Continued

<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>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>ImageJ</td><td>NIH</td><td>https://imagej.nih.gov/ij/download.html</td></tr><tr><td>Prism 9</td><td>GraphPad software</td><td>N/A</td></tr><tr><td>HALO</td><td>Indica labs</td><td>N/A</td></tr><tr><td>BioRender</td><td>BioRender</td><td>https://biorender.com/</td></tr></tbody></table>

RESOURCE AVAILABILITY

Lead contact

Further queries and reagent requests may be directed and will be fulfilled by the lead contact, Andrew C. Hsieh (ahsieh@fredhutch.org).

Materials availability

This study did not generate new unique reagents.

Data and code availability

* The CUT&Tag, RNA, and polysome sequencing data files are available at the GEO database. Accession number is listed in the key resources table. Mass spectrometry data and proteomics result files are available at the MassIVE database. Accession number is listed in the key resources table. All deposited data are publicly available as of the date of publication.

* The code used to process and analyze the data is available at Zenodo and is publicly available as of the date of publication. DOI is listed in the key resources table.

* All other data associated with this study are present in supplemental information and supplementary tables. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODELS AND SUBJECT DETAILS

Mice

Arid1a<sup>LoxP/LoxP</sup> (https://www.jax.org/strain/027717, stock number: 027717), ROSA-LSL-Yfp<sup>KI/KI</sup> (https://www.jax.org/strain/007903, stock number: 007903), and UBC-Cre<sup>ERT2</sup> (https://www.jax.org/strain/008085, stock number: 008085) mice were purchased from the Jackson Laboratory. eEF2K<sup>-/-</sup> animals were kindly provided by Alexey G. Ryazanov (Rutgers University). UBC-Cre<sup>ERT2</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> animals (mixed background) were bred to Eef2k<sup>-/-</sup> animals (C57BL/6) for one generation to produce progeny heterozygous at all loci (ROSA-LSL-Yfp<sup>KI/+</sup>; Arid1a<sup>LoxP/+</sup>; eEF2K<sup>+/-</sup>) with or without UBC-Cre<sup>ERT2</sup>. Heterozygous animals without UBC-Cre<sup>ERT2</sup> were backcrossed once more to ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> animals to reintroduce homozygosity at the Arid1a and ROSA-LSL-Yfp, while maintaining heterozygosity at the Eef2k locus (ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup>; Eef2K<sup>+/-</sup>; with and without UBC-Cre<sup>ERT2</sup>). Of these progeny, animals with UBC-Cre<sup>ERT2</sup> were bred to animals without UBC-Cre<sup>ERT2</sup> to produce two parallel lineages, both in a consistent background with a single copy of UBC-Cre<sup>ERT2</sup> (UBC-Cre<sup>ERT2</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup>; Eef2K<sup>+/+</sup> and UBC-Cre<sup>ERT2</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup>; Eef2K<sup>-/-</sup>). The lineages were maintained separately with in-breeding.

K5-Cre<sup>ERT2</sup> mice (https://www.jax.org/strain/029155, stock number: 029155) were purchased from the Jackson Laboratory. ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> animals (mixed background) were bred to K5-Cre<sup>ERT2</sup> animals (congenic C57BL/6 background) for one generation to produce progeny heterozygous at all loci (K5-Cre<sup>ERT2/+</sup>; ROSA-LSL-Yfp<sup>KI/+</sup>; Arid1a<sup>LoxP/+</sup>). These animals were backcrossed once more to ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> animals to reintroduce homozygosity at the Arid1a and ROSA-LSL-Yfp loci (K5-Cre<sup>ERT2/+</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> and K5-Cre<sup>+/+</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup>). The lineage was maintained with subsequent in-breeding of these two genotypes to maintain a single copy of CreERT.

NOD-SCID γ IL-2 mice (https://www.jax.org/strain/005557, stock number: 005557) were used for the preclinical trial. PDX models were obtained Jackson Laboratory (TM01029, TM00016, TM00024). We followed protocols approved by the Fred Hutchinson Cancer Center Animal Care and Use Committee (IACUC) for all mouse studies. The number of mice used for each study is included in figure legends. All studies were conducted using 50% male and 50% female mice. All comparison were made between littermates. For survival studies, the body weight of the mouse was recorded and monitored every two days for the duration of the survival trial. Mice were euthanized when they exhibit >20% weight loss or met euthanasia requirements by exhibiting pain and distress.

Cell culture

Cell lines used for this study include HT1376, KU1919, HT1197, UMUC11, and Human Prostate Epithelial Cells (Human PrEC). Mouse bone marrow stromal cells were derived from WT mice. HT1197 and HT1376 cells were cultured in Eagle's Minimum Essential Medium (ATCC, 30-2003) supplemented with 10% fetal bovine serum (Cytiva, SH3039603s), and 1% penicillin/streptomycin (Gibco, 15140-122). KU1919 cells were cultured in RPMI1640 (Gibco, 11875-119) supplemented with 10% fetal bovine serum, and 1%
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>ImageJ</td><td>NIH</td><td>https://imagej.nih.gov/ij/download.html</td></tr><tr><td>Prism 9</td><td>GraphPad software</td><td>N/A</td></tr><tr><td>HALO</td><td>Indica labs</td><td>N/A</td></tr><tr><td>BioRender</td><td>BioRender</td><td>https://biorender.com/</td></tr></tbody></table>

RESOURCE AVAILABILITY

Lead contact

Further queries and reagent requests may be directed and will be fulfilled by the lead contact, Andrew C. Hsieh (ahsieh@fredhutch.org).

Materials availability

This study did not generate new unique reagents.

Data and code availability

* The CUT&Tag, RNA, and polysome sequencing data files are available at the GEO database. Accession number is listed in the key resources table. Mass spectrometry data and proteomics result files are available at the MassIVE database. Accession number is listed in the key resources table. All deposited data are publicly available as of the date of publication.

* The code used to process and analyze the data is available at Zenodo and is publicly available as of the date of publication. DOI is listed in the key resources table.

* All other data associated with this study are present in supplemental information and supplementary tables. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODELS AND SUBJECT DETAILS

Mice

Arid1a<sup>LoxP/LoxP</sup> (https://www.jax.org/strain/027717, stock number: 027717), ROSA-LSL-Yfp<sup>KI/KI</sup> (https://www.jax.org/strain/007903, stock number: 007903), and UBC-Cre<sup>ERT2</sup> (https://www.jax.org/strain/008085, stock number: 008085) mice were purchased from the Jackson Laboratory. eEF2K<sup>-/-</sup> animals were kindly provided by Alexey G. Ryazanov (Rutgers University). UBC-Cre<sup>ERT2</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> animals (mixed background) were bred to Eef2k<sup>-/-</sup> animals (C57BL/6) for one generation to produce progeny heterozygous at all loci (ROSA-LSL-Yfp<sup>KI/+</sup>; Arid1a<sup>LoxP/+</sup>; eEF2K<sup>+/-</sup>) with or without UBC-Cre<sup>ERT2</sup>. Heterozygous animals without UBC-Cre<sup>ERT2</sup> were backcrossed once more to ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> animals to reintroduce homozygosity at the Arid1a and ROSA-LSL-Yfp, while maintaining heterozygosity at the Eef2k locus (ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup>; Eef2K<sup>+/-</sup>; with and without UBC-Cre<sup>ERT2</sup>). Of these progeny, animals with UBC-Cre<sup>ERT2</sup> were bred to animals without UBC-Cre<sup>ERT2</sup> to produce two parallel lineages, both in a consistent background with a single copy of UBC-Cre<sup>ERT2</sup> (UBC-Cre<sup>ERT2</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup>; Eef2K<sup>+/+</sup> and UBC-Cre<sup>ERT2</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup>; Eef2K<sup>-/-</sup>). The lineages were maintained separately with in-breeding.

K5-Cre<sup>ERT2</sup> mice (https://www.jax.org/strain/029155, stock number: 029155) were purchased from the Jackson Laboratory. ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> animals (mixed background) were bred to K5-Cre<sup>ERT2</sup> animals (congenic C57BL/6 background) for one generation to produce progeny heterozygous at all loci (K5-Cre<sup>ERT2/+</sup>; ROSA-LSL-Yfp<sup>KI/+</sup>; Arid1a<sup>LoxP/+</sup>). These animals were backcrossed once more to ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> animals to reintroduce homozygosity at the Arid1a and ROSA-LSL-Yfp loci (K5-Cre<sup>ERT2/+</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup> and K5-Cre<sup>+/+</sup>; ROSA-LSL-Yfp<sup>KI/KI</sup>; Arid1a<sup>LoxP/LoxP</sup>). The lineage was maintained with subsequent in-breeding of these two genotypes to maintain a single copy of CreERT.

NOD-SCID γ IL-2 mice (https://www.jax.org/strain/005557, stock number: 005557) were used for the preclinical trial. PDX models were obtained Jackson Laboratory (TM01029, TM00016, TM00024). We followed protocols approved by the Fred Hutchinson Cancer Center Animal Care and Use Committee (IACUC) for all mouse studies. The number of mice used for each study is included in figure legends. All studies were conducted using 50% male and 50% female mice. All comparison were made between littermates. For survival studies, the body weight of the mouse was recorded and monitored every two days for the duration of the survival trial. Mice were euthanized when they exhibit >20% weight loss or met euthanasia requirements by exhibiting pain and distress.

Cell culture

Cell lines used for this study include HT1376, KU1919, HT1197, UMUC11, and Human Prostate Epithelial Cells (Human PrEC). Mouse bone marrow stromal cells were derived from WT mice. HT1197 and HT1376 cells were cultured in Eagle's Minimum Essential Medium (ATCC, 30-2003) supplemented with 10% fetal bovine serum (Cytiva, SH3039603s), and 1% penicillin/streptomycin (Gibco, 15140-122). KU1919 cells were cultured in RPMI1640 (Gibco, 11875-119) supplemented with 10% fetal bovine serum, and 1%
penicillin/streptomycin. UMUC11 cells were cultured in DMEM (Gibco, 11965-092) supplemented with 10% fetal bovine serum, and 1% penicillin/streptomycin. These cell lines were maintained in a 37°C and 5% CO₂ incubator. All these cell lines were generously provided by MacPherson Lab (Fred Hutchinson Cancer Center). Human Prostate Epithelial Cells (Lonza, CC-2555) were cultured using Lonza's BulletKit media (Lonza, CC-3166) according to manufacturer's instructions. Mouse bone marrow stromal cells were generated by the following method. Briefly, bone marrow cells were isolated by flushing the tibia and femur bones of WT mice with PBS. Cells were filtered through a cell strainer and cultured in Iscove's modified Dulbecco's medium (Fisher, 12440053) supplemented with 15% fetal calf serum, 5% horse serum (Life Technology, 26050070), 10⁻⁵ M hydrocortisone (Sigma, H4001), 1% penicillin/streptomycin, and 10⁻⁴ M β-mercaptoethanol at 33°C.

## Organoid culture

Organoid lines were generated either from normal murine bladder or bladder tumors. Minced tissues were treated with 5 mg/mL collagenase type II solution (Life Technologies, 17101015) diluted in Dulbecco's Modified Eagle's Medium for 1 h at 37°C, followed by a 5 min TrypLE (Gibco, 12604-039) treatment at 37°C. Digested tissues were further dissociated using a syringe with 18G needle, and single cells were obtained after passing through a cell strainer. Cells were plated using EHS (NIH and Corning) and cultured in organoid media for 5-7 days at 37°C. Organoid base media was prepared using Advanced DMEM/F12 (Gibco 12634010) supplemented with 20% B27 (Gibco, 17504-044), 10 mM HEPES, Glutamax (Fisher, 35050061), and 1.25 mM N-acetyl-L-cysteine (Sigma, A9165). Base media was also supplemented with 50 ng/mL EGF (Peprotech, 315-09), 100 ng/mL Noggin (conditioned media), 500 ng/mL R-spondin (conditioned media), 200 nM A83-01 (Tocris, 2939), and 10 µM Y-27632 (Sigma, Y0503). Organoids were passaged once every week.

