;14 with you the role of aneuploidy in cancer, how we think
00:01:06;18 about it, and how we're going to elucidate its role in
00:01:10;08 these terrible diseases. I will then sort of raise the
00:01:14;28 possibility that perhaps aneuploidy could be developed
00:01:18;21 as a new therapeutic target in the disease. So, in the
00:01:24;09 second part of my talk, I tried to convince you that
00:01:29;00 aneuploidy is bad news for cells. It causes them stress,
00:01:33;20 it causes imbalance in the genome, and that is
00:01:36;28 associated with a lot of detrimental features, and it
00:01:41;15 basically leads to a stress response. So I hope by now
00:01:46;03 you are actually sitting there and asking yourself, well,
00:01:48;27 if aneuploidy is so bad for cells, if it antagonizes cell
00:01:53;03 division, how come that most solid human tumors are
00:01:58;22 aneuploid? And that's really a question that the field
00:02:04;06 has grappled with a long period of time. And there's
00:02:08;12 many answers out there, and many people have
00:02:11;07 different ideas about this. Here are my two favorite
00:02:15;10 hypotheses, okay? The first one is best summarized by
00:02:21;12 a German proverb that goes: "In the land of the blind,
00:02:26;03 the one-eyed is king." Let me explain to you what that
00:02:30;09 means. Even if you're not very good at something --
00:02:34;12 for example, seeing, because you only have one eye --
00:02:38;13 if no one around you can see at all, i.e., is blind, you
00:02:44;07 will still be top dog. And that's how you can consider
00:02:49;20 effects of aneuploidy. For example, if in an organ of
00:02:54;24 terminally differentiated cells, if no cell can divide at all,
00:03:00;14 then the one cell that acquired a copy of Myc, because
00:03:04;23 it had an extra copy of the chromosome that the Myc
00:03:07;20 gene is located on, now acquired the ability to
00:03:11;18 proliferate, even though it proliferates badly because
00:03:14;18 this extra copy of Myc comes with all the baggage
00:03:18;13 that's associated with the entire extra chromosome. It
00:03:21;15 will still prevail. So one idea that a lot of researchers,
00:03:26;03 and including us, are very excited about is this idea
00:03:29;03 that, under some specific conditions, aneuploidy can be
00:03:33;23 advantageous even though it interferes with
00:03:37;04 proliferation, and there's a number of wonderful
00:03:40;12 examples for this from experimental evolution studies,
00:03:44;16 and I've given you an example before: the fluconazole
00:03:47;18 resistance in Candida albicans that I discussed in Part
00:03:51;07 2. And so you could envision that aneuploidy might be
00:03:57;01 a quick and effective way of providing new traits to
00:04:01;10 quickly adapt to a new environment, for example,
00:04:04;29 adapting to a new niche during metastasis, and so
00:04:09;25 forth. So aneuploidy could provide ways to allow you to
00:04:17;04 adapt to a new environment. The other idea that we
00:04:21;13 were intrigued by, and that we actually set out to
00:04:24;01 experimentally test, and I'm going to show you a few
00:04:26;20 data about this, is the idea that because aneuploidy
00:04:29;29 causes protein imbalances, that because proteins that
00:04:34;11 are important for, you know, copying and segregating
00:04:39;07 the genome may be imbalanced, and that could cause
00:04:44;00 defects. And it could lead to genomic instability. And so
00:04:48;14 to test this hypothesis, we asked a very simple
00:04:51;28 question. The disomic yeast strains, this series of yeast
00:04:55;01 strains that we developed that carry one particular
00:04:58;09 extra chromosome, do they show increased genome
00:05:01;25 instability? And I'm showing you here a large table, and
00:05:06;07 I don't want you to look at every single one. You can
00:05:09;07 do that later. What I want to do now is I want to just
00:05:12;12 show you that this is the series of disomic yeast strains
00:05:16;18 that we looked at. And then each column represents
00:05:21;00 one assay for genome instability. We looked for
