ON THE ROLE OF P53 IN THE CELLULAR RESPONSE TO

ANEUPLOIDY

by

Akshay Narkar

A dissertation submitted to in conformity with the

requirements for the degree of

Baltimore, Maryland

July 2020

© 2020 Akshay Narkar

All rights reserved

ABSTRACT

A majority of solid tumors are aneuploid and the tumor suppressor protein 53, has

been implicated as the guardian of euploid genomes in animal organisms. Experiments

using transformed or non-transform human lines showed that induction

leads to p53 accumulation and a consequent, p21-mediated, G1 arrest. However,

it has been controversial as to whether there is a universal signal elicited by the aneuploid

state that activates p53. My PhD thesis attempts to advance our understanding of the universality of the p53 response in different cell and tissue context post aneuploidy induction.

In this study, we found that, whereas adherent 2-dimensional (2D) cultures of human immortalized or cell lines indeed activate p53 upon aneuploidy induction as previously reported, suspension cultures of a human lymphoid cell line undergoes a p53-

independent cell cycle arrest upon aneuploidy formation. To dive deeper into the molecular

mechanisms regulating p53-independent arrest post aneuploidy in Nalm6 cells we performed a genome wide CRISPR/Cas9 knockout screen. This revealed previously uncharacterized functions of PDCD5 and LCMT1 in regulating chromosomal aberrations.

Recent studies highlight that tissue environment plays a critical role in regulating

chromosome segregation, however the role of p53 in 3D environment is not well

characterized. To our surprise, 3-dimensional (3D) organotypic cultures of cells from

human or mouse neural, intestinal or mammary epithelial tissues do not activate p53 or

undergo G1 arrest upon aneuploidy induction. Instead, TP53-/- organoids exhibit increased

ii aneuploidy production that correlates with mitotic aberrations and dysregulated cell cycle timing.

Our data suggest that p53 is not necessarily a universal surveillance factor that prohibits the proliferation of aneuploid cells, but rather, it helps ensure stable chromosome transmission through a more direct role in mitotic fidelity.

Primary Reader and Advisor: Rong Li

Secondary Reader: Ben Ho Park

iii

ACKNOWLEDGEMENTS

I am very grateful to my advisor, Professor Rong Li, for introducing me to the

amazing world of , and for her guidance along the road of my PhD study.

Rong’s profound scientific knowledge and broad vision in science have been a role model

to me. I am also thankful to her for being patient and allowing me to explore different

research ideas. Postulating multiple hypothesis and designing experiments to rigorously

test it have not only helped me to shape my independent research but also made science

enjoyable.

I am grateful to Professor Roger Reeves, Ben Ho Park and Andrew Holland for

always being supportive and serving on my thesis committee. Thanks to the Human

Genetics program - Dr. David Valle, Dr. Kirby Smith, Dr. Haig Kazazian and Sandy

Muscelli. I feel very proud to be a part of the Institute for Genetic Medicine at Hopkins. I

also want to thank my classmates and colleagues from the department to make graduate

life fun.

A big thank you to my lab mates Linhao, Blake, Jin, Alexis, Soham, Wahid, Sree,

Tiane and Albert for scientific and fun insights.

Lastly, I am very grateful to my family for their infinite love and support, it would not have been possible without you!!

iv

Dedicated to my family,

for their eternal support, love and trust.

मा�ा कुटुंबासाठी,

ां�ा शा�त समथ�नासाठी, प्रेम आिण िव�ासासाठी समिप�त.

v TABLE OF CONTENTS

ABSTRACT……………………………………………………………………………... ii

LIST OF TABLES……………………………………………………………………... vii

LIST OF FIGURES…………………………………………………………………… viii

LIST OF SYMBOLS AND ABBREVIATIONS……………………………………… ix

CHAPTER 1. Introduction…………………………………………………………… 1

CHAPTER 2. Mechanisms to generate aneuploidy………………………………….4

CHAPTER 3. Role of p53 in regulating aneuploid cell fate…………………………7

CHAPTER 4. Genome wide screen reveals novel aneuploidy regulators…………12

SUMMARY and DISCUSSION………………………………………………………. 16

APPENDIX A. Methods for Chapter 3 and 4………………………………………... 35

REFERENCES………………………………………………………………………….41

CV………………………………………………………………………………………. 56

vi LIST OF TABLES

Table 1 – KEY RESOURCE TABLE 32

vii LIST OF FIGURES

Figure 2.1 – Mechanisms to generate aneuploidy and downstream cell fate. 5

Figure 3.1 – Induction of aneuploidy with MPS1i in mammalian cell lines 21 and 3D organotypic cultures.

Figure 3.2 – Adherent RPE1 and HCT116 but not Nalm6 cells depend on 22 p53 for growth arrest post aneuploidy induction.

Figure 3.3 – 3D organotypic cultures do not activate p53 or undergo 23 growth arrest in response to aneuploidy.

Figure 3.4 – TP53-/- mCO exhibit frequent mitotic aberrations 24

Figure 4.1 – Genome wide CRISPR/Cas9 screen reveals novel aneuploidy 25 regulators

– Supplementary Figures 26

viii LIST OF SYMBOLS AND ABBREVIATIONS

P53 Tumor Suppressor Protein p53

CIN Chromosomal Instability

MPS1i Monopolar Spindle Kinase inhibitor

mCO Mouse Colon Organoids

hMO Human Mammary Organoids

EdU 5-Ethynyl-2´-deoxyuridine

MPS1i Chromosomal Instability

ix

CHAPTER 1. INTRODUCTION

Aneuploidy refers to the state of unequal chromosome copy numbers and is one of

the most prominent genomic aberrations in a majority of solid tumors (Lengauer, Kinzler

and Vogelstein, 1998; Weaver and Cleveland, 2006; Beroukhim et al., 2010; Zack et al.,

2013; Taylor et al., 2018). In unicellular , it was shown that aneuploidy, by

altering the stoichiometry of a large number of genes, can result in dramatic changes in cellular phenotypes and and confer evolutionary under selective pressure (Selmecki, Forche and Berman, 2006; Torres et al., 2007; Pavelka et al., 2010;

Sterkers et al., 2012; Yona et al., 2012; Dephoure et al., 2014; Kaya et al., 2015; Sunshine et al., 2015). Such basic insight about aneuploidy helps explain recent findings that karyotype alterations are associated with cancer initiation as well as the emergence of (Lee et al., 2011; Navin et al., 2011; Davoli et al., 2013; Lane et al., 2014; Cai et al., 2016; Graham et al., 2017; Sack et al., 2018; Stichel et al., 2018; Yang et al., 2019).

Indeed, cancer may be viewed as a disease of cellular evolution in a multicellular setting, whereby cells of metazoans turn to resembling unicellular organisms that are free to undergo evolutionary adaptation for better survival and proliferation through gross genomic alterations (Nowell, 1976; Duesberg, Stindl and Hehlmann, 2001; Chen et al.,

2015; Gerstung et al., 2020).