## METHOD DETAILS

### ARID1A re-expression

Arid1a<sup>fl/fl</sup> organoid cells were plated at a density of 10,000 cells per 15 µL EHS in 24-well ultra-low attachment plates with lentivirus expressing (dox-inducible) pLenti-puro-ARID1A and pLenti6/TR. Organoids were cultured in organoid media (see organoid culture) for 96 h. Stable lines were generated using puromycin and blasticidin treatment.

### Rasgrp1 re-expression

Arid1a<sup>fl/fl</sup> cells were transfected with Rasgrp1-pEF6 construct using Lipofectamine 3000. Stable lines were generated using blasticidin.

### Urine BCPN analysis

Urine was collected from WT, Arid1a<sup>fl/fl</sup>, and Arid1a<sup>fl/fl</sup>:Eef2k<sup>-/-</sup> mice after 9 days of 0.075% BBN treatment. 5 µL of urine was analyzed on a Xevo QToF mass spectrometer. BCPN concentrations were calculated by comparing the urine BCPN measurements to standards of known BCPN (TCI, B3185) concentrations.

### Immunoblot analysis

Cell lysates were prepared using RIPA buffer (Fisher, P189900) containing phosphatase inhibitor (Sigma, 4906845001) and protease inhibitor cocktail (Sigma, 11836153001). The lysates were incubated on ice for 30 min with periodic vortexing, and centrifuged at 13,000 x g at 4°C for 10 min. The supernatants were subjected to SDS-PAGE and transferred to a PDVF membrane (Bio-Rad, 1704273). Membranes were blocked with 5% milk for 1 h at room temperature. Primary antibodies were diluted in 5% milk and incubated overnight at 4°C. Blots were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG [Fisher, P131460] or goat anti-mouse IgG [Fisher, P131430]) in 5% milk for 1 h at room temperature. West Pico (Fisher, P134080) was used to detect immunoreactive bands on the blot using the ChemiDoc Touch Imaging System (Bio-Rad). The following primary antibodies were used: anti-Tubulin (Sigma, T8203), anti-ARID1A (Cell Signaling Technology, 12354), anti-PTEN (Cell Signaling Technology, 9559), anti-*p*-AKT(S473) (Cell Signaling Technology, 4060), anti-AKT1 (Santa Cruz Biotechnology, Sc-1618), anti-*p*-eIF2α (S51) (Cell Signaling Technology, 3597), anti-ATF4 (Cell Signaling Technology, 11815), anti-eIF4G (Cell Signaling Technology, 2469), anti-eIF4E (Santa Cruz Biotechnology, sc-271480), anti-eIF4A (Cell Signaling Technology, 2013), anti-*p*-4EBP1 (Cell Signaling Technology, 2855), anti-4EBP1 (Cell Signaling Technology, 9644), anti-*p*-eEF2 (T56) (Cell Signaling Technology, 2331), anti-eEF2 (Cell Signaling Technology, 2332), anti- AURKB (Novusbio, NB-100-294), anti-FGFR3 (Mybiosource, MBS9212039), anti-IGF2 (Fisher, MA5-17096), anti-KIF22 (Novusbio, NBP2-17053), anti-ODC1 (Fisher, PA5-21362), anti-SKA1 (Novusbio, NBP2-88277), anti-BRCA2 (Abcam, ab27976), anti-ERCC1 (Cell Signaling Technology, 3885), anti-ERCC2 (Cell Signaling Technology, 11963), anti-FANCC (Abcam, ab97575), anti-*p*-AMPK(Thr172) (Cell Signaling Technology, 2535), anti-AMPK (Cell Signaling Technology, 2532), anti-H3K27me3 (Cell Signaling Technology, 9733), anti-H3 (Cell Signaling Technology, 9715), anti-puromycin (EMD Millipore, MABE343), anti-Rasgrp1 (Origene, TA358534), anti-p70RSK (Cell Signaling Technology, 9202), anti-*p*-p70RSK (T389) (Cell Signaling Technology, 9205), anti-eEF2K (Abcam, ab46787), anti-*p*-eEF2K (S398) (ECM Bio, EP5441), anti-*p*-eEF2K (S366) (Abcam, ab51227), anti-ARID1A (Santa Cruz, SC-32761), and anti-ARID1A (Sigma, HPA005456).
penicillin/streptomycin. UMUC11 cells were cultured in DMEM (Gibco, 11965-092) supplemented with 10% fetal bovine serum, and 1% penicillin/streptomycin. These cell lines were maintained in a 37°C and 5% CO₂ incubator. All these cell lines were generously provided by MacPherson Lab (Fred Hutchinson Cancer Center). Human Prostate Epithelial Cells (Lonza, CC-2555) were cultured using Lonza's BulletKit media (Lonza, CC-3166) according to manufacturer's instructions. Mouse bone marrow stromal cells were generated by the following method. Briefly, bone marrow cells were isolated by flushing the tibia and femur bones of WT mice with PBS. Cells were filtered through a cell strainer and cultured in Iscove's modified Dulbecco's medium (Fisher, 12440053) supplemented with 15% fetal calf serum, 5% horse serum (Life Technology, 26050070), 10⁻⁵ M hydrocortisone (Sigma, H4001), 1% penicillin/streptomycin, and 10⁻⁴ M β-mercaptoethanol at 33°C.

## Organoid culture

Organoid lines were generated either from normal murine bladder or bladder tumors. Minced tissues were treated with 5 mg/mL collagenase type II solution (Life Technologies, 17101015) diluted in Dulbecco's Modified Eagle's Medium for 1 h at 37°C, followed by a 5 min TrypLE (Gibco, 12604-039) treatment at 37°C. Digested tissues were further dissociated using a syringe with 18G needle, and single cells were obtained after passing through a cell strainer. Cells were plated using EHS (NIH and Corning) and cultured in organoid media for 5-7 days at 37°C. Organoid base media was prepared using Advanced DMEM/F12 (Gibco 12634010) supplemented with 20% B27 (Gibco, 17504-044), 10 mM HEPES, Glutamax (Fisher, 35050061), and 1.25 mM N-acetyl-L-cysteine (Sigma, A9165). Base media was also supplemented with 50 ng/mL EGF (Peprotech, 315-09), 100 ng/mL Noggin (conditioned media), 500 ng/mL R-spondin (conditioned media), 200 nM A83-01 (Tocris, 2939), and 10 µM Y-27632 (Sigma, Y0503). Organoids were passaged once every week.

## METHOD DETAILS

### ARID1A re-expression

Arid1a<sup>fl/fl</sup> organoid cells were plated at a density of 10,000 cells per 15 µL EHS in 24-well ultra-low attachment plates with lentivirus expressing (dox-inducible) pLenti-puro-ARID1A and pLenti6/TR. Organoids were cultured in organoid media (see organoid culture) for 96 h. Stable lines were generated using puromycin and blasticidin treatment.

### Rasgrp1 re-expression

Arid1a<sup>fl/fl</sup> cells were transfected with Rasgrp1-pEF6 construct using Lipofectamine 3000. Stable lines were generated using blasticidin.

### Urine BCPN analysis

Urine was collected from WT, Arid1a<sup>fl/fl</sup>, and Arid1a<sup>fl/fl</sup>:Eef2k<sup>-/-</sup> mice after 9 days of 0.075% BBN treatment. 5 µL of urine was analyzed on a Xevo QToF mass spectrometer. BCPN concentrations were calculated by comparing the urine BCPN measurements to standards of known BCPN (TCI, B3185) concentrations.

### Immunoblot analysis

Cell lysates were prepared using RIPA buffer (Fisher, P189900) containing phosphatase inhibitor (Sigma, 4906845001) and protease inhibitor cocktail (Sigma, 11836153001). The lysates were incubated on ice for 30 min with periodic vortexing, and centrifuged at 13,000 x g at 4°C for 10 min. The supernatants were subjected to SDS-PAGE and transferred to a PDVF membrane (Bio-Rad, 1704273). Membranes were blocked with 5% milk for 1 h at room temperature. Primary antibodies were diluted in 5% milk and incubated overnight at 4°C. Blots were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG [Fisher, P131460] or goat anti-mouse IgG [Fisher, P131430]) in 5% milk for 1 h at room temperature. West Pico (Fisher, P134080) was used to detect immunoreactive bands on the blot using the ChemiDoc Touch Imaging System (Bio-Rad). The following primary antibodies were used: anti-Tubulin (Sigma, T8203), anti-ARID1A (Cell Signaling Technology, 12354), anti-PTEN (Cell Signaling Technology, 9559), anti-*p*-AKT(S473) (Cell Signaling Technology, 4060), anti-AKT1 (Santa Cruz Biotechnology, Sc-1618), anti-*p*-eIF2α (S51) (Cell Signaling Technology, 3597), anti-ATF4 (Cell Signaling Technology, 11815), anti-eIF4G (Cell Signaling Technology, 2469), anti-eIF4E (Santa Cruz Biotechnology, sc-271480), anti-eIF4A (Cell Signaling Technology, 2013), anti-*p*-4EBP1 (Cell Signaling Technology, 2855), anti-4EBP1 (Cell Signaling Technology, 9644), anti-*p*-eEF2 (T56) (Cell Signaling Technology, 2331), anti-eEF2 (Cell Signaling Technology, 2332), anti- AURKB (Novusbio, NB-100-294), anti-FGFR3 (Mybiosource, MBS9212039), anti-IGF2 (Fisher, MA5-17096), anti-KIF22 (Novusbio, NBP2-17053), anti-ODC1 (Fisher, PA5-21362), anti-SKA1 (Novusbio, NBP2-88277), anti-BRCA2 (Abcam, ab27976), anti-ERCC1 (Cell Signaling Technology, 3885), anti-ERCC2 (Cell Signaling Technology, 11963), anti-FANCC (Abcam, ab97575), anti-*p*-AMPK(Thr172) (Cell Signaling Technology, 2535), anti-AMPK (Cell Signaling Technology, 2532), anti-H3K27me3 (Cell Signaling Technology, 9733), anti-H3 (Cell Signaling Technology, 9715), anti-puromycin (EMD Millipore, MABE343), anti-Rasgrp1 (Origene, TA358534), anti-p70RSK (Cell Signaling Technology, 9202), anti-*p*-p70RSK (T389) (Cell Signaling Technology, 9205), anti-eEF2K (Abcam, ab46787), anti-*p*-eEF2K (S398) (ECM Bio, EP5441), anti-*p*-eEF2K (S366) (Abcam, ab51227), anti-ARID1A (Santa Cruz, SC-32761), and anti-ARID1A (Sigma, HPA005456).
## Mouse genotyping

Mouse tissue was incubated in 1.67 mg/mL of Proteinase K solution (Sigma, 3115879001) overnight at 55°C, then subsequently extracted using phenol:chloroform:isoamyl alcohol (Ambion, AM9732) followed by ethanol precipitation. Genotypes were confirmed using PCR with GoTaq Green Master Mix (Promega PRM7123) followed by agarose gel electrophoresis (Bio-Rad, 161–3102). Primers used for genotyping PCR, *Arid1a* (forward, 5'-GTAATGGGAAAGCGACTACTGGAG-3'; reverse, 5'-TGTTCGTTTTGTGGCGGGAG-3'), *UBC-Cre*<sup>ERT</sup> (forward, 5'-GCGGTCTGGCAGTAAAAACTATC-3'; reverse, 5'-GTGAAACAGCATTGCTGTCACTT-3'; internal control: forward, 5'-CTAGGCCACAGAATTGAAAGATCT-3'; reverse, 5'-GTAGGTGGAAATTCTAGCATCATCC-3'), *ROSA-LSL-Yfp*<sup>KI/KI</sup> (WT: forward, 5'-CTGGCTTCTGAGGACCG-3'; reverse, 5'-CAGGACAACGCCCACACA-3'. YFP: forward, 5'-AGGGCGAGGAGCTGTTCA-3'; reverse, 5'-TGAAGTCGATGCCCTTCAG-3'), *K5-Cre*<sup>ERT2</sup> (WT: forward, 5'-GCAAGACCCTGGTCCTCAC-3'; reverse, 5'-ACCGGCCTTATTCCAAGC-3'), *Eef2k*<sup>-/-</sup> (WT: forward, 5'-GGCCGGCTGCTAGAGAGTGTC-3'; reverse, 5'-CATCAGCTGATTGTAGTGACATC-3'. eEF2K KO: forward, 5'TGCGAGGCCAGAGGCCACTTGTGTAGC-3'; reverse, 5'-CAGGGCCTGCTTTCTTGGTGGCAG-3').