00:05:24;16 increased chromosome missegregation, mutation rates,
00:05:28;06 and various signs of DNA damage, and I'll come back to
00:05:31;14 those in a minute. And what I would like you to take in
00:05:35;06 is that there is a ton of pluses on this table. And what
00:05:39;27 that tells is that every single disomic yeast strain at
00:05:44;14 least shows one form of genomic instability. Okay? So
00:05:50;00 this idea that whole chromosomal aneuploidy basically
00:05:53;27 induces genome instability raises the very exciting
00:05:57;08 possibility to us that, basically, aneuploidy could be a
00:06:01;20 driver of evolution. It basically allows the cell to roll the
00:06:05;29 dice more often. One particular form of genome
00:06:10;27 instability we were particularly excited about, and I
00:06:14;15 want to highlight here, and that is this realization that
00:06:18;27 virtually every single disomic yeast strain that we
00:06:21;27 looked at had an increased number of Rad52 foci.
00:06:27;15 What is Rad52? Rad52 is a protein that is important for
00:06:33;00 DNA repair, specifically it's important for homologous
00:06:37;18 recombination. And when you start seeing foci in the
00:06:41;24 cell of that particular protein, that tells you that there's
00:06:46;28 ongoing DNA repair. And so, these increasing Rad52
00:06:52;28 foci was not again specific to the disomic yeast strains
00:06:57;10 that we originally observed. We also saw that again
00:07:02;02 these more complex aneuploid cells that we generate
00:07:06;08 through inducing meiosis of triploid yeast cells, we also
00:07:10;06 saw that there was an increase of cells with Rad52
00:07:13;26 foci. So a critical question that sort of comes from
00:07:19;25 these particular studies is sort of: Are there more
00:07:23;03 breaks, or is the repair defective? We're working on
00:07:26;25 this, our data right now suggest that there's actually
00:07:29;22 more breaks being made. We're trying to understand
00:07:32;14 why they're being made. And so while we are still going
00:07:36;23 into the details of trying to understand the molecular
00:07:40;15 mechanisms underlying this genome instability, I want
00:07:46;09 to highlight another very exciting finding to us. This
00:07:49;23 was work done by my colleague, Osami Niwa in Japan,
00:07:53;21 and he for a long time has been studying the effects of
00:07:56;15 aneuploidy in fission yeast. And his data too suggest
00:08:01;21 that aneuploid fission yeast cells shown here on the
00:08:04;21 right, versus euploid cells shown here on the left, they
00:08:08;27 too have a dramatic increase in number of Rad52 foci,
00:08:14;04 raising the exciting possibility that these genome
00:08:17;18 instability-inducing events of aneuploidy could be
00:08:22;05 conserved at least across yeast species. So obviously a
00:08:27;29 very important question that we haven't addressed yet
00:08:30;23 but that are critically important: Are these genome
00:08:33;29 instability-inducing events due to single gene
00:08:36;29 imbalances that either interfere with DNA metabolism or
00:08:40;13 chromosome segregation? There are examples for this
00:08:43;21 in the literature. We know that when you change the
00:08:47;07 stoichiometries of centrosome components that that
00:08:49;26 can lead to chromosome missegregation. Or, is this yet
00:08:53;28 another facet of the general effects of aneuploidy,
00:08:57;23 causing cellular stress? There are examples for that in
00:09:01;23 the literature, also. We know that heat shock causes
00:09:05;17 sensitivity to DNA-damaging agents, so the general
00:09:09;07 proteotoxic stress that we in the aneuploid cells could
00:09:12;21 also lead to increased genomic instability. So clearly,
00:09:19;10 one way of thinking about how aneuploidy could impact
00:09:22;24 tumorigenesis, as I said, by inducing genome instability,
00:09:27;22 it may allow the cells to roll the dice more often, and so
00:09:32;04 we are now basically interested in trying to understand
00:09:35;23 if aneuploid cells basically are more quick to adapt to
00:09:42;07 various stresses that they're imposed on. So to