What might differentiate a genomically stable animal organism from the free- adapting unicellular eukaryotes is the presence of p53, acting as the guardian of genome stability by playing key regulatory roles in such processes as the DNA damage response,

1 senescence, and apoptosis (Aylon and Oren, 2011; Reinhardt and Schumacher, 2014;

Kastenhuber and Lowe, 2017; Mello and Attardi, 2018; Hafner et al., 2019; Mijit et al.,

2020). The loss of functional p53 has been associated with the onset of many metastatic

with heightened genomic instability especially on the chromosomal level, whereas

an increased p53 gene copy number is thought to be chemopreventive (Sulak et al., 2016;

Wasylishen and Lozano, 2016; Bykov et al., 2018; Donehower et al., 2019). Studies in

recent years have further suggested roles for p53 in limiting the proliferation of aneuploid

cells, however, these studies were limited towards established human cell lines that were

chromosomally stable and near diploid, such as RPE1, an hTERT-immortalized retinal

pigmented epithelial cell line, HCT116, a colon carcinoma cell line along with a few other

cell lines (Cianchi et al., 1999; Li et al., 2010; Thompson and Compton, 2010; Janssen et

al., 2011; Kurinna et al., 2013; Hinchcliffe et al., 2016; Potapova et al., 2016; Santaguida

et al., 2017; Soto et al., 2017; Giam et al., 2019). Recent studies also revealed complex

interplays between p53 and several other genome-protective proteins, such as p38, H3.3 and BCL9L (Hinchcliffe et al., 2016; López-García et al., 2017; Simões-Sousa et al.,

2018). However, it has been unclear whether a universal signal elicited by abnormal karyotypes may be sensed by the p53 regulatory mechanism, or karyotype-specific stress states are sensed through diverse mechanisms and converge upon p53 activation. It was also unknown whether cell type or growth environment could contribute to the p53- mediated response to aneuploidy.

In my PhD studies, I investigated p53 regulation and downstream cell fate after aneuploidy induction in diverse cell culture models. We set out to answer four questions: 1) How universal is the relation between aneuploidy induction and p53 activation? 2) What are the

2 downstream cell fate consequences after acute aneuploidy induction in different types of cell culture models? 3) Does p53 play a direct role in faithful mitosis rather than sensing aneuploid cells after erroneous mitosis? 4) Identify novel aneuploidy regulators using genome wide CRISR/Cas9 knockout screens? While we confirm that, upon acute aneuploidy induction by treating cells with an inhibitor of the spindle assembly checkpoint

(SAC) kinase MPS1 (MPS1i), p53 and p21 were upregulated and cause growth arrest in

RPE1 and HCT116 cell lines, this response was not conserved in 3D organotypic cultures of primary cells from mouse and human tissues. Live imaging suggests that p53’s importance in preventing aneuploidy may be attributed to a direct role in mitotic regulation.

The genome wide screen revealed previously not well characterized roles of LCMT1 and

PDCD5 in regulating Nalm6 cell fate post aneuploidy induction.

3 CHAPTER 2. MECHANISMS THAT GENERATE ANEUPLOIDY

AND CELL FATE

Chromosome segregation is a dynamic process that relies on the structural integrity of the microtubule spindle apparatus and a checkpoint signaling pathway to ensure high fidelity (Compton, 2011). Bipolar orientation is critical for faithful chromosome segregation to daughter cells. However, in certain cases microtubules are organized into monopolar or multipolar spindle configurations which can lead to aneuploidy, cell death or growth arrest. The mechanics and structural components of kinetochore and microtubules play a vital role in accurate chromosome segregation (Compton, 2011). Due to stochastic of kinetochore-microtubule (kMT) attachments if the kinetochore is attached to microtubules from both ends of the pole it will result in satisfying the SAC and this merotelic attachment may generate lagging chromosomes or breakage (Cimini et al.,

2001; Gregan et al., 2011). FISH analyses show that the mis-segregation rate of chromosomes in aneuploid tumor cells is 20- to 100-fold higher than non-transformed diploid cells (Lengauer, Kinzler and Vogelstein, 1998; Thompson and Compton, 2008).

The k-MT attachments also regulate timing of sister chromatid separation at anaphase by a checkpoint signaling pathway. Both increased and decreased time in mitosis can result in aneuploidy and cell arrest.

Other events like cell fusion, abortive cell cycle and endoreplication generate polyploidy which acts as precursor to aneuploid and tetraploid progeny (Storchova and Pellman,

2004). It was shown that tetraploid cells, which were generated by inhibition of cytokinesis with an -depolymerizing drug, also arrested in G1 phase through a p53-dependent mechanism (Andreassen et al., 2001). Another major player required for the normal completion of cytokinesis is the centrosome. Polyploid cells with supernumerary

4 centrosomes can experience several fates including centrosome clustering or p53 dependent cell cycle arrest (Nigg, 2002; Tarapore and Fukasawa, 2002). Other pathways including but not limited to chromothripsis, telomere damage, cytokinesis failure, replication defects, cohesion defects and weakened SAC may contribute to aneuploidy and genome perturbations (Bennett, Forche and Berman, 2014;

Orr, Godek and Compton, 2015).

Figure 2.1. Mechanisms to generate aneuploidy and downstream cell fate.

Literature review during my early PhD career helped me identify the use of MPS1i as a central upstream route to induce aneuploidy in different cellular

5 models. One of the critical mitotic kinases is monopolar spindle kinase 1 Mps1.

Mps1 was identified in a budding yeast mutant that harbors a defect in the spindle pole body (yeast centrosome) duplication process, resulting in a monopolar spindle. Mps1 phosphorylates serine, threonine and tyrosine in vitro; thus, it is a dual specificity protein kinase TTK. MPS1 is essential for chromosome alignment and for the spindle assembly checkpoint (SAC)(Kollu et al., 2015;

Choi et al., 2017). Using different dose titrations and timing we optimized drug concentrations and washout timing for recovery of maximum heterogenous aneuploid populations.

6 CHAPTER 3. ROLE OF P53 POST ANEUPLOIDY IN

DIFFERENT CELLULAR MODELS.

The contents are adapted from:

On the Role of p53 in the Cellular Response to Aneuploidy. Akshay Narkar, Blake Johnson, Pandurang Bharne, Jin Zhu, Veena Padmanaban, Debojyoti Biswas, Andrew

Fraser, Pablo Iglesias, Andrew J Ewald, Rong Li “Under Review Developmental Cell”

Induction of aneuploidy with MPS1i in mammalian cell lines and 3D organotypic cultures.

To investigate the response to aneuploidy in a broadly representative panel of cell models, we not only included the previously used RPE1 and HCT116 cell lines, but also Nalm6, a chromosomally stable pre-B cell lymphoma line that grows in suspension (Hurwitz et al.,

1979), and 3D organotypic cultures of primary cells. Potentially influential parameters also

included different stages of development, for which we included embryonic (eNPCs) and

adult (aNPCs) mouse neural progenitor cells. For 3D cultures of primary cells that better

mimic in-vivo tissues, we also included human mammary organoids (hMOs), mouse colon

organoids (mCOs). To induce aneuploidy, each type of culture was treated with the MPS1i

NMS-P715 for 24 hours, followed by drug washout and recovery growth for 24 or 48 hours

in drug-free media , to alleviate any direct drug effect before various analyses (Figure

3.1A). We first quantified the percent numerical aneuploidy by chromosome counting in

metaphase spreads after the above treatment. For RPE1, HCT116, Nalm6, hMOs and

mCOs, the basal whole-chromosome aneuploidy levels were low in untreated cultures, but

7 a significant increase in aneuploidy percentages, to 45-55%, in each MPS1i-treated culture

(Figure 3.1B). NPCs also showed increased aneuploidy after MPS1i treatment, but the

basal aneuploidy percentages were higher, consistent with earlier reports (Yang et al.,

2003; Rehen et al., 2005). The chromosome number distributions showed both gains and

losses of chromosomes across all cell models, consistent with the production of random

karyotypes as a result of mitotic errors (Figure 3.1C and 3.1D). Note that our method of

inducing aneuploid cell populations did not involve cell synchronization as some agents

altering cell cycle progression may cause DNA damage (Darzynkiewicz et al., 2011;

Halicka et al., 2016), which could activate p53. As expected, pH2AX immunoblots showed that MPS1i treatments in our experiments did not induce DNA damage (Figure S1A and

S1B).