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

Bladder samples were collected, fixed in 4% PFA, dehydrated with ethanol, and embedded in paraffin wax. Paraffin blocks were sectioned (5 µm) using a rotary microtome and H&E staining were performed at the Fred Hutchinson Cancer Center Histopathology Core. IHC staining was performed after rehydrating the tissue. In brief, antigen retrieval was performed by preheating in sodium citrate buffer (pH 6.0 or pH 9) solution for 7–30 min at 95°C–125°C in a pressure cooker. Hydrogen peroxidase (Vector Laboratories, SP-6000) treatment was done for 5 min to quench endogenous peroxides. Tissue was blocked in 1% BSA for 1 h. Primary antibodies were applied and incubated at room temperature for 1 h in a humidified chamber followed by secondary antibody (Envision system HRP-labeled polymer, Dako, K4003). Color development was achieved by applying DAB solution (Agilent Technology, K3467) for 2–5 min, depending on the primary antibody. The sections were counterstained with hematoxylin (Agilent technology, S3309), washed, and cover-slipped using aqueous-based mounting medium (Agilent Technology, S302580). All IHC slides were scanned using Aperio ScanScope AT Turbo (Leica Biosystems). Following primary antibodies were used for this study: anti-ARID1A (Sigma, HPA005456), anti-eEF2 (Abcam, ab33523), anti-*p*-eEF2 (T56) (Abcam, ab53114), anti-eEF2K (Aviva systems biology, OAAF00684), anti-ERK1/2 (Cell Signaling Technology, 9102), anti-*p*-ERK1/2 (Cell Signaling Technology, 9101), anti-MEK1/2 (Cell Signaling Technology, 9122), anti-*p*-MEK1/2 (Cell Signaling Technology, 9121), anti-*p*-p90RSK (Abcam, ab32114), anti-*p*-p90RSK (Cell Signaling Technology, 11989), anti-RASGRP1 (Fisher, PA5-25750), anti-Ki67 (Cell Signaling Technology, 12202), anti-γH2AX (Cell Signaling Technology, 9718), anti-CC3 (Cell Signaling Technology, 9579).

IF staining was performed in the following manner. Antigen retrieval was conducted by preheating in sodium citrate buffer (pH 6.0) for 30 min at 95°C in a pressure cooker. Tissue was blocked in 1% BSA solution for 2 h or in M.O.M blocking solution (Vector Laboratories, MKB 2213) for 1 h at room temperature, followed by blocking in 1% BSA solution for 2 h. Primary antibodies were applied overnight at 4°C in a humidified chamber, and secondary antibodies (Alexa Fluor 594: Invitrogen, A11032; Alexa Fluor 488: Invitrogen, A11034; Alexa Fluor 594: Invitrogen, A11037; Alexa 647: Invitrogen, A-21244) were applied for 1 h at room temperature. All IF slides were mounted using Pro-Long gold mounting media with DAPI (Fisher Scientific, P36935). IF slides were scanned using an Aperio Scanscope FL (Leica Biosystems). The following primary antibodies were used for IF study: anti-cytokeratin 5 (Biolegend Inc, PRB-160P), anti-puromycin (EMD Millipore, MABE343), anti-*p*-eEF2K(S366) (Cell Signaling Technology, 3691), anti-ARID1A (Sigma, HPA005456), anti-Cytokeratin 5 (Abcam, ab52635). Image analysis was done using semiautomated image analysis software (HALO, Indica Labs).

## ChIP qPCR

5 x 10⁶ cells were used per immunoprecipitation reaction using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9003S) as per manufacturers' instruction. Chromatin was cross-linked with 1% formaldehyde in PBS for 10 min at room temperature and quenched by the addition of 10x Glycine and incubation for 5 min at room temperature, followed by a wash with PBS. Crosslinked chromatin was fractionated by digestion with micrococcal nuclease (0.2 uL per IP) for 20 minutes at 37°C. This was followed by 6 cycles of sonication (20 seconds on, 30 seconds off, 50% amplitude) using a cup horn sonicator (Qsonica Q500). IPs were performed using 1:100 anti-ARID1A (D2A8U) (Cell Signaling Technology, 12354) and equivalent concentration of rabbit anti-IgG control (Cell Signaling Technology, 2729) at 4°C overnight. Crosslinks were reversed with Proteinase K at 65°C for 2 h and DNA was purified using the SimpleChIP Enzymatic Chromatin IP Kit. ChIP-qPCR was performed as per manufacturers' instructions using Bio-Rad CFX384 Real-Time System using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 172-5272). Primer sequences against ARID1A target regions used for ChIP-qPCR (forward, 5'- CTT CGA ATC CTG CCC CCA TT-3'; reverse, 5'- TCT TGG GCT GGG AGA GAT GA-3').

## In vivo and in vitro puromycin incorporation assay

For the *in vivo* puromycin incorporation assay, mice were injected i.p. with 200 µL of 2.5 mM puromycin (Fisher, BP2956100) and euthanized after 1 hour. For *in vitro* puromycin incorporation assay, cells were treated with 1 µM puromycin for 30 minutes at 37°C.
## Mouse genotyping

Mouse tissue was incubated in 1.67 mg/mL of Proteinase K solution (Sigma, 3115879001) overnight at 55°C, then subsequently extracted using phenol:chloroform:isoamyl alcohol (Ambion, AM9732) followed by ethanol precipitation. Genotypes were confirmed using PCR with GoTaq Green Master Mix (Promega PRM7123) followed by agarose gel electrophoresis (Bio-Rad, 161–3102). Primers used for genotyping PCR, *Arid1a* (forward, 5'-GTAATGGGAAAGCGACTACTGGAG-3'; reverse, 5'-TGTTCGTTTTGTGGCGGGAG-3'), *UBC-Cre*<sup>ERT</sup> (forward, 5'-GCGGTCTGGCAGTAAAAACTATC-3'; reverse, 5'-GTGAAACAGCATTGCTGTCACTT-3'; internal control: forward, 5'-CTAGGCCACAGAATTGAAAGATCT-3'; reverse, 5'-GTAGGTGGAAATTCTAGCATCATCC-3'), *ROSA-LSL-Yfp*<sup>KI/KI</sup> (WT: forward, 5'-CTGGCTTCTGAGGACCG-3'; reverse, 5'-CAGGACAACGCCCACACA-3'. YFP: forward, 5'-AGGGCGAGGAGCTGTTCA-3'; reverse, 5'-TGAAGTCGATGCCCTTCAG-3'), *K5-Cre*<sup>ERT2</sup> (WT: forward, 5'-GCAAGACCCTGGTCCTCAC-3'; reverse, 5'-ACCGGCCTTATTCCAAGC-3'), *Eef2k*<sup>-/-</sup> (WT: forward, 5'-GGCCGGCTGCTAGAGAGTGTC-3'; reverse, 5'-CATCAGCTGATTGTAGTGACATC-3'. eEF2K KO: forward, 5'TGCGAGGCCAGAGGCCACTTGTGTAGC-3'; reverse, 5'-CAGGGCCTGCTTTCTTGGTGGCAG-3').

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

Bladder samples were collected, fixed in 4% PFA, dehydrated with ethanol, and embedded in paraffin wax. Paraffin blocks were sectioned (5 µm) using a rotary microtome and H&E staining were performed at the Fred Hutchinson Cancer Center Histopathology Core. IHC staining was performed after rehydrating the tissue. In brief, antigen retrieval was performed by preheating in sodium citrate buffer (pH 6.0 or pH 9) solution for 7–30 min at 95°C–125°C in a pressure cooker. Hydrogen peroxidase (Vector Laboratories, SP-6000) treatment was done for 5 min to quench endogenous peroxides. Tissue was blocked in 1% BSA for 1 h. Primary antibodies were applied and incubated at room temperature for 1 h in a humidified chamber followed by secondary antibody (Envision system HRP-labeled polymer, Dako, K4003). Color development was achieved by applying DAB solution (Agilent Technology, K3467) for 2–5 min, depending on the primary antibody. The sections were counterstained with hematoxylin (Agilent technology, S3309), washed, and cover-slipped using aqueous-based mounting medium (Agilent Technology, S302580). All IHC slides were scanned using Aperio ScanScope AT Turbo (Leica Biosystems). Following primary antibodies were used for this study: anti-ARID1A (Sigma, HPA005456), anti-eEF2 (Abcam, ab33523), anti-*p*-eEF2 (T56) (Abcam, ab53114), anti-eEF2K (Aviva systems biology, OAAF00684), anti-ERK1/2 (Cell Signaling Technology, 9102), anti-*p*-ERK1/2 (Cell Signaling Technology, 9101), anti-MEK1/2 (Cell Signaling Technology, 9122), anti-*p*-MEK1/2 (Cell Signaling Technology, 9121), anti-*p*-p90RSK (Abcam, ab32114), anti-*p*-p90RSK (Cell Signaling Technology, 11989), anti-RASGRP1 (Fisher, PA5-25750), anti-Ki67 (Cell Signaling Technology, 12202), anti-γH2AX (Cell Signaling Technology, 9718), anti-CC3 (Cell Signaling Technology, 9579).

IF staining was performed in the following manner. Antigen retrieval was conducted by preheating in sodium citrate buffer (pH 6.0) for 30 min at 95°C in a pressure cooker. Tissue was blocked in 1% BSA solution for 2 h or in M.O.M blocking solution (Vector Laboratories, MKB 2213) for 1 h at room temperature, followed by blocking in 1% BSA solution for 2 h. Primary antibodies were applied overnight at 4°C in a humidified chamber, and secondary antibodies (Alexa Fluor 594: Invitrogen, A11032; Alexa Fluor 488: Invitrogen, A11034; Alexa Fluor 594: Invitrogen, A11037; Alexa 647: Invitrogen, A-21244) were applied for 1 h at room temperature. All IF slides were mounted using Pro-Long gold mounting media with DAPI (Fisher Scientific, P36935). IF slides were scanned using an Aperio Scanscope FL (Leica Biosystems). The following primary antibodies were used for IF study: anti-cytokeratin 5 (Biolegend Inc, PRB-160P), anti-puromycin (EMD Millipore, MABE343), anti-*p*-eEF2K(S366) (Cell Signaling Technology, 3691), anti-ARID1A (Sigma, HPA005456), anti-Cytokeratin 5 (Abcam, ab52635). Image analysis was done using semiautomated image analysis software (HALO, Indica Labs).

