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Supplementary Figure S1 shows the strategy and result of co-essentiality module identificationfrom the DepMap CRISPR screen dataset and further characterization of the BIRC6 module. Supplementary Figure S2 shows the viability effect of BIRC6 depletion in BIRC6-dependent and -nondependent cancer cells lines as well asin nontransformed cells. Supplementary Figure S3 shows in vitro confirmations of the inducible BIRC6 depletion (by RNAi and CRISPR) systems as well as the effect of BIRC6 depletion on metastasis in xenograft models. Supplementary Figure S4 shows detailed results for the allele competition assay to evaluate the role of the BIR and UBC domains of BIRC6 and further evidence for the physical assembly of BIRC6 ubiquitin ligase complex. Supplementary Figure S5 shows evidence for the selective activation of the p-eIF2alpha/ATF4 arm of UPR (or ISR) upon BIRC6 depletion. Supplementary Figure S6 shows evidence supporting the importance of HRI-mediated ISR activation as a mechanism underlying the loss of viability caused by BIRC6 complex suppression. Supplementary Figure S7 shows evidence for the direct regulation of HRI ubiquitination and degradation by the BIRC6 complex Supplementary Figure S8 shows elevated expression of HRI mRNA in the tumor samples compared to the normal samples in large-scale datasets (TCGA, TARGET and GTEx). Supplementary Figure S9 shows the inefficiency of the gene expression and copy number of the functionally related genes in predicting BIRC6 dependency. Supplementary Table S1 lists the top 50 co-essentiality modules identified in the analysis explained in Supplementary Figure S1A. Supplementary Table S2 lists the key DNA and RNA sequences used in this study.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy.Significance:We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells.See related commentary Leli and Koumenis, p. 535.This article is highlighted in the In This Issue feature, p. 517

Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.

Targeting of mutated oncogenes has led to the identification of new targeted therapies. However, druggable oncogenes do not occur in most cancers. Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 793 cancer cell lines to identify selective co-essentiality modules and found that a ubiquitination ligase complex composed of UBA6, BIRC6, KCMF1 and UBR4, which encode an E1, E2 and two heterodimeric E3 subunits, respectively, is required for the survival of a subset of epithelial tumors, particularly subtypes of breast cancer. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization and upregulation of the heme-regulated inhibitor (HRI), a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy. Citation Format: Lisa D Cervia, Tsukasa Shibue, Benjamin Gaeta, Ashir A Borah, Lisa Leung, Naomi Li, Nancy Dumont, Alfredo Gonzalez, Nolan Bick, Mariya Kazachkova, Joshua M Dempster, John M Krill-Burger, Federica Piccioni, Namrata D Udeshi, Meagan E Olive, Steven A Carr, David E Root, James M McFarland, Francisca Vazquez, William C Hahn. A ubiquitination cascade regulating the integrated stress response and survival in carcinomas [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-01.

Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/CAT1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a post-translational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors--by inhibition of deoxyhypusine synthase--abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This impacted pathway is critical for eIF5A-regulated erythropoiesis as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins were especially sensitive to the loss of hypusine and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in del(5q) myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPC, synchronizing mitochondrial metabolism with erythroid differentiation.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy.Significance:We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells.See related commentary Leli and Koumenis, p. 535.This article is highlighted in the In This Issue feature, p. 517

Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. We discovered that SLC7A1/CAT1-dependent arginine uptake and its catabolism to spermidine control the erythroid specification of HSPCs via activation of eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on the metabolism of spermidine to hypusine and inhibiting hypusine synthesis abrogates erythropoiesis and diverts EPO-stimulated HSPCs to a myeloid fate. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A and induction of mitochondrial function partially rescues erythropoiesis in the absence of hypusine. Within the hypusine network, ribosomal proteins are highly enriched and we identify defective eIF5A hypusination in erythroid pathologies caused by abnormal ribosome biogenesis. Thus, eIF5A-dependent protein synthesis is critical in the branching of erythro-myeloid differentiation and attenuated eIF5A activity characterizes ribosomal protein-linked disorders of ineffective erythropoiesis.

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy., SigPath is a targeted, quantitative mass spectrometry assay that measures 284 phosphosites spanning 200 phosphoproteins with high throughput and quantitative precision across a broad swath of signaling biology of known interest.

The trafficking of specific protein cohorts to correct subcellular locations at correct times is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or fluorescence-activated cell sorting (FACS). To empower the study of protein trafficking processes with gene perturbation, we developed a genetically encoded molecular tool named HiLITR (High-throughput Localization Indicator with Transcriptional Readout). HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identified genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in mislocalization and destabilization of many mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.

