Genomic binding of NF-Y in mouse and human cells.

NF-Y is a Transcription Factor that regulates transcription through binding to the CCAAT-box. To understand its strategy, we analyzed 16 ChIP-seq datasets from human and mouse cells. Shared loci, mostly located in promoters of expressed genes of cell cycle, metabolism and gene expression pathways, are associated with histone marks of active chromatin and specific modules of TFs. Other peaks are in enhancers and Transposable Elements -TE- of retroviral origin in human and mouse. We evaluated the relationship with USF1, a common synergistic partner in promoters and MLT1 TEs, upon NF-YB inactivation: USF1 binding decreases in promoters, modestly in MLT1, suggesting a pioneering role of NF-Y in formers, not in the latters. These data define a common set of NF-Y functional targets across different mammalian cell types, suggesting a pioneering role in promoters with respect to TEs.

Combination Targeted Therapy in Relapsed Diffuse Large B-Cell Lymphoma.

BACKGROUND: The identification of oncogenic mutations in diffuse large B-cell lymphoma (DLBCL) has led to the development of drugs that target essential survival pathways, but whether targeting multiple survival pathways may be curative in DLBCL is unknown. METHODS: We performed a single-center, phase 1b-2 study of a regimen of venetoclax, ibrutinib, prednisone, obinutuzumab, and lenalidomide (ViPOR) in relapsed or refractory DLBCL. In phase 1b, which included patients with DLBCL and indolent lymphomas, four dose levels of venetoclax were evaluated to identify the recommended phase 2 dose, with fixed doses of the other four drugs. A phase 2 expansion in patients with germinal-center B-cell (GCB) and non-GCB DLBCL was performed. ViPOR was administered every 21 days for six cycles. RESULTS: In phase 1b of the study, involving 20 patients (10 with DLBCL), a single dose-limiting toxic effect of grade 3 intracranial hemorrhage occurred, a result that established venetoclax at a dose of 800 mg as the recommended phase 2 dose. Phase 2 included 40 patients with DLBCL. Toxic effects that were observed among all the patients included grade 3 or 4 neutropenia (in 24% of the cycles), thrombocytopenia (in 23%), anemia (in 7%), and febrile neutropenia (in 1%). Objective responses occurred in 54% of 48 evaluable patients with DLBCL, and complete responses occurred in 38%; complete responses were exclusively in patients with non-GCB DLBCL and high-grade B-cell lymphoma with rearrangements of MYC and BCL2 or BCL6 (or both). Circulating tumor DNA was undetectable in 33% of the patients at the end of ViPOR therapy. With a median follow-up of 40 months, 2-year progression-free survival and overall survival were 34% (95% confidence interval [CI], 21 to 47) and 36% (95% CI, 23 to 49), respectively. CONCLUSIONS: Treatment with ViPOR was associated with durable remissions in patients with specific molecular DLBCL subtypes and was associated with mainly reversible adverse events. (Funded by the Intramural Research Program of the National Cancer Institute and the National Center for Advancing Translational Sciences of the National Institutes of Health and others; ClinicalTrials.gov number, NCT03223610.).

IRF4 requires ARID1A to establish plasma cell identity in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy that exploits transcriptional networks driven by IRF4. We employ a multi-omics approach to discover IRF4 vulnerabilities, integrating functional genomics screening, spatial proteomics, and global chromatin mapping. ARID1A, a member of the SWI/SNF chromatin remodeling complex, is required for IRF4 expression and functionally associates with IRF4 protein on chromatin. Deleting Arid1a in activated murine B cells disrupts IRF4-dependent transcriptional networks and blocks plasma cell differentiation. Targeting SWI/SNF activity leads to rapid loss of IRF4-target gene expression and quenches global amplification of oncogenic gene expression by MYC, resulting in profound toxicity to MM cells. Notably, MM patients with aggressive disease bear the signature of SWI/SNF activity, and SMARCA2/4 inhibitors remain effective in immunomodulatory drug (IMiD)-resistant MM cells. Moreover, combinations of SWI/SNF and MEK inhibitors demonstrate synergistic toxicity to MM cells, providing a promising strategy for relapsed/refractory disease.

