Class I PI3K Isoforms Exert a Differential Role On Survival and Cell Trafficking In Multiple Myeloma
Authors: Sahin Ilyas , Azab Feda , Moschetta Michele , Mishima Yuji , Tsang Brian , Maiso Patricia , Sacco Antonio , Roccaro Aldo M , Azab Abdel Kareem , Ghobrial Irene M. 
Published: October 2019

Abstract

Background
The phosphatidylinositol-3 kinase (PI3K) pathway is a critical regulator of tumor progression, protein translation and cytoskeletal dynamics, collectively required for cell proliferation, survival, adhesion and migration in many malignancies including multiple myeloma (MM). Despite the absence of mutations in the PI3K/Akt genes, many studies have demonstrated that this pathway is constitutively activated in MM cells. In this study, we investigated the role of inhibition of class I PI3K isoforms known as p110α, p110β, p110γ and p110δ in cell trafficking of MM cells using isoform-specific knockdown (KD). We have also evaluated the effect of pan-PI3K inhibitior, NVP-BKM120, on survival, adhesion and migration of MM cells both in vitro and in vivo.

Methods
The baseline expression of class I PI3K isoforms in MM cell lines (MM.1S, OPM1, OPM2, H929, RPMI, INA6, U266, and U266LR7) has been evaluated by immnunobloting. MM tumor cells (MM.1S-GFP+/luc+) were infected with lentivirus mediated shRNA targeting class I PI3K isoforms. RT-qPCR and immunoblotting were performed to show infection efficiency. In vivo tumor growth of isoform specific KDs were assessed by using in vivo bioluminescence (BLI) in SCID mice. Detection of circulating MM-GFP+ cells ex vivo was performed by flow cytometry. Analysis of circulating tumor cells for each isoform-specific KD cells against relative tumor volume was performed by lineer regression using GraphPad software. Survival, adhesion and migration of KD cells were tested by MTT, adhesion and migration in vitro assay, respectively. NVP-BKM120, a pan-PI3K-inhibitor (Novartis, MA) has been tested both in vitro and in vivo. Ex vivo detection of mobilization and tumor growth of MM cells (MM.1S-GFP+/luc+) treated with 1) vehicle; 2) NVP-BKM120 in SCID mice were assessed by using flow cytometry and in vivo BLI. Homing of MM cells to the BM of mice pre-treated with NVP-BKM120 was evaluated by in vivo confocal. Increased concentrations of NVP-BKM120 have been tested on survival, cell cycle and apoptotic pathways in MM cells, by using MTT, PI staining in flow cytometry and immunoblotting, respectively. NVP-BKM120 induced dose-dependent effect on chemotaxis and adhesion of MM.1s to BM stromal cells (BMSCs) and fibronectin were tested by migration and adhesion assays.

Results
PI3K-p110β was highly expressed in all cell lines; while other isoforms were expressed in some of the MM cell lines tested. Of note, MM.1S expressed all isoforms. Mice injected with PI3K isoform specific knockdown MM.1S cells presented with different tumor burdens; p110β and p110δ mice showed significantly slower tumor progression compared to scramble control cell line (P<.05), whereas tumor growth was similar in p110α and p110γ to control mice. We next compared the number of circulating tumor cells (CTCs) at the same tumor burden between groups, which showed only p110β presented with a higher number of CTCs compared to the scramble group (P=0.01). In vitro, we observed reduced adhesion and enhanced migration of KD cells compared to control with no cell survival difference. The effect of pan-inhibition of PI3K with NVP-BKM120 induced MM cell mobilization from the BM to the circulation (Vehicle: 0.002 % vs NVP-BKM120: 0.023%; P<.05). This was supported by the inhibition of homing of MM cells to the BM (84% decrease) in the mice pre-treated with NVP-BKM120 (P<.05). Furthermore, treatment of mice with 50mg/kg of NVP-BKM120 once a day by oral gavage for five weeks significantly decreased the rate of tumor progression in MM compared to the vehicle treated group, as shown by BLI (69% decrease, P<.01). NVP-BKM120 decreased the activation of adhesion-related signaling in MM cells induced by co-culture with stroma, including pFAK, pSrc, pCoffilin and pMLC, as shown by immunoblotting. Moreover, it caused cell cycle arrest, as detected by PI staining and analyzed by flow cytometry.

Conclusion
This study suggests that inhibition of Class I PI3K isoforms, particularly p110β and p110δ, can play an important role in the regulation of cell trafficking in MM by disrupting adhesion of MM cells to the BM and inducing mobilization. Thus, pan-PI3K inhibition by NVP-BKM120 is a promising approach, which may enhance therapeutic response and overcome resistance in the treatment of MM.

Disclosures:
Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.