# Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression

Sujata Jana, Sandipan Brahma, Sonali Arora, Cynthia Wladyka, Patrick Hoang, Steven Blinka, Rowan Hough, Jessie Horn, Yuzhen Liu, Li-Jie Wang, et al.

► To cite this version:

Sujata Jana, Sandipan Brahma, Sonali Arora, Cynthia Wladyka, Patrick Hoang, et al. Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression. Cancer Cell, 2023, 41 (5), pp.853-870.e13. 10.1016/j.ccell.2023.03.021. hal-04603609

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# Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression

Sujata Jana, Sandipan Brahma, Sonali Arora, Cynthia Wladyka, Patrick Hoang, Steven Blinka, Rowan Hough, Jessie Horn, Yuzhen Liu, Li-Jie Wang, et al.

<figure><img src="image_2.png" /></figure>

► To cite this version:

Sujata Jana, Sandipan Brahma, Sonali Arora, Cynthia Wladyka, Patrick Hoang, et al. Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression. Cancer Cell, 2023, 41 (5), pp.853-870.e13. 10.1016/j.ccell.2023.03.021. hal-04603609

HAL Id: hal-04603609

https://hal.science/hal-04603609v1

Submitted on 6 Jun 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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Distributed under a Creative Commons Attribution 4.0 International License
# Cancer Cell

## Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression

**Graphical abstract**

<figure><img src="image_2.png" /></figure>

**Authors**

Sujata Jana, Sandipan Brahma, Sonali Arora, ..., Arvind R. Subramaniam, Steven Henikoff, Andrew C. Hsieh

**Correspondence**

ahsieh@fredhutch.org

**In brief**

Jana et al. reveal that ARID1A orchestrates a molecular conflict between gene-specific DNA transcription and mRNA translation in the bladder epithelium that is tumor suppressive. Transcriptional-translational conflict represents a new molecular stress that protects cells from transformation and must be overcome to enable cancer phenotypes.

**Highlights**

* Transcriptional-translational conflict is tumor suppressive in bladder epithelium

* De-repression of translation elongation enables oncogenic phenotypes

* ARID1A-low tumors are sensitive to drug-induced transcriptional-translational conflict

* Impaired DNA damage response is a collateral effect of gene expression conflict

<figure><img src="image_3.png" /></figure>
# Cancer Cell

## Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression

**Graphical abstract**

<figure><img src="image_4.png" /></figure>

**Authors**

Sujata Jana, Sandipan Brahma, Sonali Arora, ..., Arvind R. Subramaniam, Steven Henikoff, Andrew C. Hsieh

**Correspondence**

ahsieh@fredhutch.org

**In brief**

Jana et al. reveal that ARID1A orchestrates a molecular conflict between gene-specific DNA transcription and mRNA translation in the bladder epithelium that is tumor suppressive. Transcriptional-translational conflict represents a new molecular stress that protects cells from transformation and must be overcome to enable cancer phenotypes.

**Highlights**

* Transcriptional-translational conflict is tumor suppressive in bladder epithelium

* De-repression of translation elongation enables oncogenic phenotypes

* ARID1A-low tumors are sensitive to drug-induced transcriptional-translational conflict

* Impaired DNA damage response is a collateral effect of gene expression conflict

<figure><img src="image_5.png" /></figure>
Article

# Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression

Sujata Jana,<fnref n="1" /> Sandipan Brahma,<fnref n="2" /> Sonali Arora,<fnref n="1" /> Cynthia L. Wladyka,<fnref n="1" /> Patrick Hoang,<fnref n="1" /> Steven Blinka,<fnref n="1" />,<fnref n="3" /> Rowan Hough,<fnref n="1" />
Jessie L. Horn,<fnref n="1" /> Yuzhen Liu,<fnref n="1" /> Li-Jie Wang,<fnref n="1" /> Philippe Depeille,<fnref n="4" /> Eric Smith,<fnref n="4" /> Robert B. Montgomery,<fnref n="3" /> John K. Lee,<fnref n="1" />,<fnref n="3" />,<fnref n="5" />
Michael C. Haffner,<fnref n="1" />,<fnref n="5" /> Funda Vakar-Lopez,<fnref n="5" /> Petros Grivas,<fnref n="3" /> Jonathan L. Wright,<fnref n="6" /> Hung-Ming Lam,<fnref n="6" /> Peter C. Black,<fnref n="7" />
Jeroen P. Roosevelt,<fnref n="4" /> Alexey G. Ryazanov,<fnref n="8" /> Arvind R. Subramaniam,<fnref n="2" /> Steven Henikoff,<fnref n="2" />,<fnref n="9" /> and Andrew C. Hsieh<fnref n="1" />,<fnref n="3" />,<fnref n="10" />,<fnref n="11" />,*

## SUMMARY

We uncover a tumor-suppressive process in urothelium called transcriptional-translational conflict caused by deregulation of the central chromatin remodeling component ARID1A. Loss of *Arid1a* triggers an increase in a nexus of pro-proliferation transcripts, but a simultaneous inhibition of the eukaryotic elongation factor 2 (eEF2), which results in tumor suppression. Resolution of this conflict through enhancing translation elongation speed enables the efficient and precise synthesis of a network of poised mRNAs resulting in uncontrolled proliferation, clonogenic growth, and bladder cancer progression. We observe a similar phenomenon in patients with ARID1A-low tumors, which also exhibit increased translation elongation activity through eEF2. These findings have important clinical implications because ARID1A-deficient, but not ARID1A-proficient, tumors are sensitive to pharmacologic inhibition of protein synthesis. These discoveries reveal an oncogenic stress created by transcriptional-translational conflict and provide a unified gene expression model that unveils the importance of the crosstalk between transcription and translation in promoting cancer.

## INTRODUCTION

Major tumor suppressors such as TP53, PTEN, APC, and PDCD4 are negative regulators of mRNA translation. Downregulation of each gene leads to an increase in mRNA-specific translation through cap-dependent and cap-independent mechanisms enabling cancer pathogenesis.<fnref n="1" />⁻<fnref n="4" /> These findings suggest that a major function of tumor suppression is to downregulate the protein abundance of oncogenic gene networks. Recently, pan-cancer genome sequencing coupled with mechanistic work has uncovered a new class of tumor suppressors involved in chromatin remodeling.<fnref n="5" />⁻<fnref n="8" /> In particular, components of ATP-dependent SWI/SNF family nucleosome remodeling complexes are mutated in 20% of human cancers.<fnref n="9" /> Of these, the defining component of the BAF ATP-dependent SWI/SNF nucleosome remodeler called AT-rich interactive domain-containing protein 1A (ARID1A) is the

most frequently mutated.<fnref n="10" /> ARID1A is considered a tumor suppressor because genetic deletion leads to the transcriptional activation of oncogenic mRNAs, impairments in double-stranded DNA break repair and decatenation, and tumor progression.<fnref n="5" />⁻<fnref n="7" />,<fnref n="11" />,¹² It remains to be determined how ARID1A regulates protein synthesis in normal tissues.

We uncover a relationship between ARID1A and mRNA translation that challenges the prevailing dogma that tumor suppressors inhibit protein synthesis and introduces a tumor-suppressive process in urothelium called transcriptional-translational conflict. Although deletion of *Arid1a* promotes the transcription of a regulon of potentially oncogenic genes, this is countered by a paradoxical decrease in translation elongation rates. The resulting transcriptional-translational conflict specifically restrains the synthesis of pro-proliferation and mitogenic proteins and collateral DNA damage response (DDR) genes thereby

<footnote n="1">¹Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA</footnote>

<footnote n="2">²Basic Science Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA</footnote>

<footnote n="3">³Department of Medicine, University of Washington, Seattle, WA 98195, USA</footnote>

<footnote n="4">⁴Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA</footnote>

<footnote n="5">⁵Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, USA</footnote>

<footnote n="6">⁶Department of Urology, University of Washington, Seattle, WA 98915, USA</footnote>

<footnote n="7">⁷Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada</footnote>

<footnote n="8">⁸Department of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA</footnote>

<footnote n="9">⁹Howard Hughes Medical Institute, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA</footnote>

<footnote n="10">¹⁰Genome Sciences, University of Washington, Seattle, WA 98915, USA</footnote>

<footnote n="11">¹¹Lead contact</footnote>

<footnote>*Correspondence: ahsieh@fredhutch.org</footnote>

<footnote>https://doi.org/10.1016/j.ccell.2023.03.021</footnote>
Article

# Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression

Sujata Jana,<fnref n="1" /> Sandipan Brahma,<fnref n="2" /> Sonali Arora,<fnref n="1" /> Cynthia L. Wladyka,<fnref n="1" /> Patrick Hoang,<fnref n="1" /> Steven Blinka,<fnref n="1" />,<fnref n="3" /> Rowan Hough,<fnref n="1" />
Jessie L. Horn,<fnref n="1" /> Yuzhen Liu,<fnref n="1" /> Li-Jie Wang,<fnref n="1" /> Philippe Depeille,<fnref n="4" /> Eric Smith,<fnref n="4" /> Robert B. Montgomery,<fnref n="3" /> John K. Lee,<fnref n="1" />,<fnref n="3" />,<fnref n="5" />
Michael C. Haffner,<fnref n="1" />,<fnref n="5" /> Funda Vakar-Lopez,<fnref n="5" /> Petros Grivas,<fnref n="3" /> Jonathan L. Wright,<fnref n="6" /> Hung-Ming Lam,<fnref n="6" /> Peter C. Black,<fnref n="7" />
Jeroen P. Roosevelt,<fnref n="4" /> Alexey G. Ryazanov,<fnref n="8" /> Arvind R. Subramaniam,<fnref n="2" /> Steven Henikoff,<fnref n="2" />,<fnref n="9" /> and Andrew C. Hsieh<fnref n="1" />,<fnref n="3" />,<fnref n="10" />,<fnref n="11" />,*

## SUMMARY

We uncover a tumor-suppressive process in urothelium called transcriptional-translational conflict caused by deregulation of the central chromatin remodeling component ARID1A. Loss of *Arid1a* triggers an increase in a nexus of pro-proliferation transcripts, but a simultaneous inhibition of the eukaryotic elongation factor 2 (eEF2), which results in tumor suppression. Resolution of this conflict through enhancing translation elongation speed enables the efficient and precise synthesis of a network of poised mRNAs resulting in uncontrolled proliferation, clonogenic growth, and bladder cancer progression. We observe a similar phenomenon in patients with ARID1A-low tumors, which also exhibit increased translation elongation activity through eEF2. These findings have important clinical implications because ARID1A-deficient, but not ARID1A-proficient, tumors are sensitive to pharmacologic inhibition of protein synthesis. These discoveries reveal an oncogenic stress created by transcriptional-translational conflict and provide a unified gene expression model that unveils the importance of the crosstalk between transcription and translation in promoting cancer.

## INTRODUCTION

Major tumor suppressors such as TP53, PTEN, APC, and PDCD4 are negative regulators of mRNA translation. Downregulation of each gene leads to an increase in mRNA-specific translation through cap-dependent and cap-independent mechanisms enabling cancer pathogenesis.<fnref n="1" />⁻<fnref n="4" /> These findings suggest that a major function of tumor suppression is to downregulate the protein abundance of oncogenic gene networks. Recently, pan-cancer genome sequencing coupled with mechanistic work has uncovered a new class of tumor suppressors involved in chromatin remodeling.<fnref n="5" />⁻<fnref n="8" /> In particular, components of ATP-dependent SWI/SNF family nucleosome remodeling complexes are mutated in 20% of human cancers.<fnref n="9" /> Of these, the defining component of the BAF ATP-dependent SWI/SNF nucleosome remodeler called AT-rich interactive domain-containing protein 1A (ARID1A) is the

most frequently mutated.<fnref n="10" /> ARID1A is considered a tumor suppressor because genetic deletion leads to the transcriptional activation of oncogenic mRNAs, impairments in double-stranded DNA break repair and decatenation, and tumor progression.<fnref n="5" />⁻<fnref n="7" />,<fnref n="11" />,¹² It remains to be determined how ARID1A regulates protein synthesis in normal tissues.

We uncover a relationship between ARID1A and mRNA translation that challenges the prevailing dogma that tumor suppressors inhibit protein synthesis and introduces a tumor-suppressive process in urothelium called transcriptional-translational conflict. Although deletion of *Arid1a* promotes the transcription of a regulon of potentially oncogenic genes, this is countered by a paradoxical decrease in translation elongation rates. The resulting transcriptional-translational conflict specifically restrains the synthesis of pro-proliferation and mitogenic proteins and collateral DNA damage response (DDR) genes thereby

<footnote n="1">¹Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA</footnote>

<footnote n="2">²Basic Science Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA</footnote>

<footnote n="3">³Department of Medicine, University of Washington, Seattle, WA 98195, USA</footnote>

<footnote n="4">⁴Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA</footnote>

<footnote n="5">⁵Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, USA</footnote>

<footnote n="6">⁶Department of Urology, University of Washington, Seattle, WA 98915, USA</footnote>

<footnote n="7">⁷Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada</footnote>

<footnote n="8">⁸Department of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA</footnote>

<footnote n="9">⁹Howard Hughes Medical Institute, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA</footnote>

<footnote n="10">¹⁰Genome Sciences, University of Washington, Seattle, WA 98915, USA</footnote>

<footnote n="11">¹¹Lead contact</footnote>

<footnote>*Correspondence: ahsieh@fredhutch.org</footnote>

<footnote>https://doi.org/10.1016/j.ccell.2023.03.021</footnote>
A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

Pathway enrichment analysis of 262 upregulated mRNAs

<figure><img src="image_6.png" /></figure>

C

<figure><img src="image_7.png" /></figure>

D

<figure><img src="image_8.png" /></figure>

Bladder urothelium thickness

<figure><img src="image_9.png" /></figure>

E

<figure><img src="image_10.png" /></figure>

F

<figure><img src="image_11.png" /></figure>

G

Polysome profiling

<figure><img src="image_12.png" /></figure>

H

<figure><img src="image_13.png" /></figure>

I

<figure><img src="image_14.png" /><figcaption>**Figure 1.** Loss of ARID1A leads to gene-specific transcriptional-translational conflict</figcaption></figure>

<figure><img src="image_15.png" /></figure>

For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.ccell.2023.03.021.

(A) Schema of UBC-CreERT and K5-CreERT Arid1a flox/flox models.

(legend continued on next page)
A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

Pathway enrichment analysis of 262 upregulated mRNAs

<figure><img src="image_8.png" /></figure>

C

<figure><img src="image_9.png" /></figure>

D

<figure><img src="image_10.png" /></figure>

E

<figure><img src="image_11.png" /></figure>

F

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

G

Polysome profiling

<figure><img src="image_15.png" /></figure>

H

<figure><img src="image_16.png" /></figure>

I

<figure><img src="image_17.png" /><figcaption>**Figure 1.** Loss of ARID1A leads to gene-specific transcriptional-translational conflict</figcaption></figure>

<figure><img src="image_18.png" /></figure>

For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.ccell.2023.03.021.

(A) Schema of UBC-CreERT and K5-CreERT Arid1a flox/flox models.

(legend continued on next page)
preventing uncontrolled cell growth and cancer progression. Restoring translation elongation rates is needed to drive the tumor promoting properties of ARID1A loss. Remarkably, ARID1A-null tumors retain a memory of this translational dependence, which represents a selective therapeutic vulnerability. These findings reveal an oncogenic stress created by transcriptional-translational conflict that is a gatekeeper of urothelial homeostasis.

## RESULTS

### Loss of Arid1a leads to a gene-specific transcriptional-translational conflict in urothelium

To study the physiologic impact of deleting *Arid1a* on gene expression, we used a UBC-CreERT2;ROSA-LSL-Yfp<sup>KI/KI</sup>; *Arid1a*<sup>LoxP/LoxP</sup> mouse model (herein referred to as *Arid1a*<sup>fl/fl</sup>) in which the administration of tamoxifen leads to deletion of ARID1A and expression of YFP in multiple tissues (Figure 1A). Within the urothelium, we observed a recombination efficiency of 83% (Figure S1A). We focused on the urothelium because bladder cancer is the 6th most common malignancy in humans,¹³ and 25% of urothelial carcinoma patients exhibit *Arid1a* loss-of-function mutations.¹⁴⁻¹⁶ Given that *Arid1a* loss has been shown to increase transcript levels of oncogenic genes in other tissues,⁵ we sought to determine if the same is true within urothelial cells. We isolated basal cells, a cell of origin of invasive urothelial malignancies,¹⁷,¹⁸ from wild-type (WT) and *Arid1a*<sup>fl/fl</sup> bladders and conducted RNA sequencing (RNA-seq) (Figure S1B). We observed 262 upregulated mRNAs in *Arid1a*<sup>fl/fl</sup> cells, and 80% of these were direct targets of *Arid1a* (Figure S1C; Table S1). Most overexpressed mRNAs were classified as regulators of cell proliferation (Figure 1B). Furthermore, these genes included aurora kinase B (*Aurkb*), insulin-like growth factor 2 (*Igf2*), fibroblast growth factor receptor 3 (*Fgfr3*), and ornithine decarboxylase 1 (*Odc1*), all of which are cancer drivers (Figure 1C).¹⁹⁻²² Overexpression of *Aurkb*, *Igf2*, or *Fgfr3* in transgenic models is sufficient to drive uncontrolled cell growth and tumor formation.²³⁻²⁵ Given these findings, we subsequently aged *Arid1a*<sup>fl/fl</sup> mice for 400 days to determine if they develop bladder tumors. Despite the transcriptional activation of these gene networks, *Arid1a*<sup>fl/fl</sup> mice exhibited a decrease in urothelial thickness characterized by fewer basal cells, and 0 of 9 mice developed tumors (Figure 1D).

These findings raise the important question of what might counter this gene expression program to prevent tumor initiation.

One possibility is that *Arid1a* simultaneously controls protein synthesis. To measure protein synthesis rates *in vivo* we conducted a puromycin incorporation assay²⁶ and observed a 60% decrease in *de novo* protein synthesis in ARID1A-deficient urothelium (Figure 1E). We also generated an inducible K5-CreERT2;ROSA-LSL-Yfp<sup>KI/KI</sup>; *Arid1a*<sup>LoxP/LoxP</sup> model (referred to as K5 *Arid1a*<sup>fl/fl</sup>) with 81%-87% recombination efficiency in basal cells in which we observed the same reduction in protein synthesis with striking cell type specificity (Figures S1D and S1E). To determine if this decrease was cell autonomous, we measured protein synthesis rates in organoids grown from *Arid1a*<sup>fl/fl</sup> basal urothelium. Puromycin incorporation and ³⁵S-methionine incorporation were decreased in *Arid1a*-deficient organoids (Figures 1F and S1F). Although ARID1A loss increases a distinct network of pro-proliferative and potentially oncogenic network of mRNAs (Figure 1C), it is possible that it can also cause widespread transcriptional attenuation that would result in the perception of decreased protein synthesis.²⁷ We conducted RNA-seq of spike-in normalized²⁸ WT and *Arid1a*<sup>fl/fl</sup> basal urothelial organoids. No widespread decrease in mRNA levels across the transcriptome were observed (Figure S1G). Last, we added back *Arid1a* in *Arid1a*-deficient organoids, which restored protein synthesis rates (Figure S1H). These findings demonstrate that *Arid1a* loss leads to opposing effects on transcription and translation, which is associated with an absence of tumor formation.

Next, we sought to determine how decreased *de novo* protein synthesis affects ribosome abundance on mRNA by polysome analysis.²⁹ We observed that *Arid1a*<sup>fl/fl</sup> urothelial cells exhibited a significant increase in poly-ribosome abundance normalized to the sub-polysomal fraction (P/S ratio) (Figure 1G). Increased P/S ratio in the context of decreased protein synthesis can be caused by slower elongation rates.³ To determine if ARID1A loss slows down translation elongation, we measured the ribosome half transit time.³⁰ We observed a half transit time of 85.5 ± 13 s in WT organoids and 143 ± 42 s in *Arid1a*<sup>fl/fl</sup> organoids, which supports the idea that protein synthesis is disrupted at the level of elongation (Figure S1I).

These results raise the question of how decreased ribosome transit time affects the translation of distinct mRNA species, particularly the 262 mRNAs that are upregulated upon loss of *Arid1a* (Figure 1C). We sequenced ribosome bound mRNA to measure P/S ratios transcriptome-wide (Figure S1J). Strikingly, 70% of the 262 upregulated mRNAs in *Arid1a*<sup>fl/fl</sup> cells exhibited increases in P/S ratio, providing genome-level evidence of a

<footnote>(B) Pathway enrichment analysis of 262 mRNAs upregulated in the context of ARID1A loss.</footnote>

<footnote>(C) Heatmap showing a subset of pro-proliferation upregulated mRNAs from *Arid1a*<sup>fl/fl</sup> organoids (>1.2 log₂ fold change, FDR < 0.05).</footnote>

<footnote>(D) H&E staining (left with keratin 5, inset) and quantification (right) of mouse bladder urothelial thickness (total [n = 4/genotype] and basal cells [n = 3/genotype]) in 400-day-old WT and *Arid1a*<sup>fl/fl</sup> mice. p < 0.0001 (t test).</footnote>

<footnote>(E) Immunohistochemistry (IHC) of ARID1A (left) and parallel sectioning and immunofluorescence (IF) of puromycin incorporation (right) in WT and *Arid1a*<sup>fl/fl</sup> urothelium (DAPI, nuclei). n > 6/genotype, >8,900 cells/genotype. p < 0.0001 (t test).</footnote>

<footnote>(F) Puromycin immunoblot of WT and *Arid1a*<sup>fl/fl</sup> urothelial organoids (replicate of 3).</footnote>

<footnote>(G) Schema of the polysome profiling assay (top). Polysome tracing showing an increase in polysome accumulation (left) and quantification showing an increase in the polysome (P) to sub-polysome (S) ratio (mean ± SEM). n = 2/genotype, t test.</footnote>

<footnote>(H) Waterfall plot showing polysome (P) to sub-polysome (S) ratio of 262 upregulated oncogenic mRNAs in *Arid1a*<sup>fl/fl</sup> mice.</footnote>

<footnote>(I) Volcano plot of TMT mass spectrometry showing that 70% of upregulated mRNAs (105 of 150; only 150 of the 262 genes were detected by TMT mass spectrometry) identified by RNA-seq (Figure 1C; Table S1) do not increase in protein abundance (<0.67 log₂ fold change and/or FDR > 0.05, green dots) in *Arid1a*<sup>fl/fl</sup> mice. Vertical lines demarcate log₂ fold change ± 0.67, and horizontal line demarcates FDR < 0.05 or >0.05.</footnote>

<footnote>All scale bars, 100 µm. Also see Figure S1.</footnote>
preventing uncontrolled cell growth and cancer progression. Restoring translation elongation rates is needed to drive the tumor promoting properties of ARID1A loss. Remarkably, ARID1A-null tumors retain a memory of this translational dependence, which represents a selective therapeutic vulnerability. These findings reveal an oncogenic stress created by transcriptional-translational conflict that is a gatekeeper of urothelial homeostasis.