## ChIP qPCR

5 x 10⁶ cells were used per immunoprecipitation reaction using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9003S) as per manufacturers' instruction. Chromatin was cross-linked with 1% formaldehyde in PBS for 10 min at room temperature and quenched by the addition of 10x Glycine and incubation for 5 min at room temperature, followed by a wash with PBS. Crosslinked chromatin was fractionated by digestion with micrococcal nuclease (0.2 uL per IP) for 20 minutes at 37°C. This was followed by 6 cycles of sonication (20 seconds on, 30 seconds off, 50% amplitude) using a cup horn sonicator (Qsonica Q500). IPs were performed using 1:100 anti-ARID1A (D2A8U) (Cell Signaling Technology, 12354) and equivalent concentration of rabbit anti-IgG control (Cell Signaling Technology, 2729) at 4°C overnight. Crosslinks were reversed with Proteinase K at 65°C for 2 h and DNA was purified using the SimpleChIP Enzymatic Chromatin IP Kit. ChIP-qPCR was performed as per manufacturers' instructions using Bio-Rad CFX384 Real-Time System using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 172-5272). Primer sequences against ARID1A target regions used for ChIP-qPCR (forward, 5'- CTT CGA ATC CTG CCC CCA TT-3'; reverse, 5'- TCT TGG GCT GGG AGA GAT GA-3').

## In vivo and in vitro puromycin incorporation assay

For the *in vivo* puromycin incorporation assay, mice were injected i.p. with 200 µL of 2.5 mM puromycin (Fisher, BP2956100) and euthanized after 1 hour. For *in vitro* puromycin incorporation assay, cells were treated with 1 µM puromycin for 30 minutes at 37°C.
RNA sequencing and polysome-bound RNA sequencing

Organoids were cultured for 96 hour before harvesting for RNA extraction or polysome profiling (see polysome profiling). The library preparation and sequencing were performed at the Fred Hutchinson Cancer Center Genomics Core. Polysome fractions were mixed 1:1 with TRIzol Reagent (Invitrogen, 15596-018) and stored at -80C until processing. The sub-polysome and polysome fractions were individually pooled and processed for sequencing. RNA was extracted from each pool using Direct-zol Miniprep Plus kits (Zymo, ZR2070) and concentrations were assessed by Qubit. ERCC RNA Spike-In Control Mix (Fisher, 4456740) was serial diluted (1:1000) and 2 µL was used for every 100ng of RNA. RNA-seq libraries were constructed using TruSeq Stranded mRNA Library Prep Kit (Illumina, 20020594) with the IDT for Illumina (TruSeq RNA UD Indexes: Illumina 20022371) following manufacturer's instructions. The sequencing was done on the Illumina NovaSeq 6000 using the SP-100 flow cell sequencing kit (Illumina, 20027464), 50PE run configuration.

In vitro [³⁵S]-methionine labeling assay

Cells were plated at a density of 10,000 cells per 50 µL EHS (NIH) and cultured with organoid media (see organoid culture). Cells were treated with 0.05 mCi/mL [³⁵S]-methionine (Perkin Elmer, NEG772002MC) at 37°C for 1 hour. Total cell lysates were prepared and subjected to immunoblot analysis. X-ray film was used to capture the radioactive signal. To check de novo protein synthesis in ARID1A re-expressing organoids, cells were treated with Doxycycline (0.1 µg/mL) for 48 h before [³⁵S]-methionine labeling.

Cell viability assay

WT bladder tumor organoids were established using following methods. Briefly, WT bladder tumor tissue chunks for *Arid1a* non-recombined *Arid1a*<sup>fl/fl</sup> mice were incubated in 5 mg/mL collagenase type II (Life Technologies, 17101015) diluted in Dulbecco's Modified Eagle's Medium for 1 hour at 37°C, followed by a 5 minute digestion with TrypLE (Gibco, 12604-039). Digested tissues were dissociated using a syringe with 18G needle, and single cells were obtained after passing through a cell strainer. Cells were plated at a density of 20,000 cells per 50 µL of EHS (NIH) on 24-well ultra-low attachment plates (Corning, 3473) and cultured in organoid media (see organoid culture) to establish WT bladder tumor organoids. *Arid1a*<sup>fl/fl</sup> bladder tumor organoids were generated from the WT parental line using lentivirus expressing Cre-recombinase (Addgene, 25997). To measure the differential cytotoxicity of homoharringtonine (HHT), the CellTiter-Glo 2.0 Assay (Promega, G9242) was used. Two thousand WT and *Arid1a*<sup>fl/fl</sup> tumor cells were embedded in 5 µL of Matrigel and plated on a 96-well plate in organoid media. One day post seeding, media was replaced with media containing either 100 nM or 1 µM of homoharringtonine (HHT). After a 48 hour HHT treatment, the CellTiter-Glo 2.0 Assay was performed according to manufacturer's instructions. HT1376, KU1919, HT1197, and UMUC11 cells (5000 cells/well) were plated on a 96-well plate. Cells were treated with various concentration of HHT (1, and 10 µM) at 48 hours after plating. After 24 hours of HHT treatment, the CellTiter-Glo 2.0 Assay was performed according to manufacturer's instructions.

siRNA knockdown

siARID1A (ON-TARGETplus SMARTpool siRNA: Dharmacon, J-017263-05) were transfected into human PrEC cells according to manufacturer's protocol. Briefly, human PrEC cells were plated in 6-well plate and siRNAs were transfected using lipofectamine 2000 transfection reagents (Fisher, 11668019). Cells were harvested 72 h after transfection and subjected to immunoblot to verify knockdown.

Polysome profiling

WT or *Arid1a*<sup>fl/fl</sup> cells were plated at a density of 10,000 cells per 50 µL EHS (NIH) and cultured with organoid media for 96 hours (see *organoid culture*). Cells were treated with 100 µg/mL (final concentration) cycloheximide (Sigma, C7698) for 10 minutes at 37°C. Cells were extracted from EHS by incubating in cell recovery solution (Corning, 354253) for 1 hour at 4°C. Cell pellets were lysed on ice in 350 µL of polysome lysis buffer (10 mM Tris-HCl pH 7.4 [Ambion, AM9851 (pH 7.0) and AM9856 (pH 8.0)]), 132 mM NaCl (Ambion, AM9760G), 1.4 mM MgCl2 (Ambion, AM9530G), 19 mM DTT (Sigma, 43815), 142 µg/mL cycloheximide (Sigma, C7698), 0.1% Triton X-100 (Fisher, BP151), 0.2% NP-40 (Pierce, P128324), 607 U/mL SUPERase-In RNase Inhibitor (Life Technologies, AM2696) with periodic vortex mixing. Lysates were clarified by centrifugation at 9300 x g for 5 minutes and supernatants were transferred to fresh tubes. Protein quantification was performed on each lysate by Bradford assay (Bio-Rad, 5000006) and analyzed using a BioTek Epoch Microplate Spectrophotometer. A portion of each lysate was saved as an input total RNA sample. For each lysate, 850 µg protein in 265 µL of the supernatant was layered onto 10% to 50% (w/v) linear sucrose gradients (Fisher, BP22010) containing 2 mM DTT (Sigma, 43815) and 100 µg/mL heparin (Sigma, H3149). The gradients were centrifuged at 37,000 rpm for 2.5 hours at 4°C in a Beckman SW41Ti rotor in Seton 7030 ultracentrifuge tubes. After centrifugation, samples were fractionated using a Biocomp Gradient Station (Biocomp) by upward displacement into collection tubes, through a Bio-Rad EM-1 UV monitor (Bio-Rad) for continuous measurement of the absorbance at 254 nm.

Ribosome half-transit time

Cells were plated at a density of 10,000 cells per 50 µL EHS in 24-well ultra-low attachment plates and cultured with organoid media (see [organoid culture](#)) for 96 hours. Cells were treated with 10 µCi/mL [³⁵S]-methionine (Perkin Elmer, NEG772002MC) and incubated at 37°C. At indicated time points (5, 10 and 15 minutes) after labeling, cells were harvested in ice-cold PBS containing 100 µg/mL cycloheximide. Cells were extracted from EHS using TrypLE treatment and resuspended in 250 µL of RBS buffer (10 mM NaCl,
RNA sequencing and polysome-bound RNA sequencing

Organoids were cultured for 96 hour before harvesting for RNA extraction or polysome profiling (see polysome profiling). The library preparation and sequencing were performed at the Fred Hutchinson Cancer Center Genomics Core. Polysome fractions were mixed 1:1 with TRIzol Reagent (Invitrogen, 15596-018) and stored at -80C until processing. The sub-polysome and polysome fractions were individually pooled and processed for sequencing. RNA was extracted from each pool using Direct-zol Miniprep Plus kits (Zymo, ZR2070) and concentrations were assessed by Qubit. ERCC RNA Spike-In Control Mix (Fisher, 4456740) was serial diluted (1:1000) and 2 µL was used for every 100ng of RNA. RNA-seq libraries were constructed using TruSeq Stranded mRNA Library Prep Kit (Illumina, 20020594) with the IDT for Illumina (TruSeq RNA UD Indexes: Illumina 20022371) following manufacturer's instructions. The sequencing was done on the Illumina NovaSeq 6000 using the SP-100 flow cell sequencing kit (Illumina, 20027464), 50PE run configuration.

In vitro [³⁵S]-methionine labeling assay

Cells were plated at a density of 10,000 cells per 50 µL EHS (NIH) and cultured with organoid media (see organoid culture). Cells were treated with 0.05 mCi/mL [³⁵S]-methionine (Perkin Elmer, NEG772002MC) at 37°C for 1 hour. Total cell lysates were prepared and subjected to immunoblot analysis. X-ray film was used to capture the radioactive signal. To check de novo protein synthesis in ARID1A re-expressing organoids, cells were treated with Doxycycline (0.1 µg/mL) for 48 h before [³⁵S]-methionine labeling.

Cell viability assay

WT bladder tumor organoids were established using following methods. Briefly, WT bladder tumor tissue chunks for *Arid1a* non-recombined *Arid1a*<sup>fl/fl</sup> mice were incubated in 5 mg/mL collagenase type II (Life Technologies, 17101015) diluted in Dulbecco's Modified Eagle's Medium for 1 hour at 37°C, followed by a 5 minute digestion with TrypLE (Gibco, 12604-039). Digested tissues were dissociated using a syringe with 18G needle, and single cells were obtained after passing through a cell strainer. Cells were plated at a density of 20,000 cells per 50 µL of EHS (NIH) on 24-well ultra-low attachment plates (Corning, 3473) and cultured in organoid media (see organoid culture) to establish WT bladder tumor organoids. *Arid1a*<sup>fl/fl</sup> bladder tumor organoids were generated from the WT parental line using lentivirus expressing Cre-recombinase (Addgene, 25997). To measure the differential cytotoxicity of homoharringtonine (HHT), the CellTiter-Glo 2.0 Assay (Promega, G9242) was used. Two thousand WT and *Arid1a*<sup>fl/fl</sup> tumor cells were embedded in 5 µL of Matrigel and plated on a 96-well plate in organoid media. One day post seeding, media was replaced with media containing either 100 nM or 1 µM of homoharringtonine (HHT). After a 48 hour HHT treatment, the CellTiter-Glo 2.0 Assay was performed according to manufacturer's instructions. HT1376, KU1919, HT1197, and UMUC11 cells (5000 cells/well) were plated on a 96-well plate. Cells were treated with various concentration of HHT (1, and 10 µM) at 48 hours after plating. After 24 hours of HHT treatment, the CellTiter-Glo 2.0 Assay was performed according to manufacturer's instructions.

siRNA knockdown

siARID1A (ON-TARGETplus SMARTpool siRNA: Dharmacon, J-017263-05) were transfected into human PrEC cells according to manufacturer's protocol. Briefly, human PrEC cells were plated in 6-well plate and siRNAs were transfected using lipofectamine 2000 transfection reagents (Fisher, 11668019). Cells were harvested 72 h after transfection and subjected to immunoblot to verify knockdown.