Molecular targets of glucocorticoids that elucidate their therapeutic efficacy in aggressive lymphomas.

Glucocorticoids have been used for decades to treat lymphomas without an established mechanism of action. Using functional genomic, proteomic, and chemical screens, we discover that glucocorticoids inhibit oncogenic signaling by the B cell receptor (BCR), a recurrent feature of aggressive B cell malignancies, including diffuse large B cell lymphoma and Burkitt lymphoma. Glucocorticoids induce the glucocorticoid receptor (GR) to directly transactivate genes encoding negative regulators of BCR stability (LAPTM5; KLHL14) and the PI3 kinase pathway (INPP5D; DDIT4). GR directly represses transcription of CSK, a kinase that limits the activity of BCR-proximal Src-family kinases. CSK inhibition attenuates the constitutive BCR signaling of lymphomas by hyperactivating Src-family kinases, triggering their ubiquitination and degradation. With the knowledge that glucocorticoids disable oncogenic BCR signaling, they can now be deployed rationally to treat BCR-dependent aggressive lymphomas and used to construct mechanistically sound combination regimens with inhibitors of BTK, PI3 kinase, BCL2, and CSK.

Activity of the Ubiquitin-activating Enzyme Inhibitor TAK-243 in Adrenocortical Carcinoma Cell Lines, Patient-derived Organoids, and Murine Xenografts.

Current treatment options for metastatic adrenocortical carcinoma (ACC) have limited efficacy, despite the common use of mitotane and cytotoxic agents. This study aimed to identify novel therapeutic options for ACC. An extensive drug screen was conducted to identify compounds with potential activity against ACC cell lines. We further investigated the mechanism of action of the identified compound, TAK-243, its synergistic effects with current ACC therapeutics, and its efficacy in ACC models including patient-derived organoids and mouse xenografts. TAK-243, a clinical ubiquitin-activating enzyme (UAE) inhibitor, showed potent activity in ACC cell lines. TAK-243 inhibited protein ubiquitination in ACC cells, leading to the accumulation of free ubiquitin, activation of the unfolded protein response, and induction of apoptosis. TAK-243 was found to be effluxed out of cells by MDR1, a drug efflux pump, and did not require Schlafen 11 (SLFN11) expression for its activity. Combination of TAK-243 with current ACC therapies (e.g., mitotane, etoposide, cisplatin) produced synergistic or additive effects. In addition, TAK-243 was highly synergistic with BCL2 inhibitors (Navitoclax and Venetoclax) in preclinical ACC models including patient-derived organoids. The tumor suppressive effects of TAK-243 and its synergistic effects with Venetoclax were further confirmed in a mouse xenograft model. These findings provide preclinical evidence to support the initiation of a clinical trial of TAK-243 in patients with advanced-stage ACC. TAK-243 is a promising potential treatment option for ACC, either as monotherapy or in combination with existing therapies or BCL2 inhibitors. SIGNIFICANCE: ACC is a rare endocrine cancer with poor prognosis and limited therapeutic options. We report that TAK-243 is active alone and in combination with currently used therapies and with BCL2 and mTOR inhibitors in ACC preclinical models. Our results suggest implementation of TAK-243 in clinical trials for patients with advanced and metastatic ACC.

Comparative Assessment and High-Throughput Drug-Combination Profiling of TEAD-Palmitoylation Inhibitors in Hippo Pathway Deficient Mesothelioma.

