Inhibition Of CXCR2 As a Therapeutic Strategy In AML and MDS

Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS) arise from accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and/or committed progenitors. A series of transforming events can initially give rise to pre-leukemia stem cells (pre-LSC) as well as fully transformed leukemia stem cells (LSC), both of which need to be targeted in strategies aimed at curing these diseases. We conducted parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations in individual patients of MDS and AML (N=16) and identified candidate genes that are consistently dysregulated at multiple immature stem and progenitor cell stages. Interleukin 8 (IL8), was one of the most consistently overexpressed genes in MDS/AML Hematolpoetic Stem Cells (HSCs) and progenitors when compared to healthy control HSCs and progenitors. IL8 is a pro-inflammatory chemokine, which is able to activate multiple intracellular signaling pathways after binding to its surface receptor CXCR2. Even though increased IL8-CXCR2 signaling has been shown to promote angiogenesis, metastasis and chemotherapy resistance in many solid tumors, its role in AML and MDS is not well elucidated. We further analyzed gene expression profiles of CD34+ cells from 183 MDS patients and found significant increased expression of CXCR2 in MDS when compared to healthy controls (FDR<0.1). Most importantly, analysis of The Cancer Genome Atlas (TCGA) AML (n=200) dataset showed that CXCR2 expression was predictive of significantly adverse prognosis (log rank P value=0.0182; median survival of 245 days in cxcr2 high vs 607 days in cxcr2 low) in patients, further pointing to a critical role of IL8-CXCR2 signaling in AML/MDS.

Next, we studied the functional role of IL8 and CXCR2 in AML. A panel of leukemic cell lines (THP-1, U937, KG-1, MOLM13, HL-60, K532) were screened for CXCR2 expression and revealed significantly higher expression when compared to healthy CD34+ control cells. SB-332235, a specific inhibitor of CXCR2 was used for functional studies. CXCR2 inhibition led to significant, (p<0.05) reduction in proliferation in all 6 cell lines tested and an effect was seen as early as 24 hrs of exposure. CXCR2 inhibition was found to lead to G0/G1 cell cycle arrest and trigged apoptosis in THP-1 and U937 cells (p-value 0.004 and 0.02 respectively). Incubation of primary AML/MDS bone marrow samples with SB-332235 similarly lead to significantly reduced proliferation at 24hrs, when compared to healthy CD34+ cells. Selective, and highly significant inhibition of leukemic cell growth was also seen in colony assays from primary MDS/AML samples (mean leukemic colonies in AML/MDS= 73 vs 313 in controls, P < 0.001). Interestingly, inhibition of CXCR2 in primary AML marrow samples led to induction of apoptosis in immature CD34+/CD38- cells when compared to healthy controls. Lastly, xenografting studies with THP-1 leukemic cells revealed that CXCR2 inhibitor treatment led to decreased leukemic burden and organ infiltration when compared to placebo controls in vivo.

In summary we have found significantly increased expression of IL8 and its receptor CXCR2 in sorted HSCs and progenitors from AML and MDS patients. High CXCR2 expression was a marker of adverse prognosis in a large cohort of AML patients. Most importantly, in vitro and in vivo functional studies showed that CXCR2 is a potential therapeutic target in AML/MDS and is able to selectively target immature, LSC-enriched cell fractions in AML.