Cytokine-Mediated Inflammatory Pathways Promote Clonal Evolution and Disease Progression in Acute Myeloid Leukemia

Background: Inflammatory cytokines secreted in the bone marrow microenvironment play important roles in modulating cell survival, proliferation, differentiation, and immune responses in cancer. Perhaps not surprisingly, there is also an association between chronic inflammation and tumor progression. We recently used an ex vivo functional screen of 94 cytokines to show that the pro-inflammatory cytokines IL1α and IL1β promoted the expansion of AML progenitors in 70% (40/60) of primary samples. We therefore hypothesized that inflammatory cytokines are crucial to clonal expansion and disease progression in AML and that therapeutic targeting of these pathways may circumvent disease heterogeneity. Here we provide in vitro and in vivo evidence that IL1-mediated signaling elicits profound expansion of leukemia progenitors in AML patients harboring various genetic mutations and promotes in vivo clonal expansion and disease progression in a murine AML model. Further, these effects are reversed by targeting IL1 signaling.

Methods: We validated the role of IL1 signaling using a shRNA approach and in murine competitive repopulation and bone marrow transplantation models. We evaluated the influence of these cytokines on inflammatory markers using immunoblotting, flow cytometry, and Luminex assays, and assessed strategies to target these pathways using small-molecule inhibitors.

Results: IL1 stimulation promoted a 3- to 20-fold increase in growth, survival, and clonogenic potential of AML CD34+ cells, while paradoxically suppressing growth of healthy CD34+ cells. To identify the influence of IL1 on in vivo clonal expansion of healthy and leukemic progenitors simultaneously, we established an in vivo murine competitive repopulation study utilizing TET2-null mice. IL1 treatment promoted clonal expansion of TET2-null myeloid cells over wild-type cells during 6 weeks of IL1β treatment. Consistent with this, both flow cytometry analysis and blood differential counts showed an increased percentage of granulocytes and reduced percentage of lymphocytes in IL1-treated mice. In this model, TET2-null monocytes have greater expression of IL1 than wild-type cells, suggesting IL1 promotes clonal growth of TET2-mutated early leukemic progenitors. Similarly, IL1β and IL1 receptors (IL1R1 and IL1RAP) were overexpressed in IL1-sensitive AML bone marrow and peripheral blood samples compared to nonsensitive AML samples and normal samples. Intracellular FACS showed that the majority of IL1β was secreted by monocytes and to some extent by myeloid progenitors. Accordingly, IL1-sensitive AML samples exhibited trends towards monocytic and myelomonocytic clinical features. Reduced survival of AML cells after monocyte depletion was rescued by IL1 treatment, suggesting that IL1 mediates paracrine regulation of AML cell growth. Silencing of the IL1 receptor, IL1R1, reduced the clonogenic potential of AML primary samples and oncogene (AML1-ETO9a, NRAS^G12D, and MLL-ENL)-transduced mouse bone marrow. In a murine bone marrow transplantation model, recipients of IL1R1^-/- marrow transduced with AML1-ETO9a/NRAS^G12D survived significantly longer than did recipients of wild-type marrow. We also found that IL1β increased phosphorylation of p38MAPK and MK2, as well as secretion of multiple downstream inflammatory cytokines (IL6, IL8, MCP1, MIP1α, and MIP1β) from CD34+ progenitors, in IL1-sensitive AML samples compared to IL1-nonsensitive progenitors. Conversely, treating AML cells with p38MAPK inhibitors such as doramapimod andralimetinib reduced the growth of AML cells by decreasing p38MAPK and MK2 phosphorylation and reducing secretion of inflammatory cytokines from AML progenitors. Clinical and demographic analyses suggest that AML patients are dependent on IL1 signaling irrespective of mutation status and clinical features. Targeting this unifying mechanism of IL1-mediated clonal expansion may thus have application across heterogeneous AML subtypes.

Conclusion: We demonstrate that IL1 promotes in vitro and in vivo clonal expansion of leukemic cells and promotes disease progression in AML. As IL1 signaling is active across heterogeneous disease subtypes, AML patients may therefore benefit from drugs targeting IL1/p38MAPK signaling because of their potential to inhibit AML while enhancing normal hematopoiesis, a significant clinical advantage over traditional chemotherapy.