Adherent RPE1 and HCT116 but not suspension Nalm6 cells depend on p53 for growth arrest post aneuploidy induction.

We next determined the effect of acute aneuploidy induction on p53 and CDK inhibitor

CDKN1A (p21), a well-studied direct transcriptional target of p53 that controls cell cycle progression (El-Deiry et al., 1993; Dulić et al., 1994). p53 is known to be regulated predominantly by post translational modifications that affect its stability or nuclear accumulation. (Haupt et al., 1997; Oren, 1999; Kruse and Gu, 2009a). Acute induction of aneuploidy by using drugs that perturb mitosis or SAC led to increased p53 stabilization and a consequent G1 arrest upon transcriptional activation of the p21 (Li et al., 2010; Kollu et al., 2015). We observed significant increases in p53 levels in the adherent RPE1 and

HCT116 cells and suspension Nalm6 cells, but not in the NPCs or 3D organotypic cultures

(presented further below), after aneuploidy induction using MPS1i (Figure 3.2A and 3.2B).

8 Additionally, nuclear localization of p53 was confirmed in HCT116 and RPE1 cells by

florescence microscopy (Figure S1C and S1D). All cell lines showed increased p53 protein

abundance upon treatment with nutlin, which stabilizes p53 by disrupting p53-MDM2

complex, and DNA damaging agents such as doxorubicin and bleomycin (Figure 3.1A,

3.1B, 3.2A, 3.2B, S1C and S1D).

p53 regulates many cellular processes and plays a critical role in modulating the phenotypic

severity of a wide variety of cell-cycle defects (Kastenhuber and Lowe, 2017; McKinley

and Cheeseman, 2017). We examined whether p53 induction was associated with either

cell cycle arrest or apoptosis by assaying DNA synthesis using the 5-ethynyl-2′-

deoxyuridine (EdU) incorporation assay (Salic and Mitchison, 2008) and apoptosis using

Annexin V as a marker along with propidium iodide. In MPS1i-treated RPE1, HCT116

and Nalm6 cells, there was a significant reduction in EdU positive cells (Figure 3.2C and

3.2D), consistent with a suppression of proliferation, but these populations did not display

a significant increase in apoptotic cells (Figure S2E). To test the requirement for p53 in

mediating this arrest, we used TP53-/- HCT116 (a gift from Vogelstein lab), RPE1 TP53-/-

(a gift from Holland lab) and TP53-/- Nalm6 cells were generated using CRISPR/Cas9. p53 knockouts in RPE1, HCT116 and Nalm6 cell lines were confirmed by immunoblotting

(Figure S2A). Additional genotyping validation was performed for Nalm6 (Figure S2B).

Deviation from the diploid chromosome number in these cells lacking p53 was observed

even without MPS1i treatment (Figure S2C). p53 knockout rescued cell proliferation, as

indicated by the increased EdU-positive cells, in MPS1i-treated HCT116 and RPE1 populations (Figure 3.2C and 3.2D). However, this rescue was not observed in Nalm6 TP53

9 -/- suspension cells (Figure 3.2D), suggesting that, although p53 was induced after

aneuploidy, it was not required for the reduced proliferation in Nalm6 cells.

3D organotypic cultures do not activate p53 or undergo growth arrest in response to

aneuploidy

Whereas the three established cell lines (RPE1, HCT116 and Nalm6) responded to aneuploidy as previously reported despite a lack of p53 dependence for the growth arrest in Nalm6 cells, we did not observe an increase in p53 or p21 protein abundance after aneuploidy induction in 3D cultures of NPC, mCO, or hMO (Figure 3.3A, 3.3B and 3.2B).

As a positive control, treatment with nutlin or DNA damaging agent doxorubicin or bleomycin increased p53 and p21 protein abundance in these cells, suggesting that p53 is generally functional in these models. Consistent with a lack of p53 activation in 3D organotypic cultures after aneuploidy induction, EdU incorporation assay showed unimpeded cell proliferation, in contrast to reduced proliferation of the same cultures treated with nutlin or DNA damaging agents (Figure 3.3C, 3.3D and S2D). A recent study demonstrates that disrupting tissue architecture has profound impacts on chromosome segregation fidelity (Knouse et al., 2018). To rule out the possibility that the differential

responses observed between the adherent epithelial cell lines vs organotypic cultures was

due to 2D versus 3D culture environment, we cultured HCT116 cells in 3D matrigel (Figure

S3A). These cells grew into spherical structures and high-level EdU incorporation

indicated their healthy proliferation (Figure S3C). Upon aneuploidy induction, HCT116

cells cultured in 3D exhibited similar levels of p53 and p21 increase and growth arrest as

observed for those cultured in 2D (Figure S3B, S3C and S3D). This suggests that the

10 difference in aneuploidy responses observed among different cell types tested was unlikely

attributed to 2D vs 3D culture environment.

TP53-/- mouse colon organoids exhibit frequent mitotic aberrations.

Although mCO did not exhibit an elevated p53 and G1 arrest post aneuploidy, TP53-/- mCO

showed increased aneuploidy (Figure S4), suggesting that p53 may have a direct role in

preventing aneuploidy occurrence instead of a surveillance role against aneuploidy. To test

this, TP53+/+ and TP53-/- mouse colon organoids were transduced with H2B-mNeon and mitotic cell divisions were observed using live 3D confocal imaging. Several classes of mitotic aberrations including multipolar divisions, lagging chromosomes and metaphase plate misalignment were quantified from the live recordings. The fraction of cells exhibiting lagging chromosomes were most drastically increased in TP53-/- when compared

to TP53+/+ colon organoids, and there was also an increase in multipolar divisions (Figure

3.4A, 3.4B). In the presence of low-dose nocodazole, the time in mitosis (from nuclear envelope breakdown (NEBD) to anaphase onset) was lengthened significantly in both

TP53+/+ and TP53-/- mCOs, suggesting that TP53-/- did not disrupt the SAC (Figure 3.4C).

Interestingly, induction of SAC by low-dose nocodazole reduced the frequency of lagging

chromosomes in TP53-/- mCOs suggesting that lengthening mitotic duration facilitates error correction in the p53-deficient background.

11 CHAPTER 4. GENOME WIDE CRISPR/CAS9 KNOCKOUT

SCREEN REVEALS NOVEL ANEUPLOIDY REGULATORS

Our observation that loss of p53 was not sufficient to rescue the EdU arrest in Nalm6 cells

led us to investigate p53 independent pathways regulating aneuploidy. To address this in a

comprehensive way we collaborated with Mike Tyers group at Montreal to perform a

genome-wide screen. CRISPR/Cas9 technology has been recently adapted to perform

large-scale functional gene knockout screens in human cells and overcomes limitations of

previous RNAi-based gene knockdown approaches (Shalem et al., 2014; Shalem, Sanjana and Zhang, 2015). To interrogate genes essential for cell growth, proliferation and survival in human cells Tyer’s group performed a screen using extended knockout library of

278,754 single guide RNAs that targeted 19,084 RefSeq genes, 20,852 alternatively spliced exons, and 3,872 hypothetical genes (Bertomeu et al., 2018). We used their statistical analysis tool called robust analytics and normalization for knockout screens (RANKS) for scoring top hits that may be regulating aneuploidy.

