The PI3K-δ Inhibitor TGR-1202 in Combination with Brentuximab Vedotin (SGN-35) Synergistically Inhibits Tubulin Polymerization and Exerts Potent Antitumor Effects in NOD/SCID Mice with Hodgkin Lymphoma Cell Line Xenografts
Authors: Locatelli Silvia L , Tartari Silvia , Castagna Luca , Mazza Rita , Viswanadha Srikant , Sportelli Peter , Vakkalanka Swaroop , Santoro Armando , Carlo-Stella Carmelo 
Published: October 2019

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 δ isoform of PI3K (PI3K-δ) in clinical development for a variety of hematologic malignancies, might represent an attractive therapeutic option. The anti-CD30 monoclonal antibody, Brentuximab Vedotin (BV), a conjugate of Brentuximab and the microtubule-disrupting agent, monomethyl auristatin E (MMAE), induced a 75% objective response rate with limited duration of response in relapsed/refractory HL. Combination therapies aimed at enhancing the anti-tumor activity of BV may have the potential for significant clinical impact in the treatment of relapsed/refractory HL. Therefore, 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.

METHODS: Three HL cell lines, including L-540, KM-H2 and L-428, were used to investigate in vitro cell growth and cell survival. The activity of TGR-1202 and BV, each as single agents and in combination, on tubulin polymerisation and microtubule distribution across cell membrane was investigated by means of a tubulin polymerisation assay and a three-dimensional volume rendering technique. The efficacy of TGR-1202/BV in combination was finally analyzed in NOD/SCID mice with HL cell line xenografts.

RESULTS: As compared to single agents, exposure of L-540, KM-H2, and L-428 cell lines to the TGR-1202 (10 µM) and BV (10 ng/ml) combination resulted in a synergistic inhibition of mean (±SEM) cell growth (TGR-1202: 40 ± 4%; BV: 30 ± 2%; TGR-1202/BV: 85 ± 1%, P ≤.0001) and a marked increase of cells in G2/M phase (TGR-1202/BV: 72 ± 3%). 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%) and a marked Cyclin B1 and p21 overexpression. Upon TGR-1202/BV exposure, HL cell lines showed a 3-fold increase in apoptosis over that observed with single agents (TGR-1202: 27 ± 2%; BV: 27 ± 2%; TGR-1202/BV: 75 ± 2%, P ≤.0001). Activation of caspase-8, -9, -3, and cleavage of PARP were reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-dependent apoptosis. Analysis of α-tubulin by immunofluorescence showed a synergistic microtubule disruption induced by TGR-1202/BV treatment with a strong α-tubulin inhibition (40%, P ≤.0001) and a low diffuse staining with irregular microtubule fragments throughout the cytosol. In addition, TGR-1202/BV in combination strongly inhibited tubulin polymerization in a time-dependent manner, suggesting that TGR-1202/BV treatment abrogates microtubule assembly and disrupts microtubules. In NOD/SCID mice bearing human HL xenografts, TGR-1202 (150 mg/Kg) and BV (0.5 mg/Kg) combined treatment significantly reduced the growth of L-540 and L-428 nodules, resulting in an average 50% tumor growth inhibition (P ≤.0001) compared to single agent treatments. No systemic toxicity was observed in mice receiving the combination therapy. Interestingly, a significant increase of microtubule disruption resulting in a marked tumor necrosis (5-fold increase, P ≤.0001) detected in mice receiving TGR-1202/BV combination as compared to mice receiving single agents. Finally, TGR-1202/BV was found to interfere with the mitotic spindle integrity, which may suggest that the cytotoxicity of the combined TGR-1202/BV treatment primarily arises from the inhibition of tubulin polymerization.

CONCLUSIONS: The novel PI3K-δ inhibitor TGR-1202 synergistically enhances the anti-tumor activity of BV by increasing drug-induced cell death and tubulin disruption in HL cell line xenografts. These data provide a strong rationale for clinical studies using TGR-1202/BV in combination in refractory/relapsed HL patients. A Phase I study of the combination of TGR-1202 and BV is ongoing in patients with relapsed/refractory HL.

Disclosures

Viswanadha: Incozen: Employment. Sportelli:TG Therapeutics: Employment, Equity Ownership. Vakkalanka:Rhizen: Employment, Equity Ownership.