Abstract: Background Deferasirox (DFX) is widely employed as iron chelation therapy (ICT) in the current clinical practice in patients with myelodysplastic syndromes (MDS) and chronic transfusion need. The efficacy of DFX in reducing median ferritin levels in different cohorts of these patients has been reported in many trials, but the lack of worldwide accepted criteria of individual response to ICT makes it difficult to appreciate its clinical relevance for any single patient. Aim To highlight the clinical impact of ICT with DFX in a large real-life cohort of MDS patients, based on different individual ferritin variation during treatment. Methods A retrospective cohort of 301 consecutive MDS patients [M/F 187/114 (62.1%/37.9%)] of any age followed in 20 hematological Centers in Italy was analyzed: the main features at diagnosis are reported in the Table 1. Individual response to ICT was categorized as complete response (CR) (ferritin levels 〈 500 ng/ml), partial response (PR) (ferritin levels 〈 1,000 ng/ml), ferritin improvement (FI) (ferritin reduction 〉 50% of baseline value but with levels 〉 1,000 ng/ml), ferritin stability (FS) (ferritin levels without changes from baseline during ICT) or no ferritin response (NR) (ferritin levels increasing during ICT). Results ICT was started after a median period from diagnosis and from transfusion start of 21.0 months [interquartile range (IQR) 8.9 - 44.3] and 11.3 months (IQR 7.1 - 21.7), respectively, with a median burden of red cell transfusions at baseline of 22 units (IQR 14 - 35). The main features of patients at baseline of ICT are reported in the Table 1. Starting DFX dose was 〈 10 mg/Kg in 38 patients (12.7%), 10 - 14 mg/Kg in 110 patients (36.6%), 15 - 19 mg/Kg in 57 patients (18.9%) and ≥ 20 mg/Kg in 96 patients (31.9%). As to individual response, 4 patients (1.3%) were too early for evaluation ( 〈 6 months of DFX treatment): in addition, 16 patients (5.4%) discontinued ICT behind 6 months from start, due to early toxicity (10 patients, 7 for gastro-intestinal toxicity and 3 for skin toxicity) or other reasons (unrelated death, AML evolution, transplant procedure). Among the remaining 281 patients, 37 (12.3%) achieved a CR, 65 (21.6%) a PR, 23 (7.6%) a FI, 112 (37.2%) a FS and 44 (14.6%) a NR. Five-year overall survival (OS) of the whole cohort from ICT start was 43.9% (95%CI 37.1 - 50.7). Five-year OS according to ICT response was 74.8% (95%CI 57.9 - 91.7) in patients with CR, 51.7% (95%CI 37.6 - 65.8) in patients with PR, 50.6% (95%CI 28.2 - 73.0) in patients with FI, 38.6% (95%CI 27.0 - 50.2) in patients with FS and 21.1% (95%CI 5.2 - 37.0) in patients with NR (p=0.002) (Figure 1). Five-year cumulative incidence of AML evolution (CIE) of the whole cohort from ICT start was 27.1% (95%CI 20.3 - 33.9). Five-year CIE according to ICT response was 7.6% (95%CI 0 - 18.0) in patients with CR, 27.0% (95%CI 13.0 - 40.5) in patients with PR, 38.3% (95%CI 15.5 - 61.7) in patients with FI, 20.8% (95%CI 10.4 - 31.2) in patients with FS and 57.7% (95%CI 31.9 - 83.5) in patients with NR (p=0.003) (Figure 2). Notably, no statistical difference was observed for both OS and CIE among patients achieving PR, FI or FS. Conclusions Present data highlight the clinical relevance of individual response in MDS patients receiving ICT with DFX. In particular, achievement of CR seemed related to a better OS and a lower CIE, while patients with NR had a significant worst OS and CIE: furthermore, the achievement of stable ferritin levels was associated with similar OS and CIE than PR and FI and thus should be considered as a response. Disclosures Latagliata: Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Oliva:Novartis: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy. Pilo:Novartis: Other: Advisory board. Molteni:Celgene: Membership on an entity's Board of Directors or advisory committees. Balleari:Celgene: Membership on an entity's Board of Directors or advisory committees. Breccia:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Celgene: Honoraria. Foà:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Finelli:Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding, Speakers Bureau.