Overview of the screen methodology is highlighted in (Figure 4.1A). Post NGS sgRNA frequencies between untreated and MPS1i treated samples were compared to plot sgRNA enriched frequencies using RANKS statistics. It was interesting and reassuring to find cell cycle genes like ANAPC13, ANAPC15, CCNF, UBE2C and MAD2L1BP as top positive hits (Figure 4.1C). These known candidates acted as internal controls. Homozygous knockout of ANAPC13 and UBE2C in monoclonal cells showed partial rescue of EdU and pseudodiploid chromosome numbers. The mechanisms of cell cycle related genes are well established and thus I focused on non-canonical targets. It was interesting to find novel

12 regulators like PDCD5, SRSF1, LCMT1 pass the statistical cutoff. Programmed cell death

5 (PDCD5) was originally identified as an apoptosis-accelerating protein that is conserved during the process of evolution (Liu et al., 1999). PDCD5 has complex biological functions, including programmed cell death and immune regulation. Based on upstream stimuli it can accelerate apoptosis in different type of cells and rapidly translocate from the cytoplasm to the nucleus. PDCD5 regulates the activities of TIP60, HDAC3, MDM2 and

TP53 transcription factors (Li, Ma and Chen, 2016). The other candidate leucine carboxyl methyltransferase (LCMT1)-dependent PP2A methylation is associated with down- regulation of PP2A holoenzymes containing the Bα subunit (PP2A/Bα) and subsequent accumulation of phosphorylated Tau in N2a cells, in vivo and in Alzheimer’s Disease

(Sontag, Nunbhakdi-Craig and Sontag, 2013). LCMT1 has additional roles in neuroblast differentiation and regulating mitotic spindle size (Sontag et al., 2010; Xia et al., 2015).

We generated monoclonal knockouts of top hits and validated using genotyping and immunoblotting (Figure 4.1B). LCMT1-/- line showed increased basal aneuploidy levels and PDCD5-/- lines showed significant tetraploidy and polyploidy as analyzed by chromosome counting and FACS analysis. For functional validation of novel candidates, we subjected these two lines to EdU rescue post aneuploidy induction. It was interesting to find LCMT1-/- and PDCD5-/- knockouts significantly rescued aneuploidy induced growth arrest (Figure 4.1D). It would be of great interest to follow up on these two hits to understand the molecular mechanism and pathways regulating the proliferation of cells with a perturbed genome. An interesting avenue is to understand the relation of these novel candidates and p53 post aneuploidy. Here, we demonstrated how preB lymphocytes may

13 circumvent the response to aneuploidy which may help tumors evade surveillance to attain malignant transformations.

Potential Pitfalls and Alternative approaches:

We anticipated that many of these experimental designs would require rigorous optimization across multiple cell and organoid models. Thus, we titrated MPS1i to obtain random karyotypes with gains and losses of few chromosomes. This helps maintain a degree of karyotypic complexity that allows experimental investigation as opposed to mitotic catastrophe and cell death. Another area to tread carefully is the genome wide

CRISPR/Cas9 knockout screen. Limited efficiency of sgRNAs poses a difficulting in targeting genes to infer phenotype causality. To overcome this, we used at least 10sgRNA’s targeting each gene to achieve maximum knockout efficiency and minimal off target effect.

Post screen stringent statistical cutoff’s were imposed using p-Value, FDR, RANKS score

to identify true hits for functional validation.

Another avenue to control p53 stability is by post translational modifications particularly

phosphorylation (Kruse and Gu, 2009b). To investigate if there is a universal kinase

governing p53 response to aneuploidy we performed a kinase inhibitor screen of 418

compounds targeting multiple kinase pathways post aneuploidy induction in RPE1 and

HCT116 cells (data not shown). We found variable dependence on pathways like mTOR,

c-Src in both cell lines tested. This also supports our finding that it is not necessarily a

universal response of p53 post aneuploidy but a multitude of factors that govern cell fate.

P53 transcriptional activity depends largely on its nuclear localization. We used

Immunofluorescence to confirm its nuclear signal after aneuploidy and also performed live

14 cell imaging using endogenous tagged p53 protein (eFlut) toolkit (Stewart-Ornstein and

Lahav, 2016). This allowed us to track the dynamic changes in p53 and its downstream target p21 localization (data not shown). An interesting observation was cytoplasmic sequestration of p21 in cytoplasm in aNPCs post aneuploidy induction. This could potentially be a factor that these spheroids do not arrest significantly post missegregation.

Finally, to understand the effect of aneuploidy on cell and nuclear area in a p53 null background we exposed Nalm6 cells to MPS1i. To our surprise basal levels of aneuploidy and tetraploidy were increased in p53 null Nalm6 cells. Post aneuploidy induction karyotypes were extremely complex and cells demonstrated significantly larger cell and nuclear area and rate of apoptosis (data not shown). These observations warrant careful examination of the link between p53 and other candidates like PDCD5 and LCMT1 to restrict the proliferation of genome damaged cells post aneuploidy.

15 DISCUSSION:

We have examined the generality of p53 up-regulation and cellular responses after acute induction of aneuploidy in a variety of cell models linked to different tissue origins and growth environments. Based on earlier findings in immortalized and cancer cell lines such as RPE1 and HCT116, our original interest was to identify aneuploidy-associated signals that induce p53 activation in these cells. However, our inability to generate a similar response in NPC or non-p53-dependent response in Nalm6 pre-B lymphocytes suggests that p53 activation post aneuploidy induction is not necessarily a universal response but may be more specific towards certain adherent cell lines. This led us to conduct a more comprehensive investigation of cellular responses in 3D organoid environment. We found that induction of aneuploidy does not result in p53 accumulation or cell proliferation arrest in organotypic cultures from diverse tissues including neural tissue, intestinal epithelium and mammary epithelium from mouse or human. Recent study in Drosophila also demonstrates that the neural tissue exhibits delayed p53 stress response to developmental aneuploidy (Mirkovic et al., 2019). Our observed lack of p53 response was unlikely due to the 3D culturing system because 3D cultures of HCT116 cells showed a similar p53 response to those cultured in 2D. Given that 3D organotypic cultures of primary cells are better mimics of native tissues than immortalized or cancer cell lines (Schutgens and

Clevers, 2020), our findings do not support the notion that p53 acts in a surveillance mechanism against aneuploidy in the physiological context. It is possible that certain in- vitro stress associated with those artificially immortalized or cancer cell lines compounded with the stress caused by aneuploidy can trigger p53 accumulation and the resultant cell cycle arrest.

16 While our data do not support a role for p53 in aneuploidy surveillance in organotypic

cultures, there is strong evidence indicating that loss of p53 is associated with chromosome

instability and poor prognosis in the development of several cancers (Fujiwara et al., 2005;

Watson and Elledge, 2017; Donehower et al., 2019) and a majority of tumors show varied

TP53 (Clausen et al., 1998; Muller and Vousden, 2013). Certain hematological cancers however, show lower p53 mutational rates (Olivier, Hollstein and Hainaut, 2010;

Robles, Jen and Harris, 2016; Prokocimer, Molchadsky and Rotter, 2017) and our EdU data from the Nalm6 pre B lymphocyte cell line lacking p53 warrant further investigation

of other regulators of aneuploidy in blood cell lineages. Functional validation of

CRISPR/Cas9 screen hits LCMT1 and PDCD5 by generating monoclonal knockouts in

Nalm6 cells reveal p53 independent pathways that regulate cell fate post aneuploidy induction.