The hippo signaling pathway is a central tumor suppressor cascade frequently inactivated in selected human cancers, leading to the aberrant activation of TEAD transcription factors. Whereas several TEAD auto-palmitoylation inhibitors are currently in development, a comprehensive assessment of this novel drug-modality is missing. Here, we report a comparative analysis among six TEADi(s) using cell-based and biochemical assays in Hippo pathway deficient mesothelioma. Our analysis revealed varying potency and selectivity across TEADi, also highlighting their limited efficacy. To overcome this limitation, we performed an unbiased, quantitative high-throughput drug screening by combining the TEADi VT-103 with a library of approximately 3000 oncology-focused drugs. By exploiting this library's mechanistic redundancy, we identified several drug-classes robustly synergized with TEADi. These included glucocorticoid-receptor (GR) agonists, Mek1/2 inhibitors, mTOR inhibitors, and PI3K inhibitors, among others. Altogether, we report a coherent single-agent dataset informing on potency and selectivity of TEAD-palmitoylation inhibitors as single-agents. We also describe a rational pipeline enabling the systematic identification of TEAD druggable co-dependencies. This data should support the pre-clinical development of drug combination strategies for the treatment of Hippo-deficient mesothelioma, and more broadly, for other cancers dependent on the oncogenic activity of YAP/TEAD.

High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma.

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

Preclinical Evaluation of the FGFR-Family Inhibitor Futibatinib for Pediatric Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Despite decades of clinical trials, the overall survival rate for patients with relapsed and metastatic disease remains below 30%, underscoring the need for novel treatments. FGFR4, a receptor tyrosine kinase that is overexpressed in RMS and mutationally activated in 10% of cases, is a promising target for treatment. Here, we show that futibatinib, an irreversible pan-FGFR inhibitor, inhibits the growth of RMS cell lines in vitro by inhibiting phosphorylation of FGFR4 and its downstream targets. Moreover, we provide evidence that the combination of futibatinib with currently used chemotherapies such as irinotecan and vincristine has a synergistic effect against RMS in vitro. However, in RMS xenograft models, futibatinib monotherapy and combination treatment have limited efficacy in delaying tumor growth and prolonging survival. Moreover, limited efficacy is only observed in a PAX3-FOXO1 fusion-negative (FN) RMS cell line with mutationally activated FGFR4, whereas little or no efficacy is observed in PAX3-FOXO1 fusion-positive (FP) RMS cell lines with FGFR4 overexpression. Alternative treatment modalities such as combining futibatinib with other kinase inhibitors or targeting FGFR4 with CAR T cells or antibody-drug conjugate may be more effective than the approaches tested in this study.

A landscape of response to drug combinations in non-small cell lung cancer.

Combination of anti-cancer drugs is broadly seen as way to overcome the often-limited efficacy of single agents. The design and testing of combinations are however very challenging. Here we present a uniquely large dataset screening over 5000 targeted agent combinations across 81 non-small cell lung cancer cell lines. Our analysis reveals a profound heterogeneity of response across the tumor models. Notably, combinations very rarely result in a strong gain in efficacy over the range of response observable with single agents. Importantly, gain of activity over single agents is more often seen when co-targeting functionally proximal genes, offering a strategy for designing more efficient combinations. Because combinatorial effect is strongly context specific, tumor specificity should be achievable. The resource provided, together with an additional validation screen sheds light on major challenges and opportunities in building efficacious combinations against cancer and provides an opportunity for training computational models for synergy prediction.

Positional influence on cellular transcriptional identity revealed through spatially segmented single-cell transcriptomics.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.

CellMinerCDB: NCATS Is a Web-Based Portal Integrating Public Cancer Cell Line Databases for Pharmacogenomic Explorations.