Abstract: KRAS is the most commonly mutated oncogenes and is a major driver of tumor initiation and progression. Understanding the functional consequences of cancer-associated KRAS variants may have important clinical implications. For example, KRAS mutation status defines those that are likely to respond to EGFR-directed therapy in KRAS-mutant metastatic colorectal cancer. A compendium of all possible oncogenic KRAS alleles would serve as a roadmap for future therapeutic strategies directed at KRAS itself or downstream signaling effectors. Comprehensive mutagenesis of KRAS may also elucidate structure-function relationships that reveal novel biochemical properties that may be exploited for therapeutic gain. We performed saturation mutagenesis of both a wild-type (WT) and a G12D mutant form of KRAS cDNA and generated lentiviral expression libraries of 3,553 and 3,534 single amino acid substitution mutants of each backbone. We utilized these WT and G12D mutagenesis libraries for functional genetic screening to identify gain- and loss-of-function missense variants that alter critical oncogenic properties of KRAS. First, we sought to comprehensively identify all possible oncogenic missense mutations in KRAS that mediate oncogenic transformation. We stably transduced the WT library into immortalized human epithelial cells and evaluated growth in low attachment (GILA), an assay that is highly correlated with in vivo tumor formation. We identified all previously known hotspot oncogenic alleles of KRAS as well as many functionally relevant alleles that are also discovered at lower frequency in human tumors. Moreover, we also discovered a group of transforming KRAS variants that have not been well described in human tumors, thus revealing potentially novel activating mechanisms for oncogenic KRAS. In parallel, we utilized the G12D mutagenesis library to perform second-site suppressor screening to identify loss-of-function single amino acid changes that abrogate the transforming ability of oncogenic KRAS. We performed positive-selection screening in primary cell lines for variants that enable bypass of oncogene-induced senescence. Additionally, we conducted a negative-selection screen with the G12D library in a KRAS-dependent cancer cell line with inducible suppression of endogenous KRAS, thus identifying all possible second-site mutations that abolish KRAS-driven signaling necessary for maintenance of cellular proliferation and viability. Structure-function analysis of these data may reveal novel patterns of amino-acid changes that result in inactivation of oncogenic KRAS. In summary, this comprehensive dictionary of gain- and loss-of-function KRAS mutants will facilitate understanding of clinically important mutations and also yield novel insights into structure-function relationships that may improve our understanding of the KRAS oncogene. Citation Format: Eejung Kim, Seav Huong Ly, Nicole S. Persky, Belinda Wang, Xiaoping Yang, Federica Piccioni, Katherine Labella, Mihir Doshi, Robert E. Lintner, Cong Zhu, Scott Steelman, David E. Root, Cory M. Johannessen, Alex B. Burgin, Laura E. MacConaill, William C. Hahn, Andrew J. Aguirre. Saturation mutagenesis of KRAS reveals the functional landscape of missense variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2017-394

