The Pro-Inflammatory Cytokine Interleukin-1 Is Essential for Clonal Expansion and Disease Progression in Acute Myeloid Leukemia

Background: Recent 'omics'-based approaches have revealed that acute myeloid leukemia (AML) has significant genetic heterogeneity, and that clonal evolution in AML occurs through stepwise acquisition of somatic mutations. However, extrinsic factors may also influence this process. Here we provide strong evidence that inflammatory cytokines secreted in the AML microenvironment play a critical role in clonal expansion and disease progression. Specifically, we show that the proinflammatory cytokine IL-1 promotes in vitro clonal expansion in a large subset of primary AML samples and in vivo disease progression in a murine AML model.

Methods: We used several functional assays to determine which extrinsic cytokine-activated pathways are essential for AML cell survival. We analyzed 90 primary AML samples ex vivo in the absence and presence of dose gradients of 98 distinct cytokines. We used stromal cells conditioned media and a recombinant cytokine cocktail (IL-3, IL-6, SCF, GCSF, FLT3L) for positive controls. The distributions for the positive and negative control allowed us to derive rigorous empirical thresholds to define cytokine dependence for individual samples. We also performed siRNA screens targeting 188 cytokine and growth factor receptors, and measured 30 different cytokines secreted in the plasma of peripheral blood and bone marrow samples using a multiplex Luminex assay. Functionally relevant pathways were validated using shRNA and genetic mouse models.

Results: Several of the 98 cytokines promoted the growth of primary AML cells, including IL-1, GM-CSF, G-CSF, TPO, CXCL-1, IL-3, M-CSF, MCPs, TNF-α, and BMP-4. Many of these are known to induce an inflammatory response and cluster with the growth response to IL-1, a master mediator of innate immunity and inflammation. IL-1α and IL-1β had the most profound effect on the clonal expansion of myeloid progenitors, leading to 3- to 20-fold growth increase in 70% (63/90) of primary AML samples. Consistent with our findings from the cytokine screen, treatment of CD34+ AML cells with IL-1 led to increases in cell growth, survival, and clonogenic potential. Paradoxically, IL-1 suppressed both growth and colony formation in normal CD34+ cells. In support of these ex vivo findings, IL-1β was overexpressed in IL-1-sensitive AML bone marrow and peripheral blood samples compared to non-sensitive AML samples and normal samples. Intracellular FACS showed that 80% of the total IL-1β is secreted by monocytes, and to some extent by myeloid progenitors and stromal cells, but not by B or T cells in the AML bone marrow microenvironment. Consistent with this, most of the IL-1-sensitive AML samples exhibited monocytic and myelomonocytic features. These results suggest that IL-1 secreted in the bone marrow microenvironment regulates AML cell growth in a paracrine manner. Silencing of the IL-1 receptor, IL1R1, reduced the viability of AML primary samples by 60-80% and led to a significant ablation of clonogenic potential (80% reduction) of oncogene-induced leukemic cells (AML1-ETO9a, NRAS^G12D and MLL-ENL) in mouse bone marrow. In a murine bone marrow transplantation model, recipients of IL1R1^-/- marrow transduced with AML1-ETO9a/NRAS^G12D survived significantly longer (39 days; range: 28-118) than did recipients of wild-type marrow (30 days; range: 27-61; p=0.012). Mechanistically, IL-1β regulates phosphorylation of p38MAPK, a downstream component of the IL-1 pathway, in AML progenitors and IL-1-sensitive AML samples exhibit more p38 phosphorylation as compared to normal cells. Conversely, knocking down IL1R1 or treating AML cells with p38MAPK inhibitors such as doramapimod reduced the growth of AML cells by decreasing p38MAPK phosphorylation.

Conclusion: These results demonstrate a novel role for IL-1 and its receptor in promoting clonal growth and disease progression in a large subset of AML patients. Our findings suggest that AML patients may benefit from drugs targeting IL-1/IL1R1/p38MAPK signaling because of their potential to enhance normal hematopoiesis while inhibiting AML -- a significant clinical advantage over traditional chemotherapy, which kills both normal and leukemic cells.Since IL-1 and its receptors are not mutated in these patients, our data also highlight the importance of ex vivo functional screening for microenvironmental stimuli for the identification of novel therapeutic targets.