## RESULTS

### Loss of Arid1a leads to a gene-specific transcriptional-translational conflict in urothelium

To study the physiologic impact of deleting *Arid1a* on gene expression, we used a UBC-Cre<sup>ERT2</sup>;ROSA-LSL-Yfp<sup>KI/KI</sup>; *Arid1a<sup>LoxP/LoxP</sup>* mouse model (herein referred to as *Arid1a<sup>fl/fl</sup>* ) in which the administration of tamoxifen leads to deletion of ARID1A and expression of YFP in multiple tissues (Figure 1A). Within the urothelium, we observed a recombination efficiency of 83% (Figure S1A). We focused on the urothelium because bladder cancer is the 6th most common malignancy in humans,<sup>13</sup> and 25% of urothelial carcinoma patients exhibit *Arid1a* loss-of-function mutations.<sup>14–16</sup> Given that *Arid1a* loss has been shown to increase transcript levels of oncogenic genes in other tissues,<sup>5</sup> we sought to determine if the same is true within urothelial cells. We isolated basal cells, a cell of origin of invasive urothelial malignancies,<sup>17,18</sup> from wild-type (WT) and *Arid1a<sup>fl/fl</sup>* bladders and conducted RNA sequencing (RNA-seq) (Figure S1B). We observed 262 upregulated mRNAs in *Arid1a<sup>fl/fl</sup>* cells, and 80% of these were direct targets of *Arid1a* (Figure S1C; Table S1). Most overexpressed mRNAs were classified as regulators of cell proliferation (Figure 1B). Furthermore, these genes included aurora kinase B (*Aurkb*), insulin-like growth factor 2 (*Igf2*), fibroblast growth factor receptor 3 (*Fgfr3*), and ornithine decarboxylase 1 (*Odc1*), all of which are cancer drivers (Figure 1C).<sup>19–22</sup> Overexpression of *Aurkb*, *Igf2*, or *Fgfr3* in transgenic models is sufficient to drive uncontrolled cell growth and tumor formation.<sup>23–25</sup> Given these findings, we subsequently aged *Arid1a<sup>fl/fl</sup>* mice for 400 days to determine if they develop bladder tumors. Despite the transcriptional activation of these gene networks, *Arid1a<sup>fl/fl</sup>* mice exhibited a decrease in urothelial thickness characterized by fewer basal cells, and 0 of 9 mice developed tumors (Figure 1D).

These findings raise the important question of what might counter this gene expression program to prevent tumor initiation.

One possibility is that *Arid1a* simultaneously controls protein synthesis. To measure protein synthesis rates *in vivo* we conducted a puromycin incorporation assay<sup>26</sup> and observed a 60% decrease in *de novo* protein synthesis in ARID1A-deficient urothelium (Figure 1E). We also generated an inducible K5-Cre<sup>ERT2</sup>;ROSA-LSL-Yfp<sup>KI/KI</sup>;*Arid1a<sup>LoxP/LoxP</sup>* model (referred to as K5 *Arid1a<sup>fl/fl</sup>*) with 81%-87% recombination efficiency in basal cells in which we observed the same reduction in protein synthesis with striking cell type specificity (Figures S1D and S1E). To determine if this decrease was cell autonomous, we measured protein synthesis rates in organoids grown from *Arid1a<sup>fl/fl</sup>* basal urothelium. Puromycin incorporation and <sup>35</sup>S-methionine incorporation were decreased in *Arid1a*-deficient organoids (Figures 1F and S1F). Although ARID1A loss increases a distinct network of pro-proliferative and potentially oncogenic network of mRNAs (Figure 1C), it is possible that it can also cause widespread transcriptional attenuation that would result in the perception of decreased protein synthesis.<sup>27</sup> We conducted RNA-seq of spike-in normalized<sup>28</sup> WT and *Arid1a<sup>fl/fl</sup>* basal urothelial organoids. No widespread decrease in mRNA levels across the transcriptome were observed (Figure S1G). Last, we added back *Arid1a* in *Arid1a*-deficient organoids, which restored protein synthesis rates (Figure S1H). These findings demonstrate that *Arid1a* loss leads to opposing effects on transcription and translation, which is associated with an absence of tumor formation.

Next, we sought to determine how decreased *de novo* protein synthesis affects ribosome abundance on mRNA by polysome analysis.<sup>29</sup> We observed that *Arid1a<sup>fl/fl</sup>* urothelial cells exhibited a significant increase in poly-ribosome abundance normalized to the sub-polysomal fraction (P/S ratio) (Figure 1G). Increased P/S ratio in the context of decreased protein synthesis can be caused by slower elongation rates.<sup>3</sup> To determine if ARID1A loss slows down translation elongation, we measured the ribosome half transit time.<sup>30</sup> We observed a half transit time of 85.5 ± 13 s in WT organoids and 143 ± 42 s in *Arid1a<sup>fl/fl</sup>* organoids, which supports the idea that protein synthesis is disrupted at the level of elongation (Figure S1I).

These results raise the question of how decreased ribosome transit time affects the translation of distinct mRNA species, particularly the 262 mRNAs that are upregulated upon loss of *Arid1a* (Figure 1C). We sequenced ribosome bound mRNA to measure P/S ratios transcriptome-wide (Figure S1J). Strikingly, 70% of the 262 upregulated mRNAs in *Arid1a<sup>fl/fl</sup>* cells exhibited increases in P/S ratio, providing genome-level evidence of a

(B) Pathway enrichment analysis of 262 mRNAs upregulated in the context of ARID1A loss.

(C) Heatmap showing a subset of pro-proliferation upregulated mRNAs from *Arid1a<sup>fl/fl</sup>* organoids (>1.2 log₂ fold change, FDR < 0.05).

(D) H&E staining (left with keratin 5, inset) and quantification (right) of mouse bladder urothelial thickness (total [n = 4/genotype] and basal cells [n = 3/genotype]) in 400-day-old WT and *Arid1a<sup>fl/fl</sup>* mice. p < 0.0001 (t test).

(E) Immunohistochemistry (IHC) of ARID1A (left) and parallel sectioning and immunofluorescence (IF) of puromycin incorporation (right) in WT and *Arid1a<sup>fl/fl</sup>* urothelium (DAPI, nuclei). n > 6/genotype, >8,900 cells/genotype. p < 0.0001 (t test).

(F) Puromycin immunoblot of WT and *Arid1a<sup>fl/fl</sup>* urothelial organoids (replicate of 3).

(G) Schema of the polysome profiling assay (top). Polysome tracing showing an increase in polysome accumulation (left) and quantification showing an increase in the polysome (P) to sub-polysome (S) ratio (mean ± SEM). n = 2/genotype, t test.

(H) Waterfall plot showing polysome (P) to sub-polysome (S) ratio of 262 upregulated oncogenic mRNAs in *Arid1a<sup>fl/fl</sup>* mice.

(I) Volcano plot of TMT mass spectrometry showing that 70% of upregulated mRNAs (105 of 150; only 150 of the 262 genes were detected by TMT mass spectrometry) identified by RNA-seq (Figure 1C; Table S1) do not increase in protein abundance (<0.67 log₂ fold change and/or FDR > 0.05, green dots) in *Arid1a<sup>fl/fl</sup>* mice. Vertical lines demarcate log₂ fold change ± 0.67, and horizontal line demarcates FDR < 0.05 or >0.05.

All scale bars, 100 µm. Also see Figure S1.
<figure><img src="image_16.png" /></figure>

A

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B

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C

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eEF2K

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D

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G

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<figure><img src="image_27.png" /><figcaption>Figure 2. ARID1A is a positive regulator of mRNA translation elongation</figcaption></figure>

(A) Immunoblots of regulators of mRNA translation in WT and *Arid1a*<sup>fl/fl</sup> urothelial organoids (replicate of 3).

(B) IHC and quantification of phospho-eEF2 (T56) and total eEF2 levels in WT and *Arid1a*<sup>fl/fl</sup> bladder urothelium. n ≥ 3/genotype. p = 0.02 (t test).

(C) IHC or IF and quantification of total eEF2K and phospho-eEF2K (S366) levels in WT and *Arid1a*<sup>fl/fl</sup> bladder urothelium. Violin plot represents >9,500 cells per genotype. n ≥ 4/genotype. p = 0.04 (left), p < 0.0001 (right) (t test).

(D and E) IHC analysis of phospho-eEF2 (T56) and IF analysis of puromycin incorporation after treatment with A-484954 in *Arid1a*<sup>fl/fl</sup> mice. Violin plot represents >7,700 cells/genotype. n ≥ 4/treatment arm p = 0.0006 (D), p < 0.0001 (E) (t test).

(F and G) IHC analysis of phospho-eEF2 (T56) and IF analysis of puromycin incorporation in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mice. Violin plot represents >6,000 cells/genotype. n ≥ 5/genotype. p = 0.02 (F), p < 0.0001 (G) (t test).
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eEF2K

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D

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<figure><img src="image_29.png" /><figcaption>Figure 2. ARID1A is a positive regulator of mRNA translation elongation</figcaption></figure>

(A) Immunoblots of regulators of mRNA translation in WT and *Arid1a*<sup>fl/fl</sup> urothelial organoids (replicate of 3).

(B) IHC and quantification of phospho-eEF2 (T56) and total eEF2 levels in WT and *Arid1a*<sup>fl/fl</sup> bladder urothelium. n ≥ 3/genotype. p = 0.02 (t test).

(C) IHC or IF and quantification of total eEF2K and phospho-eEF2K (S366) levels in WT and *Arid1a*<sup>fl/fl</sup> bladder urothelium. Violin plot represents >9,500 cells per genotype. n ≥ 4/genotype. p = 0.04 (left), p < 0.0001 (right) (t test).

(D and E) IHC analysis of phospho-eEF2 (T56) and IF analysis of puromycin incorporation after treatment with A-484954 in *Arid1a*<sup>fl/fl</sup> mice. Violin plot represents >7,700 cells/genotype. n ≥ 4/treatment arm p = 0.0006 (D), p < 0.0001 (E) (t test).

(F and G) IHC analysis of phospho-eEF2 (T56) and IF analysis of puromycin incorporation in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mice. Violin plot represents >6,000 cells/genotype. n ≥ 5/genotype. p = 0.02 (F), p < 0.0001 (G) (t test).
conflict between transcription of these genes and their translation (Figure 1H). Likewise, using tandem-mass-tag (TMT) mass spectrometry and immunoblot analysis, we found that 70% of the upregulated mRNAs also exhibited no increase in protein levels (Figures 1I and S1K; Table S2). These conflicted genes were enriched for guanine and cytosine nucleotides within their coding sequences (CDS), which is consistent with previous findings that higher guanine and cytosine content in the CDS is associated with slower codons (Figure S1L).³¹ Thus, loss of ARID1A prevents the efficient translation of the majority of upregulated mRNAs, leading to transcriptional-translational conflict. These findings raise the important questions of how ARID1A regulates translation elongation.

## ARID1A is a central regulator of eukaryotic elongation factor 2

Next, we sought to determine the mechanism by which ARID1A regulates translation elongation by conducting a candidate gene analysis of regulators of protein synthesis. We found no differences in 5.8S and 5S rRNA levels (Figure S2A). Next, we considered the PI3K signaling pathway (PTEN and AKT [total and phospho-S473]), the integrated stress response (phospho-eIF2α-S51 and ATF4), the eIF4F translation initiation complex (4EBP1 [total and phospho-T37/46], eIF4G, eIF4E, and eIF4A), and a translation elongation regulator (eEF2 [total and phospho-T56]).³²⁻³⁴ Of these, only eEF2 phosphorylation was significantly increased upon Arid1a loss in vitro and in vivo (Figures 2A and 2B). eEF2 catalyzes GTP-dependent ribosome translocation during translation elongation, and its phosphorylation at T56 decreases protein synthesis rates.³⁵ The primary kinase responsible for eEF2 phosphorylation is the eukaryotic elongation factor 2 kinase (eEF2K), which regulates the cellular response to nutrient deprivation.³⁵⁻³⁷ We measured eEF2K mRNA and protein levels and observed increases in both in Arid1a<sup>fl/fl</sup> urothelium, coinciding with a significant reduction in its inactive S366 phosphorylated form (Figures 2C, S2B, and S2C).

We used a pharmacogenetic strategy to determine if eEF2K activity regulates protein synthesis and elongation speed in Arid1a<sup>fl/fl</sup> urothelium. First, we treated Arid1a<sup>fl/fl</sup> mice with the eEF2K small-molecule inhibitor A-484954.³⁸ We observed an attenuation of eEF2 phosphorylation concomitant to restoration of protein synthesis in Arid1a<sup>fl/fl</sup> bladders (Figures 2D, 2E, and S2D). Next, we intercrossed Arid1a<sup>fl/fl</sup> and Eef2k<sup>-/-</sup> mice³⁹ to develop the Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mouse model in which eEF2 can no longer be phosphorylated (Figures 2F and S2E). We found that loss of Eef2k restores protein synthesis in Arid1a-deficient urothelium (Figure 2G). Furthermore, we determined that loss of Eef2k restores translation elongation rates in Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> organoids back to WT levels in a cell-autonomous manner (ribosome half transit time of 80.2 ± 28 s) (Figures 2H and S1I). These data show that ARID1A maintains mRNA translation elongation by promoting eEF2 activity through downregulation of eEF2K. However, in the context of ARID1A loss, eEF2 activity decreases leading to slower ribosome transit

time and a conflict between upregulated transcripts and their subsequent translation.

## RASGRP1 is necessary to maintain eEF2 activity

Next, we sought to determine how ARID1A negatively regulates eEF2K activity. We have shown that ARID1A deficiency leads to a decrease in eEF2K phosphorylation at serine 366 (Figure 2C). The decrease of this inhibitory post-translational modification of eEF2K leads to an increase in eEF2 phosphorylation.⁴⁰ The MAP kinase (MAPK) and PI3K-AKT-mTOR signaling pathways converge on eEF2K S366 (Figure 3A).⁴⁰ In addition, an AMP kinase (AMPK) activating eEF2K phosphorylation site at serine 398 has also been described.⁴¹ We found that ARID1A loss in urothelial organoids does not decrease PI3K-AKT-mTOR signaling or increase AMPK activity, suggesting these pathways are not responsible for the increase in eEF2 phosphorylation caused by ARID1A deficiency (Figures 2A and S3A). However, MEK1/2 (S217/221), ERK (T202/Y204), and p90RSK (S380) phosphorylation levels decreased in Arid1a-deficient urothelium without affecting total protein abundance or transcript levels (Figures 3B, S3B, and S3C). This finding suggests that the MAPK pathway is downregulated in Arid1a-deficient bladder urothelium which may affect eEF2K activity.

To determine how ARID1A controls the MAPK pathway, we measured mRNA abundance of MAPK regulators in WT and Arid1a<sup>fl/fl</sup> urothelial organoids. Surveying components in the Ras-MAPK pathway, we found no difference in expression of Hras, Kras, Nras, Shc1, Src, Braf, Craf, or Sos1 (Figure 3C). However, Rasgrp1 transcript levels were decreased by 80%, and RASGRP1 protein abundance was also decreased in Arid1a-deficient urothelium (Figures 3C, 3D, and S3D). Furthermore, ARID1A binds to the Rasgrp1 promoter in WT urothelial cells (Figure 3E), and Rasgrp1 mRNA and protein levels were restored by adding back ARID1A in Arid1a<sup>fl/fl</sup> organoids (Figures S3E and S3F). RASGRP1 is a diacylglycerol-regulated RasGEF, which activates the MAPK pathway with functions in T cells, T cell leukemia, and the intestinal epithelium.⁴²⁻⁴⁵ However, its role in the urothelium is unknown. To determine if the decrease in RASGRP1 can increase eEF2K activity, we measured eEF2 phosphorylation in Rasgrp1<sup>-/-</sup> urothelium. Strikingly, Rasgrp1<sup>-/-</sup> urothelium exhibited a significant increase in eEF2 phosphorylation (Figures 3F and 3G), independent of total eEF2 protein levels (Figure S3G), which is consistent with Arid1a<sup>fl/fl</sup> mice.

We next sought to delineate how ARID1A regulates Rasgrp1 expression. We conducted CUT&Tag chromatin profiling of histone H3 trimethylation of lysine 27 (H3K27me3) because downregulation of SWI/SNF activity is associated with H3K27me3 accumulation and repression of distinct genes.⁴⁶,⁴⁷ We observed that 2,558 gene promoters exhibited greater than 2-fold increases in the H3K27me3 in Arid1a<sup>fl/fl</sup> organoids (Figure S3H). Moreover, on a gene-specific level, H3K27me3 at the Rasgrp1 promoter was enriched 4.5-fold in Arid1a-deficient urothelial organoids and decreased in ARID1A add-back

<footnote>(H) Ribosome half-transit time in Arid1a<sup>fl/fl</sup> and Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> organoids (linear regression, left panels). Bar graph (right) represents the average ribosome half-transit time of 3 independent experiments. PMS, post-mitochondrial supernatants (complete + nascent proteins); PRS, post-ribosomal supernatant (complete proteins). p = 0.02 (t test).</footnote>

<footnote>n.s., not significant. Scale bar, 100 µm; mean ± SEM. Also see Figure S2.</footnote>
conflict between transcription of these genes and their translation (Figure 1H). Likewise, using tandem-mass-tag (TMT) mass spectrometry and immunoblot analysis, we found that 70% of the upregulated mRNAs also exhibited no increase in protein levels (Figures 1I and S1K; Table S2). These conflicted genes were enriched for guanine and cytosine nucleotides within their coding sequences (CDS), which is consistent with previous findings that higher guanine and cytosine content in the CDS is associated with slower codons (Figure S1L).31 Thus, loss of ARID1A prevents the efficient translation of the majority of upregulated mRNAs, leading to transcriptional-translational conflict. These findings raise the important questions of how ARID1A regulates translation elongation.

## ARID1A is a central regulator of eukaryotic elongation factor 2

Next, we sought to determine the mechanism by which ARID1A regulates translation elongation by conducting a candidate gene analysis of regulators of protein synthesis. We found no differences in 5.8S and 5S rRNA levels (Figure S2A). Next, we considered the PI3K signaling pathway (PTEN and AKT [total and phospho-S473]), the integrated stress response (phospho-eIF2α-S51 and ATF4), the eIF4F translation initiation complex (4EBP1 [total and phospho-T37/46], eIF4G, eIF4E, and eIF4A), and a translation elongation regulator (eEF2 [total and phospho-T56]).32–34 Of these, only eEF2 phosphorylation was significantly increased upon Arid1a loss in vitro and in vivo (Figures 2A and 2B). eEF2 catalyzes GTP-dependent ribosome translocation during translation elongation, and its phosphorylation at T56 decreases protein synthesis rates.35 The primary kinase responsible for eEF2 phosphorylation is the eukaryotic elongation factor 2 kinase (eEF2K), which regulates the cellular response to nutrient deprivation.35–37 We measured eEF2K mRNA and protein levels and observed increases in both in Arid1a<sup>fl/fl</sup> urothelium, coinciding with a significant reduction in its inactive S366 phosphorylated form (Figures 2C, S2B, and S2C).

We used a pharmacogenetic strategy to determine if eEF2K activity regulates protein synthesis and elongation speed in Arid1a<sup>fl/fl</sup> urothelium. First, we treated Arid1a<sup>fl/fl</sup> mice with the eEF2K small-molecule inhibitor A-484954.38 We observed an attenuation of eEF2 phosphorylation concomitant to restoration of protein synthesis in Arid1a<sup>fl/fl</sup> bladders (Figures 2D, 2E, and S2D). Next, we intercrossed Arid1a<sup>fl/fl</sup> and Eef2k<sup>-/-</sup> mice39 to develop the Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mouse model in which eEF2 can no longer be phosphorylated (Figures 2F and S2E). We found that loss of Eef2k restores protein synthesis in Arid1a-deficient urothelium (Figure 2G). Furthermore, we determined that loss of Eef2k restores translation elongation rates in Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> organoids back to WT levels in a cell-autonomous manner (ribosome half transit time of 80.2 ± 28 s) (Figures 2H and S1I). These data show that ARID1A maintains mRNA translation elongation by promoting eEF2 activity through downregulation of eEF2K. However, in the context of ARID1A loss, eEF2 activity decreases leading to slower ribosome transit

time and a conflict between upregulated transcripts and their subsequent translation.

## RASGRP1 is necessary to maintain eEF2 activity

Next, we sought to determine how ARID1A negatively regulates eEF2K activity. We have shown that ARID1A deficiency leads to a decrease in eEF2K phosphorylation at serine 366 (Figure 2C). The decrease of this inhibitory post-translational modification of eEF2K leads to an increase in eEF2 phosphorylation.40 The MAP kinase (MAPK) and PI3K-AKT-mTOR signaling pathways converge on eEF2K S366 (Figure 3A).40 In addition, an AMP kinase (AMPK) activating eEF2K phosphorylation site at serine 398 has also been described.41 We found that ARID1A loss in urothelial organoids does not decrease PI3K-AKT-mTOR signaling or increase AMPK activity, suggesting these pathways are not responsible for the increase in eEF2 phosphorylation caused by ARID1A deficiency (Figures 2A and S3A). However, MEK1/2 (S217/221), ERK (T202/Y204), and p90RSK (S380) phosphorylation levels decreased in Arid1a-deficient urothelium without affecting total protein abundance or transcript levels (Figures 3B, S3B, and S3C). This finding suggests that the MAPK pathway is downregulated in Arid1a-deficient bladder urothelium which may affect eEF2K activity.

To determine how ARID1A controls the MAPK pathway, we measured mRNA abundance of MAPK regulators in WT and Arid1a<sup>fl/fl</sup> urothelial organoids. Surveying components in the Ras-MAPK pathway, we found no difference in expression of Hras, Kras, Nras, Shc1, Src, Braf, Craf, or Sos1 (Figure 3C). However, Rasgrp1 transcript levels were decreased by 80%, and RASGRP1 protein abundance was also decreased in Arid1a-deficient urothelium (Figures 3C, 3D, and S3D). Furthermore, ARID1A binds to the Rasgrp1 promoter in WT urothelial cells (Figure 3E), and Rasgrp1 mRNA and protein levels were restored by adding back ARID1A in Arid1a<sup>fl/fl</sup> organoids (Figures S3E and S3F). RASGRP1 is a diacylglycerol-regulated RasGEF, which activates the MAPK pathway with functions in T cells, T cell leukemia, and the intestinal epithelium.42–45 However, its role in the urothelium is unknown. To determine if the decrease in RASGRP1 can increase eEF2K activity, we measured eEF2 phosphorylation in Rasgrp1<sup>-/-</sup> urothelium. strikingly, Rasgrp1<sup>-/-</sup> urothelium exhibited a significant increase in eEF2 phosphorylation (Figures 3F and 3G), independent of total eEF2 protein levels (Figure S3G), which is consistent with Arid1a<sup>fl/fl</sup> mice.

