Comparison of the PI3K-δ Inhibitors TGR1202 and GS-1101 in Inducing Cytotoxicity and Inhibiting Phosphorylation of Akt in CLL Cells in Vitro

The PI3K pathway is a central pro-survival mechanism in chronic lymphocytic leukemia (CLL). Expression of the delta isoform of PI3K is largely restricted to lymphocytes. Inhibition of PI3K activity in vitro induces CLL cell apoptosis and death. Clinical evaluation of PI3K-d inhibitors, such as GS-1101, has been promising, with responses seen in relapsed and/or refractory CLL patients. TGR-1202 is a novel PI3K-d specific inhibitor previously demonstrated to inhibit Akt phosphorylation and induce apoptosis in B-cell lymphoma cell lines. Given the efficacy of other PI3K-d inhibitors observed in CLL, we assessed the ability of TGR-1202 to induce cytotoxicity and apoptosis, and inhibit Akt phosphorylation in primary CLL lymphocytes.

We collected blood from CLL patients seen at the Duke Center for CLL and enrolled in IRB approved protocols at the Duke University and Durham VA Medical Centers. CLL lymphocytes were isolated using negative selection yielding greater than 95% purity of CLL lymphocytes. Primary CLL cells were incubated with serial dilutions of TGR-1202 or GS-1101 for 48 hours and tested for apoptosis by activated caspase-3 and 7AAD staining measured by flow cytometry. After 72 hours of incubation with either compound, CLL cells were evaluated for cytotoxicity using the colorimetric MTS reagent. Phosphorylated Akt (S473) was measured by flow cytometry after one hour of incubation of either compound and ten minutes of incubation with anti-IgM or anti-IgD. Akt phosphorylation was quantified by median fluorescent intensity (MFI).

We evaluated TGR-1202 and GS-1101 in CLL lymphocytes collected from seven patients. Five had mutated IGHV, five had 13q deletion or normal cytogenetics determined by fluorescent in situ hybridization, three were ZAP-70 negative, and seven were CD38 negative. IgM expression ranged between 13% and 90%, whereas IgD expression was uniformly elevated. Both TGR-1202 and GS-1101 significantly induced apoptosis (caspase-3+/7AAD+) and cytotoxicity in a dose-dependent manner in concentrations between 0.1 and 25.6 μM (p < 0.05 in pairwise Wilcoxon signed rank tests). There was no significant difference observed between the compounds in terms of induction of apoptosis or cytotoxicity at any of the tested concentrations tested (0.1 – 25.6 μM) except 0.4 μM, where GS-1101 induced more apoptosis and cytotoxicity (median of 18.6 vs. 13.7% caspase-3+/7AAD+; median of 55.2 vs. 48.6% cytotoxicity, p = 0.03 for both, Wilcoxon signed rank test). Incubation with anti-surface immunoglobulin significantly induced Akt phosphorylation compared to media alone (median MFI 1011.5 and 369, respectively, p = 0.03, Wilcoxon rank sum test). The addition of either TGR-1202 or GS-1101 significantly abrogated this effect (p = 0.03 for 0.1, 0.4, and 1.6 uM, Wilcoxon rank sum test) and returned Akt phosphorylation to baseline (p > 0.05 comparing drug treatment to media control, Wilcoxon rank sum test).

TGR-1202 is a potent PI3K-δ inhibitor that suppresses Akt phosphorylation and induces apoptosis-dependent cytotoxicity in primary CLL lymphocytes. These effects are comparable to those seen with GS-1101, another PI3K-δ inhibitor that has demonstrated efficacy in CLL clinical trials. Differences seen at the 0.4 μM concentration were not observed at higher or lower concentrations. Evaluating additional CLL patients' cells would help clarify these findings. Given the improved selectivity for the delta isoform of PI3K seen with TGR-1202 compared to GS-1101, these results suggest that TGR-1202 may have benefit in treating CLL, while inducing fewer off-target effects and toxicities. These findings provide the rationale for future clinical trials evaluating TGR-1202 in CLL.

Friedman:Rhizen Pharmaceuticals: Research Funding. Miskin:TG Therapeutics: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals: Employment, Equity Ownership.