Polysome profiling

WT or *Arid1a*<sup>fl/fl</sup> cells were plated at a density of 10,000 cells per 50 µL EHS (NIH) and cultured with organoid media for 96 hours (see *organoid culture*). Cells were treated with 100 µg/mL (final concentration) cycloheximide (Sigma, C7698) for 10 minutes at 37°C. Cells were extracted from EHS by incubating in cell recovery solution (Corning, 354253) for 1 hour at 4°C. Cell pellets were lysed on ice in 350 µL of polysome lysis buffer (10 mM Tris-HCl pH 7.4 [Ambion, AM9851 (pH 7.0) and AM9856 (pH 8.0)]), 132 mM NaCl (Ambion, AM9760G), 1.4 mM MgCl2 (Ambion, AM9530G), 19 mM DTT (Sigma, 43815), 142 µg/mL cycloheximide (Sigma, C7698), 0.1% Triton X-100 (Fisher, BP151), 0.2% NP-40 (Pierce, P128324), 607 U/mL SUPERase-In RNase Inhibitor (Life Technologies, AM2696) with periodic vortex mixing. Lysates were clarified by centrifugation at 9300 x g for 5 minutes and supernatants were transferred to fresh tubes. Protein quantification was performed on each lysate by Bradford assay (Bio-Rad, 5000006) and analyzed using a BioTek Epoch Microplate Spectrophotometer. A portion of each lysate was saved as an input total RNA sample. For each lysate, 850 µg protein in 265 µL of the supernatant was layered onto 10% to 50% (w/v) linear sucrose gradients (Fisher, BP22010) containing 2 mM DTT (Sigma, 43815) and 100 µg/mL heparin (Sigma, H3149). The gradients were centrifuged at 37,000 rpm for 2.5 hours at 4°C in a Beckman SW41Ti rotor in Seton 7030 ultracentrifuge tubes. After centrifugation, samples were fractionated using a Biocomp Gradient Station (Biocomp) by upward displacement into collection tubes, through a Bio-Rad EM-1 UV monitor (Bio-Rad) for continuous measurement of the absorbance at 254 nm.

Ribosome half-transit time

Cells were plated at a density of 10,000 cells per 50 µL EHS in 24-well ultra-low attachment plates and cultured with organoid media (see [organoid culture](#)) for 96 hours. Cells were treated with 10 µCi/mL [³⁵S]-methionine (Perkin Elmer, NEG772002MC) and incubated at 37°C. At indicated time points (5, 10 and 15 minutes) after labeling, cells were harvested in ice-cold PBS containing 100 µg/mL cycloheximide. Cells were extracted from EHS using TrypLE treatment and resuspended in 250 µL of RBS buffer (10 mM NaCl,
<figure><img src="image_75.png" /></figure>

10 mM Tris-HCl at pH 7.4, 15 mM MgCl2, 100 µg/mL heparin and protease inhibitor). 35 µL of lysis buffer (10% Triton X-100, 10% deoxycholate) was also added to lyse the cells. Cell suspension was incubated on ice for 10 minutes with periodic vortexing. Nuclei and mitochondria were separated by centrifugation for 5 minutes at 10,000 x g at 4°C. Supernatant (PMS) was removed and equal volume of polysome buffer (25 mM NaCl, 10 mM MgCl2, 25 mM Tris-HCl at pH 7.4, 0.05% Triton X-100, 0.14 M sucrose, 500 µg/mL heparin) was added. Polysomes were pelleted by centrifugation of half of the volume at 87,000 rpm for 2 hours using the TLA100 rotor and the supernatant (PRS) was removed. PMS represents nascent and completed proteins while PRS represents only completed proteins. Equal volume of PMS and PRS samples were precipitated on glass microfiber filters (Sigma, WHA1822024) using 20% TCA for 20 minutes on ice. Filters were washed with 10% TCA twice and once with ice-cold ethanol. Filters were air dried before taking counts. Liquid scintillation cocktail (Fisher, 50-904-0129) was added prior to liquid scintillation counting.

## TMT mass spectrometry

**Disulfide bond reduction/alkylation:** Protein solutions (120 µg) were diluted to 2.4 µg/µL in 50 mM HEPES pH 8.5. Disulfide bonds within the proteins were reduced by adding tris (2-carboxyethyl) phosphine to a concentration of 5 mM and mixing at room temperature for 15 minutes. The reduced proteins were alkylated by adding 10 mM 2-chloroacetamide to a concentration of 10 mM and mixed in the dark at room temperature for 30 minutes. Excess 2-chloroacetamide was quenched by adding dithiothreitol to a concentration of 10 mM and mixing at room temperature for 15 minutes.

**Protein precipitation and protease digestion:** Samples (120 µg) were diluted to 1 µg/µL with 100 mM ammonium bicarbonate in a 1.5 mL Eppendorf low-bind tube. Protein precipitation was carried out as follows: 400 µL of methanol was added to the sample and vortexed for 5 seconds, 100 µL of chloroform was added to the sample and vortexed for 5 seconds, 300 µL of water was added to the sample and vortexed for 5 seconds, the samples were centrifuged for 1 minute at 14,000 x g, and the aqueous and organic phases were removed, leaving a protein wafer in the tube. The protein wafers were washed with 400 µL of methanol and centrifuged at 21,000 x g at room temperature for 2 minutes. The supernatants were removed and the pellets were allowed to air dry. The samples were resuspended in 70 µL 100 mM HEPES (pH 8.5) and digested with rLys-C protease (100:1, protein to protease ratio) with mixing at 37°C for 4 hours. Trypsin protease (100:1, protein to protease ratio) was added and the reaction was mixed overnight at 37°C.

**TMTpro18plex labeling:** Each TMTpro18plex labeling reagent (Fisher, A44521 and A52048) was brought up in 30 µL acetonitrile and added to their assigned digested peptide solution (100 µg), yielding a final organic concentration of 30% (v/v), and mixed at room temperature for 1 hour. A 2-µg aliquot from each sample was combined, dried to remove the acetonitrile, processed with a C18 Zip-tip, and analyzed via LC/MS as a "label check". The label check was used to check that the labeling efficiency of the TMT reagent exceeded 97% and to determine the volumes from each sample to be used for equalizing the protein amounts when combining the samples. After the label check, the reactions were quenched with hydroxylamine to a final concentration of 0.3% (v/v) and mixing for 15 minutes. The TMTpro18plex labeled samples were pooled at 1:1 ratio based on the equalization volumes determined in the label check and concentrated in a Speedvac to remove acetonitrile. Half of the material was desalted with an Oasis HLB 3cc cartridge and dried with a Speedvac.

**bRP Fractionation:** The desalted TMT sample was resuspended in 100 µL of 10 mM ammonium bicarbonate pH 8. The material was loaded on to a Zorbax 2.1 cm x 150 mm (5-µm particle size) Extend-C18 column connected to a HPLC equipped with a diode array detector and fraction collector. The sample was gradient-eluted from the column at a flowrate of 250 µL/min over 55 minutes using a combination of solvents "A" (10 mM ammonium bicarbonate) and "B" (acetonitrile). The elution profile used was as follows: from 0 to 5 minutes "B" was held at 1%, from 5 to 55 minutes "B" varied from 5% to 40% followed by an increase to 90% "B" over 5 minutes, and then a hold for an additional 5 minutes at 90% "B". The UV signal was monitored at 210 nm and fractions were collected every 50 seconds, resulting in 96 fractions. The fractions were combined into 24 pools by concatenating every 24th fraction into a pool (pool 1 = fractions 1, 25, 49, and 73, pool 2 = fractions 2, 26, 50, and 74, etc). The pools were taken to dryness by vacuum centrifugation and subsequently analyzed by LC/MS.

**LC/MS:** The dried basic reverse phase fractions were brought up in 2% acetonitrile in 0.1% formic acid (20 µL) and analyzed (2.5 µL) by LC/ESI MS/MS with a Easy1200 nLC coupled to a tribrid Orbitrap Eclipse with FAIMS pro mass spectrometer. In-line de-salting was accomplished using a reversed-phase trap column (100 µm x 20 mm) packed with Magic C₁₈AQ (5-µm, 200Å resin) followed by peptide separations on a reversed-phase column (75 µm x 270 mm) packed with ReproSil-Pur C₁₈AQ (3-µm, 120Å resin) directly mounted on the electrospray ion source. A 120-minute gradient from 4% to 44% B (80% acetonitrile in 0.1% formic acid/water) at a flow rate of 300 nL/min was used for chromatographic separations. The temperature of the chromatographic column was maintained at 40°C using a heating blanket. A spray voltage of 2300 V was applied to the electrospray tip in-line with a FAIMS pro source using varied compensation voltages of -40 V, -60 V, -80 V while the Orbitrap Eclipse instrument was operated in the data-dependent mode. MS survey scans were in the Orbitrap (Normalized AGC target value 300%, resolution 120,000, and max injection time 50 ms) using a 3 s cycle time and MS/MS spectra acquisition were also detected in the Orbitrap (Normalized AGC target value of 250%, resolution 50,000 and max injection time 100 ms) using higher energy collision-induced dissociation (HCD) activation with HCD collision energy of 38%.

## Preclinical trials

Tumor pieces (1 × 1 × 1 mm) were implanted into the flank of 4-6 weeks old NOD-SCID γ IL-2 mice. Xenografts were measured every 2 days and tumor volume was calculated using the formula (L x W x W)/2, where L is the length of the tumor and W its width. When
10 mM Tris-HCl at pH 7.4, 15 mM MgCl2, 100 µg/mL heparin and protease inhibitor). 35 µL of lysis buffer (10% Triton X-100, 10% deoxycholate) was also added to lyse the cells. Cell suspension was incubated on ice for 10 minutes with periodic vortexing. Nuclei and mitochondria were separated by centrifugation for 5 minutes at 10,000 x g at 4°C. Supernatant (PMS) was removed and equal volume of polysome buffer (25 mM NaCl, 10 mM MgCl2, 25 mM Tris-HCl at pH 7.4, 0.05% Triton X-100, 0.14 M sucrose, 500 µg/mL heparin) was added. Polysomes were pelleted by centrifugation of half of the volume at 87,000 rpm for 2 hours using the TLA100 rotor and the supernatant (PRS) was removed. PMS represents nascent and completed proteins while PRS represents only completed proteins. Equal volume of PMS and PRS samples were precipitated on glass microfiber filters (Sigma, WHA1822024) using 20% TCA for 20 minutes on ice. Filters were washed with 10% TCA twice and once with ice-cold ethanol. Filters were air dried before taking counts. Liquid scintillation cocktail (Fisher, 50-904-0129) was added prior to liquid scintillation counting.

## TMT mass spectrometry

**Disulfide bond reduction/alkylation:** Protein solutions (120 µg) were diluted to 2.4 µg/µL in 50 mM HEPES pH 8.5. Disulfide bonds within the proteins were reduced by adding tris (2-carboxyethyl) phosphine to a concentration of 5 mM and mixing at room temperature for 15 minutes. The reduced proteins were alkylated by adding 10 mM 2-chloroacetamide to a concentration of 10 mM and mixed in the dark at room temperature for 30 minutes. Excess 2-chloroacetamide was quenched by adding dithiothreitol to a concentration of 10 mM and mixing at room temperature for 15 minutes.

**Protein precipitation and protease digestion:** Samples (120 µg) were diluted to 1 µg/µL with 100 mM ammonium bicarbonate in a 1.5 mL Eppendorf low-bind tube. Protein precipitation was carried out as follows: 400 µL of methanol was added to the sample and vortexed for 5 seconds, 100 µL of chloroform was added to the sample and vortexed for 5 seconds, 300 µL of water was added to the sample and vortexed for 5 seconds, the samples were centrifuged for 1 minute at 14,000 x g, and the aqueous and organic phases were removed, leaving a protein wafer in the tube. The protein wafers were washed with 400 µL of methanol and centrifuged at 21,000 x g at room temperature for 2 minutes. The supernatants were removed and the pellets were allowed to air dry. The samples were resuspended in 70 µL 100 mM HEPES (pH 8.5) and digested with rLys-C protease (100:1, protein to protease ratio) with mixing at 37°C for 4 hours. Trypsin protease (100:1, protein to protease ratio) was added and the reaction was mixed overnight at 37°C.