We next sought to delineate how ARID1A regulates Rasgrp1 expression. We conducted CUT&Tag chromatin profiling of histone H3 trimethylation of lysine 27 (H3K27me3) because downregulation of SWI/SNF activity is associated with H3K27me3 accumulation and repression of distinct genes.46,47 We observed that 2,558 gene promoters exhibited greater than 2-fold increases in the H3K27me3 in Arid1a<sup>fl/fl</sup> organoids (Figure S3H). Moreover, on a gene-specific level, H3K27me3 at the Rasgrp1 promoter was enriched 4.5-fold in Arid1a-deficient urothelial organoids and decreased in ARID1A add-back

<footnote>(H) Ribosome half-transit time in Arid1a<sup>fl/fl</sup> and Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> organoids (linear regression, left panels). Bar graph (right) represents the average ribosome half-transit time of 3 independent experiments. PMS, post-mitochondrial supernatants (complete + nascent proteins); PRS, post-ribosomal supernatant (complete proteins). p = 0.02 (t test).</footnote>

<footnote>n.s., not significant. Scale bar, 100 µm; mean ± SEM. Also see Figure S2.</footnote>
<figure><img src="image_28.png" /><figcaption>Figure 3. ARID1A regulates mRNA translation elongation through RASGRP1</figcaption></figure>

(A) Schematic of upstream signaling pathways that activate (AMPK pathway) or deactivate (PI3K and MAPK pathways) eEF2K.

(B) IHC and quantification of phospho-MEK1/2, phospho-ERK1/2, and phospho-p90RSK in WT and *Arid1a*<sup>fl/fl</sup> urothelium. n ≥ 4/genotype. p = 0.02 (left), p = 0.009 (middle), p < 0.0001 (t test).

(C) RNA-seq mRNA expression of upstream MAPK regulators in WT and *Arid1a*<sup>fl/fl</sup> urothelial cells. n = 3/genotype. p = 0.003 (t test).

(D) RASGRP1 IHC and quantification in WT and *Arid1a*<sup>fl/fl</sup> mice. n ≥ 5/genotype. p = 0.005 (t test).

(E) *Arid1a* chromatin immunoprecipitation (ChIP) qPCR of the Rasgrp1 promoter in WT and *Arid1a*<sup>fl/fl</sup> organoids. n = 2/genotype. p = 0.04 (t test).

(F and G) IHC of RASGRP1 (top) and phospho-eEF2 (T56) (bottom) in WT and Rasgrp1<sup>-/-</sup> mice. n ≥ 3/genotype. p = 0.0006 (top), p = 0.001 (bottom) (t test).

(H) H3K27me3 CUT&Tag from WT (blue) and *Arid1a*<sup>fl/fl</sup> (red) organoids. Black arrows, Rasgrp1 promoter.

(legend continued on next page)
<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /><figcaption>Figure 3. ARID1A regulates mRNA translation elongation through RASGRP1</figcaption></figure>

(A) Schematic of upstream signaling pathways that activate (AMPK pathway) or deactivate (PI3K and MAPK pathways) eEF2K.

(B) IHC and quantification of phospho-MEK1/2, phospho-ERK1/2, and phospho-p90RSK in WT and *Arid1a*<sup>fl/fl</sup> urothelium. n ≥ 4/genotype. p = 0.02 (left), p = 0.009 (middle), p < 0.0001 (t test).

(C) RNA-seq mRNA expression of upstream MAPK regulators in WT and *Arid1a*<sup>fl/fl</sup> urothelial cells. n = 3/genotype. p = 0.003 (t test).

(D) RASGRP1 IHC and quantification in WT and *Arid1a*<sup>fl/fl</sup> mice. n ≥ 5/genotype. p = 0.005 (t test).

(E) *Arid1a* chromatin immunoprecipitation (ChIP) qPCR of the Rasgrp1 promoter in WT and *Arid1a*<sup>fl/fl</sup> organoids. n = 2/genotype. p = 0.04 (t test).

(F and G) IHC of RASGRP1 (top) and phospho-eEF2 (T56) (bottom) in WT and Rasgrp1<sup>-/-</sup> mice. n ≥ 3/genotype. p = 0.0006 (top), p = 0.001 (bottom) (t test).

(H) H3K27me3 CUT&Tag from WT (blue) and *Arid1a*<sup>fl/fl</sup> (red) organoids. Black arrows, Rasgrp1 promoter.

(legend continued on next page)
organoids (Figures 3H and S3I). Accumulation of H3K27me3 suggests that loss of ARID1A may increase polycomb repressive complex 2 (PRC2) activity at the *Rasgrp1* promoter. PRC2 marks H3K27me3 through its catalytic subunit the enhancer of zeste 2 polycomb repressive complex 2 (EZH2). To demonstrate that EZH2 catalytic activity was responsible for downregulation of *Rasgrp1*, we treated *Arid1a*<sup>fl/fl</sup> organoids with GSK126, a selective EZH2 inhibitor.⁴⁸ Pharmacologic inhibition of EZH2 decreased H3K27me3 on the *Rasgrp1* promoter, which restored *Rasgrp1* mRNA and protein levels in *Arid1a*<sup>fl/fl</sup> cells (Figures 3I, 3J, and S3J–S3L). This resulted in a decrease in eEF2 phosphorylation (Figure 3K). These findings provide a mechanism by which ARID1A controls translation elongation through modulating histone dynamics on the *Rasgrp1* promoter.

Transcriptional-translational conflict is a tumor-suppressive barrier in the urothelium

These findings raise the important question of how transcriptional-
translational conflict is tumor suppressive in the urothelium. Trans-
formation is a complex process that requires a cell to generate its
own mitogenic signals, proliferate without limits, resist exogenous
growth-inhibitory signals, evade apoptosis, and acquire vascula-
ture.⁴⁹ We observed that successive passaging of primary Ari-
d1a<sup>fl/fl</sup> urothelial organoids led to the loss of ARID1A-negative cells,
which is consistent with limited replicative potential (Figure 4A).
However, Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> organoids remained ARID1A nega-
tive even after 9 passages, had higher clonogenic potential, and
proliferated faster than Arid1a<sup>fl/fl</sup> cells (Figures 4A, S4A, and
S4B). To determine if this aspect of transformation can be
observed in vivo, we aged Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mice for 400 days.
Remarkably, Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup>, but not WT, Arid1a<sup>fl/fl</sup>, or
Eef2k<sup>-/-</sup> urothelium exhibited unusually thickened urothelium
consistent with uncontrolled cell growth (Figures 4B and S4C).
To determine if this phenotype was mediated by de-repression
of conflicted mRNA, we treated Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> cells with either
the ODC1 inhibitor difluoromethylornithine (DFMO) or an anti-
FGFR3 antibody to target ODC1 or FGFR3, respectively, and
measured clonogenic growth. ODC1 and FGFR3 inhibition signif-
icantly decreased the uncontrolled growth of Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup>
cells (Figure 4C). These findings demonstrate that de-repression
of conflicted mRNAs can unmask the oncogenic properties of
ARID1A loss. Furthermore, they raise the question of the role of
translation restoration in tumor progression.

To test this hypothesis, we needed an experimental model
where we could first increase translation elongation rates while
forming tumors, then delete *Arid1a*. One of the main carcinogens
found in cigarettes is N-butyl-N-(4-hydroxybutyl)nitrosamine
(BBN).⁵⁰ BBN is a bladder-tropic pro-carcinogen that is metab-
olized into BCPN (N-butyl-N-[3-carboxypropyl]nitrosamine) and
concentrated in the bladder, which causes DNA damage and
bladder tumors in mice but does not cause cancer in other tis-
sues.⁵¹,⁵² We found that the process of carcinogenesis by
BBN markedly increased eEF2 protein levels by 4-fold, with

only a 2-fold change in eEF2 phosphorylation and no change in
eEF2K levels (Figures S4D-S4F). This was associated with an in-
crease in puromycin incorporation in bladder tumors compared
with WT urothelium (Figure S4G).⁵³ We treated WT and non-re-
combined *Arid1a*<sup>fl/fl</sup> mice with BBN for 18 weeks to form tumors
first, then dosed mice with tamoxifen to delete *Arid1a* and moni-
tored their survival (Figure 4D). *Arid1a*<sup>fl/fl</sup> mice developed larger
tumors, succumbed to bladder cancer at a much higher rate
than WT mice (68.7% vs. 23% dead 100 days after last tamox-
ifen treatment, respectively), and maintained the increase in pro-
tein synthesis caused by carcinogenesis (Figures 4D, 4E, and
S4H). Moreover, *Arid1a*<sup>fl/fl</sup> bladder tumors proliferated faster
than WT tumors (Figure 4F). At a molecular level, previously
conflicted mRNAs, including *Aurkb*, *Kif22*, *Odc1*, and *Ska1*,
were more efficiently translated in *Arid1a*<sup>fl/fl</sup> tumors compared
with WT tumors (Figure 4G).

Next, we sought to determine the drivers that increase protein synthesis to resolve transcriptional-translational conflict in *Arid1a*-deficient tumors. We developed a primary organoid line from a non-recombined BBN-induced *Arid1a*<sup>fl/fl</sup> bladder tumor (WT tumor) and used this to generate an isogenic *Arid1a*-deficient line using lentiviral Cre recombinase. We conducted pairwise RNA-seq analysis comparing normal and cancer urothelial cells in both the WT and *Arid1a*<sup>fl/fl</sup> backgrounds (Figure S4I). We focused our analysis on up- or downregulated mRNAs (red and blue, respectively) unique to *Arid1a* loss (Figure 4H; Table S3). We found that *Akt3*, *Fgfr1*, and *Kras* were upregulated in *Arid1a*<sup>fl/fl</sup> tumors. These genes have been shown to increase mRNA translation initiation and/or elongation rates.<sup>3,33,54,55</sup> The only protein synthesis regulator that was downregulated in *Arid1a*<sup>fl/fl</sup> tumors was *Eef2k* (Figure 4H; Table S3). This was associated with a significant decrease in total eEF2K and eEF2 phosphorylation in *Arid1a*<sup>fl/fl</sup> tumors (Figures 4I, S4J, and S4K). It has been shown that NF-kB is a negative regulator of Eef2k mRNA levels.<sup>56</sup> We measured a NF-kB mRNA signature in *Arid1a*<sup>fl/fl</sup> tumors and found that they had high NF-kB activity compared with normal *Arid1a*<sup>fl/fl</sup> cells, suggesting a potential mechanism for down-regulation of eEF2K mRNA (Figure S4L).

These discoveries show that a specific threshold of protein
synthesis must be achieved to overcome transcriptional-trans-
lational conflict and unlock the oncogenic potential of ARID1A
loss. To determine if these findings can be observed in pa-
tients, we measured ARID1A levels and eEF2 activity levels in
two independent international cohorts of muscle-invasive
bladder cancer. Patient tumors with low levels of ARID1A pro-
tein were more likely to have decreased eEF2 phosphorylation,
an indication of increased eEF2 activity (Figures 4J and 4K).
Furthermore, ARID1A and phospho-eEF2 low patients were
more likely to have high-T stage disease at diagnosis and
even after neoadjuvant chemotherapy (Figures 4L and 4M).
Together, our findings mechanistically show how ARID1A is a
context-dependent tumor suppressor and raises the possibility
that overcoming transcriptional-translational conflict through

<footnote>(I) qPCR of *Rasgrp1* in *Arid1a*<sup>fl/fl</sup> organoids after treatment with GSK126 (12.5 µM). p = 0.0001 (t test).</footnote>

<footnote>(J) RASGRP1 IF in *Arid1a*<sup>fl/fl</sup> organoids after treatment with GSK126. n = 4/genotype, >50,000 cells/genotype. p < 0.0001 (t test).</footnote>

<footnote>(K) Immunoblot analysis of phospho-eEF2 and total eEF2 in *Arid1a*<sup>fl/fl</sup> organoids ± GSK126. Each blot is representative of three biological replicates. n.s., not significant. Scale bar, 100 µm; mean ± SEM. Also see Figure S3.</footnote>
organoids (Figures 3H and S3I). Accumulation of H3K27me3 suggests that loss of ARID1A may increase polycomb repressive complex 2 (PRC2) activity at the *Rasgrp1* promoter. PRC2 marks H3K27me3 through its catalytic subunit the enhancer of zeste 2 polycomb repressive complex 2 (EZH2). To demonstrate that EZH2 catalytic activity was responsible for downregulation of *Rasgrp1*, we treated *Arid1a*<sup>fl/fl</sup> organoids with GSK126, a selective EZH2 inhibitor.⁴⁸ Pharmacologic inhibition of EZH2 decreased H3K27me3 on the *Rasgrp1* promoter, which restored *Rasgrp1* mRNA and protein levels in *Arid1a*<sup>fl/fl</sup> cells (Figures 3I, 3J, and S3J–S3L). This resulted in a decrease in eEF2 phosphorylation (Figure 3K). These findings provide a mechanism by which ARID1A controls translation elongation through modulating histone dynamics on the *Rasgrp1* promoter.

Transcriptional-translational conflict is a tumor-suppressive barrier in the urothelium

These findings raise the important question of how transcriptional-
translational conflict is tumor suppressive in the urothelium. Trans-
formation is a complex process that requires a cell to generate its
own mitogenic signals, proliferate without limits, resist exogenous
growth-inhibitory signals, evade apoptosis, and acquire vascula-
ture.⁴⁹ We observed that successive passaging of primary Ari-
d1a<sup>fl/fl</sup> urothelial organoids led to the loss of ARID1A-negative cells,
which is consistent with limited replicative potential (Figure 4A).
However, Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> organoids remained ARID1A nega-
tive even after 9 passages, had higher clonogenic potential, and
proliferated faster than Arid1a<sup>fl/fl</sup> cells (Figures 4A, S4A, and
S4B). To determine if this aspect of transformation can be
observed in vivo, we aged Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mice for 400 days.
Remarkably, Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup>, but not WT, Arid1a<sup>fl/fl</sup>, or
Eef2k<sup>-/-</sup> urothelium exhibited unusually thickened urothelium
consistent with uncontrolled cell growth (Figures 4B and S4C).
To determine if this phenotype was mediated by de-repression
of conflicted mRNA, we treated Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> cells with either
the ODC1 inhibitor difluoromethylornithine (DFMO) or an anti-
FGFR3 antibody to target ODC1 or FGFR3, respectively, and
measured clonogenic growth. ODC1 and FGFR3 inhibition signif-
icantly decreased the uncontrolled growth of Arid1a<sup>fl/fl</sup>;Eef2k<sup>-/-</sup>
cells (Figure 4C). These findings demonstrate that de-repression
of conflicted mRNAs can unmask the oncogenic properties of
ARID1A loss. Furthermore, they raise the question of the role of
translation restoration in tumor progression.

To test this hypothesis, we needed an experimental model
where we could first increase translation elongation rates while
forming tumors, then delete *Arid1a*. One of the main carcinogens
found in cigarettes is N-butyl-N-(4-hydroxybutyl)nitrosamine
(BBN).⁵⁰ BBN is a bladder-tropic pro-carcinogen that is metab-
olized into BCPN (N-butyl-N-[3-carboxypropyl]nitrosamine) and
concentrated in the bladder, which causes DNA damage and
bladder tumors in mice but does not cause cancer in other tis-
sues.⁵¹,⁵² We found that the process of carcinogenesis by
BBN markedly increased eEF2 protein levels by 4-fold, with

only a 2-fold change in eEF2 phosphorylation and no change in
eEF2K levels (Figures S4D-S4F). This was associated with an in-
crease in puromycin incorporation in bladder tumors compared
with WT urothelium (Figure S4G).⁵³ We treated WT and non-re-
combined *Arid1a*<sup>fl/fl</sup> mice with BBN for 18 weeks to form tumors
first, then dosed mice with tamoxifen to delete *Arid1a* and moni-
tored their survival (Figure 4D). *Arid1a*<sup>fl/fl</sup> mice developed larger
tumors, succumbed to bladder cancer at a much higher rate
than WT mice (68.7% vs. 23% dead 100 days after last tamox-
ifen treatment, respectively), and maintained the increase in pro-
tein synthesis caused by carcinogenesis (Figures 4D, 4E, and
S4H). Moreover, *Arid1a*<sup>fl/fl</sup> bladder tumors proliferated faster
than WT tumors (Figure 4F). At a molecular level, previously
conflicted mRNAs, including *Aurkb*, *Kif22*, *Odc1*, and *Ska1*,
were more efficiently translated in *Arid1a*<sup>fl/fl</sup> tumors compared
with WT tumors (Figure 4G).

Next, we sought to determine the drivers that increase protein synthesis to resolve transcriptional-translational conflict in *Arid1a*-deficient tumors. We developed a primary organoid line from a non-recombined BBN-induced *Arid1a*<sup>fl/fl</sup> bladder tumor (WT tumor) and used this to generate an isogenic *Arid1a*-deficient line using lentiviral Cre recombinase. We conducted pairwise RNA-seq analysis comparing normal and cancer urothelial cells in both the WT and *Arid1a*<sup>fl/fl</sup> backgrounds (Figure S4I). We focused our analysis on up- or downregulated mRNAs (red and blue, respectively) unique to *Arid1a* loss (Figure 4H; Table S3). We found that *Akt3*, *Fgfr1*, and *Kras* were upregulated in *Arid1a*<sup>fl/fl</sup> tumors. These genes have been shown to increase mRNA translation initiation and/or elongation rates.<sup>3,33,54,55</sup> The only protein synthesis regulator that was downregulated in *Arid1a*<sup>fl/fl</sup> tumors was *Eef2k* (Figure 4H; Table S3). This was associated with a significant decrease in total eEF2K and eEF2 phosphorylation in *Arid1a*<sup>fl/fl</sup> tumors (Figures 4I, S4J, and S4K). It has been shown that NF-kB is a negative regulator of Eef2k mRNA levels.<sup>56</sup> We measured a NF-kB mRNA signature in *Arid1a*<sup>fl/fl</sup> tumors and found that they had high NF-kB activity compared with normal *Arid1a*<sup>fl/fl</sup> cells, suggesting a potential mechanism for down-regulation of eEF2K mRNA (Figure S4L).

These discoveries show that a specific threshold of protein
synthesis must be achieved to overcome transcriptional-trans-
lational conflict and unlock the oncogenic potential of ARID1A
loss. To determine if these findings can be observed in pa-
tients, we measured ARID1A levels and eEF2 activity levels in
two independent international cohorts of muscle-invasive
bladder cancer. Patient tumors with low levels of ARID1A pro-
tein were more likely to have decreased eEF2 phosphorylation,
an indication of increased eEF2 activity (Figures 4J and 4K).
Furthermore, ARID1A and phospho-eEF2 low patients were
more likely to have high-T stage disease at diagnosis and
even after neoadjuvant chemotherapy (Figures 4L and 4M).
Together, our findings mechanistically show how ARID1A is a
context-dependent tumor suppressor and raises the possibility
that overcoming transcriptional-translational conflict through

<footnote>(I) qPCR of *Rasgrp1* in *Arid1a*<sup>fl/fl</sup> organoids after treatment with GSK126 (12.5 µM). p = 0.0001 (t test).</footnote>

<footnote>(J) RASGRP1 IF in *Arid1a*<sup>fl/fl</sup> organoids after treatment with GSK126. n = 4/genotype, >50,000 cells/genotype. p < 0.0001 (t test).</footnote>

<footnote>(K) Immunoblot analysis of phospho-eEF2 and total eEF2 in *Arid1a*<sup>fl/fl</sup> organoids ± GSK126. Each blot is representative of three biological replicates. n.s., not significant. Scale bar, 100 µm; mean ± SEM. Also see Figure S3.</footnote>
A

<figure><img src="image_29.png" /></figure>

Arid1a<sup>nn</sup>

<figure><img src="image_30.png" /></figure>

Arid1a<sup>nn</sup>;Eef2k<sup>c</sup>

B

<figure><img src="image_31.png" /></figure>

Arid1a<sup>nn</sup>, Eef2k<sup>c</sup>
Arid1a<sup>nn</sup>;Eef2k<sup>c</sup>

Bladder urothelium
thickness

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

C

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

D

<figure><img src="image_39.png" /></figure>

E

<figure><img src="image_40.png" /></figure>

F

Mouse tumor

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

G

<figure><img src="image_43.png" /></figure>

H

RNA-seq (Cancer vs Normal)

<figure><img src="image_44.png" /></figure>

(Colored sections are DEGs unique to *Arid1a*<sup>nn</sup> tumors)

I

Mouse tumor

<figure><img src="image_45.png" /></figure>

J

Human tumor (Fred Hutch/UW)

<figure><img src="image_46.png" /></figure>

K

<figure><img src="image_47.png" /></figure>

L

Clinical staging of bladder cancer (Human)

<figure><img src="image_48.png" /></figure>

M

Pathologic staging of bladder cancer (Human)

<figure><img src="image_49.png" /></figure>

(legend on next page)
A

<figure><img src="image_32.png" /></figure>

Arid1a<sup>nn</sup>

<figure><img src="image_33.png" /></figure>

Arid1a<sup>nn</sup>;Eef2k<sup>c</sup>

B

<figure><img src="image_34.png" /></figure>

Arid1a<sup>nn</sup>, Eef2k<sup>c</sup>
Arid1a<sup>nn</sup>;Eef2k<sup>c</sup>

Bladder urothelium
thickness

<figure><img src="image_35.png" /></figure>

Arid1a<sup>nn</sup> Eef2k<sup>c</sup>

<figure><img src="image_36.png" /></figure>

WT

Eef2k<sup>c</sup>

<figure><img src="image_37.png" /></figure>

P<0.0001

C

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

D

<figure><img src="image_40.png" /></figure>

E

<figure><img src="image_41.png" /></figure>

F

Mouse tumor

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

G

<figure><img src="image_44.png" /></figure>

H

RNA-seq (Cancer vs Normal)

<figure><img src="image_45.png" /></figure>

(Colored sections are DEGs unique to *Arid1a*<sup>nn</sup> tumors)

I

Mouse tumor

<figure><img src="image_46.png" /></figure>

J

Human tumor (Fred Hutch/UW)

<figure><img src="image_47.png" /></figure>

K

<figure><img src="image_48.png" /></figure>

L

Clinical staging of bladder cancer (Human)

<figure><img src="image_49.png" /></figure>

M

Pathologic staging of bladder cancer (Human)

<figure><img src="image_50.png" /></figure>

(legend on next page)
restored mRNA translation may be a linchpin to drive ARID1A-deficient tumor growth.

## Drug-induced transcriptional-translational conflict is a therapeutic vulnerability in ARID1A-deficient bladder cancer

Our findings demonstrate that transcriptional-translational conflict is a barrier to cancer progression. It is possible that inducing conflict by inhibiting protein synthesis may be a strategy to target cancer growth in genotypes that have overcome this barrier to thrive. To address this hypothesis, we treated three human and murine models of bladder cancer with the translation inhibitor homoharringtonine (HHT),⁵⁷ which we chose because it is U.S. Food and Drug Administration (FDA) approved for refractory CML.⁵⁸ HHT functions by preventing ribosomes from moving past the ATG start codon, thereby stalling the initiation of elongation⁵⁷,⁵⁹ (Figure 5A). First, we treated isogenic WT and *Arid1a*<sup>fl/fl</sup> BBN-induced cancer organoids. *Arid1a*<sup>fl/fl</sup> tumor organoids were sensitive to HHT at concentrations that had minimal impact on WT bladder cancer organoid growth (Figures 5B and S5A). Next, we identified 2 human ARID1A-deficient bladder cancer cell lines (HT1376 and KU1919) and 2 ARID1A-proficient bladder cancer cell lines (HT1197 and UMUC11). Interestingly, ARID1A-null cell lines were significantly more sensitive to HHT compared with cell lines that expressed ARID1A (Figures 5C and S5B).

