POL5551, a Novel and Potent CXCR4 Antagonist, Enhances Sensitivity to Chemotherapy in an in Vivo Model of High-Risk (HR) Pediatric Acute Lymphoblastic Leukemia (ALL)

Abstract

Background: Interaction between surface receptor CXCR4 (s-CXCR4) and chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow (BM) microenvironment. We previously demonstrated: 1) chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) and ALL enhances stromal protection from chemotherapy-induced apoptosis; 2) the FDA-approved CXCR4 inhibitor plerixafor reverses stromal protection and chemotherapy resistance both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged (MLL-R) ALL; 3) the novel Protein Epitope Mimetic POL5551, a selective and potent antagonist of CXCR4, blocks the SDF-1-binding site of CXCR4, inhibits SDF-1-induced chemotaxis, and reverses stromal-mediated protection from chemotherapy in vitro in pre-B and T ALL cell lines. Here, we further characterize the effects of POL5551 (POL) on surface adhesion molecule expression in ALL, and its in vivo effects in a xenograft model of HR pediatric ALL.In Vitro Methods/Results: We have previously shown that POL inhibits 12G5 antibody binding to s-CXCR4 in ALL cell lines, suggesting an overlapping of the two binding sites. We sought to verify these results using primary samples of pediatric ALL. We treated 3 pre B and 3 T cell ALL primary samples with a dose range of POL and measured s-CXCR4 by FACS at multiple time points. POL inhibition of 12G5 binding was potent (average IC50 at 2 hours pre B 8.3 nM, T 1.4 nM), rapid (<1 hour) and sustained (>24 hours). Further, POL was significantly more potent at inhibiting 12G5 binding than plerixafor (average IC50 at 2 hours pre B 18.4 nM, T 8.4 nM). To further characterize POL’s effects in ALL, we treated 2 pre B and 2 T cell ALL cell lines with POL or vehicle control and then treated with SDF-1α or vehicle control. Treatment with POL inhibited SDF-1α-induced phosphorylation of ERK1/2 in a dose-dependent manner. In parallel, we measured POL-induced compensatory upregulation of the alternative surface adhesion molecules CXCR7 and VLA-4 (CD49d), and found that POL led to increased CXCR7 expression at early time points that began to decrease after 24 hours. We did not find a consistent effect of POL on CD49d surface expression.Xenograft Methods/Results: Infant MLL-R ALL primary samples (n=4) were transplanted into sublethally irradiated NSG mice. After 2 weeks, mice were treated on days 1-3 of 2 consecutive weeks with 1) vehicle control (C), 2) POL (5 mg/kg SC), 3) AraC (200 mg/kg IP), or 4) POL followed by AraC 4 hours later (POL+AraC). One week after treatment, cells were harvested from BM, spleen, and peripheral blood (PB). Leukemic blasts were defined as human CD19+ and CD45+. Overall leukemic burden (average % blasts in BM+spleen+PB) did not differ between mice treated with either C (56.2%) or POL (49.5%, p=0.12). However, treatment with AraC (36.7%, p=3E-07) or POL+AraC (26.3%, p=4E-15) significantly decreased total leukemic burden compared to C. Notably, POL+AraC significantly decreased total leukemic burden compared to AraC alone (p=0.001), demonstrating that POL increased overall sensitivity to AraC. When analyzed by organ-specific leukemic burden, POL+AraC resulted in decreased leukemic burden compared to AraC alone in BM (42.8 vs. 49.8%, p=0.27), spleen (16.9 vs. 30.8%, p=0.002), and PB (19.3 vs. 29.6%, p=0.008). Interestingly, AraC and POL+AraC led to significantly increased CXCR7 expression (blasts from BM p<0.01, spleen p<0.04, and PB p<0.03 vs. C) as well as a trend toward increased CD49d expression compared to C.Conclusions: POL5551 is a potent antagonist of s-CXCR4 in pediatric ALL cell lines and primary samples. Surface expression of adhesion molecules in ALL blasts is dynamic and affected by anti-leukemic therapy. Importantly, POL5551 enhances sensitivity to AraC in a xenograft model of infant MLL-R ALL, a HR subtype of pediatric ALL. Our findings support the continued development of BM microenvironment-targeted agents as a therapeutic strategy for pediatric ALL. DisclosuresRomagnoli:Polyphor Ltd.: Employment. Douglas:Polyphor Ltd.: Employment. Tuffin:Polyphor Ltd.: Employment. Zimmermann:Polyphor Ltd.: Employment.