Summary Secreted proteins in the bone marrow microenvironment play critical roles in acute myeloid leukemia (AML). Through an ex vivo functional screen of 94 cytokines, we identified that the pro-inflammatory cytokine interleukin-1 (IL-1) elicited profound expansion of myeloid progenitors in ~67% of AML patients while suppressing the growth of normal progenitors. Levels of IL-1β and IL-1 receptors were increased in AML patients, and silencing of the IL-1 receptor led to significant suppression of clonogenicity and in vivo disease progression. IL-1 promoted AML cell growth by enhancing p38MAPK phosphorylation and promoting secretion of various other growth factors and inflammatory cytokines. Treatment with p38MAPK inhibitors reversed these effects and recovered normal CD34+ cells from IL-1-mediated growth suppression. These results highlight the importance of ex vivo functional screening to identify common and actionable extrinsic pathways in genetically heterogeneous malignancies and provide impetus for clinical development of IL-1/IL1R1/p38MAPK pathway-targeted therapies in AML.

Key Points Coordinated BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. Oncogenic functions of p27 that persist despite effective BCR-ABL1 inhibition may contribute to resistance to tyrosine kinase inhibitors.

Background: p38 mitogen-activated protein kinase (p38MAPK) is activated by various pro-inflammatory and stress-related stimuli, and has been an attractive therapeutic target for autoimmune diseases. p38MAPK (hereafter referred to as p38) signaling is also involved in cell proliferation, differentiation, apoptosis, and invasion, suggesting that it may be a potential therapeutic target for cancer. We found that inflammatory cytokines, including interleukin-1 (IL-1), promote growth and survival of more than half of the acute myeloid leukemia (AML) patient samples we tested. Since p38 is a downstream mediator of inflammatory pathways, we hypothesized that targeting p38 might be an effective therapeutic strategy in AML and other hematologic malignancies. To test this hypothesis, we evaluated the effectiveness of three p38 inhibitors using in vitro studies in primary AML patient samples. We found that targeting p38 blocks IL-1-activated extrinsic signaling and is a critical therapeutic target in a large subset of AML patients. Methods: We screened ~1000 primary leukemia patient samples for sensitivity to p38 inhibition using varying concentrations of doramapimod (BIRB 796) in a cell growth assay. We compared the sensitivity profile of doramapimod with 2 other small-molecule p38 inhibitors currently in clinical trials: ARRY 614, a dual p38/Tie2 inhibitor, and ralimetinib, which blocks activation of p38 by its substrate MK2. We determined cell viability, survival, and downstream signaling in the presence of 10 ng/ml IL-1α or IL-1β. Patient samples with IC50 < 1000nM were considered drug responsive. Results: In our patient population, we observed response rates of 31% in AML (109/350), 27% in myelodysplastic syndromes (MDS; 25/93), 19% in myeloproliferative neoplasms (23/123), 13% in mature B-cell neoplasms (30/232), and 10% in precursor lymphoid neoplasms (19/182). Focusing on AML, we compared the sensitivity profile of doramapimod with two other small-molecule p38 inhibitors, ralimetinib and ARRY 614. These inhibitors showed strikingly similar sensitivity profiles to doramapimod when tested in an additional 25 primary AML samples, with ~25% responsive and median IC50 of 11 nM for ARRY 614 (range: 7-650nM), 105 nM for ralimetinib (range: 7-850nM), and 18 nM for BIRB 796 (range: 13-40nM). Because IL-1 is known to stimulate p38 signaling, we compared the response rates for these three p38 inhibitors with or without IL-1 in a dose-response study. IL-1 increased the percent of AML samples responding to p38 inhibition from 25% to 60%, indicating a potentially important role of extrinsic inflammatory stimuli in p38 inhibitor sensitivity. Consistent with this all three p38 inhibitors were similarly effective in blocking the growth of primary AML CD34+ progenitors, suggesting that targeting p38 might reduce early progenitor AML cells. Further, we compared doramapimod, ralimetinib, and ARRY 614 for their ability to inhibit p38 phosphorylation in primary AML samples using flow cytometry and immunoblot analysis; all three inhibitors blocked p38 pathway activation in AML cells. Notably, in clinical studies of ARRY 614 in MDS patients, preliminary biomarker analyses demonstrated persistent inhibition of phospho-p38 in the bone marrow during the treatment. Also, consistent with functional inhibition of p38, there was a profound decrease in plasma cytokine concentrations, most significantly IL-1, during ARRY 614 treatment. In 250 primary AML samples, we observed no correlation between BIRB 796 sensitivity in vitro and clinical metrics such as white blood cell count, blast percentage in peripheral blood or bone marrow, karyotype, or tumor genotype. This suggests that IL-1 and p38 activation might be independent biomarkers of drug sensitivity. Conclusions: These data underscore the importance of the p38MAPK pathway in the pathobiology of AML and provide strong preclinical evidence to support p38MAPK as a therapeutic target. Targeting p38MAPK might also block tumor-extrinsic signaling, as indicated by IL-1-activated signaling. That all three p38MAPK inhibitors showed comparable sensitivity profiles holds promise for ARRY614, which showed the lowest median IC50 and is currently in clinical development. In addition, with further study these findings may be extended to hematologic malignancies other than AML. Disclosures Winski: Array BioPharma Inc.: Employment. Cable:Array BioPharma Inc.: Employment. Tyner:Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding. Agarwal:CTI BioPharma: Research Funding.