Abstract: PDE3A-SLFN12 complex formation is induced by a class of compounds, now called “velcrins”, exemplified by the small molecule, DNMDP. Cancer cells that express elevated levels of PDE3A and SLFN12 are sensitive to a velcrin-mediated cytotoxic response, which is independent of PDE3A inhibition. However, the details of complex formation have not yet been revealed. We solved the crystal structure of PDE3A with a series of ligands bound to the active site and found that PDE3A exists as a dimer, and velcrin binding does not cause any obvious structural changes in the PDE3A protein structure. Hydrogen-deuterium exchange (HDX-MS) experiments with velcrin-bound PDE3A in the absence and presence of SLFN12 identified three regions of PDE3A that are shielded from solvent as a result of velcrin-induced SLFN12 binding. Two of these regions are near the velcrin binding site, and the third region lies at the PDE3A homodimerization interface. In order to further investigate the structural relationship between PDE3A, DNMDP, and SLFN12, we took a deep-mutation scanning (DMS) approach to identify residues of PDE3A that impact DNMDP sensitivity. A library of PDE3A alleles was developed in which the sequence encoding amino acids 668-1141, including the PDE3A catalytic domain, was substituted with a codon for every other possible amino acid or a stop codon in the context of the full-length cDNA. The library was transduced into PDE3A-knockout GB1 glioblastoma cells and assessed for survival in the presence of DMSO or DNMDP. Corroborating the HDX-MS data, we identified three regions of PDE3A in which missense mutations abrogated DNMDP response: the active site, the homodimerization surface, and an alpha helix containing amino acid F914. We confirmed that mutations of F914 and the homodimer interface retain the ability to bind resin-conjugated compound but fail to complex with SLFN12. Taken together, the HDX and DMS results suggest that PDE3A dimerization is required to stabilize velcrin-induced SLFN12 binding and implicate the alpha helix containing F914 as the SLFN12 binding interface of PDE3A. Citation Format: Xiaoyun Wu, Malvina Papanastasiou, Gavin Schnitzler, Colin Garvie, Stephanie Hoyt, Terry Zhang, James Mullahoo, Andrew Baker, Joseph McGaunn, Bethany Kaplan, Sooncheol Lee, Martin Lange, Steven Carr, Xiaoping Yang, Federica Piccioni, Andrew Cherniack, Matthew Meyerson, Heidi Greulich. Deep mutational scanning of PDE3A identifies residues required for DNMDP response [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 1219.

Abstract: To identify clinically relevant mechanisms of resistance to ER-directed therapies in ER+ breast cancer. Experimental Design: We conducted a genome-scale functional screen spanning 10,135 genes to investigate genes whose overexpression confer resistance to selective estrogen receptor degraders. In parallel, we performed whole-exome sequencing in paired pretreatment and postresistance biopsies from 60 patients with ER+ metastatic breast cancer who had developed resistance to ER-targeted therapy. Furthermore, we performed experiments to validate resistance genes/pathways and to identify drug combinations to overcome resistance. Results: Pathway analysis of candidate resistance genes demonstrated that the FGFR, ERBB, insulin receptor, and MAPK pathways represented key modalities of resistance. The FGFR pathway was altered via FGFR1, FGFR2, or FGF3 amplifications or FGFR2 mutations in 24 (40%) of the postresistance biopsies. In 12 of the 24 postresistance tumors exhibiting FGFR/FGF alterations, these alterations were acquired or enriched under the selective pressure of ER-directed therapy. In vitro experiments in ER+ breast cancer cells confirmed that FGFR/FGF alterations led to fulvestrant resistance as well as cross-resistance to the CDK4/6 inhibitor palbociclib. RNA sequencing of resistant cell lines demonstrated that FGFR/FGF induced resistance through ER reprogramming and activation of the MAPK pathway. The resistance phenotypes were reversed by FGFR inhibitors, a MEK inhibitor, and/or a SHP2 inhibitor. Conclusions: Our results suggest that FGFR pathway is a distinct mechanism of acquired resistance to ER-directed therapy that can be overcome by FGFR and/or MAPK pathway inhibitors.

