Targeted Suppression of Interleukin-1 Signaling Attenuates Leukemic Cellular Growth and Disease Progression in Primary Human Cells and Murine Models of Acute Myeloid leukemia­­

Background: Despite the great strides that have been made in the treatment of AML, resistance to treatment is common and signaling pathways influencing leukemic cellular proliferation have not been comprehensively defined. Cytokines and growth factors play important roles in cell survival, proliferation, differentiation, senesence, and immune response in both normal and cancer cells. Recent studies have demonstrated that certain inflammatory cytokines known to suppress normal hematopoiesis can have the opposite effect (i.e. enhancement of proliferation) on cell growth of AML cells. To test the importance of this mechanism, we systematically assessed the functional relevance of 98 cytokines and their receptors in primary human AML cells and identified interleukin-1 (IL-1) and its receptor as critical determinants in aberrant regulation of AML cell growth.

Methods: To identify functionally important cytokine signaling pathways in AML pathogenesis we first quantified the proliferation of 50 primary AML patient samples in the presence of graded concentrations of 98 cytokines. We then employed a functional siRNA screen targeting 188 cytokine and growth factor receptors found to be highly expressed in 140 primary leukemia samples by gene expression analysis. Cytokine pathways of interest were functionally tested for their role as potential therapeutic targets utilizing primary patient samples and in vivo murine models.

Results: We found that 40% of primary AML samples exhibited a 3- to 20-fold increase in cellular growth and a 2-fold decrease in apoptosis in the presence of IL-1α/β. Paradoxically, IL-1 suppressed the growth of normal CD34+ cells. Silencing of the IL-1 receptor, IL1R1, reduced the viability of these AML primary samples by 60-80%. Notably, most of the IL-1-sensitive AML samples exhibited monocytic and myelomonocytic features. To demonstrate the importance of IL-1 signaling in the survival of AML cells, we utilized IL1R1-/- mice and oncogene-induced leukemic cells in vitro and in vivo. We observed that IL1R1-/- mice and wild-type mice showed a normal distribution of stem and progenitor populations in their marrow by FACS analysis. Further, the absence of IL1R1 in murine bone marrow leads to a significant ablation of clonogenic potential (80% reduction) of oncogene-induced leukemic cells (AML1-ETO9a, NRAS^G12D and MLL-ENL) when compared to oncogene-induced leukemic cells from wild-type mice in a ligand-dependent manner. No difference in colony growth was observed with empty vector-transduced marrow cells. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRAS^G12D survived significantly longer for a median of 39 days (range: 28-118) compared to 30 days (range: 27-61) for recipients of wild-type marrow (p=0.012). Mice transplanted with IL1R1-/- marrow or wild-type marrow showed comparable white blood cell and platelet counts. However, histopathological analysis showed a significant reduction in myeloid infiltrates in liver and lungs as well as reduced marrow cellularity and reticulin fibrosis in IL1R1-/- leukemic mice as compared to wild-type leukemic mice. To exclude the possibility that this difference was related to differences in homing and engraftment of wild-type and IL1R1-/- bone marrow cells, we compared short-term homing and long-term engraftment of empty vector-transduced wild-type and IL1R1-/- marrow cells and found no significant differences. These results demonstrate an in vivo role of IL1-receptor in AML cell proliferation and progression. Mechanistically, exogenous IL-1 promotes the growth and survival of primary CD34+ AML cells and AML cell lines by increasing p38 MAPK phosphorylation. Conversely, knocking down IL1R1 or treating AML cells with p38 kinase inhibitors such as doramapimod (n=10; median IC₅₀: 70 nM) reduced the growth of AML cells by decreasing phosphorylation of p38 kinase. No toxic effect of doramapimod was observed on normal cell growth, suggesting that targeting p38 kinase might be therapeutically beneficial for a subset of AML patients dependent on IL-1 signaling.

Conclusion: These results demonstrate a novel in vitro and in vivo role for IL-1 and its receptor in promoting leukemic cellular growth and progression in a large subset of AML patients and warrant further investigation of this pathway as a therapeutic opportunity.