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, NRASG12D 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/NRASG12D 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. Disclosures No relevant conflicts of interest to declare.

Background: Acute myeloid leukemia (AML) represents the most common type of leukemia in adults and is associated with high morbidity and mortality. Targeted therapy represents a promising approach in AML with the potential for increased efficacy and reduced toxicity. Because recent studies have demonstrated that certain inflammatory cytokines known to suppress normal hematopoiesis have the opposite effect (ie. enhancing proliferation) on cell growth of AML cells, we systematically assessed the functional relevance of 90 cytokines and their receptors in primary human AML cells. Here we present a novel role of Interleukin-1 (IL-1) signaling in aberrant regulation of AML cell growth. Methods and Results: To identify functionally important cytokine signaling pathways in leukemia pathogenesis we took a two pronged approach. First, we quantified the growth of 45 primary AML patient samples against graded concentrations of 90 cytokines. Second, we employed a functional siRNA screen targeting 188 cytokine receptors that were found highly expressed in 140 primary leukemia samples by gene expression analysis. Using this approach we identified that IL-1 signaling had the most dramatic effect. Forty percent of AML samples exhibited a 5-25 fold increase in growth in the presence of IL-1α/β. Paradoxically, IL-1 suppressed growth of normal CD34+ cells. Silencing the IL-1 receptor, IL1R1, reduced the viability of these AML primary samples by 60%. Most IL-1 sensitive AML samples exhibit monocytic and myelomonocytic features; however, no correlation with specific somatic mutations has thus far been observed. IL-1 responsive samples are from patients with abnormally increased serum IL-1β levels (5 fold). Consistent with this, conditioned media from the mononuclear cells of these samples promote the growth of AML CD34+ cells. Deep sequencing of primary AML samples indicated that genes encoding signaling molecules of IL-1 pathway (IL1R1, IL1RAP, MYD88, TRAF6, IRAK, IKK, cJUN, NFκB and p38) are normal. We confirmed the importance of IL1R1 using a genetic model. We found that the absence of IL1R1 in murine bone marrow leads to significant ablation of clonogenic potential (80% reduction) of oncogene-induced leukemic cells (AML1-ETO9a, NRASG12D and MLL-ENL) when compared to oncogene-induced leukemic cells from wild type mice. However, no effect on colony growth was observed with empty vector controls. These results suggest that multiple genetic drivers of leukemia share a dysfunctional IL-1 pathway. Mechanistically, IL-1 promotes CD34+ AML cell survival by increasing p38 phosphorylation that is significantly inhibited with IL1R1 knockdown or p38 kinase inhibitors such as Doramapimod. No toxic effect of Doramapimod was observed on normal cell growth. Conclusion: These results demonstrate a novel role for IL-1 signaling in promoting oncogenesis in AML and provide evidence that targeting this pathway might be beneficial to AML patients. Citation Format: Anupriya Agarwal, Alyssa Carey, Elie Traer, Jeffrey Tyner, Grover C. Bagby, Brian J. Druker. Targeted suppression of interleukin-1 signaling inhibits growth of primary human acute myeloid leukemia cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5248. doi:10.1158/1538-7445.AM2014-5248