We also conducted preclinical trials of patient-derived xenografts (PDXs) that expressed low (PDX1), medium (PDX2), or high (PDX3) ARID1A protein levels (Figure 5D), dosing HHT twice daily at a concentration physiologically achievable in human patients without any toxicity.⁶⁰,⁶¹ We observed a response that correlated with ARID1A levels. HHT decreased tumor growth by 59.3% in the ARID1A-low model, but only decreased tumor growth by 35.9% in the ARID1A-medium model (Figures 5E, 5F, and S5C). Strikingly, the ARID1A-high model was completely insensitive to HHT (Figures 5G and S5C). In addition, we found that the ARID1A-low PDX exhibited the most significant improvement in survival when treated with HHT (Figures 5H

and S5D). At a cellular level, HHT increased apoptosis and decreased cell proliferation in the ARID1A-deficient but not ARID1A-proficient PDXs (Figures 5I, 5J, and S5E-S5H). These findings demonstrate that translation inhibition may reverse the gains of cancers that overcome transcriptional-translational conflict and represents a new therapeutic paradigm.

## eEF2 phosphorylation leads to a collateral decrease in the translation of DNA damage response genes

Transcriptional-translational conflict is a barrier to bladder cancer pathogenesis mediated by eEF2 phosphorylation. However, the effects of slower translation elongation caused by *Arid1a* loss likely affects the protein synthesis of genes beyond the 262 upregulated oncogenic mRNAs. Indeed, analyzing mRNAs that exhibit no transcriptional difference, we found 278 additional stalled mRNAs in *Arid1a*<sup>fl/fl</sup> organoids (>1.35-fold change in P/S ratio, false discovery rate [FDR] < 0.05) (Figure 6A; Table S4). These stalled mRNAs also exhibited a high GC content within their coding sequences, similar to the 262 upregulated mRNAs in *Arid1a*<sup>fl/fl</sup> organoids (Figures S1L and S6A). Interestingly, the top Reactome pathway was DNA double-strand break repair (FDR = 5.02e-4) and included genes such as BRCA2 DNA repair-associated protein (Brca2), ERCC excision repair 1, endonuclease non-catalytic subunit (Ercc1), ERCC excision repair 2, TFIIH core complex helicase subunit (Ercc2), and FA complementation group C (Fancc) (Figure 6A). These proteins maintain genome stability enabling DNA repair and cell survival in the context of DNA damage.⁶² BRCA2 plays a primary role in homology-directed repair of double-strand DNA breaks.⁶³ ERCC1 and ERCC2 are critical for identification, excision, and sealing of damaged DNA through nucleotide excision repair.⁶⁴ FANCC is a core component of the Fanconi anemia pathway that is required to repair interstrand crosslinks in DNA.⁶⁵ Protein levels of BRCA2, ERCC1, ERCC2, and FANCC were significantly decreased in *Arid1a*<sup>fl/fl</sup> organoids without concomitant changes in mRNA levels (Figures 6B, 6C, and S6B). Furthermore, genetic de-repression of eEF2 phosphorylation restored the protein levels of each of

<caption>**Figure 4.** Transcriptional-translational conflict is a tumor-suppressive barrier</caption>

(A) Fluorescence-activated cell sorting (FACS) analysis of *Arid1a* recombinant cells (YFP+) in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> urothelial organoids over 9 successive passages (P2–P9). Representative ARID1A immunoblot from *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> urothelial organoids after two passages (P2) and 9 passages (P9).
(B) H&E (left) and urothelial thickness (right) in WT, *Eef2k*<sup>-/-</sup>, *Arid1a*<sup>fl/fl</sup>, and *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> mice 400 days after tamoxifen. n ≥ 4/genotype. p < 0.0001 (t test).
(C) Clonogenic assay of *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> urothelial cells treated with an anti-FGFR3 antibody or the ODC1 inhibitor DFMO. n = 8 biological replicates. p < 0.0001 (t test).
(D) Kaplan-Meier survival curve of WT and *Arid1a*<sup>fl/fl</sup> mice treated with BBN followed by tamoxifen. WT, n = 13 mice; *Arid1a*<sup>fl/fl</sup>, n = 16 mice. p = 0.047 (Log-rank test).
(E) IF and quantification of puromycin incorporation in WT and *Arid1a*<sup>fl/fl</sup> mice treated with BBN followed by tamoxifen (D). n = 4/genotype, >30,000 cells/genotype.
(F) Ki67 staining and quantification in WT and *Arid1a*<sup>fl/fl</sup> tumors (D). n = 4/genotype. p = 0.01 (t test).
(G) Immunoblots of AURKB, KIF22, ODC1, and SKA1 in WT and *Arid1a*<sup>fl/fl</sup> tumors (replicate of 3).
(H) RNA-seq analysis of normal and cancer urothelial cells in WT and *Arid1a*<sup>fl/fl</sup> backgrounds. Upregulated (red) or downregulated (blue) mRNAs are unique to ARID1A loss (DEGs, differentially expressed genes).
(I) IHC and quantification of phospho-eEF2 (T56) in WT and *Arid1a*<sup>fl/fl</sup> tumors. n ≥ 5/genotype. p = 0.05 (t test).
(J) Representative images and quantification of human muscle-invasive bladder cancer (MIBC) from the University of Washington showing high and low ARID1A protein levels (left panel) and corresponding phospho-eEF2 (T56). n(ARID1A high) = 26, n(ARID1A low) = 15. p = 0.008 (t test).
(K) Representative images and quantification of MIBC obtained from the University of British Columbia showing high and low ARID1A protein levels (left panel) and corresponding phospho-eEF2 (T56). n(ARID1A high) = 17, n(ARID1A low) = 16. p = 0.02 (t test).
(L and M) Clinical staging and post-neoadjuvant chemotherapy pathologic staging of patients in (J) and (K) separated by ARID1A and p-eEF2 levels (n-low = 31, n-high = 43). p = 0.04 (L), p < 0.0001 (M) (Chi-square test).
n.s., not significant. Scale bar, 100 µm; mean ± SEM. Also see Figure S4.
restored mRNA translation may be a linchpin to drive ARID1A-deficient tumor growth.

## Drug-induced transcriptional-translational conflict is a therapeutic vulnerability in ARID1A-deficient bladder cancer

Our findings demonstrate that transcriptional-translational conflict is a barrier to cancer progression. It is possible that inducing conflict by inhibiting protein synthesis may be a strategy to target cancer growth in genotypes that have overcome this barrier to thrive. To address this hypothesis, we treated three human and murine models of bladder cancer with the translation inhibitor homoharringtonine (HHT),⁵⁷ which we chose because it is U.S. Food and Drug Administration (FDA) approved for refractory CML.⁵⁸ HHT functions by preventing ribosomes from moving past the ATG start codon, thereby stalling the initiation of elongation⁵⁷,⁵⁹ (Figure 5A). First, we treated isogenic WT and *Arid1a*<sup>fl/fl</sup> BBN-induced cancer organoids. *Arid1a*<sup>fl/fl</sup> tumor organoids were sensitive to HHT at concentrations that had minimal impact on WT bladder cancer organoid growth (Figures 5B and S5A). Next, we identified 2 human ARID1A-deficient bladder cancer cell lines (HT1376 and KU1919) and 2 ARID1A-proficient bladder cancer cell lines (HT1197 and UMUC11). Interestingly, ARID1A-null cell lines were significantly more sensitive to HHT compared with cell lines that expressed ARID1A (Figures 5C and S5B).

We also conducted preclinical trials of patient-derived xenografts (PDXs) that expressed low (PDX1), medium (PDX2), or high (PDX3) ARID1A protein levels (Figure 5D), dosing HHT twice daily at a concentration physiologically achievable in human patients without any toxicity.⁶⁰,⁶¹ We observed a response that correlated with ARID1A levels. HHT decreased tumor growth by 59.3% in the ARID1A-low model, but only decreased tumor growth by 35.9% in the ARID1A-medium model (Figures 5E, 5F, and S5C). Strikingly, the ARID1A-high model was completely insensitive to HHT (Figures 5G and S5C). In addition, we found that the ARID1A-low PDX exhibited the most significant improvement in survival when treated with HHT (Figures 5H

and S5D). At a cellular level, HHT increased apoptosis and decreased cell proliferation in the ARID1A-deficient but not ARID1A-proficient PDXs (Figures 5I, 5J, and S5E-S5H). These findings demonstrate that translation inhibition may reverse the gains of cancers that overcome transcriptional-translational conflict and represents a new therapeutic paradigm.

## eEF2 phosphorylation leads to a collateral decrease in the translation of DNA damage response genes

Transcriptional-translational conflict is a barrier to bladder cancer pathogenesis mediated by eEF2 phosphorylation. However, the effects of slower translation elongation caused by *Arid1a* loss likely affects the protein synthesis of genes beyond the 262 upregulated oncogenic mRNAs. Indeed, analyzing mRNAs that exhibit no transcriptional difference, we found 278 additional stalled mRNAs in *Arid1a*<sup>fl/fl</sup> organoids (>1.35-fold change in P/S ratio, false discovery rate [FDR] < 0.05) (Figure 6A; Table S4). These stalled mRNAs also exhibited a high GC content within their coding sequences, similar to the 262 upregulated mRNAs in *Arid1a*<sup>fl/fl</sup> organoids (Figures S1L and S6A). Interestingly, the top Reactome pathway was DNA double-strand break repair (FDR = 5.02e-4) and included genes such as BRCA2 DNA repair-associated protein (Brca2), ERCC excision repair 1, endonuclease non-catalytic subunit (Ercc1), ERCC excision repair 2, TFIIH core complex helicase subunit (Ercc2), and FA complementation group C (Fancc) (Figure 6A). These proteins maintain genome stability enabling DNA repair and cell survival in the context of DNA damage.⁶² BRCA2 plays a primary role in homology-directed repair of double-strand DNA breaks.⁶³ ERCC1 and ERCC2 are critical for identification, excision, and sealing of damaged DNA through nucleotide excision repair.⁶⁴ FANCC is a core component of the Fanconi anemia pathway that is required to repair interstrand crosslinks in DNA.⁶⁵ Protein levels of BRCA2, ERCC1, ERCC2, and FANCC were significantly decreased in *Arid1a*<sup>fl/fl</sup> organoids without concomitant changes in mRNA levels (Figures 6B, 6C, and S6B). Furthermore, genetic de-repression of eEF2 phosphorylation restored the protein levels of each of

<caption>**Figure 4.** Transcriptional-translational conflict is a tumor-suppressive barrier</caption>

(A) Fluorescence-activated cell sorting (FACS) analysis of *Arid1a* recombinant cells (YFP+) in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>;*Eef2k*<sup>-/-</sup> urothelial organoids over 9 successive passages (P2–P9). Representative ARID1A immunoblot from *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>;*Eef2k*<sup>-/-</sup> urothelial organoids after two passages (P2) and 9 passages (P9).

(B) H&E (left) and urothelial thickness (right) in WT, *Eef2k*<sup>-/-</sup>, *Arid1a*<sup>fl/fl</sup>, and *Arid1a*<sup>fl/fl</sup>;*Eef2k*<sup>-/-</sup> mice 400 days after tamoxifen. n ≥ 4/genotype. p < 0.0001 (t test).

(C) Clonogenic assay of *Arid1a*<sup>fl/fl</sup>;*Eef2k*<sup>-/-</sup> urothelial cells treated with an anti-FGFR3 antibody or the ODC1 inhibitor DFMO. n = 8 biological replicates. p < 0.0001 (t test).

(D) Kaplan-Meier survival curve of WT and *Arid1a*<sup>fl/fl</sup> mice treated with BBN followed by tamoxifen. WT, n = 13 mice; *Arid1a*<sup>fl/fl</sup>, n = 16 mice. p = 0.047 (Log-rank test).

(E) IF and quantification of puromycin incorporation in WT and *Arid1a*<sup>fl/fl</sup> mice treated with BBN followed by tamoxifen (D). n = 4/genotype, >30,000 cells/genotype.

(F) Ki67 staining and quantification in WT and *Arid1a*<sup>fl/fl</sup> tumors (D). n = 4/genotype. p = 0.01 (t test).

(G) Immunoblots of AURKB, KIF22, ODC1, and SKA1 in WT and *Arid1a*<sup>fl/fl</sup> tumors (replicate of 3).

(H) RNA-seq analysis of normal and cancer urothelial cells in WT and *Arid1a*<sup>fl/fl</sup> backgrounds. Upregulated (red) or downregulated (blue) mRNAs are unique to ARID1A loss (DEGs, differentially expressed genes).

(I) IHC and quantification of phospho-eEF2 (T56) in WT and *Arid1a*<sup>fl/fl</sup> tumors. n ≥ 5/genotype. p = 0.05 (t test).

(J) Representative images and quantification of human muscle-invasive bladder cancer (MIBC) from the University of Washington showing high and low ARID1A protein levels (left panel) and corresponding phospho-eEF2 (T56). n(ARID1A high) = 26, n(ARID1A low) = 15. p = 0.008 (t test).

(K) Representative images and quantification of MIBC obtained from the University of British Columbia showing high and low ARID1A protein levels (left panel) and corresponding phospho-eEF2 (T56). n(ARID1A high) = 17, n(ARID1A low) = 16. p = 0.02 (t test).

(L and M) Clinical staging and post-neoadjuvant chemotherapy pathologic staging of patients in (J) and (K) separated by ARID1A and p-eEF2 levels (n-low = 31, n-high = 43). p = 0.04 (L), p < 0.0001 (M) (Chi-square test).

n.s., not significant. Scale bar, 100 µm; mean ± SEM. Also see Figure S4.
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<figure><img src="image_61.png" /><figcaption>**Figure 5. Pharmacologic inhibition of translation elongation initiation inhibits growth of ARID1A-deficient but not ARID1A-proficient tumors**</figcaption></figure>

(A) Mechanism of action of homoharringtonine (HHT).

(B) WT and *Arid1a*<sup>n/n</sup> tumor organoids cell viability after treatment with HHT (CellTiter-Glo 2.0). n ≥ 3/genotype. p < 0.0001 (t test).

(C) Cell viability of ARID1A-proficient and ARID1A-deficient human bladder cancer cell lines treated with HHT. n = 3/genotype. p = 0.001 (left), p = 0.0005 (right) (t test).

(D) Schematic of patient-derived xenograft (PDX) model generation. Representative ARID1A IHC from PDX1 (ARID1A-low), PDX2 (ARID1A-medium), and PDX3 (ARID1A-high) tumor tissues.

(E–G) Tumor growth rate in PDX1, PDX2, and PDX3 models treated with HHT (0.7 mg/kg; twice/day). n ≥ 10/treatment arm, p < 0.05.

(H) HHT or vehicle (PBS) PDX1 Kaplan-Meier survival curve. n = 10/arm. p = 0.0003 (log-rank test).

(I) Percentage CC3-positive cells from the PDX1 group treated with HHT or vehicle (PBS). n = 6/arm. p = 0.04 (t test).

(J) Percentage Ki67-positive cells from the PDX1 group treated with HHT or vehicle (PBS). n = 6/treatment arm. p = 0.03 (t test). n.s., not significant. Mean ± SEM. Also see Figure S5.
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<caption>Figure 5. Pharmacologic inhibition of translation elongation initiation inhibits growth of ARID1A-deficient but not ARID1A-proficient tumors</caption>

(A) Mechanism of action of homoharringtonine (HHT).

(B) WT and *Arid1a*<sup>n/n</sup> tumor organoids cell viability after treatment with HHT (CellTiter-Glo 2.0). n ≥ 3/genotype. p < 0.0001 (t test).

(C) Cell viability of ARID1A-proficient and ARID1A-deficient human bladder cancer cell lines treated with HHT. n = 3/genotype. p = 0.001 (left), p = 0.0005 (right) (t test).

(D) Schematic of patient-derived xenograft (PDX) model generation. Representative ARID1A IHC from PDX1 (ARID1A-low), PDX2 (ARID1A-medium), and PDX3 (ARID1A-high) tumor tissues.

(E–G) Tumor growth rate in PDX1, PDX2, and PDX3 models treated with HHT (0.7 mg/kg; twice/day). n ≥ 10/treatment arm, p < 0.05.

(H) HHT or vehicle (PBS) PDX1 Kaplan-Meier survival curve. n = 10/arm. p = 0.0003 (log-rank test).

(I) Percentage CC3-positive cells from the PDX1 group treated with HHT or vehicle (PBS). n = 6/arm. p = 0.04 (t test).

(J) Percentage Ki67-positive cells from the PDX1 group treated with HHT or vehicle (PBS). n = 6/treatment arm. p = 0.03 (t test). n.s., not significant. Mean ± SEM. Also see Figure S5.
<figure><img src="image_62.png" /><figcaption>Figure 6. ARID1A loss leads to a decrease in the translation of DNA damage response mRNAs</figcaption></figure>

(A) Volcano plot showing 278 translationally stalled mRNAs (orange dots, increased P/S ratio), which include *Brca2*, *Erc1*, *Erc2*, and *Fanc* (FDR < 0.05).

(B and C) Protein (replicate of 2) and mRNA (replicate of 3) levels of DNA damage response genes in WT and *Arid1afl/fl* urothelial cells.

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<figure><img src="image_61.png" /><figcaption>**Figure 6.** ARID1A loss leads to a decrease in the translation of DNA damage response mRNAs</figcaption></figure>

(A) Volcano plot showing 278 translationally stalled mRNAs (orange dots, increased P/S ratio), which include *Brca2*, *Erc1*, *Erc2*, and *Fanc* (FDR < 0.05).

(B and C) Protein (replicate of 2) and mRNA (replicate of 3) levels of DNA damage response genes in WT and *Arid1afl/fl* urothelial cells.

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these genes (Figure 6D). Therefore, transcriptional-translational conflict leads to the collateral decrease of gene networks critical for DNA damage repair.

To determine if the decreased protein synthesis of DNA damage response genes creates a vulnerability to DNA damage in *Arid1a*-deficient urothelium, we treated WT and *Arid1a*<sup>fl/fl</sup> mice with BBN for 9 days and measured γH2AX foci (a marker for double-stranded DNA breaks) and cleaved caspase-3 (CC3; a marker for apoptosis). *Arid1a*-deficient mice exhibited a significant increase in urothelial γH2AX and CC3 (Figures 6E and 6F). This was associated with increased DNA damage, as measured using the comet assay (Figure 6G). Mass spectrometry for BCPN in WT and *Arid1a*<sup>fl/fl</sup> mouse urine showed no difference in carcinogen exposure (Figure 6H). These findings suggest that *Arid1a*<sup>fl/fl</sup> cells may exhibit impaired transformation because of an inability to efficiently repair damaged DNA. To test this concept, WT and *Arid1a*-deficient mice were dosed with BBN for 150 days. Although both WT and *Arid1a*<sup>fl/fl</sup> mice developed the same number of tumors, *Arid1a*<sup>fl/fl</sup> tumors were smaller compared with their WT counterparts (Figures 6I and 6J). Given the mosaic nature of the *Arid1a*<sup>fl/fl</sup> model (Figure S1A) we reasoned that if loss of ARID1A is an impediment toward transformation, then the WT urothelium should more readily form tumors. Indeed, all tumors in *Arid1a*<sup>fl/fl</sup> mice still expressed ARID1A (Figure 6K). Thus, ARID1A is necessary to enable the survival of BBN-treated urothelium raising the question of the role of translation elongation in this process.

## eEF2 activity is necessary for carcinogenesis in the context of ARID1A loss

ARID1A directly interacts with ataxia telangiectasia and RAD3-related protein (ATR) to promote double-strand break end resection and ATR activation to maintain chromosomal stability in response to radiation.¹¹ Our data suggest that mRNA-specific translation elongation downstream of ARID1A may also promote DNA damage repair and cell survival. To determine if the decrease in translation elongation which occurs upon ARID1A loss is responsible for the impaired DDR independent of ARID1A's role as an ATR interactor, we treated *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> mice with BBN for 9 days. We found that genetic restoration of translation elongation, which restored BRCA2, ERCC1, ERCC2, and FANCC protein abundance, was sufficient to decrease DNA damage and apoptosis in *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> mice (Figures 6D, 7A, and 7B). Carcinogen exposure in both mouse models was equivalent (Figure S7A). To determine if this rescue was mediated by the catalytic activity of eEF2K, we pre-treated *Arid1a*<sup>fl/fl</sup> mice with A-484954 before a 9-day course of BBN. Like the genetic

model, inhibition of eEF2K activity markedly decreased DNA damage and apoptosis (Figures 7C, 7D, and S7B).

These short-term studies raise the possibility that mRNA-specific impairments in translation elongation form a protective barrier that prevent *Arid1a*-deficient cells from carcinogen-mediated transformation. To directly address this question, *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> mice were treated with BBN for 150 days. *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> mice formed significantly larger bladder tumors compared with *Arid1a*<sup>fl/fl</sup> mice (Figures 7E and S7C). Remarkably, unlike all tumors in *Arid1a*<sup>fl/fl</sup> mice which continue to express ARID1A protein, only 1 of 7 *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> tumors was positive for ARID1A (Figures 6K, 7F, and 7G). As such, restoring translation elongation and mRNA-specific translation is sufficient to drive carcinogenesis and the formation of *Arid1a*-deficient tumors.

## DISCUSSION

Here we demonstrate that a tumor suppressor can function as a positive regulator of translation elongation. This has historical precedence because major tumor suppressors have been shown to negatively, but not positively regulate the protein-synthetic capacity of cells. For example, TP53 binds to and inhibits the rRNA methyl-transferase fibrillarin which prevents IRES-mediated translation of cancer genes.¹ Loss of PTEN within prostate epithelium promotes cancer progression through hyperactivation of the mTOR-eIF4F signaling pathway and translation of pro-metastasis mRNAs.² APC loss leads to intestinal tumorigenesis driven by enhanced translation elongation of cyclin D mRNA.³ Contrary to these tumor suppressors, we show that ARID1A is necessary for the maintenance of translation elongation through eEF2 (Figure S7D). Therefore, loss of ARID1A has significant negative implications for the ability of upregulated proliferation and oncogenic mRNA networks to be synthesized into proteins. This conflict between transcription and translation forms a functional barrier to uncontrolled cell growth and cancer progression, which we propose is a source of oncogenic stress that occurs when chromatin remodeling is deregulated in urothelium.