**TMTpro18plex labeling:** Each TMTpro18plex labeling reagent (Fisher, A44521 and A52048) was brought up in 30 µL acetonitrile and added to their assigned digested peptide solution (100 µg), yielding a final organic concentration of 30% (v/v), and mixed at room temperature for 1 hour. A 2-µg aliquot from each sample was combined, dried to remove the acetonitrile, processed with a C18 Zip-tip, and analyzed via LC/MS as a "label check". The label check was used to check that the labeling efficiency of the TMT reagent exceeded 97% and to determine the volumes from each sample to be used for equalizing the protein amounts when combining the samples. After the label check, the reactions were quenched with hydroxylamine to a final concentration of 0.3% (v/v) and mixing for 15 minutes. The TMTpro18plex labeled samples were pooled at 1:1 ratio based on the equalization volumes determined in the label check and concentrated in a Speedvac to remove acetonitrile. Half of the material was desalted with an Oasis HLB 3cc cartridge and dried with a Speedvac.

**bRP Fractionation:** The desalted TMT sample was resuspended in 100 µL of 10 mM ammonium bicarbonate pH 8. The material was loaded on to a Zorbax 2.1 cm x 150 mm (5-µm particle size) Extend-C18 column connected to a HPLC equipped with a diode array detector and fraction collector. The sample was gradient-eluted from the column at a flowrate of 250 µL/min over 55 minutes using a combination of solvents "A" (10 mM ammonium bicarbonate) and "B" (acetonitrile). The elution profile used was as follows: from 0 to 5 minutes "B" was held at 1%, from 5 to 55 minutes "B" varied from 5% to 40% followed by an increase to 90% "B" over 5 minutes, and then a hold for an additional 5 minutes at 90% "B". The UV signal was monitored at 210 nm and fractions were collected every 50 seconds, resulting in 96 fractions. The fractions were combined into 24 pools by concatenating every 24th fraction into a pool (pool 1 = fractions 1, 25, 49, and 73, pool 2 = fractions 2, 26, 50, and 74, etc). The pools were taken to dryness by vacuum centrifugation and subsequently analyzed by LC/MS.

**LC/MS:** The dried basic reverse phase fractions were brought up in 2% acetonitrile in 0.1% formic acid (20 µL) and analyzed (2.5 µL) by LC/ESI MS/MS with a Easy1200 nLC coupled to a tribrid Orbitrap Eclipse with FAIMS pro mass spectrometer. In-line de-salting was accomplished using a reversed-phase trap column (100 µm x 20 mm) packed with Magic C₁₈AQ (5-µm, 200Å resin) followed by peptide separations on a reversed-phase column (75 µm x 270 mm) packed with ReproSil-Pur C₁₈AQ (3-µm, 120Å resin) directly mounted on the electrospray ion source. A 120-minute gradient from 4% to 44% B (80% acetonitrile in 0.1% formic acid/water) at a flow rate of 300 nL/min was used for chromatographic separations. The temperature of the chromatographic column was maintained at 40°C using a heating blanket. A spray voltage of 2300 V was applied to the electrospray tip in-line with a FAIMS pro source using varied compensation voltages of -40 V, -60 V, -80 V while the Orbitrap Eclipse instrument was operated in the data-dependent mode. MS survey scans were in the Orbitrap (Normalized AGC target value 300%, resolution 120,000, and max injection time 50 ms) using a 3 s cycle time and MS/MS spectra acquisition were also detected in the Orbitrap (Normalized AGC target value of 250%, resolution 50,000 and max injection time 100 ms) using higher energy collision-induced dissociation (HCD) activation with HCD collision energy of 38%.

## Preclinical trials

Tumor pieces (1 × 1 × 1 mm) were implanted into the flank of 4-6 weeks old NOD-SCID γ IL-2 mice. Xenografts were measured every 2 days and tumor volume was calculated using the formula (L x W x W)/2, where L is the length of the tumor and W its width. When
tumors reached a total volume of 100 mm³, animals were randomly selected for one of two treatment arms: Homoharringtonine (Carbosynth, FH15974) at 0.7 mg/kg dissolved in PBS or vehicle (PBS) twice daily by oral gavage.

## CUT&Tag chromatin profiling

CUT&Tag was performed following the published protocol,⁷⁸ with some modifications. Briefly, cells were harvested and prepared into homogenous suspension in PBS, aliquoted with 10% DMSO and slow-frozen to -80°C in isopropanol freezing chambers. CUT&Tag was performed with native nuclei.⁷⁹ Cells were thawed at 37°C for 1 minute, washed twice with PBS and centrifugation at 600 x g for 3 minutes, and resuspended into ice-cold NE1 buffer (20mM K-HEPES pH 7.9, 10mM KCl, 10% Triton X-100, 20% Glycerol, 0.5 mM spermidine, and EDTA-free protease inhibitor). Cells were kept on ice for 10 minutes, centrifuged at 1400 x g for 3 minutes using a swinging-bucket rotor, pellets were resuspended in Wash buffer (20 mM Na-HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and EDTA-free protease inhibitor), nuclei were counted using Vi-CELL Cell Viability Analyzer (Beckman Coulter) and aliquoted into 0.6 mL low-binding flip-cap tubes for CUT&Tag experiments.

500,000 nuclei were used for each CUT&Tag experiment targeting a histone posttranslational modification, while 200,000 nuclei were used for ARID1A. Bio-Mag Plus Concanavalin A coated magnetic beads (Bangs laboratories, BP531) were equilibrated with binding buffer (20 mM K-HEPES pH7.9, 10 mM KCl, and 1 mM each CaCl₂ and MnCl₂). To each sample, 10 µL of activated beads were added and held at room temperature for 5 minutes with occasional gentle mixing. Beads (with bound nuclei) were magnetized, supernatants were removed, beads were washed once with 400 µL antibody buffer (Wash buffer supplemented with 2 mM EDTA and 0.05% Digitonin), and resuspended in 200 µL antibody buffer containing the respective primary antibody in 1:100 v/v dilution (H3K27me3-Cell Signaling Technology, 9733 H3K27ac - Millipore-Sigma, MABE647; ARID1A - Cell Signaling Technology, 12354). Primary antibody incubations were performed on a rotating platform overnight at 4°C. Beads were magnetized, supernatant removed, washed once with 400 µL Dig-Wash (Wash buffer supplemented with 0.05% Digitonin), resuspended in 200 µL Dig-Wash containing guinea pig α-rabbit IgG secondary antibody (Antibodies-online.com, ABIN101961) at 1:100 dilution. Secondary antibody incubations were performed on a rotating platform for 30 minutes at room temperature. Beads were magnetized, supernatant removed, washed twice with 400 µL Dig-Wash to remove unbound antibodies, and resuspended in 200 µL Dig-Med buffer (Dig-Wash buffer, except containing 300 mM NaCl) with 1:200 dilution (~0.04 µM) of proteinA-Tn5 transposase fusion protein (pA-Tn5) pre-loaded with double-stranded adapters with 19-mer mosaic ends.⁷⁸ pA-Tn5 incubations were performed on a rotating platform for 1 hour at room temperature. Beads were magnetized, supernatant removed, washed three times with 400 µL Dig-Med to remove unbound pA-Tn5, and resuspended in 300 µL Tagmentation buffer (Dig-Med supplemented with 10 mM MgCl₂). Tagmentation reactions were performed by incubating samples at 37°C on a rotating platform for 1 hour. Tagmentation reactions were stopped by sequentially adding 10 µL of 0.5 mM EDTA, 3.1 µL of 10% SDS (1% final), and 2 µL of 20 mg/mL Proteinase K, mixed well, and incubated in 50°C water bath for 1 hour. DNA was extracted using phenol-chloroform-isoamyl alcohol (25:24:1), re-extracted with one volume CHCl₃, aqueous phase (~300 µL) was transferred to fresh tube, and DNA was precipitated by adding 900 µL ethanol overnight at -20°C followed by centrifugation for 45 minutes at 13,000 rpm and 4°C. Pellets were rinsed with 100% ice-cold ethanol, air-dried, dissolved in 30 µL 0.1 x TE (1 mM Tris pH 8, 0.1 mM EDTA) supplemented with RNase A (1:400 dilution of 10 mg/mL), and incubated in 37°C water bath for 15 minutes.

To amplify libraries, 21 µL DNA was mixed with 2 µL each of a universal i5 primer and a uniquely barcoded i7 primer,⁸⁰ using a different barcode for each sample, and 25 µL of NEB Next HiFi 2x PCR Master mix. Samples were placed in a thermocycler with heated lid using the following cycling conditions: 72°C for 5 minutes (gap filling); 98°C for 30 seconds (denaturation); 13 cycles of 98°C for 10 seconds and 63°C for 10 seconds (combined annealing and extension); final extension at 72°C for 1 minute, and held at 8°C. Post-PCR clean-up was performed using a double-sided clean-up method. 25 µL (0.5 X) of Ampure XP beads (Beckman Counter) was mixed with the libraries and incubated for 10 minutes at room temperature to remove large DNA fragments (>1300 bp). Beads were magnetized, supernatants were removed to fresh tubes and mixed with 45 µL (1.4 X - 0.5 X = 0.9 X) of Ampure XP beads and incubated for 10 minutes at room temperature. Beads were magnetized and the supernatants containing unused PCR primers were discarded. Beads were washed three times with 80% ethanol and eluted in 30 µL 10 mM Tris pH 8.0. Libraries were sequenced for 25 cycles in 25bp paired-end mode on the Illumina HiSeq 2500 platform or 50 bp paired-end mode on Illumina NextSeq 2000 at the Fred Hutchinson Cancer Center Genomics Shared Resource.

## Flow cytometry

To isolate basal urothelial cells, bladder tissue was digested with collagenase II and generated single cell suspension (for details see organoid culture). Fluorescence-conjugated antibodies were then added to cell suspensions and stained for 30 minutes on ice. Live basal cells were separated by flow cytometry after staining with DAPI, EPCAM-APC (epithelial marker) and CD49f-PE (basal cell marker). 0.125 µg of EPCAM-APC (Invitrogen, 17-5791-80) and CD49f-PE (Fisher, 12-0495-82) were used respectively per sample containing 1x10⁸ cells.

## Cell cycle analysis

Cells were fixed using pre-cooled 66% ethanol at 4°C for at 2 hours. Propidium iodide staining was performed following manufacturer's instruction (Abcam, ab139418). The cells were read using flow cytometry (Fortessa X50) and cell cycle phases were analyzed using FlowJo software.
tumors reached a total volume of 100 mm³, animals were randomly selected for one of two treatment arms: Homoharringtonine (Carbosynth, FH15974) at 0.7 mg/kg dissolved in PBS or vehicle (PBS) twice daily by oral gavage.

## CUT&Tag chromatin profiling

CUT&Tag was performed following the published protocol,⁷⁸ with some modifications. Briefly, cells were harvested and prepared into homogenous suspension in PBS, aliquoted with 10% DMSO and slow-frozen to -80°C in isopropanol freezing chambers. CUT&Tag was performed with native nuclei.⁷⁹ Cells were thawed at 37°C for 1 minute, washed twice with PBS and centrifugation at 600 x g for 3 minutes, and resuspended into ice-cold NE1 buffer (20mM K-HEPES pH 7.9, 10mM KCl, 10% Triton X-100, 20% Glycerol, 0.5 mM spermidine, and EDTA-free protease inhibitor). Cells were kept on ice for 10 minutes, centrifuged at 1400 x g for 3 minutes using a swinging-bucket rotor, pellets were resuspended in Wash buffer (20 mM Na-HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and EDTA-free protease inhibitor), nuclei were counted using Vi-CELL Cell Viability Analyzer (Beckman Coulter) and aliquoted into 0.6 mL low-binding flip-cap tubes for CUT&Tag experiments.