Abstract: Somatic copy number alterations that result in loss of tumor suppressor gene function are important drivers of tumorigenesis. However, few existing therapeutic options to target oncogenic processes evoked by tumor suppressor gene inactivation exist. The discovery of synthetic lethal interactions with genetic drivers of cancer may yield new therapeutic strategies with cancer selective potential. We examined genome-scale CRISPR-SpCas9 and RNA interference screens to uncover new synthetic lethal vulnerabilities associated with the loss of common tumor suppressor genes (TSGs). Vacuolar protein sorting 4 homolog A (VPS4A) scored as a strong, selective dependency in cancer cells with genomic loss of the SMAD4 tumor suppressor due to co-deletion of VPS4A's paralog gene, VPS4B. VPS4B resides 12.3 Mb away from the SMAD4 TSG on chromosome 18q and is lost in approximately 33% of all cancers, suggesting broad clinical applicability. VPS4A and VPS4B function as AAA ATPases forming a multimeric protein complex within the endosomal sorting complex required for transport (ESCRT) pathway to regulate membrane remodeling in a range of cellular processes. VPS4A suppression in cells with VPS4B/SMAD4 loss led to accumulation of ESCRT-III filaments, cytokinesis defects, nuclear deformation and micronucleation, which ultimately resulted in G2/M cell cycle arrest and apoptosis. Furthermore, upon VPS4A suppression, we observerd potent in vivo tumor regression, which led to extended survival, in mouse subcutaneous xenograft models with human cancer cell lines harboring VPS4B loss. Finally, genome-scale CRISPR-SpCas9 loss-of-function screening revealed other ESCRT pathway members and regulators of cellular abscission as modifiers of VPS4A dependency. Using the most comprehensive available CRISPR-SpCas9 and RNA-interference screening datasets to date, we provide a compendium of synthetic lethal vulnerabilities with TSG loss and credential VPS4A as a new and promising therapeutic target in cancers with VPS4B/SMAD4 deletion. Citation Format: Jasper E. Neggers, Brenton R. Paolella, Adhana Asfaw, Michael V. Rothberg, Thomas A. Skipper, Radha L. Kalekar, John M. Krill-Burger, Andrew L. Hong, Guillaume Kugener, Jeremie Kalfon, Annan Yang, Chen Yuan, Nancy Dumont, Alfredo Gonzalez, Mai Abdusamad, Yvonne Y. Li, Liam F. Spurr, Westley W. Wu, Federica Piccioni, Brian M. Wolpin, David E. Root, Jesse S. Boehm, Andrew D. Cherniack, Aviad Tsherniak, Todd R. Golub, Francisca Vazquez, Andrew J. Aguirre. VPS4A is a synthetic lethal target in VPS4B-deficient cancers due to co-deletion with SMAD4 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-053.

Abstract: Somatic copy number alterations that result in loss of tumor suppressor gene function are important drivers of tumorigenesis. However, few existing therapeutic options to target oncogenic processes evoked by tumor suppressor gene inactivation exist. The discovery of synthetic lethal interactions with genetic drivers of cancer may yield new therapeutic strategies with cancer selective potential. We examined genome-scale CRISPR-SpCas9 and RNA interference screens to uncover new synthetic lethal vulnerabilities associated with the loss of common tumor suppressor genes (TSGs). The ATPases Vacuolar protein sorting 4 homolog A (VPS4A) and B (VPS4B) scored as strong synthetic lethal dependencies, with VPS4A selectively essential in cancers harboring loss of VPS4B adjacent to SMAD4 and VPS4B required in tumors with co-deletion of VPS4A and CDH1 (encoding E-cadherin). VPS4B resides 12.3 Mb away from the SMAD4 TSG on chromosome 18q and is lost in approximately 33% of all cancers, suggesting broad clinical applicability. Moreover, VPS4B is commonly lost in pancreatic cancer due to the frequent loss of SMAD4, highlighting VPS4A represents a promising target for this deadly cancer. VPS4A and VPS4B function as AAA ATPases forming a multimeric protein complex within the endosomal sorting complex required for transport (ESCRT) pathway to regulate membrane remodeling in a range of cellular processes. VPS4A suppression in cells with VPS4B/SMAD4 loss led to accumulation of ESCRT-III filaments, cytokinesis defects, nuclear deformation and micronucleation, which ultimately resulted in G2/M cell cycle arrest and apoptosis. Furthermore, upon VPS4A suppression, we observed potent in vivo tumor regression, which led to extended survival, in mouse subcutaneous xenograft models utilizing a pancreatic or rhabdomyosarcoma cancer cell line harboring VPS4B loss. CRISPR-SpCas9 screening and integrative genomic analysis revealed other ESCRT members, regulators of abscission and interferon signaling as modifiers of VPS4A dependency. Using the most comprehensive available CRISPR-SpCas9 and RNA-interference screening datasets to date, we provide a compendium of synthetic lethal vulnerabilities with TSG loss and credential VPS4A as a new and promising therapeutic target in cancers with VPS4B/SMAD4 deletion. Citation Format: Jasper E. Neggers, Brenton R. Paolella, Adhana Asfaw, Michael V. Rothberg, Thomas A. Skipper, Radha L. Kalekar, Michael J. Krill-Burger, Neekesh V. Dharia, Guillaume Kugener, Adam D. Durbin, Annan Yang, Nancy Dumont, Yvonne Y. Li, Brian M. Wolpin, Federica Piccioni, David E. Root, Jesse S. Boehm, Andrew D. Cherniack, Aviad Tsherniak, Andrew L. Hong, William C. Hahn, Kimberly Stegmaier, Todd R. Golub, Francisca Vazquez, Andrew J. Aguirre. Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in SMAD4 or CDH1-deleted cancers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-011.