00:09:45;12 address this question of the importance of rolling the
00:09:49;05 dice more frequently. So, irrespective of whether and
00:09:55;24 how aneuploidy contributes to tumorigenesis (the
00:09:59;16 gene-specific effects, the genome instability-inducing
00:10:02;13 effects, and many other possibilities), the fact remains
00:10:06;11 the vast majority of human cancers are aneuploid. And
00:10:11;24 so, with that statement, there are two questions that
00:10:17;06 immediately arise from this. The first one is, these
00:10:21;24 aneuploid cancer cells proliferate really well. So they
00:10:25;27 must have learned ways in which they can tolerate the
00:10:30;17 adverse effects of aneuploidy in order to take
00:10:34;01 advantage of potential beneficial effects. So the
00:10:37;29 question is, they must have some mutations that allow
00:10:40;17 them to tolerate aneuploidy. And if we understand
00:10:44;01 what kind of mutations allow you to tolerate
00:10:47;07 aneuploidy, that might actually might help us to
00:10:50;04 understand how tumors arise, and how tumors evolve.
00:10:55;08 So one question we're particularly interested in is, is it
00:10:58;19 actually possible to identify suppressors of the adverse
00:11:02;27 effects of aneuploidy? And I'm going to show you a
00:11:05;21 brief example in budding yeast that suggests that that
00:11:08;24 is actually the case. And a flip side to this question is,
00:11:12;19 can we identify enhancers of the aneuploid state? If
00:11:17;06 we find drugs or genetic manipulations that specifically
00:11:21;29 target either gene-specific effects in aneuploidy or the
00:11:25;24 general effects of aneuploidy, well maybe then that
00:11:29;22 would be a way for us to develop new cancer
00:11:32;02 therapeutics. And after we'll talk about the
00:11:35;13 suppressors, I'm going to tell you about study in
00:11:37;29 mammalian cells that we've taken as a proof of principle
00:11:41;27 that suggests that indeed such compounds do exist. So
00:11:49;11 basically, we were interested in understanding the
00:11:52;05 question, can we identify suppressors that allow
00:11:58;11 aneuploid cells to grow better? The way we did this is,
00:12:01;21 very simply, we took some aneuploid yeast strains.
00:12:04;18 They grow really badly, and we keep growing them
00:12:07;20 until they grow better, and then we simply ask, what
00:12:11;04 has changed, okay? So this is growth doubling times,
00:12:17;06 that's a graph that shows how quickly various strains
00:12:20;10 divide. And in red, you see the parental aneuploid
00:12:24;07 strain, and in blue right next to it, for example, those
00:12:28;02 are basically evolved isolates that clearly now grow a
00:12:32;06 lot better than the parental strain. So what we then do
00:12:35;25 we take these strains, we submit them to whole
00:12:39;24 genome sequencing, and then identify the mutations.
00:12:42;23 And then we've generated a large list of mutations that
00:12:45;26 allow various aneuploid strains to grow better. What
00:12:50;07 was interesting is that among all these evolved
00:12:54;05 isolates, we actually found three mutations multiple
00:12:57;25 times in various different aneuploids. For example, we
00:13:01;08 found a truncation mutation in UBP6 in both strains that
00:13:05;28 had an extra copy of chromosome 5, or an extra copy
00:13:09;06 of chromosome 9. We found mutations in VPS64,
00:13:14;10 excuse me, in two strains. And we also found a
00:13:17;12 segmental duplication in multiple ones of them. So we
00:13:20;18 decided to pick one of these and look at it in more
00:13:23;26 detail. And so we started with UBP6 mainly because it's
00:13:28;05 the first one we found. And so wonderful work by Dan
00:13:31;27 Finley at Harvard Medical School had previously
00:13:34;18 characterized this protein. They found that it is a
00:13:38;13 deubiquitinating enzyme that's associated with the
00:13:40;18 proteasome. The way you think about UBP6 is that is
00:13:43;24 competes with the proteasome. So it antagonizes
00:13:46;17 proteasome function. And so the first question we
00:13:50;19 wanted to ask is, does deleting UBP6 actually indeed