Observations via live cell imaging in mCO support a direct role for p53 in mitosis, the absence of which leads to a variety of mitotic aberrations, notably lagging chromosomes

and multipolar mitosis. An increase in lagging chromosomes was also reported for p53

deficient human liver and colon organoids. (Drost et al., 2015; Artegiani et al., 2020). Our results further suggest that the mitotic defect was not due to an inability to activate SAC, and in fact SAC activation reduced the occurrence of lagging chromosomes. It is thus possible that the loss of p53 leads to mitotic spindle errors that can be corrected with a longer mitotic duration produced by SAC. The multipolar mitosis phenotype may be associated with cytokinesis defects, which had been reported in p53 deficient cells

(Fujiwara et al., 2005). A better understanding of the precise role for p53 in suppressing

17 aneuploidy may allow therapeutic preventions of mitotic abnormalities and metastatic diseases associated with p53 loss.

18 Main Figure Legends

Figure 3.1. Induction of aneuploidy with MPS1i in mammalian cell lines and 3D organotypic cultures.

(A) Schematic of the workflow to induce aneuploidy and detect TP53 regulation in RPE1,

HCT116, Nalm6, NPC, Organoids and downstream cell fate analysis. (B) Representative

images of untreated and MPS1i treated RPE1 cells along with quantification of percent

numerical aneuploidy by metaphase spreads. Cell lines RPE1, HCT116, Nalm6, NPCs,

human mammary organoid (hMO) and mouse colon organoid (mCO) were treated with

400nM-1µM MPS1i for 24 hours followed by drug washout and recovery growth in drug-

free media for 24 hours (24r) before sample collection. Plots show percent numerical

aneuploidy with n≥3 biological replicates, n≥30 mitosis counted for each condition. Scale

bar 10µm. Error bars represent mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 one-

tailed Fisher’s exact test. (C) Histograms represent number of spreads with indicated

number of chromosomes by metaphase spreads of cells as described in (B). n≥30 mitotic

spreads counted each condition, representative results from three independent experiments.

(D) Chromosome number analysis and histogram representation for organoid culture mCO

and hMO treated similarly as described in (B).

Figure 3.2. Adherent RPE1 and HCT116 but not Nalm6 cells depend on p53 for

growth arrest post aneuploidy induction. (A) Levels of p53 and p21 as determined by

immunoblot analysis. Mammalian cells RPE1, HCT116, Nalm6, NPCs unsynchronized

populations either untreated or MPS1i treated for 24 hours followed by drug washout and

recovery growth in drug-free media for 24 hours (24r) and 48 hours (48r). Bleomycin,

doxorubicin and nutlin were used as positive controls for p53 and p21 induction. GAPDH

19 served as loading control. (B) Quantification of p53 fold change by densitometry analysis,

n≥3 biological replicates of the data in (A). (C) Representative FACS profiles of EdU

incorporation in untreated, MPS1i-treated and drug free recovery, nutlin-treated TP53+/+

or TP53-/- RPE1 cells. (D) Quantification of the percentages of EdU+ cells from the FACS

data, n≥3 biological replicates in the same experiment as in (C). Graphs show mean ± SEM.

* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 by one-tailed Fisher’s exact test, n.s.: not significant.

Figure 3.3. 3D organotypic cultures do not activate p53 or undergo growth arrest in response to aneuploidy. (A) Representative immunoblots from mouse colon organoids

(mCO) and human mammary organoids (hMO) showing p53 and p21 protein abundance

post aneuploidy induction with MPS1i and positive controls with cells treated with

bleomycin, nutlin or doxorubicin. (B) Quantification of p53 and p21 fold change by

densitometry analysis; n≥3 biological repeats of the data in (A). (C) Quantification of the

percentages of EdU+ cells from the FACS data, n≥3 biological replicates in TP53+/+ and

TP53-/- mCO either untreated or treated with MPS1i, followed by EdU incorporation for

16 hrs. (D) 3D reconstruction of a human mammary organoid showing EdU + cells along

with quantification, n≥3 biological replicates. Graphs show mean ± SEM. * p ≤ 0.05; ** p

≤ 0.01; *** p ≤ 0.001 by one-tailed Fisher’s exact test, n.s.: not significant.

Figure 3.4. TP53-/- mCO exhibit frequent mitotic aberrations. (A) Representative

frames from time-lapse movies of mCO with chromosome labeled with H2B-mNeon

undergoing normal mitosis (top row), mitosis with lagging chromosomes (middle row),

and multipolar mitosis (bottom row). Arrowheads indicate relevant mitotic aberrations (B)

20 Quantification of the percentage of mitoses with a mitotic error. Each data point represents

one organoid. Percentage was obtained by dividing the number of mitoses with an error by

total number of mitoses observed. (C) Quantification of time from nuclear envelope breakdown to anaphase onset. Each data point represents one mitosis. Data in B and C are mean ± SEM of n≥3 independent experiments. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 by

Wilcoxon ranked-sum test, n.s.: not significant (p > 0.05). See also Supplementary video

1-4.

Figure 4.1. NAlm6 CRISPR: (A) Outline of the genome wide CRISPR/Cas9 knockout

screen in Nalm6 cells. (B) Genotyping to confirm LCMT1 disruption in Nalm6 cells by

using CRISPR/Cas9 directed genome editing. Representative sequencing segment with

highlighted box showing the frameshift indel. (C) RANKS statistics and score used to plot

sgRNA enriched hits by NGS post aneuploidy induction. Genes related to cell cycle (navy

blue), novel candidates (orange) and not significant genes (grey). (D) EdU incorporation

in untreated, MPS1i-treated and drug free recovery, nutlin-treated LCMT1-/- or PDCD5-/-

Nalm6 cells. Quantification of the percentages of EdU+ cells from the FACS data, n≥3

biological replicates in the same experiment. Graphs show mean ± SEM. * p ≤ 0.05; ** p

≤ 0.01; *** p ≤ 0.001 by one-tailed Fisher’s exact test, n.s.: not significant.

SUPPLEMENTARY TITLES AND LEGENDS:

Figure S1: Acute aneuploidy induction using MPS1i and p53 response. (related to

Figure 1 and 2)

21 (A) Levels of pH2AX were determined by immunoblot analysis. Cells were either untreated or treated with MPS1i without or with recovery for 24 or 48 hours after drug

washouts. Treatments with 2.5mg/ml bleomycin and 500nM-1µM doxorubicin were performed as positive controls for p53 response after DNA damage, and nutlin 1µM was used as an additional positive control for p53. GAPDH served as loading control. n≥3 biological replicates each condition. Similar western blots for mCO and hMO (B). Parental

RPE1, HCT116 immunofluorescence assay showing TP53 nuclear intensity by antibody and Hoechst staining along with quantification (C, D). (>50 cells per condition per experiment), n≥3 biological replicates, graph shows mean ± SEM. p value of ≤ 0.05 was considered as the cut off for significance; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 by one- tailed Fisher’s exact test, n.s.: not significant. Scale bar 10µm.

Figure S2:TP53 knockout and cell response analysis post aneuploidy induction.

(related to Figure 3.1 and 3.2)

(A) Levels of p53 were determined by immunoblot analysis. RPE1, HCT116, Nalm6, mCO

and Nalm6 along with their TP53-/- counterparts were either untreated or treated with

MPS1i without or with recovery (r) for 24 or 48 hours after drug washouts. Treatments

with 2.5mg/ml bleomycin and 500nM-1µM doxorubicin were performed as positive

controls for p53 response after DNA damage and 1µM nutlin was used as an additional

positive control for p53 elevation. GAPDH served as loading control. (B) Genotyping to

confirm TP53 disruption in Nalm6 cells by using CRISPR/Cas9 directed genome editing.

