Treatment of Hodgkin Lymphoma Xenografts with the Novel PI3K δ/γ Inhibitor RP6530 Suppresses M2 Macrophage Polarization and Results in Potent Antitumor and Antiangiogenic Effects

INTRODUCTION: Primary refractory and early-relapsed patients with Hodgkin lymphoma (HL) experience poor responses to salvage chemotherapy and dismal long-term disease control. Therefore, they represent an unmet medical need urgently requiring not only new diagnostic tools but also novel therapeutic agents to overcome the chemo-refractory phenotype.Tumor-associated macrophages (TAMs) have been implicated in the pathogenesis of HL and have been suggested to negatively impact clinical outcome. The phosphatidylinositol 3-kinase (PI3K) is a major effector pathway mediating tumor and endothelial cell survival and functions, as well as macrophage activation. As PI3Kδ and PI3Kγ are constitutively activated in several hematologic malignancies resulting in cancer development and progression, we hypothesize that the novel dual PI3K δ/γ inhibitor,RP6530, might represent a unique therapeutic opportunity for HL patients. This study aimed at investigating the activity and mechanism(s) of action of RP6530 in preclinical HL models.

METHODS: The effects of RP6530 on HL cell lines (L-540, KM-H2, L-428) were investigated by assaying cell proliferation, cell death, and gene expression profiling. Upon treatment with RP6530, human macrophages co-cultured with HL cell lines were assessed by flow cytometry for cell death and expression of M1/M2 macrophage markers. The antitumor efficacy and mechanism(s) of action of RP6530 were also analyzed in NOD/SCID mice bearing HL cell line xenografts. Tumor vasculature was analyzed by in vivo biotinylation of vascular endothelial proteins.

RESULTS: Exposure of HL cell lines to RP6530 (1.25 - 10 µM) induced a marked, early, and dose-dependent dephosphorylation of PI3K/Akt and MAPK pathways that was associated with inhibition of cell proliferation (L-540: 40 ± 4%, KM-H2: 33 ± 5% and L-428 30 ± 5%, mean ± SEM) and S phase cell cycle arrest (3-fold reduction, compared to vehicle-controls). Significant levels of caspase-dependent cell death were observed in L-540 (20µM: 61 ± 5 vs. 15 ± 2%), KM-H2 (20µM: 41 ± 4 vs. 12 ± 1%) and L-428 (20µM: 31± 4% vs. 12 ± 1%) cells that were associated with mitochondrial depolarization (up to 40%). Interestingly, microarray gene expression analysis of KM-H2 cell line showed that a 24-hour treatment with RP6530 decreased the amplitude of several gene signatures, including “PI3K signaling in B lymphocytes”, “Tumor cell proliferation”, “Tumor angiogenesis”, and “Macrophages activation”, while genes involved in positive regulation of cell death were upregulated. In KM-H2 co-culture, besides observing cell death of human macrophages (up to 40% following a 24-hour exposure), we found that RP6530 inhibited M2 macrophage differentiation, as suggested by decreased CD209 and CD163 expression, and increased M1 macrophage differentiation by upregulating CD40 and CD80 expression. Compared to vehicle-controls, analysis of RP6530-treated tumor xenografts showed a marked reduction of TAM (-44% and -50%, P ≤.0001 in L-540 and KM-H2, respectively), as well as tumor vessel density (average decrease: 80%), consistent with the role of PI3K signaling in the macrophage-dependent promotion of tumor growth and tumor-induced angiogenesis. This finding was paralleled by the histological detection of tumor vasculature disruption, and inhibition of phospho-Akt and -Erk on both endothelial and tumor cells. Finally, RP6530 treatment (150 mg/kg/BID/3wks) of NOD/SCID mice significantly reduced the growth of KM-H2 nodules (-60%, P ≤.0001) compared to vehicle-controls. Reduction of tumor growth was associated with a strong decrease in Ki-67 expression by tumor cells, increase in tumor apoptosis (1.2- and 12-fold in L-540 and KM-H2, respectively) and tumor necrosis (average increase: 7-fold, P≤.0001).

CONCLUSIONS: These results demonstrate that the therapeutic potential of RP6530 in HL is likely due to a reduction of M2 macrophages polarization and inhibition of tumor angiogenesis. Our findings warrant further investigation in future clinical trials.