00:13:53;21 help these disomic strains, right? Is mutation of UBP6
00:13:58;29 actually responsible for the phenotype you observe?
00:14:01;16 As I'm showing you these experiments here, what
00:14:04;22 we're doing is we're doing what's called competition
00:14:06;10 experiments here. So we take the, excuse me,
00:14:11;11 parental strain and we mark it in green here, and then
00:14:15;20 we take the same disomic strain that's deleted now for
00:14:18;08 UBP6. We co-inoculate them here at zero, and then we
00:14:21;22 grow them over time, and then ask, what fraction of
00:14:28;14 the co-culture is due to one strain versus the other
00:14:31;14 strain. And you see very clearly that disome 5 and
00:14:36;07 disome 9, deleting UBP6 really clearly helps them, which
00:14:40;10 was good news, because that's how we actually
00:14:42;26 identified mutations. You further might notice that
00:14:46;01 there were two other strains that were also helped by
00:14:48;06 deleting UBP6, and actually only one of the disomic
00:14:50;24 strains actually grew more poorly. So indeed, deleting
00:14:55;29 UBP6 helps four disomic yeast strains to grow better.
00:15:00;10 And so Eduardo Torres, who did this work in the lab,
00:15:03;01 developed the hypothesis that deletion UBP6 increases
00:15:07;18 the degradation of an unknown number of proteins,
00:15:11;24 and reverts the protein composition more back to that
00:15:15;08 of wild type, okay? Sort of because deleting UBP6
00:15:18;25 unantagonizes the proteasome, so now the
00:15:21;22 proteasome is more active, it degrades excess
00:15:24;16 proteins. So using SILAC mass spectrometry
00:15:27;08 experiments, we can actually test that hypothesis. And
00:15:30;21 again this was work in collaboration with Steve Gygi,
00:15:35;03 and what you see here is if you take two wild-type
00:15:37;08 strains and you compare them, you get a certain
00:15:41;22 distribution with a certain standard deviation. If you
00:15:45;01 now compare a disomic 5 strain with a wild-type strain,
00:15:48;28 obviously there's some extra proteins here, so the
00:15:51;11 distribution gets wider and the standard deviation
00:15:54;14 increases, okay? And what's really obvious just from
00:15:58;06 this bird's-eye view, ten miles up in the air, you can
00:16:02;00 see, if we now take a disome 5 strain that's deleted for
00:16:05;28 UBP6, okay, and ask what's the standard deviation of
00:16:11;11 that is, you see that compared to, excuse me, disome
00:16:16;10 5 alone, it becomes more narrow. It doesn't reach wild-
00:16:19;18 type, but clearly things happen. So Eduardo was very
00:16:22;20 curious to figure out, well, what exactly does happen
00:16:25;20 to these cells? So he did a simple experiment. He
00:16:29;12 basically rank-ordered the proteins according to
00:16:33;21 whether they're overrepresented in the disome 5
00:16:36;10 strains versus that are underrepresented in disome 5
00:16:40;05 strains. And then he created three bins. He created a
00:16:44;05 bin where nothing changes, like within one standard
00:16:47;11 deviation of the mean (obviously the vast majority of
00:16:49;13 proteins don't change). Then there's a small group that
00:16:52;21 gets upregulated in disome 5 versus wild type. And
00:16:56;13 then there's a small group that gets downregulated.
00:16:59;05 And he specifically asked, when I now look at these
00:17:02;06 three groups, what happens, when I delete UBP6, to
00:17:05;28 these three groups? And that's shown here. That
00:17:09;18 middle graph, where nothing happens, that basically
00:17:12;29 shows you that deleting UBP6 doesn't really do
00:17:16;24 anything to the proteins that didn't change in disome 5
00:17:20;07 strains in the first place, okay? It makes sense. But
00:17:23;18 what was very striking is, so these are the proteins
00:17:26;13 that are overrepresented in disome 5 strains. When we
00:17:29;18 now delete UBP6, you actually see that they get
00:17:32;29 dramatically attenuated, okay? So they're getting
00:17:36;04 downregulated. And he could that this attenuation is
00:17:40;27 both due to transcriptional and post-transcriptional