Representative sequencing segment with highlighted box showing the frameshift indel. (C)

Representative example of histogram shows the number of spreads with indicated

chromosome numbers in cell lines lacking p53. n≥3 biological replicates with n≥30 mitotic

22 spreads counted for each condition. (D) Embryonic (left) and adult (right) NPC either untreated or treated with MPS1i, followed by EdU incorporation for 16 hrs after drug washout. EdU was detected after fixation and counterstaining with Hoechst 33342, and the

percentage of EdU + cells was quantified and plotted on Y-axis. (E) Representative gated

flow images for RPE1 cells either untreated or treated with MPS1i washout, followed by

staining. Annexin V positive cells were detected after fixation and counterstaining with

Propidium iodide, and percentage of AnnexinV + cells was quantified for different cell

lines along with TP53-/- counterparts and patient derived human mammary organoids

(hMO) by flow cytometry. n≥3 biological replicates, graphs show mean ± SEM. p value of

≤ 0.05 was considered as the cut off for significance; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 by one-tailed Fisher’s exact test, n.s.: not significant. Scale bar 10µm

Figure S3: 2D and 3D cultures of HCT116 had similar p53 responses to aneuploidy induction. (related to Figure 3.1 and 3.2)

(A) Representative phase contrast images of HCT116 cells grown as either 2D monolayer or as 3D spheroids embedded in matrigel (H3D) (B) Representative examples of metaphase chromosome spread images and histogram for HCT116 3D culture untreated or treated with MPS1i for 24 hours then washed out and recovered for 24 hours in drug free media before sample collection. n≥3 biological replicates with n≥30 mitosis counted each condition. Histograms represent chromosome number plotted against total number of chromosome spreads analyzed each condition. (C) HCT116 3D culture either untreated or treated with MPS1i or with bleomycin, nutlin and doxorubicin as positive controls, followed by EdU incorporation for 16 hrs. EdU was detected after fixation with DNA counterstaining with Hoechst 33342, and percentage of EdU + cells was quantified by flow

23 cytometry and/or imaging. Graphs show mean ± SEM. p ≤ 0.05 by one-tailed Fisher’s exact test. (D) Representative immunoblots of HCT116 cultured in 3D showing p53 and p21 protein abundance post treatments with MPS1i washouts, or bleomycin, nutlin or doxorubicin as positive controls. p53 and p21 fold change was quantified by densitometry analysis using n≥3 biological repeats each condition. Graphs show mean ± SEM. p value of ≤ 0.05 was considered as the cut off for significance; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤

0.001 by one-tailed Fisher’s exact test, n.s.= not significant. Scale bar 10µm

Figure S4: Loss of p53 increases aneuploidy in mCO. (related to Figure 3.2 and 3.3)

Representative examples of metaphase chromosome spread images and histograms for mCO TP53-/- either untreated or treated with MPS1i for 24 hours then washed out and recovered for 24 hours in drug free media before sample collection. n≥3 biological replicates with n≥30 mitosis counted each condition. Histograms represent chromosome number plotted against total number of chromosome spreads analyzed each condition.

24 Figure 3.1.

25 Figure 3.2.

26 Figure 3.3

27 Figure 3.4

28 Figure 4.1.

29 Supplementary Figure S1.

30 Supplementary Figure S2.

31 Supplementary Figure S3.

32 Supplementary Figure S4.

33 KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies p53 Mouse monoclonal [DO-1] Abcam CAT # AB1101 p53 (1C12) CAT # 2524 p21 Waf1/Cip1 (12D1) Cell Signaling CAT # 2974 p21 Antibody Mouse mAb (F-5) Santa Cruz CAT # sc-6246

GAPDH (D16H11) Cell Signaling CAT # 5174

Phospho–histone H2AX (Ser-139; 20E3) Cell Signaling CAT # 9718

Chemicals

NMS-P715 EMD/Millipore 475949

Nutlin-3 Sigma Aldrich N6287

Bleomycin EMD/Millipore 203410

Nocodazole Sigma Aldrich M1404

ZM 447439 Selleckchem S1103

Doxorubicin Sigma Aldrich AMBH324A4B72

Commercial Assays Click-iT™ EdU Alexa Fluor™ 488 Invitrogen C10337, C10420

Annexin V, Alexa Fluor™ 488 conjugate Invitrogen A13201

34 Experimental Models: Cell Lines

RPE-1 hTERT ATCC CRL-4000

Gift of Andrew Holland RPE-1 TP53-/- (JHU) NA

Gift of Bert Vogelstein HCT116 (JHU) NA

Gift of Bert Vogelstein HCT116 TP53-/- (JHU) NA

Nalm6 Gift of Mike Tyers (IRIC) NA

Other sgRNA plasmids and vectors used to generate cell lines in this study are available at Addgene

35 APPENDIX A. METHODS AND MATERIALS USED

Cell culture

hTERT-RPE1 (ATCC, Manassas, VA) were grown at 37°C in 5% CO2 in DMEM

supplemented with 10% fetal bovine serum (FBS). RPE-1 TP53-/- cells were a gift from

Andrew Holland lab. HCT116 cells were grown at 37°C in 5% CO2 in McCoy’s media supplemented with 10% fetal bovine serum (FBS). HCT116 TP53-/- cells were a gift from

Bert Vogelstein lab. Nalm6 cells were a gift from Mike Tyers lab and were grown at 37°C

in 5% CO2 in RPMI supplemented with 10% fetal bovine serum (FBS). Nalm6 TP53-/-

cells were generated in-house using lentiCRISPRV2. TP53 knockout was confirmed by

sequencing and western blot validation. Neuronal Progenitor cells isolated from embryonic

mouse brain (frontal) and adult 10 week old mice SVZ and DG areas and cultured as

neurospheres on ultra-low attachment plates using DMEM and Ham’s F-12 medium in a

1:1 ratio (DMEM/F-12; Omega Scientific, cat. no. DM-25) B27 serum-free supplement

(B27; Invitrogen/Gibco, cat. no. 17504-044) Basic fibroblast growth factor-2 (FGF-2;

PeproTech, cat. no. 100-18B-B) Epidermal growth factor (EGF; PeproTech, cat. no. 100-

15) l-Glutamine (Invitrogen/Gibco, cat. no. 2503-081) Antibiotic-antimycotic (Anti-Anti;

Invitrogen/Gibco, cat. no. 15240-062) as previously described in (Guo et al., 2012).

Colonoid isolation and culture

Mouse colon organoids were generated from wild-type C57BL/6J (The Jackson

Laboratory, 000664) and p53 KO mice (The Jackson Laboratory, 002101). All mouse care and use were approved by the Institutional Animal Care and Use Committee at the Johns

36 Hopkins University. p53 KO genotype was determined using the following primers: 5’-

CCCGAGTATCTGGAAGACAG-3’ and 5’- ATAGGTCGGCGGTTCAT-3’. Murine colon organoids were generated as previously described(Sato et al., 2011).The distal third of the colon was removed and rinsed with PBS. The distal colon was placed in complete chelating solution and minced. Minced tissue was then incubated with 150 rpm shaking in

31.25 mM EDTA in chelating solution for 30 minutes-1 hours at 4˚C to release crypts.

Tissue fragments were allowed to settle and supernatant containing crypts was collected.

