A Circulating Microbial Metabolite Drives the Clonal Expansion of Pre-Leukemic Cells

Inflammatory mediators, including cytokines and microbial ligands, are implicated in the development of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A consequence of aging and various autoimmune and inflammatory conditions is loss of intestinal barrier integrity and translocation of microbial species and metabolites into circulation. More recent findings revealed that patients with inflammatory bowel disease (IBD) harbor mutant hematopoietic clones and often present with clinical manifestations of MDS/AML. Moreover, intestinal barrier dysfunction is commonly reported at diagnosis in patients with MDS and AML. These observations led us to speculate that microbial-derived signals may contribute to the expansion of pre-leukemic hematopoietic stem and progenitor cells (HSPCs) and development of myeloid malignancies.

To determine if loss of intestinal barrier integrity contributes to the expansion of pre-leukemic DNMT3a-mutant HSPCs, we generated a model of IBD by inducing gut epithelial injury using dextran sulfate sodium (DSS). Upon gut epithelial injury, we observed a significant expansion of DNMT3a-mutant HSPCs, but not of wild-type (WT) HSPCs, in vivo. Unexpectedly, DNMT3a-mutant HSPCs do not exhibit a competitive advantage over WT cells in the absence of intestinal barrier dysfunction, such as following DSS or high-dose radiation. Fecal material from DSS-treated WT mice was sufficient to induce expansion of DNMT3a-mutant HSPCs and this expansion was prevented with broad-spectrum antibiotics. These findings indicate that the competitive advantage of DNMT3a-mutant HSPCs following gut epithelial injury was due to alterations in the microbiome.

In parallel sequencing studies, we identified a significant enrichment of gram-negative bacterial phyla in stool samples from the IBD mouse model, IBD patients, and treatment-naive AML patients. All gram-negative bacteria uniquely generate a microbial intermediate, ADP-heptose, as part of the lipopolysaccharide biosynthesis pathway. ADP-heptose can enter the blood circulation and freely translocate into cells and activate the ALPK1-TIFA complex ("TIFAsome"). Alpha-protein kinase 1 (ALPK1) and tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with an FHA domain (TIFA) are overexpressed in primary human MDS and AML HSPCs as compared to healthy donors and their expression is correlated with poor prognosis. Gram negative bacteria and corresponding ADP-heptose levels were also significantly increased in the plasma of individuals with intestinal barrier injury, such with IBD, MDS, or upon aging, as compared to young healthy individuals. The circulating levels of ADP-heptose from aged individuals and IBD or MDS patients were sufficient to induce the TIFAsome in leukemic cells. Moreover, administration of disease relevant levels of ADP-heptose resulted in expansion and enhanced function of mouse DNMT3a-mutant HSPCs and of human AML cell lines and primary MDS cells in vivo. Expansion of DNMT3a-mutant HSPCs and human MDS/AML cells was directly related to overexpression of ALPK1, as WT HSPCs or ALPK1-deficient AML cells did not respond to ADP-heptose.

To gain deeper mechanistic insight, we examined global transcriptional and epigenetic changes in purified WT and DNTM3a-mutant HSCs treated with ADP-heptose. DNMT3a-mutant HSCs treated with ADP-heptose resulted in epigenetic changes and increased expression of gene programs related to non-canonical NF-kB, ERK/MAPK, cell cycle activation, cell survival, and stem cell function. Indeed, ADP-heptose caused increased cycling and self-renewal of DNMT3a-mutant HSPCs, but not WT HSPCs, via the TIFAsome in vitro and in vivo. Based on the enriched pathways in DNMT3a-mutant HSCs treated with ADP-heptose, we screened small molecule inhibitors of TIFAsome activation. We found that targeting UBE2N abrogated ADP-heptose-mediated signaling via the TIFAsome and expansion of patient-derived AML xenografts.

In summary, we report that conditions leading to intestinal barrier dysfunction and circulating ADP-heptose from gram-negative bacteria induce expansion of pre-leukemic and MDS/AML cells via ALPK1-TIFA. Lastly, we provide a potential therapeutic strategy to prevent the competitive advantage of pre-leukemic cells prior to overt malignant transformation.

Setchell:Mirum Pharmaceuticals: Consultancy; Asklepion Pharmaceuticals: Current equity holder in private company; Aliveris s.r.l.: Current equity holder in private company; Travere Therapeutics: Consultancy. Starczynowski:Sumitomo Pharma Oncology, Inc: Research Funding; Captor Therapeutics: Consultancy; Kurome Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tolero Therapeutics: Research Funding; Treeline Biosciences: Research Funding; Kymera Therapeutics: Consultancy.