00:17:43;19 mechanisms. And this kind of makes sense, right? So
00:17:47;08 the proteasome works better, so all these excess
00:17:49;27 proteins get degraded. But what was unanticipated
00:17:52;27 and exciting to us, that there, actually the
00:17:55;07 downregulated proteins got attenuated, too. And he
00:17:58;10 could show that this was due to indirect transcriptional
00:18:01;11 effects, okay? So this basically led to a very simple
00:18:07;05 hypothesis, two hypotheses. One possibility is that
00:18:12;03 UBP6 attenuates the proteome of the cells that get
00:18:17;22 improved by deleting UBP6, whose growth properties
00:18:20;27 increased. The other possibility is that deleting UBP6
00:18:26;04 attenuates the proteome of all the disomes, but only
00:18:29;27 the small number that benefits are the ones where
00:18:34;14 proteins that interfere with proliferation are sort of
00:18:38;03 cleared, and the guys that don't benefit, well, there,
00:18:41;17 the reason why they proliferate poorly, deleting UBP6
00:18:45;15 can't really help them. So there's a very simple
00:18:48;06 experiment that we can do to distinguish between
00:18:50;28 these hypotheses. We can take a disome where
00:18:55;06 deleting UBP6 doesn't make them grow better, okay?
00:19:00;15 And then prediction is, if hypothesis A is right, then
00:19:06;01 nothing should happen to the proteome in these guys.
00:19:08;28 If hypothesis B is right, they should still get
00:19:10;29 attenuated, but they are not being helped. So we take
00:19:14;22 a disome 13 strain, we do exactly what we did with
00:19:18;00 disome 5, and what we found was, like disome 5, the
00:19:22;29 proteins that were overrepresented in disome 13 were
00:19:26;10 also attenuated by deleting UBP6. Interestingly, and I
00:19:31;12 don't have time to get into this, the attenuation of the
00:19:34;12 downregulated proteins does not occur, and we
00:19:37;12 actually suspect that that's the reason why these cells
00:19:41;01 are not helped by deleting UBP6. But what this
00:19:45;02 experiment tells you is the deletion of UBP6 attenuates
00:19:50;12 the proteomes of all strains, okay? But disomes in which
00:19:54;29 UBP6 clients interfere with proliferation are rescued by
00:19:59;22 deleting UBP6, but disomes by which other proteins
00:20:04;20 interfere with proliferation that are not touched by
00:20:07;00 UBP6, obviously deleting UBP6 won't help them. Okay?
00:20:11;06 So, there's two general conclusions that I want to make
00:20:15;12 here. Again, our data suggest that the ubiquitin-
00:20:17;26 proteasome pathway is really important for the life of
00:20:22;19 aneuploid strains. And sort of a more general
00:20:25;07 conclusion: Yes, it is possible to obtain mutations that
00:20:30;01 allow aneuploid strains to grow better. And sort of
00:20:33;12 obviously, identifying such mutations in the context of
00:20:36;15 cancer will be instrumental in trying to figure out how
00:20:41;11 tumor cells evolve from benign to more and more
00:20:44;27 aggressive disease. So, in the last part of my talk, I
00:20:50;18 want to address of question of whether the aneuploid
00:20:54;11 states of cancer can be used in targeted therapy. And
00:20:58;17 Yun-Chi Tang, a postdoctoral fellow in the lab, asked a
00:21:01;24 very simple question: Is it possible to identify drugs or
00:21:06;26 compounds that preferentially impair the proliferation
00:21:11;19 of aneuploid cells versus euploid cells. And she initially
00:21:15;17 did a very small, targeted screen that again showed
00:21:19;09 that indeed such compounds exist. She identified an
00:21:23;15 energy stress-inducing compound called AICAR and a
00:21:28;00 proteotoxic stress-inducing compound, 17-AAG, as
00:21:31;13 having these properties. So initially, when we did this
00:21:35;25 screen, we sort of rather than screening sixty, a
00:21:39;26 hundred thousand compounds at the same time, Yun-
00:21:42;23 Chi sat down and asked a very simple question, well,
00:21:46;12 what have we learned about aneuploidy over the
00:21:49;04 years? We've learned that these cells are under