Crypts were centrifuged at 200 x g for 5 minutes and embedded in Matrigel and seeded on

24 well plates. After solidification of Matrigel, 500 µl WENR culture medium was added(Sato et al., 2011). Media was changed every 2 days and colon organoids were passaged using enzymatic dissociation with TrypLE Express every 7 days.

Generation of primary human mammary organoids

Normal mammary reduction samples were acquired from the Cooperative Human Tissue

Network in compliance with a study protocol (NA_00077976) that was categorized as exempt/ not human subject research by the Johns Hopkins School of Medicine Institutional

Review Board. Each sample was deidentified prior to receipt and shipped overnight in basal DMEM/ RPMI medium. Upon receipt, samples were washed with an anti-fungal solution, minced, and digested in a collagenase (Sigma C2139) solution. Normal mammary epithelial organoids were then enriched by performing a series of differential centrifugation steps, as previously described (Nguyen-Ngoc et al., 2012). Mammary organoids were then embedded in growth factor reduced Matrigel and cultured in the presence of medium containing EGF, hydrocortisone, insulin, and cholera toxin (Cheung et al., 2013).

37 Lentivirus production

Plasmids used: pMD2.G (gift from Didier Trono (Addgene plasmid # 12259 ;

http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 (a gift from Didier Trono

(Addgene plasmid # 12260 ; http://n2t.net/addgene:12260 ; RRID:Addgene_12260), pLV-

H2B-Neon-ires-Puro (a gift from the Hugo Snippert and Geert Kops labs). HEK 293FT

cells were co-transfected with the lentiviral transfer plasmid, packaging plasmid, and

envelope plasmid. Media containing lentivirus was collected 24 and 48 hours after

transfection. Lentivirus was concentrated using a centrifugal filter (Amicon Ultra-15,

100,000 NMWL). Lentiviral titer was determined by qPCR (abm qPCR Lentivirus

Titration Kit, cat. # LV900).

Organoid live imaging

Colon organoids were transduced with H2B-Neon lentivirus as described previously

(Bolhaqueiro et al., 2018, 2019). Following transduction, colon organoids were cultured in

WENR culture media for 5-7 days. Colon organoids were then dissociated to single cells

and allowed to grow for 2 days in WENR culture media. Then, cells stably expressing the

construct were selected with 1 µg/ml puromycin for 2-4 days. For live imaging, selected

colon organoids were enzymatically dissociated with TrypLE Express and seeded on 8-

well coverglass bottom chamber slides. 3-5 days after seeding, organoids were imaged

using a laser-scanning confocal microscope (Zeiss 780). The environmental chamber was maintained at 37˚C with 5% CO2. Colon organoids were imaged with a 40x water-

immersion objective. Colon organoids were imaged every 3-3.5 minutes with 15-20 2.0

µM z slices. Images were processed using a custom ImageJ macro modified from the

38 ImageJ plugin “Temporal-Color Code” (Miura, 2010). Mitotic errors were scored

manually.

Metaphase chromosome spreads

Chromosome spreads were obtained by arresting cells in mitosis in Karyomax Colcemid

solution (1:100; Life Technologies) for 4–8 h, followed by harvesting using trypsin and/or

accutase. Trypsinized cells were collected by centrifugation for 5 min at 300 × g and gently

resuspended in a small amount of medium ( 1 ml). Resuspended cells were allowed to

swell for 7–10 min in 0.4% KCl solution at room∼ temperature and prefixed by addition of

freshly prepared methanol: acetic acid (3:1) fixative solution ( 100 μl per 10 ml of total volume). Prefixed cells were collected by centrifugation and fixed∼ in methanol:acetic acid

(3:1) fixative solution. Spreads were dropped on a glass slide and incubated at 65°C for at least 1 h.For counting, spreads were mounted in Vectashield containing 4′,6-diamidino-2-

phenylindole (DAPI; Vector Laboratories, Burlingame, CA), imaged on a Nikon TiE-

Eclipse epifluorescence microscope (60× oil immersion objective), and counted using the

Cell Counter plug-in in ImageJ (National Institutes of , Bethesda, MD).

Immunoblotting

Spheroids were isolated from Matrigel by incubation with dispase (Corning) at 37°C for 1

hr. Digestion was terminated by addition of 5 mM EDTA and spheroids resuspended by

trituration. Spheroids and cells were pelleted at 200 g for 5 min and resuspended in RIPA

buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 1% NP-40, 0.5% sodium

deoxycholate, 0.1% SDS) containing Complete Mini Protease Inhibitor Cocktail (Roche).

Lysates were homogenized using a rubber policeman, incubated on ice for 20 min, and

39 Centrifuged at 13,000 rpm at 4°C for 20 min. Protein concentration of supernatants was measured using Bradford dye (Bio-Rad) on a spectrophotometer. Lysates were diluted in

5X sample buffer (250 mM Tris pH 6.8, 50% glycerol, 5% β-mercaptoethanol 0.025% bromophenol blue, 5% SDS). Samples were separated on homemade polyacrylamide gels and transferred to Immobilon-P membranes (Millipore) via wet transfer. Membranes were blocked with 5% milk in TBST (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% Tween-20) for

1 hr at room temperature. Membranes were incubated in primary antibodies diluted in 5% milk in TBST at 4°C with rocking for 16 hr and washed with TBST for 10 min thrice.

Membranes were incubated in secondary antibodies diluted in TBST at room temperature with rocking for 1 hr and washed with TBST for 10 min thrice. Membranes were incubated in ECL Prime Western Blotting Detection Reagent (GE Healthcare) for 5 min and imaged on an ImageQuant LAS 4000 luminescent image analyzer (GE Healthcare). The following primary antibodies were used: β1 integrin (clone MAB1997, 1:1,000, Millipore) and β- actin (clone AC- 74, 1:20,000, Sigma). For β1 integrin, β-mercaptoethanol was eliminated from the 5X sample buffer. All secondary antibodies were ECL horseradish peroxidase linked (1:5,000, GE Healthcare.

EdU labeling

For EdU incorporation assays, cells were typically seeded in multiwell black, optically clear bottom, tissue culture–treated plates (PerkinElmer) for imaging and treated with 10

μM EdU (Thermo Fisher Scientific) for the indicated times. Cells were fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for 15 min and then permeabilized with 0.5% Triton X-100. Fixed cells were washed with PBS and stained with 1 μM Alexa Fluor 488 or Alexa Fluor 555–conjugated azide diluted in PBS containing

40 2 mM CuSO4 and 50 mM ascorbic acid. To counterstain the DNA, Hoechst 33342 was

added to 2 μg/ml. Cells were incubated for several hours or overnight at room temperature

protected from light and evaporation and then washed three times with PBS. Later they

were subjected to imaging using Nikon TiE-Eclipse epifluorescence microscope (60× oil immersion objective) and/or Attune flow analyzer.

Antibodies

We used the following antibodies: p53 Mouse monoclonal [DO-1] (Abcam: AB1101) and p53 (1C12) Mouse mAb (#2524

Cell Signaling Technology, Danvers, MA). p21 Waf1/Cip1 (12D1) rabbit mAb, 2947 (Cell

Signaling Technology) and p21 Antibody Mouse mAb (F-5): sc-6246. GAPDH (D16H11) rabbit mAb, (#5174 Cell Signaling Technology) Phospho–histone H2AX (Ser-139; 20E3) rabbit mAb, (#9718 Cell Signaling Technology) Secondary antibodies for immunofluorescence (Alexa 488 and Alexa 594 conjugates) were obtained from Life

Technologies. Secondary HRP-conjugated antibodies for Western blotting were from Cell

Signaling Technology and typically used at 1:5000 dilution.