This concept has important parallels with other major oncogenic signaling pathways. For example, it has been shown that loss of the tumor suppressor PTEN can upregulate TP53 levels which promotes cellular senescence and prevents the formation of lethal prostate cancer. However, combined loss of PTEN and TP53 can overcome this barrier leading to uninhibited tumor growth.⁶⁶ This is also true for oncogenes such as MYC and PIK3CA. MYC which is commonly amplified in human malignancies causes apoptosis in the context of overexpression in normal cells.⁶⁷,⁶⁸

(D) Protein levels of DNA damage response genes in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> urothelial cells (replicate of 3).

(E and F) γH2AX or CC3 staining and quantification in WT and *Arid1a*<sup>fl/fl</sup> mice after tamoxifen administration followed by 9 days of BBN treatment. Urothelium is marked with red dotted lines. n ≥ 4/genotype. p = 0.008 (E), p = 0.01 (F) (t test).

(G) Representative comet assay showing increased DNA damage (tail length) in *Arid1a*<sup>fl/fl</sup> organoids compared with WT organoids treated with BCPN.

(H) Mass spectrometry measurements of urine BCPN in WT and *Arid1a*<sup>fl/fl</sup> mice after 9 days of BBN treatment (n ≥ 3/genotype).

(I) Pie chart showing tumor outcome in WT and *Arid1a*<sup>fl/fl</sup> mice treated with BBN for 150 days after ARID1A deletion (WT, n = 11; *Arid1a*<sup>fl/fl</sup>, n = 12).

(J) H&E staining of tumors from WT and *Arid1a*<sup>fl/fl</sup> mice after 150 days of BBN treatment. Tumor area is marked with yellow dotted lines. WT, n = 6 tumors; *Arid1a*<sup>fl/fl</sup>, n = 5 tumors. p = 0.01 (t test).

(K) ARID1A IHC of *Arid1a*<sup>fl/fl</sup> tumors after tamoxifen followed by 150 days of BBN treatment (I). Tumor area is marked with yellow dotted lines. n.s., not significant. Scale bar, 100 µm; mean ± SEM. Also see Figure S6.
these genes (Figure 6D). Therefore, transcriptional-translational conflict leads to the collateral decrease of gene networks critical for DNA damage repair.

To determine if the decreased protein synthesis of DNA damage response genes creates a vulnerability to DNA damage in *Arid1a*-deficient urothelium, we treated WT and *Arid1a*<sup>fl/fl</sup> mice with BBN for 9 days and measured γH2AX foci (a marker for double-stranded DNA breaks) and cleaved caspase-3 (CC3; a marker for apoptosis). *Arid1a*-deficient mice exhibited a significant increase in urothelial γH2AX and CC3 (Figures 6E and 6F). This was associated with increased DNA damage, as measured using the comet assay (Figure 6G). Mass spectrometry for BCPN in WT and *Arid1a*<sup>fl/fl</sup> mouse urine showed no difference in carcinogen exposure (Figure 6H). These findings suggest that *Arid1a*<sup>fl/fl</sup> cells may exhibit impaired transformation because of an inability to efficiently repair damaged DNA. To test this concept, WT and *Arid1a*-deficient mice were dosed with BBN for 150 days. Although both WT and *Arid1a*<sup>fl/fl</sup> mice developed the same number of tumors, *Arid1a*<sup>fl/fl</sup> tumors were smaller compared with their WT counterparts (Figures 6I and 6J). Given the mosaic nature of the *Arid1a*<sup>fl/fl</sup> model (Figure S1A) we reasoned that if loss of ARID1A is an impediment toward transformation, then the WT urothelium should more readily form tumors. Indeed, all tumors in *Arid1a*<sup>fl/fl</sup> mice still expressed ARID1A (Figure 6K). Thus, ARID1A is necessary to enable the survival of BBN-treated urothelium raising the question of the role of translation elongation in this process.

## eEF2 activity is necessary for carcinogenesis in the context of ARID1A loss

ARID1A directly interacts with ataxia telangiectasia and RAD3-related protein (ATR) to promote double-strand break end resection and ATR activation to maintain chromosomal stability in response to radiation.¹¹ Our data suggest that mRNA-specific translation elongation downstream of ARID1A may also promote DNA damage repair and cell survival. To determine if the decrease in translation elongation which occurs upon ARID1A loss is responsible for the impaired DDR independent of ARID1A's role as an ATR interactor, we treated *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> mice with BBN for 9 days. We found that genetic restoration of translation elongation, which restored BRCA2, ERCC1, ERCC2, and FANCC protein abundance, was sufficient to decrease DNA damage and apoptosis in *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> mice (Figures 6D, 7A, and 7B). Carcinogen exposure in both mouse models was equivalent (Figure S7A). To determine if this rescue was mediated by the catalytic activity of eEF2K, we pre-treated *Arid1a*<sup>fl/fl</sup> mice with A-484954 before a 9-day course of BBN. Like the genetic

model, inhibition of eEF2K activity markedly decreased DNA damage and apoptosis (Figures 7C, 7D, and S7B).

These short-term studies raise the possibility that mRNA-specific impairments in translation elongation form a protective barrier that prevent *Arid1a*-deficient cells from carcinogen-mediated transformation. To directly address this question, *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> mice were treated with BBN for 150 days. *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> mice formed significantly larger bladder tumors compared with *Arid1a*<sup>fl/fl</sup> mice (Figures 7E and S7C). Remarkably, unlike all tumors in *Arid1a*<sup>fl/fl</sup> mice which continue to express ARID1A protein, only 1 of 7 *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> tumors was positive for ARID1A (Figures 6K, 7F, and 7G). As such, restoring translation elongation and mRNA-specific translation is sufficient to drive carcinogenesis and the formation of *Arid1a*-deficient tumors.

## DISCUSSION

Here we demonstrate that a tumor suppressor can function as a positive regulator of translation elongation. This has historical precedence because major tumor suppressors have been shown to negatively, but not positively regulate the protein-synthetic capacity of cells. For example, TP53 binds to and inhibits the rRNA methyl-transferase fibrillarin which prevents IRES-mediated translation of cancer genes.¹ Loss of PTEN within prostate epithelium promotes cancer progression through hyperactivation of the mTOR-eIF4F signaling pathway and translation of pro-metastasis mRNAs.² APC loss leads to intestinal tumorigenesis driven by enhanced translation elongation of cyclin D mRNA.³ Contrary to these tumor suppressors, we show that ARID1A is necessary for the maintenance of translation elongation through eEF2 (Figure S7D). Therefore, loss of ARID1A has significant negative implications for the ability of upregulated proliferation and oncogenic mRNA networks to be synthesized into proteins. This conflict between transcription and translation forms a functional barrier to uncontrolled cell growth and cancer progression, which we propose is a source of oncogenic stress that occurs when chromatin remodeling is deregulated in urothelium.

This concept has important parallels with other major oncogenic signaling pathways. For example, it has been shown that loss of the tumor suppressor PTEN can upregulate TP53 levels which promotes cellular senescence and prevents the formation of lethal prostate cancer. However, combined loss of PTEN and TP53 can overcome this barrier leading to uninhibited tumor growth.⁶⁶ This is also true for oncogenes such as MYC and PIK3CA. MYC which is commonly amplified in human malignancies causes apoptosis in the context of overexpression in normal cells.⁶⁷,⁶⁸

(D) Protein levels of DNA damage response genes in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>; *Eef2k*<sup>-/-</sup> urothelial cells (replicate of 3).

(E and F) γH2AX or CC3 staining and quantification in WT and *Arid1a*<sup>fl/fl</sup> mice after tamoxifen administration followed by 9 days of BBN treatment. Urothelium is marked with red dotted lines. n ≥ 4/genotype. p = 0.008 (E), p = 0.01 (F) (t test).

(G) Representative comet assay showing increased DNA damage (tail length) in *Arid1a*<sup>fl/fl</sup> organoids compared with WT organoids treated with BCPN.

(H) Mass spectrometry measurements of urine BCPN in WT and *Arid1a*<sup>fl/fl</sup> mice after 9 days of BBN treatment (n ≥ 3/genotype).

(I) Pie chart showing tumor outcome in WT and *Arid1a*<sup>fl/fl</sup> mice treated with BBN for 150 days after ARID1A deletion (WT, n = 11; *Arid1a*<sup>fl/fl</sup>, n = 12).

(J) H&E staining of tumors from WT and *Arid1a*<sup>fl/fl</sup> mice after 150 days of BBN treatment. Tumor area is marked with yellow dotted lines. WT, n = 6 tumors; *Arid1a*<sup>fl/fl</sup>, n = 5 tumors. p = 0.01 (t test).

(K) ARID1A IHC of *Arid1a*<sup>fl/fl</sup> tumors after tamoxifen followed by 150 days of BBN treatment (I). Tumor area is marked with yellow dotted lines. n.s., not significant. Scale bar, 100 µm; mean ± SEM. Also see Figure S6.
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<figure><img src="image_74.png" /><figcaption>**Figure 7.** Restoration of translation elongation is necessary to enable carcinogenesis in ARID1A-deficient urothelium</figcaption></figure>

(A and B) γH2AX and CC3 staining and quantification in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>*;Eef2k*<sup>-/-</sup> mouse urothelium (outlined in red) after tamoxifen administration followed by 9 days of BBN treatment. n ≥ 6/genotype. p = 0.008 (A), p = 0.01 (B) (t test).

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<figure><img src="image_73.png" /><figcaption>**Figure 7.** Restoration of translation elongation is necessary to enable carcinogenesis in ARID1A-deficient urothelium</figcaption></figure>

(A and B) γH2AX and CC3 staining and quantification in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>*;Eef2k<sup>−/−</sup>* mouse urothelium (outlined in red) after tamoxifen administration followed by 9 days of BBN treatment. n ≥ 6/genotype. p = 0.008 (A), p = 0.01 (B) (t test).

(legend continued on next page)
This is mediated through enhanced expression of the apoptosis regulator BIM.⁶⁹ Importantly co-expression of MYC and the anti-apoptotic and inhibitor of BIM, BCL-2, leads to a significant acceleration in tumorigenesis.⁷⁰ Activated PIK3CA causes cellular differentiation in epidermal progenitors which is mediated by the AKT substrate SH3RF1. This leads to terminal differentiation of epidermal cells that possess activating mutations of PIK3CA and prevents oncogenic clonal expansion.⁷¹ In line with these oncogenic stress pathways, our data show that ARID1A loss leads to a significant reduction in eEF2 activity which promotes a conflict between the expression of oncogenic genes and their subsequent translation (Figure S7D). Restoration of translation elongation enabling gene expression parity is sufficient to unleash the oncogenic properties of ARID1A deficiency leading to uncontrolled cell growth and tumor progression.

The relationship between ARID1A and protein synthesis may be a common theme in enabling transformation or cancer progression in the context of ARID1A loss. ARID1A deficiency by itself does not drive transformation in murine ovary, liver, lung, or pancreas, and human gastric organoids.⁵⁻⁷,⁷²⁻⁷⁴ However, it has been shown in these tissues that co-deletion of the tumor suppressor PTEN,⁷ overexpression of the oncogene MYC,⁵ or expression of oncogenic KRAS or PIK3CA⁶,⁷,²,⁷³ along with ARID1A loss can drive tumorigenesis and cancer progression. All of these oncogenic signaling pathways that synergize with ARID1A loss are regulators of protein synthesis.⁷⁵ Therefore, gaining the ability to synthesize proteins efficiently is a key parameter for enabling the oncogenic properties of ARID1A loss. Our work reveals that ARID1A deficiency leads to a selective decrease in the translation of key oncogenic factors and DNA damage repair machinery. It remains to be determined if this is also true in other normal tissues. To this end, we have observed that ARID1A loss leads to a decrease in *de novo* protein synthesis within human prostate epithelial cells and murine bone marrow derived stroma (Figures S7E and S7F).

We have also identified a therapeutic vulnerability unique to ARID1A-deficient tumors. Given that rescue of translation elongation is needed to drive ARID1A-deficient tumor progression, we reasoned that this may provide a therapeutic window using an FDA approved translation inhibitor. Using 3 model systems, we found that ARID1A-deficient but not proficient cancers are exquisitely sensitive to translation inhibition. It has also been shown that SWI/SNF defective cancer cell lines and rhabdoid tumors are also sensitive to PI3K pathway inhibitors or HHT.⁷⁶,⁷⁷ These findings demonstrate how ARID1A deficiency creates a synthetic lethal relationship with mRNA translation which can be targeted to inhibit tumor growth. Given that multiple components of the SWI/SNF complex are mutated in various cancers,⁹ our findings raise the possibility that targeting

the translation apparatus may represent a new precision medicine paradigm applicable to a wide spectrum of human malignancies.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **RESOURCE AVAILABILITY**

* Lead contact

* Materials availability

* Data and code availability

* **EXPERIMENTAL MODELS AND SUBJECT DETAILS**

* Mice

* Cell culture

* Organoid culture

* **METHOD DETAILS**

* ARID1A re-expression

* Rasgrp1 re-expression

* Urine BCPN analysis

* Immunoblot analysis

* Mouse genotyping

* Hematoxylin and eosin staining (H&E), immunohistochemistry (IHC), immunofluorescence (IF)

* ChIP qPCR

* *In vivo* and *in vitro* puromycin incorporation assay

* RNA sequencing and polysome-bound RNA sequencing

* *In vitro* [³⁵S]-methionine labeling assay

* Cell viability assay

* siRNA knockdown

* Polysome profiling

* Ribosome half-transit time

* TMT mass spectrometry

* Preclinical trials

* CUT&Tag chromatin profiling

* Flow cytometry

* Cell cycle analysis

* Comet assay

* Clonogenic assay

* Organoid embedding and staining

* Drug administration

* **QUANTIFICATION AND STATISTICAL ANALYSIS**

* RNA-sequencing and polysome sequencing analysis

* CUT&Tag data analysis

* TMT mass spectrometry data analysis

* Ribosome half-transit time data analysis

* %GC content in coding sequence analysis

(C and D) γH2AX and CC3 staining and quantification in *Arid1a*<sup>fl/fl</sup> mouse urothelium (outlined in red) after tamoxifen administration and pre-treatment with A-484954 followed by a 9-day BBN treatment. n ≥ 4/arm. p < 0.0001 (C), p = 0.04 (D) (t test).

(E) Tumor size after tamoxifen followed by 150 days of BBN treatment in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mice. n ≥ 6/genotype. p < 0.0001 (t test).

(F) Percentage ARID1A (+) cells in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mice after tamoxifen followed by 150 days of BBN treatment. n ≥ 6/genotype. p < 0.0001 (t test).

(G) ARID1A IHC of *Arid1a*<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> tumors after tamoxifen followed by 150 days of BBN treatment. This demonstrates the presence of ARID1A-null tumors compared with Figure 6K.

Scale bar, 100 µm; mean ± SEM. Also see Figure S7.
This is mediated through enhanced expression of the apoptosis regulator BIM.⁶⁹ Importantly co-expression of MYC and the anti-apoptotic and inhibitor of BIM, BCL-2, leads to a significant acceleration in tumorigenesis.⁷⁰ Activated PIK3CA causes cellular differentiation in epidermal progenitors which is mediated by the AKT substrate SH3RF1. This leads to terminal differentiation of epidermal cells that possess activating mutations of PIK3CA and prevents oncogenic clonal expansion.⁷¹ In line with these oncogenic stress pathways, our data show that ARID1A loss leads to a significant reduction in eEF2 activity which promotes a conflict between the expression of oncogenic genes and their subsequent translation (Figure S7D). Restoration of translation elongation enabling gene expression parity is sufficient to unleash the oncogenic properties of ARID1A deficiency leading to uncontrolled cell growth and tumor progression.

The relationship between ARID1A and protein synthesis may be a common theme in enabling transformation or cancer progression in the context of ARID1A loss. ARID1A deficiency by itself does not drive transformation in murine ovary, liver, lung, or pancreas, and human gastric organoids.⁵⁻⁷,⁷²⁻⁷⁴ However, it has been shown in these tissues that co-deletion of the tumor suppressor PTEN,⁷ overexpression of the oncogene MYC,⁵ or expression of oncogenic KRAS or PIK3CA⁶,⁷,²²,⁷³ along with ARID1A loss can drive tumorigenesis and cancer progression. All of these oncogenic signaling pathways that synergize with ARID1A loss are regulators of protein synthesis.⁷⁵ Therefore, gaining the ability to synthesize proteins efficiently is a key parameter for enabling the oncogenic properties of ARID1A loss. Our work reveals that ARID1A deficiency leads to a selective decrease in the translation of key oncogenic factors and DNA damage repair machinery. It remains to be determined if this is also true in other normal tissues. To this end, we have observed that ARID1A loss leads to a decrease in *de novo* protein synthesis within human prostate epithelial cells and murine bone marrow derived stroma (Figures S7E and S7F).

We have also identified a therapeutic vulnerability unique to ARID1A-deficient tumors. Given that rescue of translation elongation is needed to drive ARID1A-deficient tumor progression, we reasoned that this may provide a therapeutic window using an FDA approved translation inhibitor. Using 3 model systems, we found that ARID1A-deficient but not proficient cancers are exquisitely sensitive to translation inhibition. It has also been shown that SWI/SNF defective cancer cell lines and rhabdoid tumors are also sensitive to PI3K pathway inhibitors or HHT.⁷⁶,⁷⁷ These findings demonstrate how ARID1A deficiency creates a synthetic lethal relationship with mRNA translation which can be targeted to inhibit tumor growth. Given that multiple components of the SWI/SNF complex are mutated in various cancers,⁹ our findings raise the possibility that targeting

the translation apparatus may represent a new precision medicine paradigm applicable to a wide spectrum of human malignancies.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **RESOURCE AVAILABILITY**

* Lead contact

* Materials availability

* Data and code availability

* **EXPERIMENTAL MODELS AND SUBJECT DETAILS**

* Mice

* Cell culture

* Organoid culture

* **METHOD DETAILS**

* ARID1A re-expression

* Rasgrp1 re-expression

* Urine BCPN analysis

* Immunoblot analysis

* Mouse genotyping

* Hematoxylin and eosin staining (H&E), immunohistochemistry (IHC), immunofluorescence (IF)

* ChIP qPCR

* *In vivo* and *in vitro* puromycin incorporation assay

* RNA sequencing and polysome-bound RNA sequencing

* *In vitro* [³⁵S]-methionine labeling assay

* Cell viability assay

* siRNA knockdown

* Polysome profiling

* Ribosome half-transit time

* TMT mass spectrometry

* Preclinical trials

* CUT&Tag chromatin profiling

* Flow cytometry

* Cell cycle analysis

* Comet assay

* Clonogenic assay

* Organoid embedding and staining

* Drug administration

* **QUANTIFICATION AND STATISTICAL ANALYSIS**

* RNA-sequencing and polysome sequencing analysis

* CUT&Tag data analysis

* TMT mass spectrometry data analysis

* Ribosome half-transit time data analysis

* %GC content in coding sequence analysis

(C and D) γH2AX and CC3 staining and quantification in *Arid1a*<sup>fl/fl</sup> mouse urothelium (outlined in red) after tamoxifen administration and pre-treatment with A-484954 followed by a 9-day BBN treatment. n ≥ 4/arm. p < 0.0001 (C), p = 0.04 (D) (t test).

(E) Tumor size after tamoxifen followed by 150 days of BBN treatment in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mice. n ≥ 6/genotype. p < 0.0001 (t test).

(F) Percentage ARID1A (+) cells in *Arid1a*<sup>fl/fl</sup> and *Arid1a*<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> mice after tamoxifen followed by 150 days of BBN treatment. n ≥ 6/genotype. p < 0.0001 (t test).

(G) ARID1A IHC of *Arid1a*<sup>fl/fl</sup>;Eef2k<sup>-/-</sup> tumors after tamoxifen followed by 150 days of BBN treatment. This demonstrates the presence of ARID1A-null tumors compared with Figure 6K.

Scale bar, 100 µm; mean ± SEM. Also see Figure S7.
SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.ccell.2023.03.021.

ACKNOWLEDGMENTS

We thank the patients who contributed to this study. This work was supported by the Kleberg Foundation, NIH grants R01 CA276308, R37 CA230617, R01 GM135362, and P30 CA015704, a Burroughs Wellcome Fund Career Award of Medicine Scientists (1012314.02), and grants from the Emerson Collective (691630), Seattle Translational Tumor Research, and Nancy & Dick Bernheimer, Matthews Family, Stinchcomb Family, and Thomas & Patricia Wright Memorial Funds to A.C.H. This research was also supported by the Shared Resources of the FHCC/UW (P30 CA015704 and S10 OD028685), NIH grants R01 CA187318 (J.P.R.), P01 AI091580 (J.P.R.), and the Mark Foundation For Cancer Research (P.D.). H.-M.L. is supported by U.S. Department of Defense (DOD) grant W81XWH-19-1-0624. S.J. is supported by DOD grant W81XWH-19-1-0658. S. Brahma is supported by NIH grant K99-GM138920. S. Blinka is supported by NIH grant T32-CA009515.

AUTHOR CONTRIBUTIONS

Conceptualization, S.J. and A.C.H.; Investigation, S.J., S. Brahma, C.L.W., P.H., S. Blinka, R.H., J.L.H., Y.L., S.A., L.-J.W., P.D., E.S., M.C.H., and A.R.S.; Writing - Original Draft, S.J. and A.C.H.; Writing - Review & Editing, S.J., A.C.H., R.B.M., J.K.L., P.G., J.L.W., H.-M.L., P.C.V., J.P.R., A.G.R., and S.H.; Funding Acquisition, A.C.H.; Resources, R.B.M., J.K.L., P.G., J.L.W., H.-M.L., P.C.V., J.P.R., A.G.R., and S.H.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: July 27, 2022

Revised: January 31, 2023

Accepted: March 24, 2023

Published: April 20, 2023

REFERENCES

1. Marcel, V., Ghayad, S.E., Belin, S., Therizols, G., Morel, A.P., Solano-González, E., Vendrell, J.A., Hacot, S., Mertani, H.C., Albaret, M.A., et al. (2013). p53 acts as a safeguard of translational control by regulating fibrillarin and rRNA methylation in cancer. Cancer Cell 24, 318-330. https://doi.org/10.1016/j.ccr.2013.08.013.

2. Hsieh, A.C., Liu, Y., Edlind, M.P., Ingolia, N.T., Janes, M.R., Sher, A., Shi, E.Y., Stumpf, C.R., Christensen, C., Bonham, M.J., et al. (2012). The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 485, 55-61. https://doi.org/10.1038/nature10912.

3. Faller, W.J., Jackson, T.J., Knight, J.R., Ridgway, R.A., Jamieson, T., Karim, S.A., Jones, C., Radulescu, S., Huels, D.J., Myant, K.B., et al. (2015). mTORC1-mediated translational elongation limits intestinal tumour initiation and growth. Nature 517, 497-500. https://doi.org/10.1038/nature13896.

4. Yang, H.S., Jansen, A.P., Komar, A.A., Zheng, X., Merrick, W.C., Costes, S., Lockett, S.J., Sonenberg, N., and Colburn, N.H. (2003). The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol. Cell Biol. 23, 26-37. https://doi.org/10.1128/jmb.23.1.26-37.2003.