Major advances have been made in the field of precision medicine for treating cancer. However, many open questions remain that need to be answered to realize the goal of matching every patient with cancer to the most efficacious therapy. To facilitate these efforts, we have developed CellMinerCDB: National Center for Advancing Translational Sciences (NCATS; https://discover.nci.nih.gov/rsconnect/cellminercdb_ncats/), which makes available activity information for 2,675 drugs and compounds, including multiple nononcology drugs and 1,866 drugs and compounds unique to the NCATS. CellMinerCDB: NCATS comprises 183 cancer cell lines, with 72 unique to NCATS, including some from previously understudied tissues of origin. Multiple forms of data from different institutes are integrated, including single and combination drug activity, DNA copy number, methylation and mutation, transcriptome, protein levels, histone acetylation and methylation, metabolites, CRISPR, and miscellaneous signatures. Curation of cell lines and drug names enables cross-database (CDB) analyses. Comparison of the datasets is made possible by the overlap between cell lines and drugs across databases. Multiple univariate and multivariate analysis tools are built-in, including linear regression and LASSO. Examples have been presented here for the clinical topoisomerase I (TOP1) inhibitors topotecan and irinotecan/SN-38. This web application provides both substantial new data and significant pharmacogenomic integration, allowing exploration of interrelationships. SIGNIFICANCE: CellMinerCDB: NCATS provides activity information for 2,675 drugs in 183 cancer cell lines and analysis tools to facilitate pharmacogenomic research and to identify determinants of response.

Targeting N-linked Glycosylation for the Therapy of Aggressive Lymphomas.

Diffuse large B-cell lymphoma (DLBCL) can be subdivided into the activated B-cell (ABC) and germinal center B cell-like (GCB) subtypes. Self-antigen engagement of B-cell receptors (BCR) in ABC tumors induces their clustering, thereby initiating chronic active signaling and activation of NF-κB and PI3 kinase. Constitutive BCR signaling is essential in some GCB tumors but primarily activates PI3 kinase. We devised genome-wide CRISPR-Cas9 screens to identify regulators of IRF4, a direct transcriptional target of NF-κB and an indicator of proximal BCR signaling in ABC DLBCL. Unexpectedly, inactivation of N-linked protein glycosylation by the oligosaccharyltransferase-B (OST-B) complex reduced IRF4 expression. OST-B inhibition of BCR glycosylation reduced BCR clustering and internalization while promoting its association with CD22, which attenuated PI3 kinase and NF-κB activation. By directly interfering with proximal BCR signaling, OST-B inactivation killed models of ABC and GCB DLBCL, supporting the development of selective OST-B inhibitors for the treatment of these aggressive cancers. SIGNIFICANCE: DLBCL depends on constitutive BCR activation and signaling. There are currently no therapeutics that target the BCR directly and attenuate its pathologic signaling. Here, we unraveled a therapeutically exploitable, OST-B-dependent glycosylation pathway that drives BCR organization and proximal BCR signaling. This article is highlighted in the In This Issue feature, p. 1749.

Schlafen 11 (SLFN11) Kills Cancer Cells Undergoing Unscheduled Re-replication.

Schlafen 11 (SLFN11) is an increasingly prominent predictive biomarker and a molecular sensor for a wide range of clinical drugs: topoisomerases, PARP and replication inhibitors, and platinum derivatives. To expand the spectrum of drugs and pathways targeting SLFN11, we ran a high-throughput screen with 1,978 mechanistically annotated, oncology-focused compounds in two isogenic pairs of SLFN11-proficient and -deficient cells (CCRF-CEM and K562). We identified 29 hit compounds that selectively kill SLFN11-proficient cells, including not only previously known DNA-targeting agents, but also the neddylation inhibitor pevonedistat (MLN-4924) and the DNA polymerase α inhibitor AHPN/CD437, which both induced SLFN11 chromatin recruitment. By inactivating cullin-ring E3 ligases, pevonedistat acts as an anticancer agent partly by inducing unscheduled re-replication through supraphysiologic accumulation of CDT1, an essential factor for replication initiation. Unlike the known DNA-targeting agents and AHPN/CD437 that recruit SLFN11 onto chromatin in 4 hours, pevonedistat recruited SLFN11 at late time points (24 hours). While pevonedistat induced unscheduled re-replication in SLFN11-deficient cells after 24 hours, the re-replication was largely blocked in SLFN11-proficient cells. The positive correlation between sensitivity to pevonedistat and SLFN11 expression was also observed in non-isogenic cancer cells in three independent cancer cell databases (NCI-60, CTRP: Cancer Therapeutics Response Portal and GDSC: Genomic of Drug Sensitivity in Cancer). The present study reveals that SLFN11 not only detects stressed replication but also inhibits unscheduled re-replication induced by pevonedistat, thereby enhancing its anticancer efficacy. It also suggests SLFN11 as a potential predictive biomarker for pevonedistat in ongoing and future clinical trials.

