Targeting adss overcomes venetoclax resistance in TP53-mutated acute myeloid leukemia Free

Venetoclax (VEN), when combined with hypomethylating agents such as azacitidine (AZA), is a critical FDA-approved AML therapy for older patients unfit for intensive chemotherapy. Despite its efficacy, resistance can emerge, and response duration is short, representing an unmet clinical need. The presence of TP53 mutations in 5–10% of newly diagnosed AML cases directly associate with VEN resistance (VEN-R), although underlying mechanisms are not well understood. Aberrant purine metabolism has been seen in cells tolerant to VEN treatment, but the driver gene(s) or their functional relevance remain elusive. Recently, employing a dynamic BH3-priming CRISPR screen, we identified ADSS2, an enzyme functioning in AMP biosynthesis, as a key modulator of VEN sensitivity; ADSS2 deletion re-sensitized VEN- and MCL1i-resistant AML cells by suppressing AMPK signaling (He X et al., ASH 2023). Recently, we evaluated ADSS2 expression in two VEN-R TP53 loss-of-function Molm13 cell lines: TP53-knockout (KO) and R248Q/− isogenic cells. Both models exhibited elevated ADSS2 expressions relative to TP53 wild-type counterparts. Moreover, ADSS2 KO combined with low-dose VEN induced robust apoptosis in both models, highlighting that high ADSS2 activity promotes TP53-mutant AML phenotypes.

Herein, to investigate the mechanism of ADSS2 upregulation, we first screened out 31 candidate transcription factors (TFs) associated with ADSS2 transcription through the TFBIND database. Further correlation analysis between TF and ADSS2 expression in the Beat AML cohort, followed by shRNA-mediated knockdown plus ChIP/qPCR validation, identified c-Myc as a critical regulator of ADSS2. Notably, c-Myc levels were significantly elevated in TP53-KO and TP53 R248Q/− Molm13 isogenic cells relative to respective parental counterparts. To track c-Myc-ADSS2-high cell emergence and their relationship to TP53 mutation at single-cell resolution, we utilized Genotyping of Transcriptomes (GoT) approach, which overlays TP53 mutational status onto single-cell RNA-seq data. We applied this approach to a paired TP53 mutant (c.742C>T, p.R248Q) patient-derived xenograft (PDX) sample prior- and after VEN/AZA treatment. Briefly, NSGS mice were engrafted with PDX blasts and treated with either V/A (VEN: 100mg/kg/i.g./q.d., 5 times/week, AZA: 3mg/kg/i.p./t.i.w) or vehicle until resistance developed and then bone marrow (BM) PDX cells were harvested for analysis. The frequency of genotyped blasts in either drug-naïve or post-treatment groups was approximately 83.2%, consistent with reported genotyping efficiency. Comparison of drug-naïve with resistant PDX cells revealed markedly increased ADSS2 and c-Myc expression in the resistant state. Genotype overlay on transcriptome maps showed that TP53-mutant cells expressed significantly higher levels of ADSS2 and c-Myc than did TP53-wild-type cells in drug-naïve samples. Notably, at relapse, the TP53-mut population expanded from 37.5% to 63.2%, contributing to c-Myc and ADSS2 upregulation in resistant samples. These results suggest that ADSS2 upregulation in resistant cells is associated with clonal selection of pre-existing TP53-mutant cells exhibiting elevated c-Myc and ADSS2 levels.

We next assessed the therapeutic potential of Cmpd3, our newly developed ADSS2 inhibitor, as monotherapy or combined with V/A in NSGS mice engrafted with a TP53-mutant AML PDX. After confirming engraftment, we treated mice 3 weeks with vehicle, Cmpd3 (100 mg/kg, intraperitoneally, once daily, five times per week), V/A, or Cmpd3 plus V/A. Analysis of BM samples revealed that combination therapy significantly decreased the leukemic burden compared to either monotherapy, indicating a synergistic anti-leukemic effect in this TP53-mutant AML model. We also assessed the impact of Cmpd3 on normal hematopoiesis in WT C57BL/6 mice. Mice treated for 4 weeks with Cmpd3 showed no significant changes in organ (kidney, liver, spleen) histology, body weight, or HSPC frequency, and only modestly decreased numbers of BM B and T cells, indicating relative safety of Cmpd3.

Collectively, our study reveals ADSS2 to be a contributor to TP53-associated VEN resistance and a promising target to restore drug sensitivity in this high-risk AML subtype.