The PI3K-δ Inhibitor TGR-1202 In Combination With Brentuximab Vedotin (SGN-35) Synergistically Induces G2/M Phase Arrest and Cell Death Via Inhibition Of Tubulin Polymerization In Hodgkin Lymphoma Cell Lines
Authors: Locatelli Silvia L , Tartari Silvia , Rubino Luca , Brusamolino Ercole , Castagna Luca , Sportelli Peter , Santoro Armando , Carlo-Stella Carmelo 
Published: October 2019

Abstract

Introduction
The phosphatidylinositol 3-kinase (PI3K) pathway is consistently activated in relapsed/refractory Hodgkin lymphoma (HL), suggesting that TGR-1202, a novel inhibitor of the delta isoform of PI3K (PI3K-δ), in clinical development for patients with hematologic malignancies, might represent an attractive therapeutic option. The anti-CD30 monoclonal antibody Brentuximab Vedotin (BV) conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) has recently been reported to induce an overall response rate of 75% in relapsed/refractory HL, but is associated with limited response duration. Combination therapies aimed at enhancing the anti-tumor activity of BV and reducing its side effects may have significant clinical impact in the treatment of relapsed/refractory HL. The present study was aimed at investigating the activity and mechanism(s) of action of the PI3K-δ inhibitor TGR-1202, in combination with BV in non-clinical models of HL.

Methods
Three HL cell lines, including L-540, KM-H2 and L-428, were used to test the effects of TGR-1202 alone, BV alone, or the combination of TGR-1202 with BV. Cell cycle effects and cell survival were determined by flow cytometry and Western blotting (WB). Additionally, WB was used to assess modulating effects of TGR-1202 on the PI3K/AKT pathway as well as microtubule interacting proteins. Cyclin B1, p21, and α-tubulin were detected by indirect immunofluorescence microscopy. The activity of TGR-1202 and/or BV on microtubule distribution and polymerization were quantified using a three-dimensional volume rendering technique.

Results
TGR-1202 and BV used as single agents were able to induce a time- and dose-dependent inhibition of cell proliferation and induction of cell death in all cell lines. Compared to the single agent effects, the combination of TGR-1202 (10 µM) and BV (10 ng/ml) synergistically inhibited the mean (±SEM) growth of L-540, KM-H2, and L-428 cell lines (TGR-1202: 40 ± 4%; BV: 30 ± 2%; TGR-1202/BV: 85 ± 1%). Inhibition of cell proliferation induced by the 2-drug combination was associated with a dramatic G2/M cell cycle arrest. Upon TGR-1202/BV treatment, the mean (±SEM) percentages of cells in G2/M phases were increased by 4-fold (72 ± 3%) as compared to TGR-1202 (18 ± 1%) or BV (18 ± 1%) alone. This finding was paralleled by a 3-fold reduction of cells in S phase (TGR-1202: 25 ± 1%; BV: 23 ± 1%; TGR-1202/BV: 9 ± 1%, mean ± SEM) and a marked Cyclin B1 and p21 overexpression. In comparison to each drug as a single agent, the TGR-1202/BV combination led to a synergistic cell death induction. In fact, upon TGR-1202/BV treatment, mean (±SEM) cell death values detected in L-540, KM-H2, and L-428 cell lines were increased by 3-fold over TGR-1202 or BV alone (TGR-1202: 27 ± 2%; BV: 27 ± 2%; TGR-1202/BV: 75 ± 2%). Analysis of caspase-3 and PARP cleavage and blocking experiments with the pan-caspase inhibitor Z-VAD-FMK revealed a caspase-dependent cell death mechanism. In addition, the antiproliferative and cytotoxic effects of TGR-1202 were associated with a marked time-dependent inhibition of PI3K/Akt pathway and dephosphorylation of GSK-3β, Aurora kinases, and stathmin, suggesting that modulation of molecules associated with microtubule polymerization are critically involved in TGR-1202/BV-triggered cell death. To asses potential effects on microtubule dynamics, HL cells were treated with TGR-1202, BV, or the combination for 24 hours, and the effect on microtubules was determined by α-tubulin staining. Compared with controls, TGR-1202 and BV treatment alone led to a modest loss of microtubules (TGR-1202: 11%; BV: 9%), while the combined TGR-1202/BV treatment resulted in a potent synergistic microtubule disruption (mean values of α-tubulin inhibition of 40%, P ≤.0001), supported by a diffuse stain and irregular microtubule fragments throughout the cytosol. Additionally, TGR-1202/BV was found to interfere with the mitotic spindle integrity which may suggest that the G2/M arrest and cytotoxicity of the combined TGR-1202/BV treatment primarily arises from the inhibition of tubulin polymerization.

Conclusions
Novel PI3K-δ inhibitor TGR-1202 enhances the anti-tumor activity of BV by increasing drug-induced apoptosis and tubulin disruption in all HL cell lines analyzed in the present study. Our data provides a strong rationale for evaluating TGR-1202 in combination with BV in patients with relapsed/refractory HL.

Disclosures:
Sportelli: TG Therapeutics, Inc.: Employment, Equity Ownership.