Abstract: Drug resistant mutations that arise in therapeutic targets often limit clinical responses. However, the discovery of such mutations has historically been performed one gene or mutation at a time, often over decades of experimental and clinical testing, limiting our understanding of conserved mechanisms of drug resistance. We hypothesized that deep mutational scanning of canonical kinases may expedite this process and identify novel conserved elements that cause drug resistance when mutated (similar to the well-studied “Gatekeeper” residue). To test this, we generated cDNA-expression libraries containing all possible amino acid substitutions in CDK6, CDK4, ERK2, and EGFR. We screened each library against clinically utilized, ATP-competitive small molecule inhibitors. We then mapped the phenotypic data for over 40,000 missense mutations onto the aligned crystal structures of each protein and searched for shared structural attributes associated with drug resistance. This analysis revealed 4 equivalent amino acid sites whose mutation conferred drug resistance to ATP-competitive inhibitors in all of our screens: the Gatekeeper residue, as well as three uncharacterized residues. One of these sites, which we have termed the “Keymaster”, was additionally found to cause resistance in published data sets of sub-saturation BRAF, HER2, BCR-ABL, and MEK1 mutagenesis screens against their respective inhibitors. We confirmed that drug resistant phenotypes are caused by these alterations utilizing growth assays and protein target phosphorylation detection assays. Mechanistically, we show preliminary evidence that Keymaster-mutant proteins are competent for drug binding, but may display elevated basal activity. Consistent with our findings, we additionally identified mutations at Keymaster residues in reported patient tumors in a number of oncogene kinases, suggesting that Keymaster mutations could be drivers of tumorigenesis, as well as drug resistance. These efforts may prove useful for characterizing somatic kinase mutations of unknown function, designing next-generation therapeutics and deepening our understanding of kinase regulation. Citation Format: Nicole S. Persky, Desiree Hernandez, Jonathon Cordova, Amanda Walker, Lisa Brenan, Federica Piccioni, Sasha Pantel, Yenarae Lee, Amy Goodale, Xiaoping Yang, Yoichiro Mitsuishi, Mariana Do Carmo, Cong Zhu, Aleksandr Andreev, David E. Root, Cory M. Johannessen. Massively parallel identification of conserved drug resistant mutations in kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1815.