00:21:51;17 proteotoxic stress, we've learned that they're under
00:21:53;25 energy stress. And so Yun-Chi reasoned that if she
00:21:57;09 now took drugs and compounds that also induced
00:22:01;17 energy stress or that also induced proteotoxic stress,
00:22:05;20 that that could synergize with the aneuploid state, and
00:22:08;25 could create what yeast geneticists call "synthetic
00:22:12;09 lethality." Either condition on their own won't kill the
00:22:16;04 cell, but if you combine them, you create what we call a
00:22:20;11 synthetic lethal situation. So used a small number of
00:22:24;09 compounds, around 30, and started screening them.
00:22:28;07 And I'm only showing one set of data here, where she
00:22:31;18 actually looked at this compound AICAR and its effects
00:22:36;15 on cells. So the cells that she's using here are trisomy 1
00:22:42;21 cells and she uses trisomy 13 cells, and then she
00:22:46;21 basically, excuse me, cultures these cells under various
00:22:53;07 different concentrations of AICAR. And I want to just
00:22:56;02 you focus on basically the open squares and the filled
00:23:01;02 squares. So the filled squares here are wild-type cells
00:23:06;18 treated with high concentrations of this energy stress-
00:23:10;07 inducing compound, AICAR. They still can grow, albeit
00:23:14;14 very poorly. But look what happens to the trisomy 1
00:23:17;28 cells. Not only do they stop growing in the presence of
00:23:24;03 this concentration of the drug, they actually start
00:23:26;10 dying, their numbers go down, okay? And the same is
00:23:30;02 true for trisomy 13, and then to a less extent, for
00:23:33;06 trisomy 16 and 19. These trisomies are trisomies of
00:23:38;21 smaller chromosomes, so obviously the energy stress is
00:23:41;21 predicted to be less. This data here basically shows you
00:23:47;01 the same data as this graph, except now we normalize
00:23:50;11 them to the untreated control. I told you in the second
00:23:53;25 part of my talk these trisomic cells have proliferation
00:23:56;22 defects, they proliferate poorly, so to specifically look
00:24:00;16 at the effects of the drug, what she did is she
00:24:03;25 normalized the growth to "one" for both untreated, and
00:24:07;25 then asked what is the percentage of the various
00:24:15;10 growth, respect to zero. And you see very clearly
00:24:18;06 dramatically different effects for both trisomy 1 and
00:24:21;12 trisomy 13. I'm not going to tell you all the details. We
00:24:25;07 characterized these effects in more detail, we know
00:24:28;26 that it's due to a p53-depedent stress response that
00:24:33;06 gets induced in these cells, and we don't exactly know
00:24:37;00 yet the mechanism by which trisomy induces this p53
00:24:41;18 response, but we're currently working on it. What was
00:24:44;20 more important to figure out initially is to ask, well,
00:24:47;18 these drugs that we found to be selective for aneuploid
00:24:51;29 cells, do they also work for cancer cells? Okay? And so
00:24:56;03 to address this question, what she decided to do is she
00:25:00;01 decided to take two colon cancer cell lines (groups).
00:25:06;07 Colon cancers come in two flavors. They come in "MIN"
00:25:11;09 flavor, and they come in a "CIN" flavor. The MIN flavor
00:25:15;15 has low-grade, low-complexity aneuploidies, and the
00:25:19;08 CIN flavor has high-grade, high-complexity
00:25:22;08 aneuploidies. So if our drugs indeed has some
00:25:26;03 selectivity for aneuploidy, the clear prediction is that
00:25:31;27 they should be more effective inhibiting proliferation of
00:25:36;15 CIN cells, compared to MIN cells. So that was a very
00:25:40;07 simple prediction. And so she tested this prediction, in
00:25:44;02 the data shown here. AICAR and 17-AAG alone have
00:25:49;00 not that great effects in the cancer cell lines, so she
00:25:52;14 combined the treatment, and in black you see euploid
00:25:55;17 controls; then in blue and in green, you see the MIN
00:26:00;12 cell lines; and then orange, red, and purple, you see
00:26:03;28 the CIN lines. And you can very clearly see a