High-throughput approaches to uncover synergistic drug combinations in leukemia.

We report a comprehensive drug synergy study in acute myeloid leukemia (AML). In this work, we investigate a panel of cell lines spanning both MLL-rearranged and non-rearranged subtypes. The work comprises a resource for the community, with many synergistic drug combinations that could not have been predicted a priori, and open source code for automation and analyses. We base our definitions of drug synergy on the Chou-Talalay method, which is useful for visualizations of synergy experiments in isobolograms, and median-effects plots, among other representations. Our key findings include drug synergies affecting the chromatin state, specifically in the context of regulation of the modification state of histone H3 lysine-27. We report open source high throughput methodology such that multidimensional drug screening can be accomplished with equipment that is accessible to most laboratories. This study will enable preclinical investigation of new drug combinations in a lethal blood cancer, with data analysis and automation workflows freely available to the community.

mTOR inhibition overcomes RSK3-mediated resistance to BET inhibitors in small cell lung cancer.

Small cell lung cancer (SCLC) is a recalcitrant malignancy with limited treatment options. Bromodomain and extraterminal domain inhibitors (BETis) have shown promising preclinical activity in SCLC, but the broad sensitivity spectrum limits their clinical prospects. Here, we performed unbiased high-throughput drug combination screens to identify therapeutics that could augment the antitumor activities of BETis in SCLC. We found that multiple drugs targeting the PI-3K-AKT-mTOR pathway synergize with BETis, among which mTOR inhibitors (mTORis) show the highest synergy. Using various molecular subtypes of the xenograft models derived from patients with SCLC, we confirmed that mTOR inhibition potentiates the antitumor activities of BETis in vivo without substantially increasing toxicity. Furthermore, BETis induce apoptosis in both in vitro and in vivo SCLC models, and this antitumor effect is further amplified by combining mTOR inhibition. Mechanistically, BETis induce apoptosis in SCLC by activating the intrinsic apoptotic pathway. However, BET inhibition leads to RSK3 upregulation, which promotes survival by activating the TSC2-mTOR-p70S6K1-BAD cascade. mTORis block this protective signaling and augment the apoptosis induced by BET inhibition. Our findings reveal a critical role of RSK3 induction in tumor survival upon BET inhibition and warrant further evaluation of the combination of mTORis and BETis in patients with SCLC.

Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma.

Oncogenic RAS mutations are common in multiple myeloma (MM), an incurable malignancy of plasma cells. However, the mechanisms of pathogenic RAS signaling in this disease remain enigmatic and difficult to inhibit therapeutically. We employ an unbiased proteogenomic approach to dissect RAS signaling in MM. We discover that mutant isoforms of RAS organize a signaling complex with the amino acid transporter, SLC3A2, and MTOR on endolysosomes, which directly activates mTORC1 by co-opting amino acid sensing pathways. MM tumors with high expression of mTORC1-dependent genes are more aggressive and enriched in RAS mutations, and we detect interactions between RAS and MTOR in MM patient tumors harboring mutant RAS isoforms. Inhibition of RAS-dependent mTORC1 activity synergizes with MEK and ERK inhibitors to quench pathogenic RAS signaling in MM cells. This study redefines the RAS pathway in MM and provides a mechanistic and rational basis to target this mode of RAS signaling.

