A multiparametric niche-like drug screening platform in acute myeloid leukemia.

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 (O2 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.

Cystine uptake inhibition potentiates front-line therapies in acute myeloid leukemia.

By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.

Screening of ETO2-GLIS2-induced Super Enhancers identifies targetable cooperative dependencies in acute megakaryoblastic leukemia.

Super Enhancers (SEs) are clusters of regulatory elements associated with cell identity and disease. However, whether these elements are induced by oncogenes and can regulate gene modules cooperating for cancer cell transformation or maintenance remains elusive. To address this question, we conducted a genome-wide CRISPRi-based screening of SEs in ETO2-GLIS2+ acute megakaryoblastic leukemia. This approach revealed SEs essential for leukemic cell growth and survival that are induced by ETO2-GLIS2 expression. In particular, we identified a de novo SE specific of this leukemia subtype and regulating expression of tyrosine kinase-associated receptors KIT and PDGFRA. Combined expression of these two receptors was required for leukemic cell growth, and CRISPRi-mediated inhibition of this SE or treatment with tyrosine kinase inhibitors impaired progression of leukemia in vivo in patient-derived xenografts experiments. Our results show that fusion oncogenes, such as ETO2-GLIS2, can induce activation of SEs regulating essential gene modules synergizing for leukemia progression.

Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.

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.

The Folate Cycle Enzyme MTHFR Is a Critical Regulator of Cell Response to MYC-Targeting Therapies.

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.

Using antagonistic pleiotropy to design a chemotherapy-induced evolutionary trap to target drug resistance in cancer.

Local adaptation directs populations towards environment-specific fitness maxima through acquisition of positively selected traits. However, rapid environmental changes can identify hidden fitness trade-offs that turn adaptation into maladaptation, resulting in evolutionary traps. Cancer, a disease that is prone to drug resistance, is in principle susceptible to such traps. We therefore performed pooled CRISPR-Cas9 knockout screens in acute myeloid leukemia (AML) cells treated with various chemotherapies to map the drug-dependent genetic basis of fitness trade-offs, a concept known as antagonistic pleiotropy (AP). We identified a PRC2-NSD2/3-mediated MYC regulatory axis as a drug-induced AP pathway whose ability to confer resistance to bromodomain inhibition and sensitivity to BCL-2 inhibition templates an evolutionary trap. Across diverse AML cell-line and patient-derived xenograft models, we find that acquisition of resistance to bromodomain inhibition through this pathway exposes coincident hypersensitivity to BCL-2 inhibition. Thus, drug-induced AP can be leveraged to design evolutionary traps that selectively target drug resistance in cancer.