Ewing sarcoma is an aggressive pediatric cancer for which no targeted therapies have been approved. This disease is driven by EWSR1::ETS family fusions that, like most fusion transcription factors, have proven difficult to target directly. Here, we focus on identifying actionable dependencies within the high-risk STAG2-mutant (STAG2-mut) disease subtype of Ewing sarcoma. In STAG2-mut Ewing sarcoma, STAG1 knockout is synthetically lethal but is not readily druggable. Using the Cancer Dependency Map (DepMap), we identify histone deacetylase 8 (HDAC8) as an enriched vulnerability in this molecular context. CRISPR validation and a newly developed HDAC8 degrader, XY-09-36, confirm this selective dependency and suggest increased reliance on HDAC8-mediated cohesin regulation when STAG2 is lost. These findings establish HDAC8 as a context-dependent, pharmacologically accessible vulnerability and outline a framework for discovering rational targets in tumor subtypes where primary drivers are intrinsically hard to drug.

Supplementary Data from An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML

Supplemental Materials and Methods

Figure S1. The glutaminase inhibitor CB-839 impairs glutathione metabolism in FLT3WT AML cells. Figure S2. The glutaminase inhibitor CB-839 decreases glutathione levels, but does not increase total cellular ROS levels in AML cells. Figure S3. Antioxidant vitamin E suppresses mitoROS and apoptosis induced by CB-839/pro-oxidant combination therapies. Figure S4. CB-839 cooperates with the pro-oxidant drug ATO in inducing apoptosis, mitoROS and AML cell death. Figure S5. CB-839/HHT therapy induces total cellular ROS. AML cells that survive therapy are metabolically similar to drug naive cells and are not resistant to subsequent therapy. Figure S6. CB-839/ATO and CB-839/HHT combination therapies do not exhibit toxicity in vivo in mice or toward normal human CD34+ cells. Figure S7. CB-839 cooperates with HHT in inducing mitoROS and apoptosis in primary human AML cells and cell death/apoptosis in ALL cells. Table S1. Combination index (CI values) for drug combinations tested in cell viability assays. Table S2. The clinical characteristics of the AML cohort studied are summarized.

Genome-scale CRISPR-Cas9 screens have the power to unveil the Achilles' heel of neoplastic cells. Typically, analyses of such large-scale data sets focus on single gene dependencies. An alternative strategy is to evaluate functional networks enriched in a disease or molecular subset of interest. We applied this strategy to the Broad Institute's Cancer Dependency Map (DepMap) data set consisting of genome-scale CRISPR-Cas9 screens in over 1000 cancer cell line models. We hypothesized that interrogating enriched pathways with context-specific dependencies leads to the discovery of functionally informed gene dependencies. Single sample gene set enrichment analysis (ssGSEA) of over 1000 cancer cell lines in the DepMap identified hematologic malignancies to be highly enriched for signatures associated with elevated transcriptional activity. Notably, within hematologic malignancies, acute myeloid leukemia (AML) with a rearrangement in the KMT2A gene ( KMT2Ar-AML) was the most significantly enriched for dependency on transcriptional activity-associated gene signatures. We next interrogated KMT2Ar-AML gene dependencies (excluding common essential genes) with the human kinase database KinMap (http://www.kinhub.org/kinmap/) to identify readily druggable genes involved in transcriptional activity. Cyclin-dependent kinase 13 ( CDK13) was the only gene meeting these criteria. Further DepMap data analysis confirmed that KMT2Ar-AML cell lines were indeed more strongly dependent on CDK13 than all other cancer cell lines screened. Moreover, when we performed GSEA on the gene dependencies observed in CDK13-dependent versus non-dependent cell lines, we found transcriptional activity and KMT2A-target gene sets as top hits. We next validated AML cell dependency on CDK13 with orthogonal genetic approaches (CRISPR-Cas9 knock-out and shRNA) in vitro in human AML cell lines and cells from patient-derived xenograft (PDX) models of KMT2Ar-AML. Perturbation of CDK13 induced cell death with hallmarks of apoptosis. Doxycycline-inducible shRNA directed against CDK13 also impaired AML progression in the peripheral blood and bone marrow in an MV4-11- KMT2Ar xenograft. Moreover, in an orthotopic PDX model, doxycycline inducible knock-out of CDK13 in mice with established disease significantly reduced leukemia burden in the peripheral blood and bone marrow. CDK13 is reported to regulate transcriptional elongation and the clearance of prematurely terminated RNAs. To decipher why AML cells, particularly KMT2Ar, need CDK13 to survive, we identified the CDK13 binding sites and histone marks involved in transcriptional regulation in AML using CUT and RUN and ChIP-seq: We determined that CDK13 co-localizes with H3K4me3 and H3K27ac at promoter sites. Global gene expression studies following the knock-out of CDK13 revealed that the majority of CDK13 bound genes were also differentially expressed, with significantly more genes decreased than increased in expression after CDK13 knock-out. GSEA of CDK13-bound genes with decreased expression upon CDK13 knock-out identified gene signatures associated with transcriptional activation and KMT2A-targets. We next studied the characteristics of CDK13 target genes and observed a predominance of long genes (total length of gene exons > 10 kb, GRCh38.p12). GSEA of genes ranked by size, confirmed that longer genes were enriched for CDK13 target genes, CDK13 differential AML dependencies, and for KMT2A-target transcriptional signatures. Additionally, following CDK13 perturbation in KMT2Ar-AML we observed a decrease of Pol2/pPol2Ser2 occupancy at the transcription end site of long genes. In summary, using functional pathway analyses, we identified CDK13 as a candidate gene dependency enriched in KMT2Ar-AML, a target not immediately prioritized with single gene analysis. Mechanistically, our data support that CDK13 controls Pol2 processivity, with CDK13 perturbation leading to a decreased occupancy at the 3' end of long genes. KMT2Ar, CDK13 dependent cells are also enriched for dependency on long genes, thereby suggesting a molecular basis for the CDK13 dependency in KMT2Ar-AML.