5. Sun, X., Wang, S.C., Wei, Y., Luo, X., Jia, Y., Li, L., Gopal, P., Zhu, M., Nassour, I., Chuang, J.C., et al. (2017). Arid1a has context-dependent oncogenic and tumor suppressor functions in liver cancer. Cancer Cell 32, 574-589.e6. https://doi.org/10.1016/j.ccell.2017.10.007.

6. Chandler, R.L., Damrauer, J.S., Raab, J.R., Schisler, J.C., Wilkerson, M.D., Didion, J.P., Starmer, J., Serber, D., Yee, D., Xiong, J., et al. (2015). Coexistent ARID1A-PIK3CA mutations promote ovarian clear-cell

tumorigenesis through pro-tumorigenic inflammatory cytokine signalling. Nat. Commun. 6, 6118. https://doi.org/10.1038/ncomms7118.

7. Guan, B., Rahmanto, Y.S., Wu, R.C., Wang, Y., Wang, Z., Wang, T.L., and Shih, I.M. (2014). Roles of deletion of Arid1a, a tumor suppressor, in mouse ovarian tumorigenesis. J. Natl. Cancer Inst. 106, dju146. https://doi.org/10.1093/jnci/dju146.

8. ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium (2020). Pan-cancer analysis of whole genomes. Nature 578, 82-93. https://doi.org/10.1038/s41586-020-1969-6.

9. Kadoch, C., and Crabtree, G.R. (2015). Mammalian SWI/SNF chromatin remodeling complexes and cancer: mechanistic insights gained from human genomics. Sci. Adv. 1, e1500447. https://doi.org/10.1126/sciadv.1500447.

10. Centore, R.C., Sandoval, G.J., Soares, L.M.M., Kadoch, C., and Chan, H.M. (2020). Mammalian SWI/SNF chromatin remodeling complexes: emerging mechanisms and therapeutic strategies. Trends Genet. 36, 936-950. https://doi.org/10.1016/j.tig.2020.07.011.

11. Shen, J., Peng, Y., Wei, L., Zhang, W., Yang, L., Lan, L., Kapoor, P., Ju, Z., Mo, Q., Shih, I.M., et al. (2015). ARID1A deficiency impairs the DNA damage checkpoint and sensitizes cells to PARP inhibitors. Cancer Discov. 5, 752-767. https://doi.org/10.1158/2159-8290.CD-14-0849.

12. Dykhuizen, E.C., Hargreaves, D.C., Miller, E.L., Cui, K., Korshunov, A., Kool, M., Pfister, S., Cho, Y.J., Zhao, K., and Crabtree, G.R. (2013). BAF complexes facilitate decatenation of DNA by topoisomerase IIalpha. Nature 497, 624-627. https://doi.org/10.1038/nature12146.

13. Siegel, R.L., Miller, K.D., Fuchs, H.E., and Jemal, A. (2021). Cancer statistics, 2021. CA. Cancer J. Clin. 71, 7-33. https://doi.org/10.3322/caac.21654.

14. Robertson, A.G., Kim, J., Al-Ahmadie, H., Bellmunt, J., Guo, G., Cherniack, A.D., Hinoue, T., Laird, P.W., Hoadley, K.A., Akbani, R., et al. (2017). Comprehensive molecular characterization of muscle-invasive bladder cancer. Cell 171, 540-556.e25. https://doi.org/10.1016/j.cell.2017.09.007.

15. Winters, B.R., De Sarkar, N., Arora, S., Bolouri, H., Jana, S., Vakar-Lopez, F., Cheng, H.H., Schweizer, M.T., Yu, E.Y., Grivas, P., et al. (2019). Genomic distinctions between metastatic lower and upper tract urothelial carcinoma revealed through rapid autopsy. JCI Insight 5, e128728. https://doi.org/10.1172/jci.insight.128728.

16. Faltas, B.M., Prandi, D., Tagawa, S.T., Molina, A.M., Nanus, D.M., Sternberg, C., Rosenberg, J., Mosquera, J.M., Robinson, B., Elemento, O., et al. (2016). Clonal evolution of chemotherapy-resistant urothelial carcinoma. Nat. Genet. 48, 1490-1499. https://doi.org/10.1038/ng.3692.

17. Van Batavia, J., Yamany, T., Molotkov, A., Dan, H., Mansukhani, M., Batourina, E., Schneider, K., Oyon, D., Dunlop, M., Wu, X.R., et al. (2014). Bladder cancers arise from distinct urothelial sub-populations. Nat. Cell Biol. 16, 981-985. https://doi.org/10.1038/ncb3038.

18. Shin, K., Lim, A., Odegaard, J.I., Honeycutt, J.D., Kawano, S., Hsieh, M.H., and Beachy, P.A. (2014). Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma. Nat. Cell Biol. 16, 469-478. https://doi.org/10.1038/ncb2956.

19. Chiu, Y.F., Wu, C.C., Kuo, M.H., Miao, C.C., Zheng, M.Y., Chen, P.Y., Lin, S.C., Chang, J.L., Wang, Y.H., and Chou, Y.T. (2020). Critical role of SOX2-IGF2 signaling in aggressiveness of bladder cancer. Sci. Rep. 10, 8261. https://doi.org/10.1038/s41598-020-65006-z.

20. Lin, H., Huang, Y.S., Fustin, J.M., Doi, M., Chen, H., Lai, H.H., Lin, S.H., Lee, Y.L., King, P.C., Hou, H.S., et al. (2021). Hyperpolyploidization of hematocyte initiates preneoplastic lesion formation in the liver. Nat. Commun. 12, 645. https://doi.org/10.1038/s41467-020-20572-8.

21. Erichsen, L., Seifert, H.H., Schulz, W.A., Hoffmann, M.J., Niegisch, G., Araúzo-Bravo, M.J., Bendhack, M.L., Poyet, C., Hermanns, T., Beermann, A., et al. (2020). Basic hallmarks of urothelial cancer unleashed in primary uroepithelium by interference with the epigenetic master regulator ODC1. Sci. Rep. 10, 3808. https://doi.org/10.1038/s41598-020-60796-8.
SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.ccell.2023.03.021.

ACKNOWLEDGMENTS

We thank the patients who contributed to this study. This work was supported by the Kleberg Foundation, NIH grants R01 CA276308, R37 CA230617, R01 GM135362, and P30 CA015704, a Burroughs Wellcome Fund Career Award of Medicine Scientists (1012314.02), and grants from the Emerson Collective (691630), Seattle Translational Tumor Research, and Nancy & Dick Bernheimer, Matthews Family, Stinchcomb Family, and Thomas & Patricia Wright Memorial Funds to A.C.H. This research was also supported by the Shared Resources of the FHCC/UW (P30 CA015704 and S10 OD028685), NIH grants R01 CA187318 (J.P.R.), P01 AI091580 (J.P.R.), and the Mark Foundation For Cancer Research (P.D.). H.-M.L. is supported by U.S. Department of Defense (DOD) grant W81XWH-19-1-0624. S.J. is supported by DOD grant W81XWH-19-1-0658. S. Brahma is supported by NIH grant K99-GM138920. S. Blinka is supported by NIH grant T32-CA009515.

AUTHOR CONTRIBUTIONS

Conceptualization, S.J. and A.C.H.; Investigation, S.J., S. Brahma, C.L.W., P.H., S. Blinka, R.H., J.L.H., Y.L., S.A., L.-J.W., P.D., E.S., M.C.H., and A.R.S.; Writing - Original Draft, S.J. and A.C.H.; Writing - Review & Editing, S.J., A.C.H., R.B.M., J.K.L., P.G., J.L.W., H.-M.L., P.C.V., J.P.R., A.G.R., and S.H.; Funding Acquisition, A.C.H.; Resources, R.B.M., J.K.L., P.G., J.L.W., H.-M.L., P.C.V., J.P.R., A.G.R., and S.H.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: July 27, 2022

Revised: January 31, 2023

Accepted: March 24, 2023

Published: April 20, 2023

REFERENCES

1. Marcel, V., Ghayad, S.E., Belin, S., Therizols, G., Morel, A.P., Solano-González, E., Vendrell, J.A., Hacot, S., Mertani, H.C., Albaret, M.A., et al. (2013). p53 acts as a safeguard of translational control by regulating fibrillarin and rRNA methylation in cancer. Cancer Cell 24, 318-330. https://doi.org/10.1016/j.ccr.2013.08.013.

2. Hsieh, A.C., Liu, Y., Edlind, M.P., Ingolia, N.T., Janes, M.R., Sher, A., Shi, E.Y., Stumpf, C.R., Christensen, C., Bonham, M.J., et al. (2012). The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 485, 55-61. https://doi.org/10.1038/nature10912.

3. Faller, W.J., Jackson, T.J., Knight, J.R., Ridgway, R.A., Jamieson, T., Karim, S.A., Jones, C., Radulescu, S., Huels, D.J., Myant, K.B., et al. (2015). mTORC1-mediated translational elongation limits intestinal tumour initiation and growth. Nature 517, 497-500. https://doi.org/10.1038/nature13896.

4. Yang, H.S., Jansen, A.P., Komar, A.A., Zheng, X., Merrick, W.C., Costes, S., Lockett, S.J., Sonenberg, N., and Colburn, N.H. (2003). The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol. Cell Biol. 23, 26-37. https://doi.org/10.1128/jmb.23.1.26-37.2003.

5. Sun, X., Wang, S.C., Wei, Y., Luo, X., Jia, Y., Li, L., Gopal, P., Zhu, M., Nassour, I., Chuang, J.C., et al. (2017). Arid1a has context-dependent oncogenic and tumor suppressor functions in liver cancer. Cancer Cell 32, 574-589.e6. https://doi.org/10.1016/j.ccell.2017.10.007.

6. Chandler, R.L., Damrauer, J.S., Raab, J.R., Schisler, J.C., Wilkerson, M.D., Didion, J.P., Starmer, J., Serber, D., Yee, D., Xiong, J., et al. (2015). Coexistent ARID1A-PIK3CA mutations promote ovarian clear-cell

tumorigenesis through pro-tumorigenic inflammatory cytokine signalling. Nat. Commun. 6, 6118. https://doi.org/10.1038/ncomms7118.

7. Guan, B., Rahmanto, Y.S., Wu, R.C., Wang, Y., Wang, Z., Wang, T.L., and Shih, I.M. (2014). Roles of deletion of Arid1a, a tumor suppressor, in mouse ovarian tumorigenesis. J. Natl. Cancer Inst. 106, dju146. https://doi.org/10.1093/jnci/dju146.

8. ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium (2020). Pan-cancer analysis of whole genomes. Nature 578, 82-93. https://doi.org/10.1038/s41586-020-1969-6.

9. Kadoch, C., and Crabtree, G.R. (2015). Mammalian SWI/SNF chromatin remodeling complexes and cancer: mechanistic insights gained from human genomics. Sci. Adv. 1, e1500447. https://doi.org/10.1126/sciadv.1500447.

10. Centore, R.C., Sandoval, G.J., Soares, L.M.M., Kadoch, C., and Chan, H.M. (2020). Mammalian SWI/SNF chromatin remodeling complexes: emerging mechanisms and therapeutic strategies. Trends Genet. 36, 936-950. https://doi.org/10.1016/j.tig.2020.07.011.

11. Shen, J., Peng, Y., Wei, L., Zhang, W., Yang, L., Lan, L., Kapoor, P., Ju, Z., Mo, Q., Shih, I.M., et al. (2015). ARID1A deficiency impairs the DNA damage checkpoint and sensitizes cells to PARP inhibitors. Cancer Discov. 5, 752-767. https://doi.org/10.1158/2159-8290.CD-14-0849.

12. Dykhuizen, E.C., Hargreaves, D.C., Miller, E.L., Cui, K., Korshunov, A., Kool, M., Pfister, S., Cho, Y.J., Zhao, K., and Crabtree, G.R. (2013). BAF complexes facilitate decatenation of DNA by topoisomerase IIalpha. Nature 497, 624-627. https://doi.org/10.1038/nature12146.

13. Siegel, R.L., Miller, K.D., Fuchs, H.E., and Jemal, A. (2021). Cancer statistics, 2021. CA. Cancer J. Clin. 71, 7-33. https://doi.org/10.3322/caac.21654.

14. Robertson, A.G., Kim, J., Al-Ahmadie, H., Bellmunt, J., Guo, G., Cherniack, A.D., Hinoue, T., Laird, P.W., Hoadley, K.A., Akbani, R., et al. (2017). Comprehensive molecular characterization of muscle-invasive bladder cancer. Cell 171, 540-556.e25. https://doi.org/10.1016/j.cell.2017.09.007.

15. Winters, B.R., De Sarkar, N., Arora, S., Bolouri, H., Jana, S., Vakar-Lopez, F., Cheng, H.H., Schweizer, M.T., Yu, E.Y., Grivas, P., et al. (2019). Genomic distinctions between metastatic lower and upper tract urothelial carcinoma revealed through rapid autopsy. JCI Insight 5, e128728. https://doi.org/10.1172/jci.insight.128728.

16. Faltas, B.M., Prandi, D., Tagawa, S.T., Molina, A.M., Nanus, D.M., Sternberg, C., Rosenberg, J., Mosquera, J.M., Robinson, B., Elemento, O., et al. (2016). Clonal evolution of chemotherapy-resistant urothelial carcinoma. Nat. Genet. 48, 1490-1499. https://doi.org/10.1038/ng.3692.

17. Van Batavia, J., Yamany, T., Molotkov, A., Dan, H., Mansukhani, M., Batourina, E., Schneider, K., Oyon, D., Dunlop, M., Wu, X.R., et al. (2014). Bladder cancers arise from distinct urothelial sub-populations. Nat. Cell Biol. 16, 981-985. https://doi.org/10.1038/ncb3038.

18. Shin, K., Lim, A., Odegaard, J.I., Honeycutt, J.D., Kawano, S., Hsieh, M.H., and Beachy, P.A. (2014). Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma. Nat. Cell Biol. 16, 469-478. https://doi.org/10.1038/ncb2956.

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65. Walden, H., and Deans, A.J. (2014). The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder. Annu. Rev. Biophys. 43, 257-278. https://doi.org/10.1146/annurev-bio-phys-051013-022737.

66. Chen, Z., Trotman, L.C., Shaffer, D., Lin, H.K., Dotan, Z.A., Niki, M., Koutcher, J.A., Scher, H.I., Ludwig, T., Gerald, W., et al. (2005). Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 436, 725-730. https://doi.org/10.1038/nature03918.

67. Neiman, P.E., Thomas, S.J., and Loring, G. (1991). Induction of apoptosis during normal and neoplastic B-cell development in the bursa of Fabricius. Proc. Natl. Acad. Sci. USA 88, 5857-5861. https://doi.org/10.1073/pnas.88.13.5857.

68. Wyllie, A.H., Rose, K.A., Morris, R.G., Steel, C.M., Foster, E., and Spandidos, D.A. (1987). Rodent fibroblast tumours expressing human myc and ras genes: growth, metastasis and endogenous oncogene expression. Br. J. Cancer 56, 251-259. https://doi.org/10.1038/bjc.1987.186.

69. Hemann, M.T., Bric, A., Teruya-Feldstein, J., Herbst, A., Nilsson, J.A., Cordon-Cardo, C., Cleveland, J.L., Tansey, W.P., and Lowe, S.W. (2005). Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants. Nature 436, 807-811. https://doi.org/10.1038/nature03845.

70. Strasser, A., Harris, A.W., Bath, M.L., and Cory, S. (1990). Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Nature 348, 331-333. https://doi.org/10.1038/348331a0.

71. Ying, Z., Sandoval, M., and Beronja, S. (2018). Oncogenic activation of PI3K induces progenitor cell differentiation to suppress epidermal growth. Nat. Cell Biol. 20, 1256-1266. https://doi.org/10.1038/s41556-018-0218-9.

72. Wang, S.C., Nassour, I., Xiao, S., Zhang, S., Luo, X., Lee, J., Li, L., Sun, X., Nguyen, L.H., Chuang, J.C., et al. (2019). SWI/SNF component ARID1A restrains pancreatic neoplasia formation. Gut 68, 1259-1270. https://doi.org/10.1136/gutjnl-2017-315490.

73. Liu, X., Li, Z., Wang, Z., Liu, F., Zhang, L., Ke, J., Xu, X., Zhang, Y., Yuan, Y., Wei, T., et al. (2022). Chromatin remodeling induced by ARID1A loss in lung cancer promotes glycolysis and confers JQ1 vulnerability. Cancer Res. 82, 791-804. https://doi.org/10.1158/0008-5472.CAN-21-0763.

74. Lo, Y.H., Kolahi, K.S., Du, Y., Chang, C.Y., Krokhotin, A., Nair, A., Sobba, W.D., Karlsson, K., Jones, S.J., Longacre, T.A., et al. (2021). A CRISPR/Cas9-Engineered ARID1A-deficient human gastric cancer organoid model reveals essential and nonessential modes of oncogenic transformation. Cancer Discov. 11, 1562-1581. https://doi.org/10.1158/2159-8290.CD-20-1109.

75. Robichaud, N., Sonenberg, N., Ruggero, D., and Schneider, R.J. (2019). Translational control in cancer. Cold Spring Harb. Perspect. Biol. 11, a032896. https://doi.org/10.1101/cshperspect.a032896.

76. Howard, T.P., Oberlick, E.M., Rees, M.G., Arnoff, T.E., Pham, M.T., Brenan, L., DoCarmo, M., Hong, A.L., Kugener, G., Chou, H.C., et al. (2020). Rhabdoid tumors are sensitive to the protein-translation inhibitor homoharringtonine. Clin. Cancer Res. 26, 4995-5006. https://doi.org/10.1158/1078-0432.CCR-19-2717.

77. Ulicna, L., Kimmey, S.C., Weber, C.M., Allard, G.M., Wang, A., Bui, N.Q., Bendall, S.C., Crabtree, G.R., Bean, G.R., and Van Rechem, C. (2022). The interaction of SWI/SNF with the ribosome regulates translation and confers sensitivity to translation pathway inhibitors in cancers with complex perturbations. Cancer Res. 82, 2829-2837. https://doi.org/10.1158/0008-5472.CAN-21-1360.

78. Kaya-Okur, H.S., Wu, S.J., Codomo, C.A., Pledger, E.S., Bryson, T.D., Henikoff, J.G., Ahmad, K., and Henikoff, S. (2019). CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10, 1930. https://doi.org/10.1038/s41467-019-09982-5.

79. Henikoff, S., Henikoff, J.G., and Ahmad, K. (2021). Simplified epigenome profiling using antibody-tethered tagmentation. Bio. Protoc. 11, e4043. https://doi.org/10.21769/BioProtoc.4043.

80. Buenrostro, J.D., Wu, B., Litzenburger, U.M., Ruff, D., Gonzales, M.L., Snyder, M.P., Chang, H.Y., and Greenleaf, W.J. (2015). Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-490. https://doi.org/10.1038/nature14590.

81. Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21. https://doi.org/10.1093/bioinformatics/bts635.

82. Anders, S., Pyl, P.T., and Huber, W. (2015). HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166-169. https://doi.org/10.1093/bioinformatics/btu638.

83. Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene
53. Jana, S., Deo, R., Hough, R.P., Liu, Y., Horn, J.L., Wright, J.L., Lam, H.M., Webster, K.R., Chiang, G.G., Sonenberg, N., and Hsieh, A.C. (2021). mRNA translation is a therapeutic vulnerability necessary for bladder epithelial transformation. JCI Insight 6, e144920. https://doi.org/10.1172/jci.insight.144920.

54. Nguyen, T.M., Kabotyanski, E.B., Dou, Y., Reineke, L.C., Zhang, P., Zhang, X.H.F., Malovannaya, A., Jung, S.Y., Mo, Q., Roarty, K.P., et al. (2018). FGFR1-Activated translation of WNT pathway components with structured 5' UTRs is vulnerable to inhibition of EIF4A-dependent translation initiation. Cancer Res. 78, 4229-4240. https://doi.org/10.1158/0008-5472.CAN-18-0631.

55. Truitt, M.L., Conn, C.S., Shi, Z., Pang, X., Tokuyasu, T., Coady, A.M., Seo, Y., Barna, M., and Ruggero, D. (2015). Differential requirements for eIF4E dose in normal development and cancer. Cell 162, 59-71. https://doi.org/10.1016/j.cell.2015.05.049.

56. Bianco, C., Thompson, L., and Mohr, I. (2019). Repression of eEF2K transcription by NF-kappaB tunes translation elongation to inflammation and dsDNA-sensing. Proc. Natl. Acad. Sci. USA 116, 22583-22590. https://doi.org/10.1073/pnas.1909143116.

57. Fresno, M., Jiménez, A., and Vázquez, D. (1977). Inhibition of translation in eukaryotic systems by harringtonine. Eur. J. Biochem. 72, 323-330. https://doi.org/10.1111/j.1432-1033.1977.tb11256.x.

58. Kantarjian, H.M., Talpaz, M., Smith, T.L., Cortes, J., Giles, F.J., Rios, M.B., Mallard, S., Gajewski, J., Murgo, A., Cheson, B., and O'Brien, S. (2000). Homoharringtonine and low-dose cytarabine in the management of late chronic-phase chronic myelogenous leukemia. J. Clin. Oncol. 18, 3513-3521.

59. Ingolia, N.T., Lareau, L.F., and Weissman, J.S. (2011). Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789-802. https://doi.org/10.1016/j.cell.2011.10.002.

60. Wolff, N.C., Pavía-Jiménez, A., Tcheuyap, V.T., Alexander, S., Vishwanath, M., Christie, A., Xie, X.J., Williams, N.S., Kapur, P., Posner, B., et al. (2015). High-throughput simultaneous screen and counterscreen identifies homoharringtonine as synthetic lethal with von Hippel-Lindau loss in renal cell carcinoma. Oncotarget 6, 16951-16962. https://doi.org/10.18632/oncotarget.4773.

61. Gandhi, V., Plunkett, W., and Cortes, J.E. (2014). Omacetaxine: a protein translation inhibitor for treatment of chronic myelogenous leukemia. Clin. Cancer Res. 20, 1735-1740. https://doi.org/10.1158/1078-0432.CCR-13-1283.

62. Mouw, K.W., and D'Andrea, A.D. (2014). Crosstalk between the nucleotide excision repair and Fanconi anemia/BRCA pathways. DNA Repair 19, 130-134. https://doi.org/10.1016/j.dnarep.2014.03.019.

63. Chen, C.C., Feng, W., Lim, P.X., Kass, E.M., and Jasin, M. (2018). Homology-directed repair and the role of BRCA1, BRCA2, and related proteins in genome integrity and cancer. Annu. Rev. Cancer Biol. 2, 313-336. https://doi.org/10.1146/annurev-cancerbio-030617-050502.

64. Marteijn, J.A., Lans, H., Vermeulen, W., and Hoeijmakers, J.H.J. (2014). Understanding nucleotide excision repair and its roles in cancer and ageing. Nat. Rev. Mol. Cell Biol. 15, 465-481. https://doi.org/10.1038/nrm3822.

