MDS is a generally incurable hematologic disorder associated with disease initiating stem cells that are not eliminated by conventional therapies and need to be targeted for potentially curative strategies. We recently demonstrated that aberrant hematopoietic stem cells are expanded in MDS, can persist during phenotypic remissions and can predict relapse. In preliminary studies, we demonstrate that Interleukin 8 (IL8) is consistently and selectively overexpressed in stem cells from MDS patients. The receptor for IL8, CXCR2, is also significantly increased in large MDS patient cohorts and is associated with a worse prognosis. Functionally, IL8/CXCR2 pathway inhibition by either shRNA-mediated knockdown or pharmacologic approaches abrogated proliferation in cell lines and primary MDS samples. Importantly, inhibition of the IL8/CXCR2 pathway selectively inhibited immature stem cells from MDS samples without an effect on healthy HSCs, and also had demonstrated efficacy in xenografts. To comprehensively examine the role of this pathway in MDS, Aim 1 will define the functional role of IL-8/CXCR2 pathway on growth of disease initiating stem cells in MDS and determine the efficacy of clinically relevant inhibitors of this pathway in large cohort of primary human samples. Additionally, responses to IL8/CXCR2 inhibition with small molecules and a novel humanized antibody will be correlated with clinical and mutational subtypes to identify targetable subsets that will be sensitive to IL8/CXCR2 inhibition. Patient derived MDS xenografts will also be used to determine in vivo efficacy. Aim 2 will determine the requirement for CXCR2 in initiation of dysplasia/disease progression in vivo by genetic deletion of CXCR2 in two mouse models of MDS. Along with the NUP-HOXD13 model; a novel model of MDS dysplasia and transformation which we have recently developed, induced by heterozygous PU.1 enhancer deletion, will be used to study the effect of CXCR2 deletion on disease initiating stem cells and disease progression. Aim 3 will identify the mechanisms of activation of the IL8-CXCR2 pathway and determine its downstream effectors in MDS. IL8 is a known component of innate immune signaling cascades, and we will determine whether upstream immune activators, IL1RAP, TLRs and IRAK1/4 are driving overactivation of the IL8/CXCR2 pathway in MDS. We will also evaluate the activation and functional significance of PI3Kinase and MAP kinase pathways as downstream effectors of the IL8/CXCR2 pathway in MDS. Taken together, these studies will study the role of the IL8/CXCR2 pathway in MDS pathogenesis and determine its potential as a therapeutic target against immature, disease initiating cells in MDS.