Multiomic characterization of patient tissues provides insights into the function of different biological pathways in the context of disease. Much work has been done to serialize proteome and post-translational modification (PTM) analyses to conserve precious patient samples. However, characterizing clinically relevant tissues with multi-ome workflows that have distinct sample processing requirements remains challenging. To overcome the obstacles of combining enrichment workflows that have unique input amounts and utilize both label free and chemical labeling strategies, we developed a highly-sensitive multi-omic networked tissue enrichment (MONTE) workflow for the full analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome and acetylome all from the same tissue sample. The MONTE workflow enables identification of a median of 9,000 HLA-I peptides, 6,000 HLA-II peptides, 10,000 Ub sites, 12,000 proteins, 20,000 phosphorylation sites and 15,000 acetylation sites from patient LUAD tumors. Because all omes are generated from the exact same tissue sample, there is less biological variability in the data enabling more robust integration. The information available in MONTE datasets facilitates the identification of putative immunotherapeutic targets, such as CT antigens and neoantigens presented by HLA complexes, as well as reveal insights into how disease-specific changes in protein expression, protein degradation, cell signaling, metabolic, and epigenetic pathways are involved in disease pathology and treatment.

Targeting of mutated oncogenes has led to the identification of new targeted therapies. However, druggable oncogenes do not occur in most cancers. Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 793 cancer cell lines to identify selective co-essentiality modules and found that a ubiquitination ligase complex composed of UBA6, BIRC6, KCMF1 and UBR4, which encode an E1, E2 and two heterodimeric E3 subunits, respectively, is required for the survival of a subset of epithelial tumors. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization and upregulation of the heme-regulated inhibitor (HRI), a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy. Citation Format: Lisa D. Cervia, Tsukasa Shibue, Benjamin Gaeta, Ashir A. Borah, Lisa Leung, Naomi Li, Nancy Dumont, Alfredo Gonzalez, Nolan Bick, Mariya Kazachkova, Joshua M. Dempster, John M. Krill-Burger, Federica Piccioni, Namrata D. Udeshi, Meagan E. Olive, Steven A. Carr, David E. Root, James M. McFarland, Francisca Vazquez, William C. Hahn. A ubiquitination cascade regulating the integrated stress response and survival in carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 73.

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanisms through which glucocorticoids regulate human erythropoiesis remain poorly understood. We report that the sensitivity of erythroid differentiation to dexamethasone is dependent on the developmental origin of human CD34(+) progenitor cells, specifically increasing the expansion of CD34(+) progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34(+) progenitors resulted in the expansion of a newly defined CD34(+)CD36(+)CD71(hi)CD105(med) immature colony-forming unit–erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57(Kip2), a Cip/Kip cyclin–dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57(Kip2), but not the related p27(Kip1), significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with dysregulated p57(Kip2) expression. Altogether, these data identify a unique glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.

Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples., Comprehensive protein ubiquitylation profiling by mass spectrometry typically requires large sample amounts, limiting its applicability to tissue samples. Here, the authors present an optimized proteomics method that enables multiplexed ubiquitylome analysis of cells and tumor tissue samples.

Systematic identification of signaling pathways required for the viability of cancer cells will facilitate the development of novel cancer therapies. We used gene essentiality measurements in 726 cancer cell lines to identify selective co-essentiality modules and found a functional ubiquitination cascade containing UBA6, BIRC6, KCMF1 and UBR4, which encode an E1, E2, and two heterodimeric E3 subunits, respectively, as a vulnerability in a subset of epithelial tumors. Suppressing BIRC6 in cancer cell lines that are dependent on this ubiquitination cascade led to a strong reduction in cell fitness in vitro, and to potent tumor regression and metastasis suppression in vivo. Mechanistically, BIRC6 suppression resulted in selective and robust activation of the integrated stress response (ISR) signaling via upregulation of the heme-regulated inhibitor (HRI). Using proteomic profiling, we found that HRI itself is a key degradation target of the UBA6/BIRC6/KCMF1/UBR4 cascade. These observations demonstrate a protein ubiquitination cascade regulating ISR and highlight the potential of this cascade as a novel therapeutic target for a subset of epithelial cancers. Citation Format: Lisa D. Cervia, Tsukasa Shibue, Benjamin Gaeta, Ashir Borah, Lisa Leung, Naomi Li, Nancy Dumont, Alfredo Gonzalez, Nolan Bick, Mariya Kazachkova, Joshua Dempster, John M. Krill-Burger, Namrata Udeshi, Meagan Olive, Steven A. Carr, David E. Root, Federica Piccioni, James M. McFarland, Francisca Vazquez, William C. Hahn. A ubiquitination cascade regulates the integrated stress response and epithelial cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1950.