65. Walden, H., and Deans, A.J. (2014). The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder. Annu. Rev. Biophys. 43, 257-278. https://doi.org/10.1146/annurev-bio-phys-051013-022737.

66. Chen, Z., Trotman, L.C., Shaffer, D., Lin, H.K., Dotan, Z.A., Niki, M., Koutcher, J.A., Scher, H.I., Ludwig, T., Gerald, W., et al. (2005). Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 436, 725-730. https://doi.org/10.1038/nature03918.

67. Neiman, P.E., Thomas, S.J., and Loring, G. (1991). Induction of apoptosis during normal and neoplastic B-cell development in the bursa of Fabricius. Proc. Natl. Acad. Sci. USA 88, 5857-5861. https://doi.org/10.1073/pnas.88.13.5857.

68. Wyllie, A.H., Rose, K.A., Morris, R.G., Steel, C.M., Foster, E., and Spandidos, D.A. (1987). Rodent fibroblast tumours expressing human myc and ras genes: growth, metastasis and endogenous oncogene expression. Br. J. Cancer 56, 251-259. https://doi.org/10.1038/bjc.1987.186.

69. Hemann, M.T., Bric, A., Teruya-Feldstein, J., Herbst, A., Nilsson, J.A., Cordon-Cardo, C., Cleveland, J.L., Tansey, W.P., and Lowe, S.W. (2005). Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants. Nature 436, 807-811. https://doi.org/10.1038/nature03845.

70. Strasser, A., Harris, A.W., Bath, M.L., and Cory, S. (1990). Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Nature 348, 331-333. https://doi.org/10.1038/348331a0.

71. Ying, Z., Sandoval, M., and Beronja, S. (2018). Oncogenic activation of PI3K induces progenitor cell differentiation to suppress epidermal growth. Nat. Cell Biol. 20, 1256-1266. https://doi.org/10.1038/s41556-018-0218-9.

72. Wang, S.C., Nassour, I., Xiao, S., Zhang, S., Luo, X., Lee, J., Li, L., Sun, X., Nguyen, L.H., Chuang, J.C., et al. (2019). SWI/SNF component ARID1A restraints pancreatic neoplasia formation. Gut 68, 1259-1270. https://doi.org/10.1136/gutjnl-2017-315490.

73. Liu, X., Li, Z., Wang, Z., Liu, F., Zhang, L., Ke, J., Xu, X., Zhang, Y., Yuan, Y., Wei, T., et al. (2022). Chromatin remodeling induced by ARID1A loss in lung cancer promotes glycolysis and confers JQ1 vulnerability. Cancer Res. 82, 791-804. https://doi.org/10.1158/0008-5472.CAN-21-0763.

74. Lo, Y.H., Kolahi, K.S., Du, Y., Chang, C.Y., Krokhotin, A., Nair, A., Sobba, W.D., Karlsson, K., Jones, S.J., Longacre, T.A., et al. (2021). A CRISPR/Cas9-Engineered ARID1A-deficient human gastric cancer organoid model reveals essential and nonessential modes of oncogenic transformation. Cancer Discov. 11, 1562-1581. https://doi.org/10.1158/2159-8290.CD-20-1109.

75. Robichaud, N., Sonenberg, N., Ruggero, D., and Schneider, R.J. (2019). Translational control in cancer. Cold Spring Harb. Perspect. Biol. 11, a032896. https://doi.org/10.1101/cshperspect.a032896.

76. Howard, T.P., Oberlick, E.M., Rees, M.G., Arnoff, T.E., Pham, M.T., Brenan, L., DoCarmo, M., Hong, A.L., Kugener, G., Chou, H.C., et al. (2020). Rhabdoid tumors are sensitive to the protein-translation inhibitor homoharringtonine. Clin. Cancer Res. 26, 4995-5006. https://doi.org/10.1158/1078-0432.CCR-19-2717.

77. Ulicna, L., Kimmey, S.C., Weber, C.M., Allard, G.M., Wang, A., Bui, N.Q., Bendall, S.C., Crabtree, G.R., Bean, G.R., and Van Rechem, C. (2022). The interaction of SWI/SNF with the ribosome regulates translation and confers sensitivity to translation pathway inhibitors in cancers with complex perturbations. Cancer Res. 82, 2829-2837. https://doi.org/10.1158/0008-5472.CAN-21-1360.

78. Kaya-Okur, H.S., Wu, S.J., Codomo, C.A., Pledger, E.S., Bryson, T.D., Henikoff, J.G., Ahmad, K., and Henikoff, S. (2019). CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10, 1930. https://doi.org/10.1038/s41467-019-09982-5.

79. Henikoff, S., Henikoff, J.G., and Ahmad, K. (2021). Simplified epigenome profiling using antibody-tethered tagmentation. Bio. Protoc. 11, e4043. https://doi.org/10.21769/BioProtoc.4043.

80. Buenrostro, J.D., Wu, B., Litzenburger, U.M., Ruff, D., Gonzales, M.L., Snyder, M.P., Chang, H.Y., and Greenleaf, W.J. (2015). Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-490. https://doi.org/10.1038/nature14590.

81. Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21. https://doi.org/10.1093/bioinformatics/bts635.

82. Anders, S., Pyl, P.T., and Huber, W. (2015). HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166-169. https://doi.org/10.1093/bioinformatics/btu638.

83. Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene
expression data. Bioinformatics 26, 139-140. https://doi.org/10.1093/bioinformatics/btp616.

84. Xiao, Z., Zou, Q., Liu, Y., and Yang, X. (2016). Genome-wide assessment of differential translations with ribosome profiling data. Nat. Commun. 7, 11194. https://doi.org/10.1038/ncomms11194.

85. Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag).

86. Law, C.W., Chen, Y., Shi, W., and Smyth, G.K. (2014). voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29. https://doi.org/10.1186/gb-2014-15-2-r29.

87. Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359. https://doi.org/10.1038/nmeth.1923.

88. Ramírez, F., Ryan, D.P., Grüning, B., Bhardwaj, V., Kilpert, F., Richter, A.S., Heyne, S., Dündar, F., and Manke, T. (2016). deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160-W165. https://doi.org/10.1093/nar/gkw257.

89. Eng, J.K., McCormack, A.L., and Yates, J.R. (1994). An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976-989. https://doi.org/10.1016/1044-0305(94)80016-2.
expression data. Bioinformatics 26, 139-140. https://doi.org/10.1093/bioinformatics/btp616.

84. Xiao, Z., Zou, Q., Liu, Y., and Yang, X. (2016). Genome-wide assessment of differential translations with ribosome profiling data. Nat. Commun. 7, 11194. https://doi.org/10.1038/ncomms11194.

85. Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag).

86. Law, C.W., Chen, Y., Shi, W., and Smyth, G.K. (2014). voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29. https://doi.org/10.1186/gb-2014-15-2-r29.

87. Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359. https://doi.org/10.1038/nmeth.1923.

88. Ramírez, F., Ryan, D.P., Grüning, B., Bhardwaj, V., Kilpert, F., Richter, A.S., Heyne, S., Dündar, F., and Manke, T. (2016). deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160-W165. https://doi.org/10.1093/nar/gkw257.

89. Eng, J.K., McCormack, A.L., and Yates, J.R. (1994). An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976-989. https://doi.org/10.1016/1044-0305(94)80016-2.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Antibodies</td></tr><tr><td>PTEN (138G6), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 9559; RRID: AB_390810</td></tr><tr><td>Phospho-Akt (Ser473) (D9E), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 4060; RRID: AB_2315049</td></tr><tr><td>Akt1 (C-20), polyclonal</td><td>Santa Cruz Biotechnology</td><td>Cat# SC-1618; RRID: AB_630849</td></tr><tr><td>Alpha-Tubulin, mouse monoclonal</td><td>Sigma</td><td>Cat# T8203; RRID: AB_1841230</td></tr><tr><td>phospho-eIF2alpha (S51), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 3597; RRID: AB_390740</td></tr><tr><td>ATF-4 (D4B8), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 11815; RRID: AB_2616025</td></tr><tr><td>eIF4G (C45A4), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2469; RRID: AB_2096028</td></tr><tr><td>eIF4E (A-10), mouse monoclonal</td><td>Santa Cruz Biotechnology</td><td>Cat# SC-271480; RRID: AB_10649368</td></tr><tr><td>eIF4A (C32B4), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2013; RRID: AB_2097363</td></tr><tr><td>Phospho-4E-BP1 (Thr37/46) (236B4), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2855; RRID: AB_560835</td></tr><tr><td>4E-BP1 (53H11), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 9644; RRID: AB_2097841</td></tr><tr><td>Phospho-eEF2 (Thr56), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 2331; RRID: AB_10015204</td></tr><tr><td>eEF2, rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2332; RRID: AB_10693546</td></tr><tr><td>AURKB, rabbit polyclonal</td><td>Novusbio</td><td>Cat# NB-100-294; RRID: AB_2061617</td></tr><tr><td>FGFR3, rabbit anti-Human, mouse<br>Fgfr3 Polyclonal</td><td>MyBioSource.com</td><td>Cat# MBS9212039</td></tr><tr><td>IGF2, mouse monoclonal</td><td>ThermoFisher</td><td>Cat# MA5-17096; RRID: AB_2538567</td></tr><tr><td>KIF22, rabbit polyclonal</td><td>Novusbio</td><td>Cat# NBP2-17053</td></tr><tr><td>ODC1, polyclonal</td><td>ThermoFisher</td><td>Cat# PA5-21362; RRID: AB_11154067</td></tr><tr><td>SKA1, rabbit polyclonal</td><td>Novusbio</td><td>Cat# NBP2-88277</td></tr><tr><td>BRCA2, rabbit polyclonal</td><td>Abcam</td><td>Cat# AB27976; RRID: AB_2067760</td></tr><tr><td>ERCC1, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 3885; RRID: AB_2100142</td></tr><tr><td>ERCC2, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 11963; RRID: AB_2797781</td></tr><tr><td>FANCC, rabbit polyclonal</td><td>Abcam</td><td>Cat# AB97575; RRID: AB_10679967</td></tr><tr><td>Phospho-AMPKα (Thr172) (40H9), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2535; RRID: AB_331250</td></tr><tr><td>AMPK alpha, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 2532; RRID: AB_330331</td></tr><tr><td>Tri-Methyl-Histone H3 (Lys27) (C36B11), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9733; RRID: AB_2616029</td></tr><tr><td>Histone H3, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9715; RRID: AB_331563</td></tr><tr><td>ARID1A/BAF250A (D2A8U), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 12354; RRID: AB_2637010</td></tr><tr><td>Anti-Puromycin, clone 12D10, mouse monoclonal</td><td>EMD Millipore</td><td>Cat# MABE343; RRID: AB_2566826</td></tr><tr><td>IgG (H + L), rabbit HRP</td><td>Invitrogen</td><td>Cat# PI31460</td></tr><tr><td>IgG (H + L), mouse HRP</td><td>Invitrogen</td><td>Cat# PI31430</td></tr><tr><td>ARID1A, rabbit</td><td>Sigma</td><td>Cat# HPA005456; RRID: AB_1078205</td></tr><tr><td>ARID1A, mouse monoclonal</td><td>Santa Cruz</td><td>Cat# SC-32761; RRID: AB_673396</td></tr><tr><td>p-eEF2K (S366), rabbit polyclonal</td><td>Abcam</td><td>Cat# AB51227; RRID: AB_869561</td></tr><tr><td>p-eEF2K (S398), rabbit polyclonal</td><td>ECM Bio</td><td>Cat# EP5441</td></tr></tbody></table>

(Continued on next page)
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Antibodies</td><td></td><td></td></tr><tr><td>PTEN (138G6), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 9559; RRID: AB_390810</td></tr><tr><td>Phospho-Akt (Ser473) (D9E), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 4060; RRID: AB_2315049</td></tr><tr><td>Akt1 (C-20), polyclonal</td><td>Santa Cruz Biotechnology</td><td>Cat# SC-1618; RRID: AB_630849</td></tr><tr><td>Alpha-Tubulin, mouse monoclonal</td><td>Sigma</td><td>Cat# T8203; RRID: AB_1841230</td></tr><tr><td>phospho-eIF2alpha (S51), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 3597; RRID: AB_390740</td></tr><tr><td>ATF-4 (D4B8), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 11815; RRID: AB_2616025</td></tr><tr><td>eIF4G (C45A4), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2469; RRID: AB_2096028</td></tr><tr><td>eIF4E (A-10), mouse monoclonal</td><td>Santa Cruz Biotechnology</td><td>Cat# SC-271480; RRID: AB_10649368</td></tr><tr><td>eIF4A (C32B4), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2013; RRID: AB_2097363</td></tr><tr><td>Phospho-4E-BP1 (Thr37/46) (236B4), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2855; RRID: AB_560835</td></tr><tr><td>4E-BP1 (53H11), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 9644; RRID: AB_2097841</td></tr><tr><td>Phospho-eEF2 (Thr56), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 2331; RRID: AB_10015204</td></tr><tr><td>eEF2, rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2332; RRID: AB_10693546</td></tr><tr><td>AURKB, rabbit polyclonal</td><td>Novusbio</td><td>Cat# NB-100-294; RRID: AB_2061617</td></tr><tr><td>FGFR3, rabbit anti-Human, mouse</td><td>MyBioSource.com</td><td>Cat# MBS9212039</td></tr><tr><td>Fgfr3 Polyclonal</td><td></td><td></td></tr><tr><td>IGF2, mouse monoclonal</td><td>ThermoFisher</td><td>Cat# MA5-17096; RRID: AB_2538567</td></tr><tr><td>KIF22, rabbit polyclonal</td><td>Novusbio</td><td>Cat# NBP2-17053</td></tr><tr><td>ODC1, polyclonal</td><td>ThermoFisher</td><td>Cat# PA5-21362; RRID: AB_11154067</td></tr><tr><td>SKA1, rabbit polyclonal</td><td>Novusbio</td><td>Cat# NBP2-88277</td></tr><tr><td>BRCA2, rabbit polyclonal</td><td>Abcam</td><td>Cat# AB27976; RRID: AB_2067760</td></tr><tr><td>ERCC1, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 3885; RRID: AB_2100142</td></tr><tr><td>ERCC2, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 11963; RRID: AB_2797781</td></tr><tr><td>FANCC, rabbit polyclonal</td><td>Abcam</td><td>Cat# AB97575; RRID: AB_10679967</td></tr><tr><td>Phospho-AMPKα (Thr172) (40H9), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 2535; RRID: AB_331250</td></tr><tr><td>AMPK alpha, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 2532; RRID: AB_330331</td></tr><tr><td>Tri-Methyl-Histone H3 (Lys27) (C36B11), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9733; RRID: AB_2616029</td></tr><tr><td>Histone H3, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9715; RRID: AB_331563</td></tr><tr><td>ARID1A/BAF250A (D2A8U), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 12354; RRID: AB_2637010</td></tr><tr><td>Anti-Puromycin, clone 12D10, mouse monoclonal</td><td>EMD Millipore</td><td>Cat# MABE343; RRID: AB_2566826</td></tr><tr><td>IgG (H + L), rabbit HRP</td><td>Invitrogen</td><td>Cat# PI31460</td></tr><tr><td>IgG (H + L), mouse HRP</td><td>Invitrogen</td><td>Cat# PI31430</td></tr><tr><td>ARID1A, rabbit</td><td>Sigma</td><td>Cat# HPA005456; RRID: AB_1078205</td></tr><tr><td>ARID1A, mouse monoclonal</td><td>Santa Cruz</td><td>Cat# SC-32761; RRID: AB_673396</td></tr><tr><td>p-eEF2K (S366), rabbit polyclonal</td><td>Abcam</td><td>Cat# AB51227; RRID: AB_869561</td></tr><tr><td>p-eEF2K (S398), rabbit polyclonal</td><td>ECM Bio</td><td>Cat# EP5441</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>eEF2K, rabbit monoclonal</td><td>Abcam</td><td>Cat# AB46787; RRID: AB_869559</td></tr><tr><td>p-p70RSK (T389), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9205; RRID: AB_330944</td></tr><tr><td>p70RSK, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9202; RRID: AB_331676</td></tr><tr><td>RASGRP1, rabbit polyclonal</td><td>Origene</td><td>Cat# TA358534</td></tr><tr><td>p44/42 MAPK (Erk1/2), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9102; RRID: AB_330744</td></tr><tr><td>MEK1/2, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9122; RRID: AB_823567</td></tr><tr><td>RSK1 p90 antibody [E4], rabbit monoclonal</td><td>Abcam</td><td>Cat# AB32114; RRID: AB_779068</td></tr><tr><td>Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9101; RRID: AB_331646</td></tr><tr><td>Phospho-MEK1/2 (Ser217/221), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9121; RRID: AB_331648</td></tr><tr><td>phospho-p90RSK (Ser380) (D3H11), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 11989; RRID: AB_2687613</td></tr><tr><td>RASGRP1, polyclonal</td><td>ThermoFisher</td><td>Cat# PA5-25750; RRID: AB_2543250</td></tr><tr><td>p-Histone H2A.X (S139) (20E3), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9718; RRID: AB_2118009</td></tr><tr><td>EEF2/Elongation factor 2, rabbit polyclonal</td><td>Abcam</td><td>Cat# AB33523; RRID: AB_732081</td></tr><tr><td>p-eEF2 (T56), rabbit polyclonal</td><td>Abcam</td><td>ab53114; RRID: AB_869560</td></tr><tr><td>eEF2K, rabbit polyclonal</td><td>Aviva systems biology</td><td>Cat# OAAF00684</td></tr><tr><td>Phospho-eEF2K (Ser366), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 3691; RRID: AB_2097313</td></tr><tr><td>Ki-67 (D3B5), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 12202; RRID: AB_2620142</td></tr><tr><td>Cleaved Caspase-3 (Asp175) (D3E9), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 9579; RRID: AB_10897512</td></tr><tr><td>ARID1A, rabbit</td><td>Sigma</td><td>Cat# HPA005456; RRID: AB_1078205</td></tr><tr><td>Alexa Fluor 594, mouse IgG (H + L)</td><td>Invitrogen</td><td>Cat# A-11032; RRID: AB_2534091</td></tr><tr><td>Alexa Fluor 488, rabbit IgG (H + L)</td><td>Invitrogen</td><td>Cat# A-11034; RRID: AB_2576217</td></tr><tr><td>Alexa Fluor 594, rabbit IgG (H + L)</td><td>Invitrogen</td><td>Cat# A-11037; RRID: AB_2534095</td></tr><tr><td>Alexa Fluor 647, rabbit IgG (H + L)</td><td>Invitrogen</td><td>Cat# A-21244; RRID: AB_2535812</td></tr><tr><td>HRP-labeled polymer, rabbit</td><td>Agilent</td><td>Cat# K4003; RRID: AB_2630375</td></tr><tr><td>CD326 (EpCAM) APC, rat</td><td>Invitrogen</td><td>Cat# 17-5791-80; RRID: AB_2734965</td></tr><tr><td>CD49f (Integrin alpha 6) PE, rat</td><td>ThermoFisher</td><td>Cat# 12-0495-82; RRID: AB_891474</td></tr><tr><td>anti-Cytokeratin 5, rabbit polyclonal</td><td>Biolegend Inc</td><td>Cat# PRB-160P; RRID: AB_291581</td></tr><tr><td>Tri-Methyl Histone H3 (Lys27) (C36B11), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 9733; RRID: AB_2616029</td></tr><tr><td>Guinea Pig anti-Rabbit IgG (Heavy & Light Chain)</td><td>Antibodies-online</td><td>Cat# ABIN101961; RRID: AB_10775589</td></tr><tr><td>Anti-acetyl-Histone H3 (Lys27) clone RM172, monoclonal</td><td>Millipore-Sigma</td><td>Cat# MABE647; RRID: AB_2893037</td></tr><tr><td>ARID1A, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 12354; RRID: AB_2637010</td></tr><tr><td>Rabbit anti-IgG</td><td>Cell Signaling Technology</td><td>Cat# 2729; RRID: AB_1031062</td></tr><tr><td colspan="3"><strong>Biological samples</strong></td></tr><tr><td>Patient-derived xenografts (PDX)1-3 [TM01029, TM00016, TM00024]</td><td>The Jackson Laboratory</td><td>http://www.jax.org</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>eEF2K, rabbit monoclonal</td><td>Abcam</td><td>Cat# AB46787; RRID: AB_869559</td></tr><tr><td>p-p70RSK (T389), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9205; RRID: AB_330944</td></tr><tr><td>p70RSK, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9202; RRID: AB_331676</td></tr><tr><td>RASGRP1, rabbit polyclonal</td><td>Origene</td><td>Cat# TA358534</td></tr><tr><td>p44/42 MAPK (Erk1/2), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9102; RRID: AB_330744</td></tr><tr><td>MEK1/2, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9122; RRID: AB_823567</td></tr><tr><td>RSK1 p90 antibody [E4], rabbit monoclonal</td><td>Abcam</td><td>Cat# AB32114; RRID: AB_779068</td></tr><tr><td>Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9101; RRID: AB_331646</td></tr><tr><td>Phospho-MEK1/2 (Ser217/221), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9121; RRID: AB_331648</td></tr><tr><td>phospho-p90RSK (Ser380) (D3H11), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 11989; RRID: AB_2687613</td></tr><tr><td>RASGRP1, polyclonal</td><td>ThermoFisher</td><td>Cat# PA5-25750; RRID: AB_2543250</td></tr><tr><td>p-Histone H2A.X (S139) (20E3), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 9718; RRID: AB_2118009</td></tr><tr><td>EEF2/Elongation factor 2, rabbit polyclonal</td><td>Abcam</td><td>Cat# AB33523; RRID: AB_732081</td></tr><tr><td>p-eEF2 (T56), rabbit polyclonal</td><td>Abcam</td><td>ab53114; RRID: AB_869560</td></tr><tr><td>eEF2K, rabbit polyclonal</td><td>Aviva systems biology</td><td>Cat# OAAF00684</td></tr><tr><td>Phospho-eEF2K (Ser366), rabbit</td><td>Cell Signaling Technology</td><td>Cat# 3691; RRID: AB_2097313</td></tr><tr><td>Ki-67 (D3B5), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 12202; RRID: AB_2620142</td></tr><tr><td>Cleaved Caspase-3 (Asp175) (D3E9), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 9579; RRID: AB_10897512</td></tr><tr><td>ARID1A, rabbit</td><td>Sigma</td><td>Cat# HPA005456; RRID: AB_1078205</td></tr><tr><td>Alexa Fluor 594, mouse IgG (H + L)</td><td>Invitrogen</td><td>Cat# A-11032; RRID: AB_2534091</td></tr><tr><td>Alexa Fluor 488, rabbit IgG (H + L)</td><td>Invitrogen</td><td>Cat# A-11034; RRID: AB_2576217</td></tr><tr><td>Alexa Fluor 594, rabbit IgG (H + L)</td><td>Invitrogen</td><td>Cat# A-11037; RRID: AB_2534095</td></tr><tr><td>Alexa Fluor 647, rabbit IgG (H + L)</td><td>Invitrogen</td><td>Cat# A-21244; RRID: AB_2535812</td></tr><tr><td>HRP-labeled polymer, rabbit</td><td>Agilent</td><td>Cat# K4003; RRID: AB_2630375</td></tr><tr><td>CD326 (EpCAM) APC, rat</td><td>Invitrogen</td><td>Cat# 17-5791-80; RRID: AB_2734965</td></tr><tr><td>CD49f (Integrin alpha 6) PE, rat</td><td>ThermoFisher</td><td>Cat# 12-0495-82; RRID: AB_891474</td></tr><tr><td>anti-Cytokeratin 5, rabbit polyclonal</td><td>Biolegend Inc</td><td>Cat# PRB-160P; RRID: AB_291581</td></tr><tr><td>Tri-Methyl Histone H3 (Lys27) (C36B11), rabbit monoclonal</td><td>Cell Signaling Technology</td><td>Cat# 9733; RRID: AB_2616029</td></tr><tr><td>Guinea Pig anti-Rabbit IgG (Heavy & Light Chain)</td><td>Antibodies-online</td><td>Cat# ABIN101961; RRID: AB_10775589</td></tr><tr><td>Anti-acetyl-Histone H3 (Lys27) clone RM172, monoclonal</td><td>Millipore-Sigma</td><td>Cat# MABE647; RRID: AB_2893037</td></tr><tr><td>ARID1A, rabbit</td><td>Cell Signaling Technology</td><td>Cat# 12354; RRID: AB_2637010</td></tr><tr><td>Rabbit anti-IgG</td><td>Cell Signaling Technology</td><td>Cat# 2729; RRID: AB_1031062</td></tr><tr><td colspan="3"><strong>Biological samples</strong></td></tr><tr><td>Patient-derived xenografts (PDX)1-3 [TM01029, TM00016, TM00024]</td><td>The Jackson Laboratory</td><td>http://www.jax.org</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN)</td><td>ThermoFisher</td><td>B0938</td></tr><tr><td>4-(N-Butyl-N-nitrosamino) butyric Acid (BCPN)</td><td>TCI</td><td>B3185</td></tr><tr><td>Homoharringtonine (HHT)</td><td>Carbosynth</td><td>FH15974</td></tr><tr><td>Puromycin Dihydrochloride</td><td>ThermoFisher</td><td>BP2956100</td></tr><tr><td>eEF2K inhibitor (A-484954)</td><td>Sigma</td><td>324516</td></tr><tr><td>GSK126</td><td>Selleckchem.com</td><td>S7061</td></tr><tr><td>DFMO</td><td>Tocris Bioscience</td><td>2761</td></tr><tr><td>Anti-FGFR3 Recombinant Antibody clone PRO-001</td><td>Creative bio labs</td><td>HPAB-0160-YC</td></tr><tr><td>Tamoxifen</td><td>Sigma</td><td>T5648</td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H4001</td></tr><tr><td>Collagenase type II</td><td>Life Technologies</td><td>17101015</td></tr><tr><td>Cell recovery solution</td><td>Corning</td><td>354253</td></tr><tr><td>B-27 Supplement</td><td>Gibco</td><td>17504-044</td></tr><tr><td>Murine EGF</td><td>Peprotech</td><td>315-09</td></tr><tr><td>A83-01</td><td>Tocris</td><td>2939</td></tr><tr><td>Y-27632 dihydrochloride</td><td>Sigma</td><td>Y0503</td></tr><tr><td>Doxycycline</td><td>ThermoFisher</td><td>AC446060050</td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>3115879001</td></tr><tr><td>Mouse on Mouse (M.O.M.) Blocking Reagent</td><td>Vector Laboratories</td><td>MKB-2213</td></tr><tr><td>Pro-Long Gold Antifade mountant with DAPI</td><td>ThermoFisher</td><td>P36935</td></tr><tr><td>DAB solution</td><td>Agilent</td><td>K3467</td></tr><tr><td>Faramount mounting medium, Aqueous</td><td>Agilent</td><td>S302580</td></tr><tr><td>Histo Gel</td><td>Epreedia</td><td>HG-4000-012</td></tr><tr><td>Cycloheximide</td><td>Sigma</td><td>C7698</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma</td><td>H3149</td></tr><tr><td>BioMag Plus Concanavalin A</td><td>Bangs laboratories, Inc.</td><td>BP531</td></tr><tr><td>Whatman® glass microfiber filters, Grade GF/C</td><td>Sigma</td><td>WHA1822024</td></tr><tr><td>LLC Liquid scintillation cocktail</td><td>ThermoFisher</td><td>50-904-0129</td></tr><tr><td>S35 EasyTagTM EXPRESS35S Protein Labeling Mix</td><td>Perkin Elmer</td><td>NEG772002MC</td></tr></tbody></table>

