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Effect of MTHFR impairment on response to OTX015.

List of sgRNA Guides Used in the CRISPR Screen.

Deciphering the impact of metabolic intervention on response to anticancer therapy may elucidate a path toward improved clinical responses. Here, we identify amino acid–related pathways connected to the folate cycle whose activation predicts sensitivity to MYC-targeting therapies in acute myeloid leukemia (AML). We establish that folate restriction and deficiency of the rate-limiting folate cycle enzyme MTHFR, which exhibits reduced-function polymorphisms in about 10% of Caucasians, induce resistance to MYC targeting by BET and CDK7 inhibitors in cell lines, primary patient samples, and syngeneic mouse models of AML. Furthermore, this effect is abrogated by supplementation with the MTHFR enzymatic product CH3-THF. Mechanistically, folate cycle disturbance reduces H3K27/K9 histone methylation and activates a SPI1 transcriptional program counteracting the effect of BET inhibition. Our data provide a rationale for screening MTHFR polymorphisms and folate cycle status to nominate patients most likely to benefit from MYC-targeting therapies.Significance:Although MYC-targeting therapies represent a promising strategy for cancer treatment, evidence of predictors of sensitivity to these agents is limited. We pinpoint that folate cycle disturbance and frequent polymorphisms associated with reduced MTHFR activity promote resistance to BET inhibitors. CH3-THF supplementation thus represents a low-risk intervention to enhance their effects.See related commentary by Marando and Huntly, p. 1791.This article is highlighted in the In This Issue feature, p. 1775

Supplementary Material and Methods

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

Hematopoietic stem cell (HSC) transplantation (HSCT) is often exploited to treat hematologic disease. Donor HSCs must survive, proliferate, and differentiate in the damaged environment of the reconstituting niche. Illuminating molecular mechanisms regulating the activity of transplanted HSCs will inform efforts to improve HSCT. Here, we report that G-protein–coupled receptor–associated sorting proteins (GPRASPs) function as negative regulators of HSCT. Silencing of Gprasp1 or Gprasp2 increased the survival, quiescence, migration, niche retention, and hematopoietic repopulating activity of hematopoietic stem and progenitor cells (HSPCs) posttransplant. We further show that GPRASP1 and GPRASP2 promote the degradation of CXCR4, a master regulator of HSC function during transplantation. CXCR4 accumulates in Gprasp-deficient HSPCs, boosting their function posttransplant. Thus, GPRASPs negatively regulate CXCR4 stability in HSCs. Our work reveals GPRASP proteins as negative regulators of HSCT and CXCR4 activity. Disruption of GPRASP/CXCR4 interactions could be exploited in the future to enhance the efficiency of HSCT.

Context. Functional precision medicine is gaining momentum in AML, notably through ex vivo drug sensitivity screening (DSS) of primary patient (pt) cells (Pemovska Cancer Discov 2013, Tyner Nature 2018). The DSS landscape differs across genetic AML subgroups (Tyner Nature 2016), of which NPM1mut is the most frequent (Papaemmanuil NEJM 2016). DSS in AML has mostly been done in standard conditions, with overall viability as unique endpoint. Niche signals, which can be partly mimicked in vitro, convey drug resistance in vivo. Drugs can induce a variety of cell fates in AML. Induction of differentiation rather than killing of blasts, can result in false negative results in global viability assays. Persistence of leukemic stem cells (LSC) represents a major cause of treatment failure. GPR56 is a ubiquitous surface marker enriching for LSCs and stable upon short-term ex vivo culture (Pabst Blood 2016). Objectives. To develop an ex vivo niche-like multiparametric DSS platform for primary AML cells. To validate its clinical relevance in NPM1mut pts treated with conventional DNR-AraC chemotherapy. To discover new sensitizers to DNR-AraC chemotherapy in NPM1mut AML. Results. We designed an MFC panel to count viable blasts and measure their differentiation (CD11b/CD14/CD15) and stemness (GPR56) after exclusion of residual lymphocytes (Figure 1A). We validated GPR56 expression as stemness marker based on increased retention of GPR56+ cells in niche-like coculture combining hypoxia (O2 3%) and MSC compared to standard conditions (p To further mimic in vivo conditions, we derived a MEMa-based plasma-like medium (PLM) based on targeted metabolomics (Figure 1E) and electro-chemoluminescent cytokine assays of 29 diagnostic AML bone marrow plasma samples compared to conditioned media of primary AML cells cultured in niche-like conditions (MSC, hypoxia). This instructed the design of our custom PLM with dialyzed FBS and defined low-dose (~1 ng/mL range) cytokines (CK) and amino-acid (AA) concentrations. We next investigated the contribution of MSCs, hypoxia, plasma-like AAs and CKs on blasts viability, differentiation, stemness and drug response in 3 NPM1mut AMLs exposed to fixed concentrations of 6 core AML therapies. This analysis uncovered significant interactions between these 4 niche components in dictating blast viability and stemness upon 72h ex vivo culture (Figure 1F) and revealed the distinct contribution of these niche components to drug sensitivity. RNA-seq of primary blasts cultured in niche-like, plasma-like conditions revealed marked enrichment of stemness pathways compared to ex vivo culture in standard conditions. Finally, we explored DNR-AraC (five-point serial dilution) alone or in combination with fixed, clinically relevant concentrations of 24 drugs in 49 primary AML samples (including 34 NPM1mut). Using AUCs of DNR-AraC on lymphocytes as internal control, we first validated our NEXT assay on NPM1 MRD levels in the 34 NPM1mut pts treated frontline with conventional DNR-AraC regimens (Figure 1G). Across all 49 pts, we uncovered 11 different optimal 'third-drugs', stressing the role of our NEXT assay to deploy precision medicine in daily practice. At the population level, we could nominate 3 top combinations, two of which are currently in clinical investigation (Venetoclax and Selinexor). The unpublished sensitizing effect of low dose (0.25µM) Ruxolitinib on DNR-AraC uncovered with our NEXT assay is currently being investigated in PDX models. Conclusion. We designed the NEXT assay, a multiparametric drug screening of AML viability, differentiation and stemness in niche-like culture combining hypoxia, stromal interactions and plasma-like medium. Components of the niche-like culture interact to govern leukemic viability and stemness. Our assay could predict MRD achievement in NPM1mut AML and identifies novel sensitizers to DNR-AraC in these pts. Disclosures Clappier: Amgen: Honoraria, Research Funding. Ades:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; novartis: Research Funding; Celgene/BMS: Research Funding. Itzykson:Amgen: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Stemline: Membership on an entity's Board of Directors or advisory committees; Oncoethix (now Merck): Research Funding; Janssen: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Daiichi Sankyo: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS (Celgene): Honoraria; Sanofi: Honoraria; Astellas: Honoraria.