500,000 nuclei were used for each CUT&Tag experiment targeting a histone posttranslational modification, while 200,000 nuclei were used for ARID1A. Bio-Mag Plus Concanavalin A coated magnetic beads (Bangs laboratories, BP531) were equilibrated with binding buffer (20 mM K-HEPES pH7.9, 10 mM KCl, and 1 mM each CaCl₂ and MnCl₂). To each sample, 10 µL of activated beads were added and held at room temperature for 5 minutes with occasional gentle mixing. Beads (with bound nuclei) were magnetized, supernatants were removed, beads were washed once with 400 µL antibody buffer (Wash buffer supplemented with 2 mM EDTA and 0.05% Digitonin), and resuspended in 200 µL antibody buffer containing the respective primary antibody in 1:100 v/v dilution (H3K27me3-Cell Signaling Technology, 9733 H3K27ac - Millipore-Sigma, MABE647; ARID1A - Cell Signaling Technology, 12354). Primary antibody incubations were performed on a rotating platform overnight at 4°C. Beads were magnetized, supernatant removed, washed once with 400 µL Dig-Wash (Wash buffer supplemented with 0.05% Digitonin), resuspended in 200 µL Dig-Wash containing guinea pig α-rabbit IgG secondary antibody (Antibodies-online.com, ABIN101961) at 1:100 dilution. Secondary antibody incubations were performed on a rotating platform for 30 minutes at room temperature. Beads were magnetized, supernatant removed, washed twice with 400 µL Dig-Wash to remove unbound antibodies, and resuspended in 200 µL Dig-Med buffer (Dig-Wash buffer, except containing 300 mM NaCl) with 1:200 dilution (~0.04 µM) of proteinA-Tn5 transposase fusion protein (pA-Tn5) pre-loaded with double-stranded adapters with 19-mer mosaic ends.⁷⁸ pA-Tn5 incubations were performed on a rotating platform for 1 hour at room temperature. Beads were magnetized, supernatant removed, washed three times with 400 µL Dig-Med to remove unbound pA-Tn5, and resuspended in 300 µL Tagmentation buffer (Dig-Med supplemented with 10 mM MgCl₂). Tagmentation reactions were performed by incubating samples at 37°C on a rotating platform for 1 hour. Tagmentation reactions were stopped by sequentially adding 10 µL of 0.5 mM EDTA, 3.1 µL of 10% SDS (1% final), and 2 µL of 20 mg/mL Proteinase K, mixed well, and incubated in 50°C water bath for 1 hour. DNA was extracted using phenol-chloroform-isoamyl alcohol (25:24:1), re-extracted with one volume CHCl₃, aqueous phase (~300 µL) was transferred to fresh tube, and DNA was precipitated by adding 900 µL ethanol overnight at -20°C followed by centrifugation for 45 minutes at 13,000 rpm and 4°C. Pellets were rinsed with 100% ice-cold ethanol, air-dried, dissolved in 30 µL 0.1 x TE (1 mM Tris pH 8, 0.1 mM EDTA) supplemented with RNase A (1:400 dilution of 10 mg/mL), and incubated in 37°C water bath for 15 minutes.

To amplify libraries, 21 µL DNA was mixed with 2 µL each of a universal i5 primer and a uniquely barcoded i7 primer,⁸⁰ using a different barcode for each sample, and 25 µL of NEB Next HiFi 2x PCR Master mix. Samples were placed in a thermocycler with heated lid using the following cycling conditions: 72°C for 5 minutes (gap filling); 98°C for 30 seconds (denaturation); 13 cycles of 98°C for 10 seconds and 63°C for 10 seconds (combined annealing and extension); final extension at 72°C for 1 minute, and held at 8°C. Post-PCR clean-up was performed using a double-sided clean-up method. 25 µL (0.5 X) of Ampure XP beads (Beckman Counter) was mixed with the libraries and incubated for 10 minutes at room temperature to remove large DNA fragments (>1300 bp). Beads were magnetized, supernatants were removed to fresh tubes and mixed with 45 µL (1.4 X - 0.5 X = 0.9 X) of Ampure XP beads and incubated for 10 minutes at room temperature. Beads were magnetized and the supernatants containing unused PCR primers were discarded. Beads were washed three times with 80% ethanol and eluted in 30 µL 10 mM Tris pH 8.0. Libraries were sequenced for 25 cycles in 25bp paired-end mode on the Illumina HiSeq 2500 platform or 50 bp paired-end mode on Illumina NextSeq 2000 at the Fred Hutchinson Cancer Center Genomics Shared Resource.

## Flow cytometry

To isolate basal urothelial cells, bladder tissue was digested with collagenase II and generated single cell suspension (for details see organoid culture). Fluorescence-conjugated antibodies were then added to cell suspensions and stained for 30 minutes on ice. Live basal cells were separated by flow cytometry after staining with DAPI, EPCAM-APC (epithelial marker) and CD49f-PE (basal cell marker). 0.125 µg of EPCAM-APC (Invitrogen, 17-5791-80) and CD49f-PE (Fisher, 12-0495-82) were used respectively per sample containing 1x10⁸ cells.

## Cell cycle analysis

Cells were fixed using pre-cooled 66% ethanol at 4°C for at 2 hours. Propidium iodide staining was performed following manufacturer's instruction (Abcam, ab139418). The cells were read using flow cytometry (Fortessa X50) and cell cycle phases were analyzed using FlowJo software.
## Comet assay

The alkaline comet assay was performed using CometAssay kit (Trevigen, 4250-050-K) according to manufacturer's instructions. Briefly, organoids were treated with BCPN (100 µg/mL) for 96 hours. Cells were extracted from EHS using TrypLE and resuspended in PBS (1x10⁴ cells/mL). The adjusted volume of cells was mixed with LMA (Low melting agarose) and plated onto comet slides. Comet slides were kept at 4°C for 30 minutes followed by overnight incubation in lysis buffer. The following day, comet slides were washed with water and immersed in alkaline unwinding solution (200mM NaOH, 1mM EDTA, pH>13) at 4°C for 1 hour in the dark. Slides were removed and placed into a horizontal electrophoresis tank containing alkaline electrophoresis buffer (200mM NaOH, 1mM EDTA, pH>13). Electrophoresis was performed at 21 V (300 mA) for 30 minutes. Slides were washed twice in water and kept in 70% ethanol for 5 minutes. SYBR Gold dye was used to stain the DNA and comet slides were scanned using Zeiss Axio Imager Z2 microscope (TissueGnostics, Austria). Damaged DNA molecules migrate slower than undamaged DNA leaving a characteristic appearance of a comet. Comet head represents undamaged DNA and length of the comet tails represent severity of damaged DNA.

## Clonogenic assay

Cells were plated in 24-well plates at a density of 300 cells/well using 50% Matrigel for 24 hours, prior to the addition of 20 mM DFMO (Tocris Bioscience, 2761) and 10 µg/mL FGFR3 antibody (Creative bio labs, HPAB-0160-YC). DFMO containing media was removed and replaced every 48 hours. Colonies were counted after 10 days using the Evos FL cell imaging system.

## Organoid embedding and staining

Organoids were fixed using 4% PFA for 2 hours at room temperature on a shaker followed by a 30 minute incubation on ice to achieve complete removal of EHS. Intact organoids were collected as a pellet after centrifugation at 1400 rpm for 5 min and washed with ddH₂O. The pellet was further washed with 0.2% BSA and organoids were mixed with HistoGel (Epredia, HG-4000-012). The organoid and HistoGel suspension was centrifuged at 1400 rpm for 5 minutes and kept on ice for 30 minutes. The solidified HistoGel structure was stored at 4°C until ready for processing and paraffin embedding. Organoid sections (5 µm) were deparaffinized in CitriSolv (Fisher, 4355121) for 10 minutes and rehydrated using 100% (2x), 90% (2x) and 70% (2x) ethanol for 5 minutes each. Antigen retrieval was performed by preheating in sodium citrate buffer (pH 6.0) solution for 10 minutes at 121°C in a pressure cooker. Organoid sections were blocked in 1% BSA solution for 1 hour followed by overnight incubation in primary antibody (anti-RASGRP1: Fisher, PA5-25750) at 4°C in a humidified chamber. Secondary antibody (Alexa-Fluor 594:Invitrogen, A11032) incubation was performed for 1 hour at room temperature. All slides were mounted using Pro-Long gold mounting media with DAPI (Fisher Scientific, P36935). Slides were scanned using an Aperio Scanscope FL (Leica Biosystems) and image analysis was done using semiautomated image analysis software (HALO, Indica Labs).

## Drug administration

Tamoxifen: For all in vivo experiments, mice were given a total 30 mg of tamoxifen (Sigma, T5648) divided in 6 doses by oral gavage over the span of 3 weeks (2 doses/week). Tamoxifen was prepared in corn oil.

BBN: BBN (Fisher, B0938) was administrated (0.075%) in drinking water *ad libitum* for days as required for specific experiment (see experiment schema in figures).

eEF2K inhibitor (A-484954): For all eEF2K inhibitor experiments, mice received A-484954 (Sigma, 324516) daily (10 mg/kg body weight; IP) for 15 days.

# QUANTIFICATION AND STATISTICAL ANALYSIS

## RNA-sequencing and polysome sequencing analysis

Raw sequencing reads were checked for quality using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). RNA-seq reads were aligned to the UCSC mm10 assembly using STAR2⁸¹ and counted for gene associations against the UCSC genes database with HTSeq.⁸² The normalized count data was used for subsequent Principal component analysis in R. Differential Expression analysis for RNA-Seq data was performed using R/Bioconductor package edgeR.⁸³ A log₂fold change cutoff of 0.33 or 1.2 and FDR <0.05 was used to find transcriptionally regulated genes. Genome wide polysome to sub-polysome ratio analysis was performed using xtail.⁸⁴ Heatmaps were made using R/Bioconductor package pheatmap (https://CRAN.R-project.org/package=p heatmap). Volcano plots were made using ggplot2 in R.⁸⁵ For the ERCC Spike-In analysis, Bowtie2 was used for aligning the polysome sequencing data to 92 sequences from ERCC92 sequences. HTSeq-count was further used to the number of reads for each of the 92 sequences. The counts were normalized using voom.⁸⁶ Log₂ normalized counts were used to make scatterplots in R.

## CUT&Tag data analysis

Paired-end *Mus musculus* reads were mapped to UCSC mm10 using Bowtie2 version 2.2.2⁸⁷ with parameters --end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700. Continuous-valued data tracks (bedGraph and bigWig) were generated using genomcov in bedtools v2.30.0 (-bg option) and normalized as fraction of total counts. Genomic tracks were displayed using Integrated Genome Browser (IGB). Heatmaps and average plots were generated using computeMatrix and plotHeatmap operations in deepTools v3.5.1.⁸⁸ Scores were averaged over 50bp non-overlapping bins with the transcription start sites (TSS) of genes as
## Comet assay

The alkaline comet assay was performed using CometAssay kit (Trevigen, 4250-050-K) according to manufacturer's instructions. Briefly, organoids were treated with BCPN (100 µg/mL) for 96 hours. Cells were extracted from EHS using TrypLE and resuspended in PBS (1x10⁴ cells/mL). The adjusted volume of cells was mixed with LMA (Low melting agarose) and plated onto comet slides. Comet slides were kept at 4°C for 30 minutes followed by overnight incubation in lysis buffer. The following day, comet slides were washed with water and immersed in alkaline unwinding solution (200mM NaOH, 1mM EDTA, pH>13) at 4°C for 1 hour in the dark. Slides were removed and placed into a horizontal electrophoresis tank containing alkaline electrophoresis buffer (200mM NaOH, 1mM EDTA, pH>13). Electrophoresis was performed at 21 V (300 mA) for 30 minutes. Slides were washed twice in water and kept in 70% ethanol for 5 minutes. SYBR Gold dye was used to stain the DNA and comet slides were scanned using Zeiss Axio Imager Z2 microscope (TissueGnostics, Austria). Damaged DNA molecules migrate slower than undamaged DNA leaving a characteristic appearance of a comet. Comet head represents undamaged DNA and length of the comet tails represent severity of damaged DNA.

