Abstract: 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.

Abstract: Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O 2 3%) and mesenchymal stromal cell (MSC) co-culture with multiparameter flow cytometry to enumerate lymphocytes and differentiating (CD11/CD14/CD15+) or leukemic stem cell (LSC)-enriched (GPR56+) cells within the leukemic bulk. After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically ( p  = 0.02) and molecular ( NPM1c MRD, p  = 0.04) response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model.

Abstract: Metabolic rewiring of neoplastic cells engenders metabolic liabilities that can be exploited to design innovative therapeutic strategies, including those to increase the therapeutic index of existing anticancer therapies. We hypothesized that metabolic perturbation may substantially influence cell response to therapies targeting major oncogenes which are involved in active hijacking of neoplastic cell metabolism. In that regard, MYC represents a paradigmatic oncogene as this transcription factor is deregulated in more than 50% of human cancers and reprograms many aspect of cell metabolism. MYC expression is controlled by clusters of super-enhancer genomic regions densely occupied by transcription factors and chromatin regulators ― including BET bromodomain proteins, and CDK7 and CDK9 kinases. Two cohorts of patients with Acute Myeloid Leukemia, AML (TCGA-LAML, n=198 and GSE14468, n=526) were queried with multiple gene sets in order to reveal a core of metabolic gene signatures, which are connected to the folate cycle, and whose activation was poorly correlated with an active MYC transcriptional program in AML. According to these data, we established that folate cycle disruption upon folic acid starvation consistently enhanced resistance to MYC targeting by BET or CDK7 inhibitors (JQ1, OTX015, THZ1) as well as BRD4-directed shRNAs in a large panel of human AML cell lines harboring a wide variety of genetic alterations (n=7), in MLL-translocated primary patient samples with AML (n=4), and in animals injected with MLL-AF9-positive leukemic cells. Using an shRNA-based screening approach against enzymes from the folate cycle, we revealed that the knockdown of the rate-limiting enzyme in the folate cycle, 5,10-methylenetetrahydroflate reductase (MTHFR), significantly increased resistance to OTX015 in AML cell lines (n=4) and in animals transplanted with Mthfr-depleted blasts. Previous reports have identified and extensively studied two common genetic variants in the MTHFR gene, C677T and A1298C, encoding two MTHFR enzyme variants with reduced activity in about 10% of Caucasians. We introduced in KG1a cells these two non-synonymous single nucleotide polymorphisms in MTHFR using CRISPR-Cas9, thereby generating isogenic cell lines exhibiting all combinations of variants. Although the clones which are heterozygous for any of the two variants had similar sensitivity to OTX015 as wild-type clones, 677 TT and 1298 CC homozygous KG1a clones were significantly more resistant to OTX015 than their wild-type counterpart, an effect that was alleviated by exogenous overexpression of wild-type MTHFR or supplementation of cells with the end-product metabolite synthesized by MTHFR, 5-CH3 THF. Consistent with these data, MLL-translocated patients displaying a homozygous and compound heterozygous MTHFR genotype for any of the two variants (n=8) responded significantly less to OTX015 than those with wild-type homozygous and heterozygous MTHFR genotypes (n=8). Finally, we established that the loss of a single copy of Mthfr which phenocopies in mice a partial impairment in MTHFR activity caused by non-synonymous single nucleotide polymorphisms on MTHFR, was sufficient to attenuate sensitivity to JQ1 of MLL-AF9-driven leukemias. Using metabolomics profiling, we pointed out that a major effect of folate cycle disturbance in AML cells is the intracellular accumulation of S-adenosyl-homocysteine, SAH, which is the downstream effector of MTHFR knockdown triggering BET inhibitor resistance. Given that SAH is a potent inhibitor of SAM-dependent methylation reactions, we determined that folate cycle impairment decreases H3K27 and H3K9 methyltransferase activities and subsequent methylation of H3K27 and H3K9 histone marks across the whole genome of AML cells. By combining ChIP- and RNA-sequencing approaches, we demonstrated that decreased methylation levels of H3K27 and H3K9 histone marks upon folate cycle alteration combined with BET inhibition activates SPI1 and IRF / Interferon signaling transcriptional programs. SPI1 knockdown significantly reduced the resistance to OTX015 of AML cells whose MTHFR expression was suppressed or MLL-AF9-transformed Mthfr knockout primary murine cells. Our data provide a rationale for screening MTHFR polymorphisms and the folate cycle status to exclude patients least likely and nominate those most likely to benefit from MYC-targeting therapies. Disclosures Dombret: CELGENE: Consultancy, Honoraria; AGIOS: Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Stegmaier:Rigel Pharmaceuticals: Consultancy; Novartis: Research Funding.

Abstract: 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 & lt;0.0001, Figure 1B) and limit dilution assays of residual GPR56+ cells at 72h of niche-like culture in 3 NPM1mut AMLs (Figure 1C). Using a limited panel of 14 drugs or combinations at fixed concentrations, our MFC readout after 72h of coculture with MSC+hypoxia revealed the distinct mode of action of different agents or combinations including the differentiation activity of ATO-ATRA, the LSC-sparring cytotoxicity of DNR-AraC and the anti-LSC- activity of VEN (Figure 1D). 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.

Abstract: 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

Abstract: CRISPRi screening in AMKL identified specific Super Enhancers controlling expression of targetable cooperative dependencies.