RNA Sequencing Reveals Mechanisms Underlying Modulation of Hodgkin Lymphoma Cells and Tumor Microenvironment By the PI3K δ/γ Inhibitor, RP6530

INTRODUCTION: Aberrant activation of the phosphoinositide 3-kinase (PI3K) pathway plays a central role in Hodgkin Lymphoma (HL) pathogenesis. The pleiotropic activities of the δ and γ isoforms of PI3K in regulating the interactions of Hodgkin/Reed-Sternberg (HRS) cells with the tumor microenvironment suggest thatPI3K inhibitors might exert a therapeutic potential in the treatment of HL. The dual PI3K δ/γ inhibitor RP6530 was proven to affect HL tumor cells and to exert immunosuppressive activities. However, a genome-wide search for molecular mediators of RP6530 is largely unexplored in HL. The present study was therefore aimed at evaluating the anti-lymphoma functions and the underlying genes affected by RP6530 in HL cell lines.

METHODS: We identified differentially expressed genes (DEGs) in two HL cell lines (L-540 and KM-H2) treated with RP6530 for 6 and 24 h by next generation RNA sequencing (RNA-Seq). The expression level of some of these genes was verified in HL cell lines using real time-PCR. Ingenuity Pathway Analysis (IPA) software was used to assign the DEGs to distinct canonical pathways and to predict the biological functions of the transcriptional changes. Gene silencing experiments were used to investigate and validate potential predictive biomarkers of response to RP6530 treatment. Additionally, serum levels of Thymus- and activation-regulated chemokine (TARC/CCL17) were analyzed by commercially available sandwich enzyme-linked immunosorbent assay (ELISA) in RP6530-treated HL patients enrolled in a phase 1 study.

RESULTS: RNA-Seq analysis following treatment of L-540 cells for 6 h caused an upregulation of 1544 and downregulation of 839 genes. After a 24 h treatment period, 625 genes remained upregulated while the number of genes that were downregulated increased to 1362 (FDR ≤ 0.1). In KM-H2 cells, treatment with RP6530 for 6 or 24 h upregulated 137 and 980 genes while downregulating 139 and 833 genes, respectively (FDR ≤ 0.1). We further classified modulated genes according to their canonical pathways using the IPA suite. The most significantly upregulated pathways detected in both L-540 and KM-H2 cell lines included apoptosis, p53, death receptor signaling and G1/S cell cycle checkpoint regulation. In contrast, PI3K/AKT, ERK/MAPK, JAK/Stat, CD40, IL-4, IL-6, IL-10 and iNOS signaling were downregulated. We also identified 118 downregulated genes shared between the two cell lines that were associated with glycolysis, HIF1, chemokine and cytokine signaling. Upon RP6530 incubation, RNA-seq analysis showed a strikingly reduced expression of Pyruvate kinase muscle isozyme M2 (PKM2), which is a limiting glycolytic enzyme that catalyzes the final step in glycolysis. Here we show that lactic acid produced by tumor cells, as a by-product of glycolysis, has a critical function in inducing the M2-like polarization of tumor-associated macrophages (TAMs). Furthermore, we demonstrated that this effect was mediated by PKM2 and was inhibited after RP6530 treatment.We also found that the lactate-induced expression of M2 markers by macrophages, i.e., CCL-17 and CCL-22, were markedly inhibited both by RP6530 [-30% and -50% (mean of HL cells), respectively] and PKM2 silencing [-50% and -30% (mean of HL cells), respectively]. However, RP6530 increased the expression of M1 markers, switching the activation of macrophages from an immunosuppressive M2-like phenotype to an inflammatory M1-like state. PKM2 pathway therefore appears to be a critical determinant of RP6530-mediated inhibition of M2 macrophage polarization. Finally, RP6530 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines and angiogenic factors (including, TARC/CCL17, CCL22, CCL5, IL-7, IL-13, and VEGF) known to be secreted by HRS cells to promote a pro-tumoral microenvironment. As the chemokine TARC/CCL17 is considered a biomarker of disease activity in HL patients, we observed a severe reduction of serum TARC/CCL17 levels in RP6530-treated HL patients that achieved CR or PR, but not in PD patients. Collectively, these findings suggest that RP6530 affects a mechanism of communication between HL tumor cells and tumor microenvironment.

CONCLUSIONS: Results demonstrate that RP6530 can affect both HL tumor cells and HL tumor microenvironment, suggesting that a novel therapeutic opportunity may be achievable for the treatment of HL patients.