Drug Treatments

We used the following chemical inhibitors: MPS1 Inhibitor, NMS-P715 - Calbiochem at

0.4µM-1µM, Nutlin-3 (Sigma-Aldrich) at 1-5 μM, Bleomycin (BLEOCIN, EMD

Millipore) at 2.5 μg/ml, Doxorubicin CAS Number 25316-40-9 (Sigma) at 0.5-5 μM and

ZM447439 (Selleckchem, Houston, TX) at 1μM.

41 QUANTIFICATION AND STATISTICAL ANALYSIS

General statistics

The number of biological replicates, the number of cells analyzed per biological replicate, and the statistical test performed are indicated in the figure legends. A biological replicate was defined as tissue or cells harvested from a single mouse or patient sample. Statistical

tests performed and figures generated using Prism (GraphPad) and/or R-Studio. For all

statistical tests, a cutoff of p < 0.05 was used to indicate significance.

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55 CV

EDUCATION

The Johns Hopkins School of Medicine, Baltimore MD

September 2015-July 2020

PhD, Human Genetics, Laboratory of Dr. Rong Li

Long Island University- POST, Greenvale NY

MS, Medical Biology, Laboratory of Dr. Marc Fink

December 2012

V.M.H.P.S College of Pharmacy, Mumbai, India

Bachelor of Pharmacy

May 2010

RESEARCH EXPERIENCE

PhD Student – Human Genetics, The Johns Hopkins University, Baltimore MD

September 2015 - Current

• Designed a genome wide CRISPR-Cas9 knockout screen to identify potential novel regulators of aneuploidy in different pre-B lymphocytes, performed large data mining post Next Gen Sequencing and functional validation assays in mammalian cells and organoids. • Studied TP53 differential regulation post chromosome mis-segregation using biochemical and cytogenetic assays in 2D adherent mammalian cell lines and compared it with 3D neurospheres and patient derived organoids. • Spearheaded a collaboration with Principal Investigator from Bioengineering Department at Hopkins to identify polymers that can enhance transfection efficiency of nanoparticles in euploid and aneuploid cells. • Managed and trained 1 graduate and 1 undergraduate student to complete projects.

56 Research Technician - Cancer Biology, Kansas University Medical Center, KS

January 2013 - September 2013

• Worked under the guidance of Dr. Tomoo Iwakuma in his lab which focuses on p53 mutations in osteosarcomas and osteochondromas. • Designed and assisted in projects involving targeting and degrading mutant p53 with techniques like novel drug targeting, hotspot mutation detection and siRNA. • Trained in mice handling and injection delivery.

Graduate Research Assistant - Long Island University, NY December 2011- December 2012 • Cellular Signaling Pathways focusing on PI3K/AKT cascades which involve P70S6K and related downstream substrates. Studied the effects of inhibitors on PhoshoS6 and P70S6K at Serine 240/244 and Threonine 389 under the guidance of Dr. Marc Fink at Long Island. • Worked on the regulation of mTOR signaling cascade focusing on the role of P70S6K in survival and proliferation of HER2+ breast cancer cell lines. • Conducted (WST-1) Cell Viability Assays on Ductal carcinoma cells used to determine the IC50 of novel inhibitors like PF-4708671 and also to assess the synergistic effects of combination therapy using BID1870 and PF4708671.

Undergraduate Research Intern - Sandoz Pvt. Ltd. (Subsidiary of Novartis), India May 2009 – June 2009 • Worked on Novel drug delivery system & Distilled water injection testing under guidance of Mr.Chandrashekhar Kaluskar (Head- Pharmaceutical Operations). • Assisted in the Microbiology, R&D, Production, Quality Control and Quality Assurance department. • Trained in Dissolution and Disintegration Apparatus and Organ bath.

PUBLICATIONS

• Zhou C, Slaughter BD, Unruh JR, Guo F, Yu Z, Mickey K, Narkar A, Trimble Ross R, McClain M, Li R. Organelle-Based Aggregation and Retention of Damaged Proteins in Asymmetrically Dividing Cells. Cell. 2014 • Iyer SV, Parrales A, Begani P, Narkar A, Adhikari AS, Martinez LA Iwakuma T. Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of- activities. Oncotarget. 2016 • Pei-Hsun Wu [et al, including Narkar A.] Single-Cell Morphology Encodes Metastatic Potential Science Advances. 2020 • Narkar AN, Bertomeu T., Albert Liu, Pandurang Bharne, Devin Mair, Anjali Nelliat, Mike Tyer, Rong Li. Genome-wide CRISPR/Cas9 screen reveals novel aneuploidy regulators. (Manuscript in preparation)

57 • Narkar AN, Blake Johnson, Pandurang Bharne, Jin Zhu, Veena Padmanaban, Debojyoti Biswas, Andrew Fraser, Pablo Iglesias, Andrew J Ewald, Rong Li. On p53’s role in the cellular response to aneuploidy. (Under Review at Developmental Cell)

TALKS, ABSTRACTS AND POSTER PRESENTATIONS

• Invited Speaker “Young Investigator Symposium” Cancer Early Detection Advanced Research Center (CEDAR) OHSU Knight Cancer Institute, Portland, October 2019. • Narkar AN, Blake Johnson, Debojyoti Biswas, Pandurang Bharne, Anjali Nelliat, Jin Zhu, Veena Padmanaban, Andrew Frazer, Rong Li. Understanding the role of TP53 in mediating aneuploid cell fate in different mammalian cells and patient derived organoids. 2nd Crick International Cancer Conference, London, September 2019. • Narkar AN, Pandurang Bharne, Anjali Nelliat, Jin Zhu, Blake Johnson, Debojyoti Biswas, Rong Li. Investigating how Nalm6 cells sense genome aberrations. MD- GEM Genetics Research Day, Johns Hopkins University, Maryland, April 2019. • Narkar AN, Pandurang Bharne, Anjali Nelliat, Jin Zhu, Blake Johnson, Debojyoti Biswas, Rong Li. Aneuploidy induces a significant p53 dependent cell cycle arrest in human adherent cells. American Society for Cell Biology -ASCB 2018. • Narkar AN, Yuping Wu, Andrei Kucharavy, Tamara Potapova, Jay Unruh, Rong Li. Embryonic and adult neural progenitors exhibit increased tolerance to aneuploidy. American Society for Cell Biology -ASCB 2014

GSA Travel Award Recipient Johns Hopkins University, Baltimore MD

September 2019

LEADERSHIP/MENTORSHIP EXPERIENCE

Director – Probono Consultant, Johns Hopkins Graduate Consulting Club February 2019- May 2020 • Worked with public and private consulting projects in medical and commercials fields and implemented efficient strategies for new market entry.

Teaching Assistant 2016

Institute of Genetic Medicine, The Johns Hopkins University, Baltimore MD • Selected by professors to coach Human Genetics graduate students and assist professors with lecture note preparations and paper discussions for advanced topics in Medical Genetics.

58 Intern and Student Orientation Leader 2012

International Student Services Office, Long Island University, NY • Guided over 160 students with international orientation, paperwork and appointments with office representatives. • Assisted office staff in data management, event coordination and record keeping.

“Sports Secretary” - Indian Pharmaceutical Association – MSB May 2010 • Spearheaded the inter-collegiate Rx sports festival featuring 13 Pharmaceutical colleges with 10 different sports events.

59