Session: 602. Myeloid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Biological Processes, pathogenesis
WT1 mutations are enriched in the NPM1mut subset of AML; thus we first focused our study on a cohort of 581 patients with de novo AML and NPM1 mutations enrolled on Alliance for Clinical Trials in Oncology studies. Transcriptomic analyses revealed that WT1mut patients phenocopied a distinct, aberrant gene expression signature associated with FLT3 internal tandem duplications (ITD), a mutation known to activate STAT signaling and associated with poor outcome in AML. We observed that WT1 expression levels were remarkably elevated in FLT3-ITD patients and were driven by STAT5A binding to the WT1 promoter. STAT5A binding and subsequent WT1 upregulation were blocked by small molecule FLT3 inhibitors.
These findings linking FLT3 activity with WT1 expression raise two possibilities: either WT1 is an oncogene cooperating with the FLT3-STAT pathway, or WT1 naturally functions to suppress FLT3 signaling in a negative feedback loop subverted by WT1 mutations. As WT1 interacts with TET2 to facilitate epigenetic remodeling and DNA binding, we performed genome-wide DNA methylation analysis on WT1mut AML patients and observed selective hypermethylation of WT1 binding motifs consistent with loss of function. We confirmed that WT1 mutations in AML commonly cause truncation of the C-terminal DNA binding domain in our patient cohort. Using co-immunoprecipitation, ChIP-sequencing and luciferase reporter assays, we found that mutant WT1 functions as a dominant-negative, inhibiting WT1 derived from the wild-type allele. Investigation of WT1 target genes by intersecting transcriptomic and DNA methylation profiles identified hypermethylation and downregulation of miR-193a, leading to a significant downregulation of miR-193a-3p in WT1mut AML patients. Overexpression of wild-type, but not mutated, WT1 rescued miR-193a expression in AML cell lines. Enforced expression of miR-193a significantly delayed AML onset in vivo. In addition, overexpression of miR-193a in multiple human AML cell lines and primary patient samples impaired AML cell growth and colony-forming capacity while promoting monocytic differentiation, underscoring its role as potent tumor suppressor. Therapeutic modulation of miRNA levels in cancer patients has been limited by inefficient delivery and tissue enrichment. To overcome this, we tested a novel lipid-nanoparticle (LNP) formulation of miR-193a-3p (INT-1B3), currently being investigated in a phase I clinical trial (NCT04675996). Biweekly i.v. treatments of INT-1B3 in the immunocompetent Hoxa9/Meis1 (H9M)-transduced model system prevented AML formation, highlighting the potent anti-leukemic activity of this miRNA based therapeutic. Overexpression of miR-193a-loaded LNPs downregulated FLT3 expression and suppressed STAT signaling in primary AML samples. Finally, treatment of primary AML cells with FLT3 inhibitors revealed enhanced sensitivity of WT1mut cells in the absence of FLT3-ITD, highlighting the role of wild-type FLT3 in WT1mut cells.
In summary, we uncovered a critical negative-feedback loop maintained by WT1 to suppress FLT3 activity. Loss-of-function WT1 mutations subvert the tumor suppressor function of WT1 via failure to maintain miR-193a expression, leading to increased FLT3 expression and STAT5 signaling, subsequently impairing differentiation, increasing proliferation and disease aggressiveness in AML (Figure 1). Our findings advocate for use of FLT3 inhibition and miR-193a supplementation for treatment of WT1mut patients, a subgroup with poor outcomes and no targeted treatment options.
Disclosures: Abdelbaky: Roche: Ended employment in the past 24 months. Blachly: Astellas: Consultancy; Leukemia Diagnostic Device: Patents & Royalties: Being prosecuted; Epigenetic classification of leukemia: Patents & Royalties: PCT conversion filed; AstraZeneca: Consultancy; AbbVie: Consultancy. Baer: Abbvie (Inst): Research Funding; Ascentage Pharma (Inst): Research Funding; FORMA Therapeutics (Inst): Research Funding; Kite, a Gilead company (Inst): Research Funding; Kura Oncology (Inst): Research Funding; Takeda (Inst): Research Funding. Klusmann: Boehringer Ingelheim: Consultancy; Jazz Pharmaceuticals: Consultancy. Eisfeld: Karyopharm Therapeutics: Other: spouse employment; Astra Zeneca: Honoraria, Other: CEI Advisory Board; OncLive: Honoraria.