Critical commercial assays

<table><tr><td>CellTiter-Glo® 2.0 Cell Viability Assay</td><td>Promega</td><td>G9242</td></tr><tr><td>Comet assay single cell gel electrophoresis assay kit</td><td>Trevigen</td><td>4250-050-K</td></tr><tr><td>Direct-zol Miniprep Plus kits</td><td>Zymo Research</td><td>ZR2070</td></tr><tr><td>TruSeq Stranded mRNA Library Prep Kit</td><td>Illumina</td><td>20020594</td></tr><tr><td>TruSeq RNA UD Indexes kit</td><td>Illumina</td><td>20022371</td></tr><tr><td>NovaSeq 6000 SP Reagent Kit</td><td>Illumina</td><td>20027464</td></tr><tr><td>Trans-Blot Turbo RTA Midi 0.2 µm PVDF Transfer Kit</td><td>Bio-Rad</td><td>1704273</td></tr><tr><td>Super Signal West Pico Chemiluminescent Substrate kit</td><td>ThermoFisher</td><td>PI34080</td></tr><tr><td>TMTpro18plex labeling reagent</td><td>ThermoFisher</td><td>A44521 and A52048</td></tr><tr><td>SimpleChIP Enzymatic Chromatin IP Kit</td><td>Cell Signaling Technology</td><td>9003S</td></tr><tr><td>Propidium Iodide Flow Cytometry Kit</td><td>Abcam</td><td>ab139418</td></tr><tr><td>GoTaq Green Master Mix</td><td>ThermoFisher</td><td>PRM7123</td></tr><tr><td>ERCC RNA Spike-In Mix</td><td>Invitrogen</td><td>4456740</td></tr></table>

(Continued on next page)
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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN)</td><td>ThermoFisher</td><td>B0938</td></tr><tr><td>4-(N-Butyl-N-nitrosamino) butyric Acid (BCPN)</td><td>TCI</td><td>B3185</td></tr><tr><td>Homoharringtonine (HHT)</td><td>Carbosynth</td><td>FH15974</td></tr><tr><td>Puromycin Dihydrochloride</td><td>ThermoFisher</td><td>BP2956100</td></tr><tr><td>eEF2K inhibitor (A-484954)</td><td>Sigma</td><td>324516</td></tr><tr><td>GSK126</td><td>Selleckchem.com</td><td>S7061</td></tr><tr><td>DFMO</td><td>Tocris Bioscience</td><td>2761</td></tr><tr><td>Anti-FGFR3 Recombinant Antibody clone PRO-001</td><td>Creative bio labs</td><td>HPAB-0160-YC</td></tr><tr><td>Tamoxifen</td><td>Sigma</td><td>T5648</td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H4001</td></tr><tr><td>Collagenase type II</td><td>Life Technologies</td><td>17101015</td></tr><tr><td>Cell recovery solution</td><td>Corning</td><td>354253</td></tr><tr><td>B-27 Supplement</td><td>Gibco</td><td>17504-044</td></tr><tr><td>Murine EGF</td><td>Peprotech</td><td>315-09</td></tr><tr><td>A83-01</td><td>Tocris</td><td>2939</td></tr><tr><td>Y-27632 dihydrochloride</td><td>Sigma</td><td>Y0503</td></tr><tr><td>Doxycycline</td><td>ThermoFisher</td><td>AC446060050</td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>3115879001</td></tr><tr><td>Mouse on Mouse (M.O.M.) Blocking Reagent</td><td>Vector Laboratories</td><td>MKB-2213</td></tr><tr><td>Pro-Long Gold Antifade mountant with DAPI</td><td>ThermoFisher</td><td>P36935</td></tr><tr><td>DAB solution</td><td>Agilent</td><td>K3467</td></tr><tr><td>Faramount mounting medium, Aqueous</td><td>Agilent</td><td>S302580</td></tr><tr><td>Histo Gel</td><td>Epreedia</td><td>HG-4000-012</td></tr><tr><td>Cycloheximide</td><td>Sigma</td><td>C7698</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma</td><td>H3149</td></tr><tr><td>BioMag Plus Concanavalin A</td><td>Bangs laboratories, Inc.</td><td>BP531</td></tr><tr><td>Whatman® glass microfiber filters, Grade GF/C</td><td>Sigma</td><td>WHA1822024</td></tr><tr><td>LLC Liquid scintillation cocktail</td><td>ThermoFisher</td><td>50-904-0129</td></tr><tr><td>S35 EasyTagTM EXPRESS35S Protein Labeling Mix</td><td>Perkin Elmer</td><td>NEG772002MC</td></tr></tbody></table>

Critical commercial assays

<table><tr><td>CellTiter-Glo® 2.0 Cell Viability Assay</td><td>Promega</td><td>G9242</td></tr><tr><td>Comet assay single cell gel electrophoresis assay kit</td><td>Trevigen</td><td>4250-050-K</td></tr><tr><td>Direct-zol Miniprep Plus kits</td><td>Zymo Research</td><td>ZR2070</td></tr><tr><td>TruSeq Stranded mRNA Library Prep Kit</td><td>Illumina</td><td>20020594</td></tr><tr><td>TruSeq RNA UD Indexes kit</td><td>Illumina</td><td>20022371</td></tr><tr><td>NovaSeq 6000 SP Reagent Kit</td><td>Illumina</td><td>20027464</td></tr><tr><td>Trans-Blot Turbo RTA Midi 0.2 µm PVDF Transfer Kit</td><td>Bio-Rad</td><td>1704273</td></tr><tr><td>Super Signal West Pico Chemiluminescent Substrate kit</td><td>ThermoFisher</td><td>PI34080</td></tr><tr><td>TMTpro18plex labeling reagent</td><td>ThermoFisher</td><td>A44521 and A52048</td></tr><tr><td>SimpleChIP Enzymatic Chromatin IP Kit</td><td>Cell Signaling Technology</td><td>9003S</td></tr><tr><td>Propidium Iodide Flow Cytometry Kit</td><td>Abcam</td><td>ab139418</td></tr><tr><td>GoTaq Green Master Mix</td><td>ThermoFisher</td><td>PRM7123</td></tr><tr><td>ERCC RNA Spike-In Mix</td><td>Invitrogen</td><td>4456740</td></tr></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>CUT&Tag, RNA, and polysome-bound RNA sequencing</td><td>This paper</td><td>GSE201222</td></tr><tr><td>TMT mass spectrometry data</td><td>This paper</td><td>MSV000089875</td></tr><tr><td>Pipeline and analysis code</td><td>This paper</td><td>https://doi.org/10.5281/ zenodo.7729526</td></tr><tr><td colspan="3"><strong>Experimental models: Cell lines</strong></td></tr><tr><td>Human: HT1376</td><td>Gift from David MacPherson Lab (Fred Hutchinson Cancer Center).</td><td>N/A</td></tr><tr><td>Human: UMUC11</td><td>Gift from David MacPherson Lab (Fred Hutchinson Cancer Center).</td><td>N/A</td></tr><tr><td>Human: HT1197</td><td>Gift from David MacPherson Lab (Fred Hutchinson Cancer Center).</td><td>N/A</td></tr><tr><td>Human: KU1919</td><td>Gift from David MacPherson Lab (Fred Hutchinson Cancer Center).</td><td>N/A</td></tr><tr><td>Human: PrEC (Human Prostate Epithelial Cells)</td><td>Lonza</td><td>CC-2555</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>Mouse: Arid1a<sup>LoxP/LoxP</sup></td><td>The Jackson Laboratory</td><td>Stock number: 027717</td></tr><tr><td>Mouse: UBC-Cre<sup>ERT2</sup></td><td>The Jackson Laboratory</td><td>Stock number: 008085</td></tr><tr><td>Mouse: ROSA-LSL-Yfp<sup>KI/KI</sup></td><td>The Jackson Laboratory</td><td>Stock number: 007903</td></tr><tr><td>Mouse: K5-Cre<sup>ERT2</sup></td><td>The Jackson Laboratory</td><td>Stock number: 029155</td></tr><tr><td>Mouse: Eef2k<sup>-/-</sup></td><td>A gift from Alexey G. Ryazanov (Rutgers University).</td><td>N/A</td></tr><tr><td>Mouse: Rasgrp1<sup>-/-</sup></td><td>A gift from Jeroen Roosevelt lab (University of California, San Francisco).</td><td>N/A</td></tr><tr><td>Mouse: NOD-SCID &gamma; IL-2</td><td>The Jackson Laboratory</td><td>Stock number: 005557</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>Primer for Arid1a<sup>LoxP/LoxP</sup> genotyping: Forward: 5'-GTAATGGGAAAGCGACTACTGGAG-3'<br>Reverse: 5'-TGTTCGTTTTTGTGGCGGGAG-3'</td><td>Integrated DNA Technologies</td><td>N/A</td></tr><tr><td>Primer for UBC-Cre<sup>ERT2</sup> genotyping: Forward: 5'-GCGGTCTGGCAGTAAAAAACTATC-3'<br>Reverse: 5'-GTGAAACAGCATTGCTGTCACTT-3'</td><td>Integrated DNA Technologies</td><td>N/A</td></tr><tr><td>Primer for ROSA-LSL-Yfp<sup>KI/KI</sup> genotyping: WT-Forward: 5'-CTGGCTTCTGAGGACCG-3'.<br>Reverse: 5'-CAGGACAACGCCCACACA-3'<br>YFP- Forward: 5'-AGGGCGAGGAGCTGTTCA-3'.<br>Reverse: 5'-TGAAGTCGATGCCCTTCAG-3'</td><td>Integrated DNA Technologies</td><td>N/A</td></tr><tr><td>Primer for K5-Cre<sup>ERT2</sup> genotyping: WT-Forward: 5'-GCAAGACCCTGGTCCCAC-3'<br>Reverse: 5'-GGAGGAAGTCAGAACCAGGAC-3'<br>CreERT-Forward: 5'-GCAAGACCCTGGTCCTCAC-3'<br>Reverse: 5'-ACCGGCCTTATTCCAAGC-3'</td><td>Integrated DNA Technologies</td><td>N/A</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>CUT&Tag, RNA, and polysome-bound RNA sequencing</td><td>This paper</td><td>GSE201222</td></tr><tr><td>TMT mass spectrometry data</td><td>This paper</td><td>MSV000089875</td></tr><tr><td>Pipeline and analysis code</td><td>This paper</td><td>https://doi.org/10.5281/ zenodo.7729526</td></tr><tr><td colspan="3"><strong>Experimental models: Cell lines</strong></td></tr><tr><td>Human: HT1376</td><td>Gift from David MacPherson Lab (Fred Hutchinson Cancer Center).</td><td>N/A</td></tr><tr><td>Human: UMUC11</td><td>Gift from David MacPherson Lab (Fred Hutchinson Cancer Center).</td><td>N/A</td></tr><tr><td>Human: HT1197</td><td>Gift from David MacPherson Lab (Fred Hutchinson Cancer Center).</td><td>N/A</td></tr><tr><td>Human: KU1919</td><td>Gift from David MacPherson Lab (Fred Hutchinson Cancer Center).</td><td>N/A</td></tr><tr><td>Human: PrEC (Human Prostate Epithelial Cells)</td><td>Lonza</td><td>CC-2555</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>Mouse: Arid1a<sup>LoxP/LoxP</sup></td><td>The Jackson Laboratory</td><td>Stock number: 027717</td></tr><tr><td>Mouse: UBC-Cre<sup>ERT2</sup></td><td>The Jackson Laboratory</td><td>Stock number: 008085</td></tr><tr><td>Mouse: ROSA-LSL-Yfp<sup>KI/KI</sup></td><td>The Jackson Laboratory</td><td>Stock number: 007903</td></tr><tr><td>Mouse: K5-Cre<sup>ERT2</sup></td><td>The Jackson Laboratory</td><td>Stock number: 029155</td></tr><tr><td>Mouse: Eef2k<sup>-/-</sup></td><td>A gift from Alexey G. Ryazanov (Rutgers University).</td><td>N/A</td></tr><tr><td>Mouse: Rasgrp1<sup>-/-</sup></td><td>A gift from Jeroen Roosevelt lab (University of California, San Francisco).</td><td>N/A</td></tr><tr><td>Mouse: NOD-SCID &gamma; IL-2</td><td>The Jackson Laboratory</td><td>Stock number: 005557</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>Primer for Arid1a<sup>LoxP/LoxP</sup> genotyping: Forward: 5'-GTAATGGGAAAGCGACTACTGGAG-3'<br>Reverse: 5'-TGTTCGTTTTTGTGGCGGGAG-3'</td><td>Integrated DNA Technologies</td><td>N/A</td></tr><tr><td>Primer for UBC-Cre<sup>ERT2</sup> genotyping: Forward: 5'-GCGGTCTGGCAGTAAAAAACTATC-3'<br>Reverse: 5'-GTGAAACAGCATTGCTGTCACTT-3'</td><td>Integrated DNA Technologies</td><td>N/A</td></tr><tr><td>Primer for ROSA-LSL-Yfp<sup>KI/KI</sup> genotyping: WT-Forward: 5'-CTGGCTTCTGAGGACCG-3'<br>Reverse: 5'-CAGGACAACGCCCACACA-3'<br>YFP- Forward: 5'-AGGGCGAGGAGCTGTTCA-3'<br>Reverse: 5'-TGAAGTCGATGCCCTTCAG-3'</td><td>Integrated DNA Technologies</td><td>N/A</td></tr><tr><td>Primer for K5-Cre<sup>ERT2</sup> genotyping: WT-Forward: 5'-GCAAGACCCTGGTCCCAC-3'<br>Reverse: 5'-GGAGGAAGTCAGAACCAGGAC-3'</td><td>Integrated DNA Technologies</td><td>N/A</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Primer for Eef2k<sup>-/-</sup> genotyping:<br>WT-Forward: 5'-GGCCGGCTGCTAGAGAGTGTC-3'.<br>Reverse: 5'-CATCAGCTGATTGTAGTGGACATC-3'<br>eEF2K KO-Forward: 5'TGCGAGGCCAGAGGCC<br>ACTTGTGTAGC-3'<br>Reverse:<br>5'-CAGGGCCTGCTTTCTTGGTGGCAG-3'</td><td>Integrated DNA<br>Technologies</td><td>N/A</td></tr><tr><td>Primer for Rasgrp1 qPCR: Forward: 5'-CTTCAACAC<br>GCTGATGGCTGTG-3'.<br>Reverse: 5'-GGACAGCAG<br>TTCAGTCATCTCG-3'</td><td>Integrated DNA<br>Technologies</td><td>N/A</td></tr><tr><td>GAPDH: Forward: 5'-CAATGAATACGGCTAC<br>AGCAAC-3'.<br>Reverse: 5'-AGGGAGATGCTCAGTGTTGG-3'</td><td>Integrated DNA<br>Technologies</td><td>N/A</td></tr><tr><td>Primer for ChIP qPCR (ARID1A targeted region):<br>Forward, 5'-CTTCGAATCCTGCCCCCATT-3'<br>Reverse, 5'-TCTTGGGCTGGGAGAGATGA-3'</td><td>Integrated DNA<br>Technologies</td><td>N/A</td></tr><tr><td>ON-TARGETplus SMARTpool siRNA targeting<br>Arid1a: GAAUAGGGCCUGAGGGAAA<br>AGAUGUGGGUGGACCGUUA<br>GCAACGACAUGAUUCCUAU<br>GGACCUCUAUCGCCUCUAU</td><td>Dharmacon</td><td>Cat#L-017263-00-0005</td></tr></tbody></table>

Recombinant DNA

<table><tr><td>pLenti-puro-ARID1A</td><td>Addgene</td><td>Cat#39478</td></tr><tr><td>pLenti6/TR</td><td>ThermoFisher</td><td>Cat#V48020</td></tr><tr><td>LV-Cre pLKO.1</td><td>a gift from Slobodan Beronja<br>lab (Fred Hutchinson Cancer<br>Research Center)</td><td>Addgene#25997</td></tr><tr><td>Rasgrp1-pEF6</td><td>A gift from the Jeroen Roosevelt<br>lab (University of California,<br>San Francisco).</td><td>N/A</td></tr></table>

Software and algorithms

<table><tr><td>voom</td><td>Law et al.<sup>1</sup></td><td>https://rdrr.io/bioc/limma/man/voom.html</td></tr><tr><td>xtail</td><td>Xiao et al.<sup>2</sup></td><td>https://anaconda.org/bioconda/xtail</td></tr><tr><td>R</td><td>The R Foundation</td><td>https://www.r-project.org</td></tr><tr><td>STAR2</td><td>Dobin et al.<sup>3</sup></td><td>https://code.google.com/archive/p/rna-star/</td></tr><tr><td>HTSeq</td><td>Anders et al.<sup>4</sup></td><td>https://htseq.readthedocs.io/en/master/</td></tr><tr><td>edgeR</td><td>Robinson et al.<sup>5</sup></td><td>http://bioconductor.org</td></tr><tr><td>pheatmap</td><td></td><td>https://cran.r-project.org/web/packages/<br>pheatmap/index.html</td></tr><tr><td>ggplot2</td><td>Wickham et al.<sup>6</sup></td><td>https://ggplot2.tidyverse.org</td></tr><tr><td>Bowtie2 version 2.2.2</td><td>Langmead et al.<sup>7</sup></td><td>https://bioweb.pasteur.fr/packages/<br>pack@bowtie2@2.2.2</td></tr><tr><td>deepTools v3.5.1</td><td>Ramirez et al.<sup>8</sup></td><td>https://deeptools.readthedocs.io/en/develop/</td></tr><tr><td>genomev</td><td></td><td>https://bedtools.readthedocs.io/en/latest/<br>content/tools/genomecov.html</td></tr><tr><td>computeMatrix</td><td>Ramirez et al.<sup>8</sup></td><td>https://deeptools.readthedocs.io/en/<br>develop/content/tools/computeMatrix.html</td></tr><tr><td>plotHeatmap</td><td>Ramirez et al.<sup>8</sup></td><td>https://deeptools.readthedocs.io/en/<br>develop/content/tools/plotHeatmap.html</td></tr><tr><td>Proteome Discoverer 2.5</td><td>ThemoFisher</td><td>https://thermo.flexnetoperations.com/<br>control/thmo/login</td></tr><tr><td>SEQUEST</td><td>Eng et al.<sup>9</sup></td><td>NA</td></tr></table>

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