Follicular Dendrytic Cells Deliver Angiogenesis Signaling To Follicular Lymphoma Cells That Is Hampered By The Pan-PI3K Inhibitor NVP-BKM120

Follicular Lymphoma (FL) is the paradigm of a neoplasia depending on the microenvironment for proliferation and survival. In the lymphoid follicle, FL cells are surrounded by follicular dendrytic cells (FDCs) that function as antigen presenting cells delivering survival and proliferation signaling. FDCs together with macrophages are associated to poor FL survival. Our aim was to uncover the signaling pathways underlying FL-FDC crosstalk and its validation as new targets for therapy using specific inhibitors. Global gene expression profiling of FL-FDC co-cultures yield a marked modulation of FL transcriptome by FDCs. The Principal Component Analysis (PCA) showed that HK-cocultured FL cells clustered together,independently of the patient origin. Then, pathways assignmentwas performed by DAVID and GSEA softwares, both of themuncovering an overrepresentation on genes related toangiogenesis. In the DAVID analysis, we found significant(False Discovery Rate (FDR)<5%,) angiogenesis–related GOterms such as, blood vessel development, blood vessel morphogenesis, VEGFR signaling pathway, sprouting angiogenesis, positive regulation of angiogenesis, patterning of blood vessels among others. In the GSEA analysis, our dataset was interrogated for enrichment of genesets belonging to C2 data base together with custom-derived ones. In accordance with DAVID results, genesets related to angiogenesis were enriched (FDR< 5%) in HK-cocultured FLcells, such as HumanAngiogenesis, Weston_VEGFA_targtes, PID_VEGFR1_2 and PID_lymphangiogenesis.

As PI3K pathway is known to play a determinant role in angiogenesis, we explored if NVP-BKM120, a pan-PI3K inhibitor in clinical trials for solid tumors, could interfere with this signaling. First, by using Taqman Angiogenesis Array®containing 94 probes against genes related to angiogenesis, we analyzed if the modulation of genes in FL cells cocultured with HK could be inhibited by NVP-BKM120. Effectively, we found a group of genes that were positively regulated by HK and inhibited by NVP-BKM120, among them ANGPT1, CXCL12, EPHB2, VEGFA, VEGFC, ADAMST1, NRP1, NRP2, HSPG2,COL4A1, PDGFRA and PDGFRB. In addition, by ELISA analysis of cell culture supernatants, we found that NVP-BKM120 reduced VEGFA and VEGFC secretion of FL cells alone or co-cultured with HK. Then, we analyzed if this reduction of proangiogenic factors effectively reduced angiogenesis. To this aim we performed HUVEC tube formation assay with the supernatants of FL cells co-cultured or not with HK, in the presence or absence of the PI3K inhibitor. We demonstrated that, as expected, supernatants derived fromFL-HK-cocultures increased the number of tubes formed, and NVP-BKM120 reduced these numbers. Then, we investigated the impact of NVP-BKM120 treatment on FL cell signalling and proliferation. NVP-BKM120 efficiently blocks both constitutive activation of PI3K/AKT pathway in FL cells and that derived from B-cell receptor stimulation or HK co-culture, reducing cell proliferation of FL cells and inducing apoptosis in a portion of FL cell lines and primary cells. NVP-BKM120 also impedes signaling and migration induced by the chemokine SDF1α. In vivo, NVP-BKM120 significantly (p<0.05) reduces tumor outgrowth in subcutaneous and systemic FL mouse models. We isolated RNA from mouse tumors and run TaqmanAngiogenesis array as before. NVP-BKM120 downmodulated the expresion of genes related to angiogenesis already found in vitro such a VEGFA, HSPGB2, NRP2 ,PDGFRA and PDGFRB, but also novel genes, including SERPINC1, FST,PDGFB, TGFB1, TNF, VEGFB, COL18A1, ANGPT4 and PECAM(CD31). In addition, the reduction on VEGFA and CD31 was also validated by IHC in these tumors.

These results warrant further investigation of pan-PI3K inhibitors for FL therapy in the context of microenvironment survival signaling inhibition.