00:26:06;28 differential effect between the MIN lines and the CIN
00:26:10;18 lines, okay? So down here is the chromosome number
00:26:14;15 and also whether or not p53 is functional or not. It was
00:26:18;13 very interesting to see that actually p53 status did not
00:26:22;05 appear to influence the sensitivity. So she
00:26:25;17 subsequently asked whether she also sees effects in
00:26:29;28 xenografts. How these experiments work is you take
00:26:32;18 an immunocompromised nude mouse, you inject these
00:26:36;24 cancer cells into both flanks, the MIN line in one flank,
00:26:41;12 the CIN line in the other flank. And then you treat the
00:26:44;12 mouse with either control, PBS, or with the drugs
00:26:48;20 combined. And you see both in the untreated, that
00:26:53;11 both sites, the MIN and the CIN cancers, grow equally.
00:26:57;14 But what was very clear is that in the presence of the
00:27:01;00 drug, the proliferation of the CIN cell line was
00:27:05;09 dramatically reduced compared to the MIN cell line. I
00:27:09;12 want to be very clear here, I do not mean to imply that
00:27:13;05 the only reason for this differential response is the
00:27:16;01 degree of aneuploidy. I'm sure there's lots of other
00:27:19;26 reasons why MIN versus CIN lines could respond
00:27:22;26 differently, but our data are consistent with the idea
00:27:29;01 that the degree of aneuploidy contributes to this
00:27:32;26 differential effect. And so with that, I would like to
00:27:36;12 conclude that aneuploidy could be a potential target in
00:27:40;22 tumor therapy. So with this I want to sort of end this
00:27:47;08 part on cancer, and I want to summarize and sort of
00:27:50;16 also discuss a little bit of what kind of implications our
00:27:56;24 studies have on how we think about the impact of
00:28:00;29 aneuploidy in cancer. Our data very clearly indicate
00:28:05;11 that aneuploidy causes a proliferative disadvantage.
00:28:09;00 It's bad news to have the wrong number of
00:28:11;10 chromosomes. And we propose that the proliferative
00:28:14;22 disadvantage caused by aneuploidy needs to be
00:28:17;21 overcome during transformation, during tumorigenesis,
00:28:20;27 for the cancer to take advantage of potential beneficial
00:28:24;02 effects of the condition. Our data also raise the
00:28:29;20 interesting possibility that cancer cells may be more
00:28:33;14 heavily dependent on the mechanisms that help cells
00:28:37;00 deal with the stresses associated with aneuploidy, for
00:28:40;06 example, proteotoxicity is something that's very
00:28:44;23 prevalent in these cells. We would also like to propose
00:28:49;13 that the phenotypes that are shared by aneuploid cells
00:28:52;08 may provide a new therapeutic target. I've shown you
00:28:55;07 one example, AICAR and 17-AAG. Now we're actually
00:28:59;05 in the process of screening large-scale, chemical
00:29:02;18 libraries for new compounds, unanticipated compounds,
00:29:06;08 that perhaps show this differential effect. And then
00:29:09;07 finally, I think one way to think about how aneuploidy
00:29:13;06 could promote tumorigenesis is by increasing, basically,
00:29:19;10 adaptive potential by allowing cells basically to roll the
00:29:23;03 dice more often. So on that note, I would like to again
00:29:27;12 thank you for your attention. And I want to end by
00:29:31;01 acknowledging the people who did this work. The
00:29:34;15 aneuploidy project was started by Eduardo Torres
00:29:37;29 many years ago. The transcriptional response work I
00:29:42;05 told you about was by a graduate student, Jason
00:29:44;15 Sheltzer. Ana Oromendia did the proteotoxicity and
00:29:49;16 polyQ work that I showed you at the very end. The
00:29:55;02 protein aggregation work was also by Stefano
00:29:58;04 Santaguida. And the hunt for drugs that specifically
00:30:02;14 inhibit proliferation of aneuploid mouse cells was the
00:30:08;10 work by postdoctoral fellow Yun-Chi Tang. And last but
00:30:11;27 not least, funding. Thank you, NIGMS and the Howard
00:30:16;09 Hughes Medical Institute. And thank you very much for
00:30:19;14 listening.
00:30:23;15

