Haley E. Ramsey

Targeting oxidative phosphorylation (OXPHOS) represents an attractive therapeutic strategy in acute myeloid leukemia, which exhibits exceptional dependence on mitochondrial respiration compared to normal hematopoietic cells. However, clinical attempts to exploit this vulnerability have been limited by on-target toxicity to healthy tissue. Here, we comprehensively compare the cellular consequences of inhibiting distinct nodes of the electron transport chain in AML. We demonstrate that selective inhibition of the F ₁ subunit of ATP synthase with EB2023 (ammocidin A) delivers an energetic stress to AML cells without the profound redox stress that characterizes complex I inhibition, preventing NAD⁺/NADH imbalance and allowing continued TCA cycling. Further, the duration of OXPHOS inhibition is transient in nature in vivo , a finding revealed through pharmacokinetic and serial pharmacodynamic monitoring of AMPK phosphorylation accompanied by OPA1-mediated mitochondrial structural remodeling that primes AML cells for BCL2 inhibitor synergy. EB2023 in combination with venetoclax demonstrates potent anti-AML activity across cell lines and patient-derived xenograft models at doses that spare normal hematopoietic progenitors and avoid the neuropathy and sustained detrimental systemic metabolic rewiring in healthy tissues associated with prior efforts to target OXPHOS. These findings establish F ₁ -selective ATP synthase inhibition as a clinically actionable therapeutic strategy in AML and establish the duration of OXPHOS inhibition as a critical and previously underappreciated determinant of therapeutic index.

Myelodysplastic syndrome (MDS) is characterized by progressive cytopenias and a substantial risk of transformation to acute myeloid leukemia. Immune dysregulation is central to the pathogenesis of MDS, and immune suppression is often associated with disease progression. However, immunotherapies such as anti-programmed cell death protein-1 therapy have shown limited efficacy in patients, indicating an incomplete understanding of immune suppression in MDS. To better characterize the immune features of MDS, we performed extensive immune phenotyping of MDS patient bone marrow (BM). We demonstrate that V-domain Ig suppressor of T cell activation (VISTA), an inhibitory immune checkpoint, is upregulated in MDS patients by CD34+ blasts and granulocytic myeloid-derived suppressor cells (G-MDSCs) compared to healthy controls, and G-MDSC VISTA expression positively correlates with T cell suppression. In studies employing the NUP98-HOXD13 transgenic MDS mouse model, genetic deletion of VISTA delays the progression of anemia and decreases the rate of death due to severe cytopenias. Assessment of MDS mice reveals that VISTA deletion reduces BM populations of G-MDSCs and reverses the suppression of T cells and macrophages. Together, our findings suggest that VISTA may augment G-MDSC-mediated immune suppression and significantly contribute to ineffective hematopoiesis in MDS.

CRISPR-Cas9 is a useful tool for inserting precise genetic alterations through homology-directed repair (HDR), although current methods rely on provision of an exogenous repair template. Here, we tested the possibility of repairing heterozygous single nucleotide variants (SNVs) using the cell′s own wild-type allele rather than an exogenous template. Using high-fidelity Cas9 to perform allele-specific CRISPR across multiple human leukemia cell lines as well as in primary hematopoietic cells from patients with leukemia, we find high levels of reversion to wild-type in the absence of exogenous template. Moreover, we demonstrate that bulk treatment to revert a truncating mutation in ASXL1 using CRISPR-mediated interallelic gene conversion (IGC) is sufficient to prolong survival in a human cell line-derived xenograft model (median survival 33 days vs 27.5 days; p = 0.0040). These results indicate that IGC can be applied to numerous types of leukemia and can meaningfully alter cellular phenotypes at scale. Because our method targets single-base mutations, rather than larger variants targeted by IGC in prior studies, it greatly expands the pool of risk-increasing genetic lesions which could potentially be targeted by IGC. This technique may reduce cost and complexity for experiments modeling phenotypic consequences of SNVs. The principles of SNV-specific IGC demonstrated in this proof-of-concept study could be applied to investigate the phenotypic effects of targeted clonal reduction of leukemogenic SNV driver mutations.