## Clonogenic assay

Cells were plated in 24-well plates at a density of 300 cells/well using 50% Matrigel for 24 hours, prior to the addition of 20 mM DFMO (Tocris Bioscience, 2761) and 10 µg/mL FGFR3 antibody (Creative bio labs, HPAB-0160-YC). DFMO containing media was removed and replaced every 48 hours. Colonies were counted after 10 days using the Evos FL cell imaging system.

## Organoid embedding and staining

Organoids were fixed using 4% PFA for 2 hours at room temperature on a shaker followed by a 30 minute incubation on ice to achieve complete removal of EHS. Intact organoids were collected as a pellet after centrifugation at 1400 rpm for 5 min and washed with ddH₂O. The pellet was further washed with 0.2% BSA and organoids were mixed with HistoGel (Epredia, HG-4000-012). The organoid and HistoGel suspension was centrifuged at 1400 rpm for 5 minutes and kept on ice for 30 minutes. The solidified HistoGel structure was stored at 4°C until ready for processing and paraffin embedding. Organoid sections (5 µm) were deparaffinized in CitriSolv (Fisher, 4355121) for 10 minutes and rehydrated using 100% (2x), 90% (2x) and 70% (2x) ethanol for 5 minutes each. Antigen retrieval was performed by preheating in sodium citrate buffer (pH 6.0) solution for 10 minutes at 121°C in a pressure cooker. Organoid sections were blocked in 1% BSA solution for 1 hour followed by overnight incubation in primary antibody (anti-RASGRP1: Fisher, PA5-25750) at 4°C in a humidified chamber. Secondary antibody (Alexa-Fluor 594:Invitrogen, A11032) incubation was performed for 1 hour at room temperature. All slides were mounted using Pro-Long gold mounting media with DAPI (Fisher Scientific, P36935). Slides were scanned using an Aperio Scanscope FL (Leica Biosystems) and image analysis was done using semiautomated image analysis software (HALO, Indica Labs).

## Drug administration

Tamoxifen: For all in vivo experiments, mice were given a total 30 mg of tamoxifen (Sigma, T5648) divided in 6 doses by oral gavage over the span of 3 weeks (2 doses/week). Tamoxifen was prepared in corn oil.

BBN: BBN (Fisher, B0938) was administrated (0.075%) in drinking water *ad libitum* for days as required for specific experiment (see experiment schema in figures).

eEF2K inhibitor (A-484954): For all eEF2K inhibitor experiments, mice received A-484954 (Sigma, 324516) daily (10 mg/kg body weight; IP) for 15 days.

# QUANTIFICATION AND STATISTICAL ANALYSIS

## RNA-sequencing and polysome sequencing analysis

Raw sequencing reads were checked for quality using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). RNA-seq reads were aligned to the UCSC mm10 assembly using STAR2⁸¹ and counted for gene associations against the UCSC genes database with HTSeq.⁸² The normalized count data was used for subsequent Principal component analysis in R. Differential Expression analysis for RNA-Seq data was performed using R/Bioconductor package edgeR.⁸³ A log₂fold change cutoff of 0.33 or 1.2 and FDR <0.05 was used to find transcriptionally regulated genes. Genome wide polysome to sub-polysome ratio analysis was performed using xtail.⁸⁴ Heatmaps were made using R/Bioconductor package pheatmap (https://CRAN.R-project.org/package=pheatmap). Volcano plots were made using ggplot2 in R.⁸⁵ For the ERCC Spike-In analysis, Bowtie2 was used for aligning the polysome sequencing data to 92 sequences from ERCC92 sequences. HTSeq-count was further used to the number of reads for each of the 92 sequences. The counts were normalized using voom.⁸⁶ Log₂ normalized counts were used to make scatterplots in R.

## CUT&Tag data analysis

Paired-end *Mus musculus* reads were mapped to UCSC mm10 using Bowtie2 version 2.2.2⁸⁷ with parameters --end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700. Continuous-valued data tracks (bedGraph and bigWig) were generated using genomcov in bedtools v2.30.0 (-bg option) and normalized as fraction of total counts. Genomic tracks were displayed using Integrated Genome Browser (IGB). Heatmaps and average plots were generated using computeMatrix and plotHeatmap operations in deepTools v3.5.1.⁸⁸ Scores were averaged over 50bp non-overlapping bins with the transcription start sites (TSS) of genes as
reference points and plotted as the mean. Box-and-whiskers plots were generated with GraphPad Prism. Average scores were computed for 2kb bins with TSS coordinates as the midpoint using the multi-Bigwig Summary operation in deepTools v3.5.1. Outliers (identified using the ROUT method, Q = 1%) were discarded. Boxes show the 75th and 25th percentiles and the median. The Tukey method was used to plot whiskers. CUT&Tag data were analyzed as described (https://www.protocols.io/view/cut-amp-tag-data-processing-and-analysis-tutorial-e6nvw93x7gmk/v1). Briefly, adapters were clipped and paired-end *M. musculus* reads were mapped to UCSC mm10 using Bowtie2 with parameters: -very-sensitive-local -soft-clipped-unmapped-tlen -dovetail -no-mixed -no-discordant -q -phred33 -l 10 -X 1000. Spike-in *E. coli* reads were mapped to Ensembl masked R64-1-1 with parameters: -end-to-end -very-sensitive -no-overlap -no-dovetail -no-unal -no-mixed -no-discordant -q -phred33 -l 10 -X 700.

## TMT mass spectrometry data analysis

Protein database searching and quantification of TMT reporter ions was performed using Proteome Discoverer 2.5 (Thermo Scientific, San Jose, CA). The data were searched against a Mouse database (UP00000598 Human 030721) that included common contaminants (cRAPome). Searches were performed with settings for the proteolytic enzyme trypsin. Maximum missed cleavages were set to 2. The precursor ion tolerance was set to 10 ppm and the fragment ion tolerance was set to 0.6 Da. Dynamic peptide modifications were set for oxidation on methionine (+15.995 Da) and modifications on the protein N-terminus, consisting of acetylation (+42.011 Da), Met-loss (–131.040 Da), and Met-loss+Acetyl (–89.030 Da). Static modifications were set for TMTpro on any N-terminus (+304.207 Da), TMTpro on lysine (+304.207 Da), and carbamidomethyl on cysteine (+57.021 Da). Sequest HT was used for protein database searching and Percolator was used for peptide validation.⁸⁹

Peptide to spectrum matches (PSMs) were filtered to a 1% false discovery rate and the resulting proteins were further filtered to a 1% false discovery rate. TMT channels were normalized to the largest channel intensity. Quantitative values were transformed to log2 values and missing data were imputed by 50% of global minimum intensity. To delineate differentially expressed proteins, both pairwise two-sample t-tests and Wilcoxon rank-sum tests were conducted (R package). The Benjamini-Hochberg procedure was used for controlling familywise error rate (FWER).

## Ribosome half-transit time data analysis

[³⁵S]-methionine incorporation in nascent and completed protein within the PMS and completed protein within PRS was deduced by linear regression analysis. Ribosome half-transit time was calculated by the displacement of regression lines (PMS and PRS) at time 300 s.

## %GC content in coding sequence analysis

To compare GC content of different classes of open reading frames (ORFs) (Figures S1L and S6A), *Mus musculus* UCSC genome sequence mm39 and Ensembl transcript annotations GRCm39 v106 were downloaded. Unique transcripts were identified for each protein coding gene by first checking for consensus coding sequence annotation (CCDSID) and then choosing the transcript with the highest support level. GC content of each CCDS ORF was calculated using letterFrequency function from the Biostrings R package. GC content for ORF groups that were transcriptionally up-regulated and those with high P/S ratio were plotted in comparison to the remaining ORFs using the geom_violin function from the ggplot2 R package. Statistical significance of differences in GC content between different ORF groups were calculated using the paired Wilcoxon test (wilcox.test R function).
reference points and plotted as the mean. Box-and-whiskers plots were generated with GraphPad Prism. Average scores were computed for 2kb bins with TSS coordinates as the midpoint using the multi-Bigwig Summary operation in deepTools v3.5.1. Outliers (identified using the ROUT method, Q = 1%) were discarded. Boxes show the 75th and 25th percentiles and the median. The Tukey method was used to plot whiskers. CUT&Tag data were analyzed as described (https://www.protocols.io/view/cut-amp-tag-data-processing-and-analysis-tutorial-e6nvw93x7gmk/v1). Briefly, adapters were clipped and paired-end *M. musculus* reads were mapped to UCSC mm10 using Bowtie2 with parameters: -very-sensitive-local -soft-clipped-unmapped-tlen -dovetail -no-mixed -no-discordant -q -phred33 -l 10 -X 1000. Spike-in *E. coli* reads were mapped to Ensembl masked R64-1-1 with parameters: -end-to-end -very-sensitive -no-overlap -no-dovetail -no-unal -no-mixed -no-discordant -q -phred33 -l 10 -X 700.

## TMT mass spectrometry data analysis

Protein database searching and quantification of TMT reporter ions was performed using Proteome Discoverer 2.5 (Thermo Scientific, San Jose, CA). The data were searched against a Mouse database (UP00000598 Human 030721) that included common contaminants (cRAPome). Searches were performed with settings for the proteolytic enzyme trypsin. Maximum missed cleavages were set to 2. The precursor ion tolerance was set to 10 ppm and the fragment ion tolerance was set to 0.6 Da. Dynamic peptide modifications were set for oxidation on methionine (+15.995 Da) and modifications on the protein N-terminus, consisting of acetylation (+42.011 Da), Met-loss (–131.040 Da), and Met-loss+Acetyl (–89.030 Da). Static modifications were set for TMTpro on any N-terminus (+304.207 Da), TMTpro on lysine (+304.207 Da), and carbamidomethyl on cysteine (+57.021 Da). Sequest HT was used for protein database searching and Percolator was used for peptide validation.⁸⁹

Peptide to spectrum matches (PSMs) were filtered to a 1% false discovery rate and the resulting proteins were further filtered to a 1% false discovery rate. TMT channels were normalized to the largest channel intensity. Quantitative values were transformed to log2 values and missing data were imputed by 50% of global minimum intensity. To delineate differentially expressed proteins, both pairwise two-sample t-tests and Wilcoxon rank-sum tests were conducted (R package). The Benjamini-Hochberg procedure was used for controlling familywise error rate (FWER).

## Ribosome half-transit time data analysis

[³⁵S]-methionine incorporation in nascent and completed protein within the PMS and completed protein within PRS was deduced by linear regression analysis. Ribosome half-transit time was calculated by the displacement of regression lines (PMS and PRS) at time 300 s.

## %GC content in coding sequence analysis

To compare GC content of different classes of open reading frames (ORFs) (Figures S1L and S6A), *Mus musculus* UCSC genome sequence mm39 and Ensembl transcript annotations GRCm39 v106 were downloaded. Unique transcripts were identified for each protein coding gene by first checking for consensus coding sequence annotation (CCDSID) and then choosing the transcript with the highest support level. GC content of each CCDS ORF was calculated using letterFrequency function from the Biostrings R package. GC content for ORF groups that were transcriptionally up-regulated and those with high P/S ratio were plotted in comparison to the remaining ORFs using the geom_violin function from the ggplot2 R package. Statistical significance of differences in GC content between different ORF groups were calculated using the paired Wilcoxon test (wilcox.test R function).