Videos in this Talk
  • Part 1: Review of the Study of Aneuploidy
    Part 1: Review of the Study of Aneuploidy
    Audience:
    • Student
    • Researcher
    • Educators of Adv. Undergrad / Grad
  • Part 2: Effects of Aneuploidy on Cell Physiology
    Part 2: Effects of Aneuploidy on Cell Physiology
    Audience:
    • Student
    • Researcher
    • Educators of Adv. Undergrad / Grad
  • Part 3: Disease Implications
    Part 3: Disease Implications
    Audience:
    • Student
    • Researcher
    • Educators of Adv. Undergrad / Grad
Speaker: Angelika Amon
Total Duration: 1:40:06
Recorded: July 2012
All Talks in Cell Biology

Talk Overview

Amon begins her talk by explaining what aneuploidy is and how it arises. She explains that autosomal aneuploidy is usually devastating to an organism, while aneuploidy at a cellular level may result in the unrestricted growth seen in cancer.

In Part 2, Amon provides more details about the impact of aneuploidy on individual cells. Using budding yeast and mouse lines engineered to have specific aneuploidies, Amon’s lab was able to show that aneuploid cells show gene specific effects, as well as a higher overall level of cellular stress.

Part 3 focuses on the role of aneuploidy in disease, specifically in cancer. Amon discusses how identifying enhancers and suppressors of the aneuploid condition can shed light on the effects of aneuploidy in cancer and can lead to the development of novel cancer therapeutics.

Speaker Bio

Angelika Amon

Angelika Amon received both her B.S. and Ph.D. degrees from the University of Vienna. She was a post-doctoral fellow at the Whitehead Institute and was subsequently named a Whitehead Fellow. In 1999, she joined the Massachusetts Institute of Technology where she is currently Professor of biology and Kathleen and Curtis Marble Professor in cancer research… Continue Reading

Playlist: Cancer

Related Resources

Primary papers:
Torres EM, Sokolsky T, Tucker CM, Chan LY, Boselli M, Dunham MJ, Amon A. Effects of aneuploidy on cellular physiology and cell division in haploid yeast. Science. 2007 Aug 17; 317(5840): 916-924.

Williams BR, Prabhu VR, Hunter KE, Glazier CM, Whittaker CA, Housman DE, Amon A. Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science. 2008 Oct 31; 322(5902): 703-709. PMCID: PMC2701511

Sheltzer JM, Blank HM, Pfau SJ, Tange Y, George BM, Humpton TJ, Brito IL, Hiraoka Y, Niwa O, Amon A. Aneuploidy drives genomic instability in yeast. Science. 2011 Aug 19; 333(6045): 1026-1030. PMCID: PMC Journal In Process

Torres EM, Dephoure N, Panneerselvam A, Tucker CM, Whittaker CA, Gygi SP, Dunham MJ, Amon A. Identification of Aneuploidy-Tolerating Mutations. Cell. 2010 Oct 1; 143(1): 71-83. Epub 2010 Sep 16. PMCID: PMC2993244

Tang YC, Williams BR, Siegel JJ, Amon A. Identification of aneuploidy-selective antiproliferation compounds. Cell. 2011 Feb 18; 144(4): 499-512. NIHMSID #: 276601

Oromendia AB, Dodgson SE, Amon A. (2012). Aneuploidy causes proteotoxic stress in yeast. Genes Dev. 15, 2696-708.

Reviews:
Siegel JJ, Amon A. (2012). New insights into the troubles of aneuploidy. Annu Rev Cell Dev Biol. 28:189-214.

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