Abstract: Resistance to therapy is one of the major challenges in cancer treatment today, equally applicable to conventional chemotherapy as well as targeted therapy. Malignant tumors have widespread epigenetic alterations including aberrant expression of chromatin modifiers in a wide variety of tumors and chromosomal translocations involving chromatin modifiers that can drive development of some cancers. In addition, cancer genome sequencing studies have identified frequent somatic alterations in many chromatin-regulating enzymes. Moreover, epigenetic changes have been implicated in the development of drug resistance. T cell acute lymphoblastic leukemia (T-ALL) has a high rate of treatment-refractory disease and relapse that significantly lowers survival rates compared to other forms of ALL. The identification of activating somatic NOTCH1 mutations in over 50% of patients with T-ALL led to the development of γ-secretase inhibitors (GSI) that prevent cleavage and activation of NOTCH1. Although effective in vitro, the rapid development of resistance that develops with Notch inhibition in vivo has so far prevented the translation of these inhibitors into the clinical setting. We have developed a model of therapeutic resistance to inhibition of Notch signaling in T-ALL. In this model, ‘persister’ cells readily expand in the presence of GSI and the absence of Notch signaling. Rare persister cells are pre-existing in naïve T-ALL populations. Intriguingly, in vitro resistance to NOTCH1 inhibitor therapy is reversible, suggesting that it is epigenetically mediated. When compared to GSI-sensitive cells, persisters are characterized by distinct signaling and gene expression programs, and demonstrate global chromatin compaction. Using a short-hairpin knock-down screen of ∼ 300 known chromatin regulators we identified the chromatin reader BRD4 as essential for persister T-ALL cells. BRD4 expression levels are upregulated in persister T-ALL cells. Genome-wide binding studies of BRD4 show that it is found at active regulatory elements in the genome that are associated with genes known to be important for cell proliferation, survival and signaling pathways in T-ALL, e. g. MYC and BCL2. Treatment of persisters with the BRD4 inhibitor JQ1 down-regulates expression of these target genes. Functionally, JQ1 treatment leads to growth arrest and apoptosis in persister T-ALL cells, at doses well tolerated by GSI-sensitive leukemia cells. Furthermore, combination therapy of GSI and JQ1 is significantly more effective over vehicle or single agent therapy for primary human T-ALLs in vitro and in vivo. These studies demonstrate epigenetic heterogeneity as a basis of drug resistance in leukemia. We suggest that combination therapies that include targeting of chromatin regulators may hold great therapeutic promise for prevention and treatment of resistant disease. Citation Format: Birgit Knoechel, Justine Roderick, Kaylyn Williamson, Jiang Zhu, Jens Lohr, Matthew Cotton, Shawn Gillespie, Daniel Fernandez, Manching Ku, Hongfang Wang, Federica Piccioni, Serena Silver, Mohit Jain, Daniel Pearson, Michael Kluk, Christopher Ott, Dale Greiner, Michael Brehm, Leonard Shultz, Alejandro Gutierrez, Kimberly Stegmaier, Marian Harris, Lewis Silverman, Stephen Sallan, Andrew Kung, David Root, James Bradner, Jon Aster, Michelle Kelliher, Bradley Bernstein. Epigenetic resistance to Notch inhibition in T cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4782. doi:10.1158/1538-7445.AM2014-4782

Abstract: The FDA-approval of potent targeted therapies has led to great changes in the therapeutic landscape of chronic lymphocytic leukemia (CLL). As a key example, venetoclax, a first-in-class BCL-2 inhibitor, leads to response in about 80% of patients with relapsed/refractory (R/R) CLL. Disease progression on venetoclax, however, has been increasingly observed, and better biologic understanding of resistance mechanisms to this agent is needed. To systematically discover the potential mechanisms of resistance to venetoclax, we performed both genome-scale loss- (LOF) and gain-of-function (GOF) genetic modifier screens in the BCL-2-driven OCI-Ly1 lymphoma cell line using CRISPR-Cas9 sgRNA and ORF libraries, respectively. Significant hits from both screens included the BCL-2 family: the LOF screen with pro-apoptotic genes (PMAIP1, BAX, BAK1, BCL-2L11) and the GOF screen with anti-apoptotic genes (BCL2L1, BCL2L2, BCL2, MCL1). In addition, the