Inhibition of HSP 90 is associated with potent anti-tumor activity in Papillary Renal Cell Carcinoma.

BACKGROUND: There is no universally accepted treatment for patients with advanced papillary renal cell carcinoma (PRCC). The presence of activating mutations in MET, as well as gain of chromosome 7, where the MET gene is located, are the most common genetic alterations associated with PRCC, leading to the clinical evaluation of MET tyrosine kinase inhibitors (TKIs) in this cancer. However, TKIs targeting MET selectively, as well as multitargeted TKIs with activity against MET demonstrate modest efficacy in PRCC and primary and secondary treatment failure is common; other approaches are urgently needed to improve outcomes in these patients. METHODS: High throughput screening with small molecule libraries identified HSP90 inhibitors as agents of interest based on antitumor activity against patient derived PRCC cell lines. We investigated the activity of the orally available HSP90 inhibitor, SNX2112 in vitro, using 2D/3D PRCC cell culture models and in vivo, in mice tumor xenograft models. The molecular pathways mediating antitumor activity of SNX2112 were assessed by Western blot analysis, Flow cytometry, RNA-seq analysis, Real Time qPCR and imaging approaches. RESULTS: SNX2112 significantly inhibited cellular proliferation, induced G2/M cell cycle arrest and apoptosis in PRCC lines overexpressing MET. In contrast to TKIs targeting MET, SNX2112 inhibited both MET and known downstream mediators of MET activity (AKT, pAKT1/2 and pERK1/2) in PRCC cell lines. RNAi silencing of AKT1/2 or ERK1/2 expression significantly inhibited growth in PRCC cells. Furthermore, SNX2112 inhibited a unique set of E2F and MYC targets and G2M-associated genes. Interestingly, interrogation of the TCGA papillary RCC cohort revealed that these genes were overexpressed in PRCC and portend a poor prognosis. Finally, SNX-2112 demonstrated strong antitumor activity in vivo and prolonged survival of mice bearing human PRCC xenograft. CONCLUSIONS: These results demonstrate that HSP90 inhibition is associated with potent activity in PRCC, and implicate the PI3K/AKT and MEK/ERK1/2 pathways as important mediators of tumorigenesis. These data also provide the impetus for further clinical evaluation of HSP90, AKT, MEK or E2F pathway inhibitors in PRCC.

SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

Overcoming Acquired Epigenetic Resistance to BTK Inhibitors.

The use of Bruton tyrosine kinase (BTK) inhibitors to block B-cell receptor (BCR)-dependent NF-κB activation in lymphoid malignancies has been a major clinical advance, yet acquired therapeutic resistance is a recurring problem. We modeled the development of resistance to the BTK inhibitor ibrutinib in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma, which relies on chronic active BCR signaling for survival. The primary mode of resistance was epigenetic, driven in part by the transcription factor TCF4. The resultant phenotypic shift altered BCR signaling such that the GTPase RAC2 substituted for BTK in the activation of phospholipase Cγ2, thereby sustaining NF-κB activity. The interaction of RAC2 with phospholipase Cγ2 was also increased in chronic lymphocytic leukemia cells from patients with persistent or progressive disease on BTK inhibitor treatment. We identified clinically available drugs that can treat epigenetic ibrutinib resistance, suggesting combination therapeutic strategies. SIGNIFICANCE: In diffuse large B-cell lymphoma, we show that primary resistance to BTK inhibitors is due to epigenetic rather than genetic changes that circumvent the BTK blockade. We also observed this resistance mechanism in chronic lymphocytic leukemia, suggesting that epigenetic alterations may contribute more to BTK inhibitor resistance than currently thought.See related commentary by Pasqualucci, p. 555. This article is highlighted in the In This Issue feature, p. 549.