Despite effective clinical use, the mechanistic bases for the regulation of human erythropoiesis by glucocorticoids remain poorly understood. Here, we employed an erythroid culture system to differentiate primary human CD34+cells isolated from control peripheral blood, cord blood and patients with Diamond Blackfan anemia (DBA), as model of an erythropoietic disorder treated with glucocorticoids. We found that the response to Dexamethasone is dependent on the developmental origin of the source of CD34+cells, specifically increasing the expansion of CD34+cells from peripheral blood but not cord blood. We also demonstrated that Dex treatment leads to the expansion of a novel immature colony-forming unit-erythroid (CFU-E) population in peripheral blood which is uniquely responsible for the proliferative effects observed during human adult erythropoiesis. Dex treatment of peripheral blood CFU-E cells also induced p57Kip2expression while patients with DBA resistant to steroids had altered p57Kip2expression that did not increase with Dex. Furthermore, shRNA knockdown of p57Kip2reduced proliferation and abrogated the effects of Dex. Finally, proteomics of Dex-treated CFU-E from peripheral blood and cord blood additionally revealed novel Dex targets in the erythroid system. Altogether, these results provide novel insights into the regulation of human erythropoiesis by glucocorticoids.Graphical Abstract

Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites are necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-ɛ-GG peptides are enriched with anti-K-e-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-e-GG antibody (mK-e-GG) and a magnetic particle processor. We report the identification of ∼20,000 ubiquitylation sites from a TMT10-plex with 500 μg input per sample processed in ∼2 hours. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility and significantly reduced processing time. The automated method also significantly reduced variability across process replicates compared to the manual method. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of the method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.

Glucocorticoids are widely used to increase red cell production in a number of hematological disorders including Diamond Blackfan anemia (DBA). Despite their clinical effectiveness we do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. To improve the clinical management of these patients, we studied the mechanisms of dexamethasone (Dex) on human erythroid progenitors. We employed an erythroid culture system to differentiate primary human CD34+ cells isolated from patients with DBA and healthy controls as well as cord blood samples. We found that Dex increases the proliferation of CD34+ cells derived from peripheral blood but surprisingly not of CD34+ cells from cord blood. Furthermore, by mass spectrometry proteomic analysis, we found that Dex specifically altered the induction of several targets unique to peripheral blood including the transcription factor and cell cycle regulator NR4A1. When examining the cell populations expanded with Dex treatment by immunophenotyping, we detected a specific expansion of CFU-Es. Dex increased the average size of CFU-E colonies in methylcellulose colony forming assays in the presence of Epo alone, suggesting that CFU-Es are the most Dex responsive progenitor in humans. In support of this hypothesis, reticulocyte counts from patients with DBA treated with prednisone were found to increase approximately one week after treatment, consistent with the time course of differentiation of CFU-E to reticulocytes in bone marrow. In order to further resolve the progenitor target of steroid responsive progenitor we developed a new gating scheme that allows for increased resolution of BFU-E and CFU-E subpopulations by monitoring surface expression of CD71 and CD105. Using this system, we found that Dex treatment led to the maintenance of a novel immature transitional progenitor population in peripheral blood-derived cells that we term immature CFU-E. In methylcellulose colony forming assays, these peripheral blood-derived immature CFU-E treated with Dex produced larger colonies than the untreated controls and mature CFU-E. The expansion and proliferation of Dex treated immature CFU-Es derived from peripheral blood was associated with a decreased percentage of cells in the S phase of the cell cycle. No change was noted for either Dex treated BFU-E from peripheral blood or for BFU-E or CFU-E from cord blood. Importantly, Dex responsive peripheral blood-derived CFU-Es divided at a faster rate than untreated CFU-Es, revealing a dissociation in the link between differentiation and proliferation. Mechanistically, we found that Dex treated peripheral blood CFU-E exhibited increased expression of the negative cell cycle regulator p57Kip2. Moreover, this protein was also found to be dysregulated in CD34+ cells from transfusion-dependent patients with DBA, wherein p57Kip2 expression was not significantly altered following Dex treatment, indicating a role for p57Kip2 in the erythroid progenitor defects seen in these patients. To further evaluate the role of p57Kip2 in the Dex dependent alteration in CFU-E proliferation, peripheral blood-derived CD34+ cells were transduced with p57Kip2-specific shRNA lentiviral constructs. shRNA knockdown of p57Kip2 abrogated the effects of Dex on growth and induced a premature EPO-mediated differentiation. In summary, our findings provide new insights into functional heterogeneity of human erythroid progenitors and reveal mechanistic insights into the action of Dex in normal and disordered erythropoiesis. Disclosures Vlachos: Novartis: Other: Steering committee member.