LOF screen uncovered genes in pathways relevant to lymphoid biology (i.e, NFKBIA) and lymphoid transcription factors and modulators (IKZF5, ID3, EP300, NFIA). The GOF screen also uncovered components of the energy-stress sensor PKA/AMPK signaling pathways (ADIPOQ, PRKAR2B, PRKAA2) and regulators of mitochondrial metabolism. In parallel, we performed an integrated transcriptome, whole proteome and functional characterization of an OCI-Ly1 cell line rendered resistant to venetoclax (OCI-Ly1-R) from the parental cell line (OCI-Ly1-S). RNA-seq and spectrometry-based proteomics revealed coordinated dysregulation of transcripts and proteins in the resistant line originating from genes critical to cellular metabolism, cell cycle, B-cell biology and autophagy. Of the transcripts and proteins significantly associated with the resistant cell line, only MCL-1 overlapped with the gene hits from the genome-scale screens. Treatment of the OCI-Ly-R cells with the MCL-1 inhibitor S63845 synergized with venetoclax. Given the dysregulation of proteins critical to metabolism in both the GOF screen and in OCI-Ly1-R cells, we also evaluated the role of metabolic reprogramming in venetoclax resistance. We first assessed mitochondrial respiration by measuring the oxygen consumption rate. Compared to OCI-Ly-S cells, OCI-Ly1-R cells demonstrated markedly higher respiration levels, suggesting a state of higher oxidative phosphorylation (OXPHOS). More directly, we measured oxygen consumption following venetoclax exposure. Consistent with impairment of OXPHOS by venetoclax, we observed both an immediate decrease in oxygen consumption and an immediate burst of glycolysis following venetoclax in the OCI-Ly1-S cells, but not in the OCI-Ly1-R cells. In line with these findings, the AMPK inhibitor dorsomorphin and mitochondrial electron transport chain (mETC) inhibitors synergized with venetoclax in OCI-Ly1-S cells. Transcriptome related to ID3 (identified as one of the LOF screen targets) was characterized in isogenic ID3-knockout OCI-Ly1 lines. It revealed PRKAR2B overexpression as a key effect, suggesting a role for ID3, and perhaps of other lymphoid transcription factors in regulating metabolic reprogramming associated with resistance. Indeed, exposure of ID3 knockout lines to mETC inhibitors overcame resistance to venetoclax. To determine if there is a genetic basis for the drug resistance seen in OCI-Ly1-R cells, we compared whole-exome sequencing (WES) results of DNA isolated from the OCI-Ly1-R and OCI-Ly1-S cell lines. A clear region was amplified on chromosome 1q23, which includes MCL1 and PRKAB2 (the regulatory subunit of AMPK). Similarly, a WES-based analysis of paired CLL DNA samples isolated from 6 R/R CLL patients just prior to venetoclax initiation and at time of progression on venetoclax was performed. We did not identify any non-silent somatic single nucleotide in BCL2 or its family members at baseline or at progression, despite marked clonal shifts in all patients. We confirmed the presence of the amp(1q23) as acquired at relapse after venetoclax in 3 out of 6 patients. Our study reveals that venetoclax resistance implicates changes not only for outer mitochondrial membrane (MCL-1 expression) but also for inner membrane (oxydative metabolism). Such mitochondrial reprogramming represents a new vulnerability that can potentially be exploited through combinatorial therapy with metabolic modulators to overcome resistance. Disclosures Guieze: abbvie: Honoraria; janssen: Honoraria; gilead: Honoraria. Thompson:Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Adaptive Biotechnologies: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Davids:Merck: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; MEI Pharma: Consultancy, Research Funding; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Surface Oncology: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Brown:Sun Pharmaceutical Industries: Research Funding; Abbvie: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy; Janssen: Consultancy; Sunesis: Consultancy; Roche/Genentech: Consultancy; Verastem: Consultancy, Research Funding; Boehringer: Consultancy; Loxo: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy; Genentech: Consultancy. Wierda:AbbVie, Inc: Research Funding; Genentech: Research Funding. Letai:AstraZeneca: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Vivid Biosciences: Equity Ownership. Wu:Neon Therapeutics: Equity Ownership.