Table S1: Metabolomics Raw Data

Table S2: Characteristics of AML patient samples tested with BETi.

Figure S4: UK5099 does not enhance the efficacy of BETi in preventing MOLM-13 chimerism. NSGS mice were engrafted with MOLM-13 cells and treated with vehicle control, BETi (50 mg/kg) 5 times a week, twice daily and/or UK5099 (40 mg/kg) three times a week, once daily. After 2 wks, peripheral blood (BL), spleen (SPL), and bone marrow (BM) were harvested for chimerism analysis. Each point represents a single mouse (n = 5). Two-way ANOVA with Tukey multiple comparisons test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant).

Impairing the BET family coactivator BRD4 with small-molecule inhibitors (BETi) showed encouraging preclinical activity in treating acute myeloid leukemia (AML). However, dose-limiting toxicities and limited clinical activity dampened the enthusiasm for BETi as a single agent. BETi resistance in AML myeloblasts was found to correlate with maintaining mitochondrial respiration, suggesting that identifying the metabolic pathway sustaining mitochondrial integrity could help develop approaches to improve BETi efficacy. Herein, we demonstrated that mitochondria-associated lactate dehydrogenase allows AML myeloblasts to utilize lactate as a metabolic bypass to fuel mitochondrial respiration and maintain cellular viability. Pharmacologically and genetically impairing lactate utilization rendered resistant myeloblasts susceptible to BET inhibition. Low-dose combinations of BETi and oxamate, a lactate dehydrogenase inhibitor, reduced in vivo expansion of BETi-resistant AML in cell line and patient-derived murine models. These results elucidate how AML myeloblasts metabolically adapt to BETi by consuming lactate and demonstrate that combining BETi with inhibitors of lactate utilization may be useful in AML treatment.Significance:Lactate utilization allows AML myeloblasts to maintain metabolic integrity and circumvent antileukemic therapy, which supports testing of lactate utilization inhibitors in clinical settings to overcome BET inhibitor resistance in AML.See related commentary by Boët and Sarry, p. 950

Figure S1: Glutaminolysis and fatty acid oxidation does not allow MOLM-13 to metabolically bypass BET inhibition. (A) MV-4-11 and MOLM-13 cells were cultured for 72 hr with BETi (0.15 µM) and viability quantified using a fluorescent plate reader. Viability was quantified relative to cells treated with vehicle control. Each point represents the mean (technical triplicate) value obtained from cells from an individual experiment (n=3). (B-E) MV-4-11 and MOLM-13 cells were loaded with a CellTrace dye and cultured for 72 hr with BETi and either (B-C) V-0302 (25 µM) or (D-E) etomoxir (150 µM). (B, D) To assess replication, CellTrace fluorescence intensity was quantified by flow cytometry and (C, E) viability was quantified in a fluorescent plate reader. Replication and viability were quantified relative to cells treated with vehicle control. (F) Raw CT values from qPCR experiment in Fig. 2A. Each point represents (B, D) the median fluorescent intensity (MFI) from or (C, E) the mean (technical triplicate) value obtained from cells from an individual experiment (n=3). (A) Paired t test or (B-E) Two-way ANOVA with Sidak’s multiple comparisons test (**P ≤ 0.01, ns = not significant).

Table S1: Metabolomics Raw Data

Figure S2: Maximal respiration in MOLM-13 cells is unaffected by the addition of BETi and/or lactate utilization inhibitors. MV-4-11 and MOLM-13 cells were cultured for 72 hr with indicated treatments (BETi = 0.15 µM; AZD3965, UK5099, oxamate = 0.1 µM), subjected to a mitochondrial stress test, and oxygen consumption rates (OCR) quantified by extracellular flux analysis. Maximal respiration was quantified where each point represents individual experiments (n=3). Two-way ANOVA with Sidak’s multiple comparisons test (*P ≤ 0.05, ns = not significant).