Abstract NUT carcinoma is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of progrowth genes. BET bromodomain inhibitors (BETi) are a promising treatment for NUT carcinoma that can impede BRD4-NUT's ability to activate genes, but the efficacy of BETi as monotherapy is limited. Here, we demonstrated that enhancer of zeste homolog 2 (EZH2), which silences genes through establishment of repressive chromatin, is a dependency in NUT carcinoma. Inhibition of EZH2 with the clinical compound tazemetostat potently blocked growth of NUT carcinoma cells. Epigenetic and transcriptomic analysis revealed that tazemetostat reversed the EZH2-specific H3K27me3 silencing mark and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4-NUT–regulated genes. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NUT carcinoma cells. CDKN2A was identified as the only gene among all tazemetostat-derepressed genes to confer resistance to tazemetostat in a CRISPR-Cas9 screen. Combined inhibition of EZH2 and BET synergized to downregulate cell proliferation genes, resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In preclinical models, combined tazemetostat and BETi synergistically blocked tumor growth and prolonged survival of NUT carcinoma–xenografted mice, with complete remission without relapse in one cohort. Identification of EZH2 as a dependency in NUT carcinoma substantiates the reliance of NUT carcinoma tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary, chromatin regulatory pathways to maintain NUT carcinoma growth. Significance: Repression of tumor suppressor genes, including CDKN2A, by EZH2 provides a mechanistic rationale for combining EZH2 and BET inhibitors for the clinical treatment of NUT carcinoma. See related commentary by Kazansky and Kentsis, p. 3827

Supplementary Tables S1-S6 describing patient characteristics, gene lists, LSC signatures, and reagents.

Supplementary Figures S1-S5 supporting Main Figures 1-5.

NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUT’s ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCE Identification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.

Transcription factors (TFs) are frequently mutated in cancer. Paediatric cancers exhibit few mutations genome-wide but frequently harbour sentinel mutations that affect TFs, which provides a context to precisely study the transcriptional circuits that support mutant TF-driven oncogenesis. A broadly relevant mechanism that has garnered intense focus involves the ability of mutant TFs to hijack wild-type lineage-specific TFs in self-reinforcing transcriptional circuits. However, it is not known whether this specific type of circuitry is equally crucial in all mutant TF-driven cancers. Here we describe an alternative yet central transcriptional mechanism that promotes Ewing sarcoma, wherein constraint, rather than reinforcement, of the activity of the fusion TF EWS–FLI supports cancer growth. We discover that ETV6 is a crucial TF dependency that is specific to this disease because it, counter-intuitively, represses the transcriptional output of EWS–FLI. This work discovers a previously undescribed transcriptional mechanism that promotes cancer.

Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.

The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis-chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype.

First-generation, large-scale functional genomic screens have revealed hundreds of potential genetic vulnerabilities in acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because these large-scale genetic screens were primarily performed in vitro in established AML cell lines, their translational relevance has been debated. Therefore, we established a protocol for CRISPR screening in orthotopic xenograft models of human AML, including patient-derived-xenograft (PDX) models that are tractable for CRISPR-editing.

Metabolic reprogramming contributes to tumor development and sustains cancer cell proliferation. Like other cancers, acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival, has altered metabolic features, providing new possibilities for AML treatment. Since the niche can reshape the metabolic properties of cancer cells, it is critical to validate AML metabolic vulnerabilities in a proper microenvironment. To this end, we optimized a protocol for CRISPR screening in orthotopic xenograft AML models, including patient-derived-xenograft (PDX) models tractable for CRISPR-editing, to enable the systematic evaluation of the physiological relevance of top AML dependencies. We performed in vivo screens in MV4-11 and U937 cell lines and a PDX model, which converged to reveal the sodium/myo-inositol cotransporter SLC5A3 as a top-ranked in vivo gene target.

Abstract CRISPR–Cas9-based genetic screens have successfully identified cell type–dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. Significance: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities. This article is highlighted in the In This Issue feature, p. 275

Chronic myeloid leukemia (CML) is a heterogeneous disease, initiated by reciprocal translocation of chromosome 9 and 22, resulting in the generation of a BCR-ABL fusion protein and constitutive activation of the ABL kinase. ABL tyrosine kinase inhibitors (TKIs) have been very successful in suppressing CML disease. However, TKIs may not eliminate leukemia stem cells (LSCs), as evidenced by the frequent re-emergence of the disease upon TKI discontinuation. Moreover, blast phase CML (bpCML) remains a formidable challenge in disease management. Recent clinical evidence suggests that the BCL2 inhibitor venetoclax (Ven) in combination with ABL-targeting tyrosine kinase inhibitors (TKI) can eradicate bpCML LSCs. However, the exact mechanism by which this combination may targets LSCs is not known. In this report, we confirm the efficacy and LSC-targeting capacity of Ven/TKI combination in preclinical models of bpCML and we further identify that inhibition of free fatty acid (FFA) mobilization pathways may provide enhanced efficacy against LSCs.