Simultaneous inhibition of Vps34 kinase would enhance PI3Kδ inhibitor cytotoxicity in the B-cell malignancies.

Xiaochuan Liu,Stacey M Fernandes,Qingsong Liu,Hong Wu,Beilei Wang,Shanchun Zhang,Jing Liu,Juanjuan Liu,Xin Zhang,Zheng Zhao,Feiyang Liu,Cheng Chen,Aoli Wang,Li Wang,Xiaofei Liang,Jennifer R Brown,Fengming Zou,Qi Zhou ,Ellen Weisberg ,Teck Peng Loh ,Yunfei Deng ,Ilene Galinsky ,James D Griffin ,Yun‐He Xu ,Qianqian Wang ,Richard Stone

doi:10.18632/oncotarget.10650

Abstract

PI3Kδ has been found to be over-expressed in B-Cell-related malignancies. Despite the clinical success of the first selective PI3Kδ inhibitor, CAL-101, inhibition of PI3Kδ itself did not show too much cytotoxic efficacy against cancer cells. One possible reason is that PI3Kδ inhibition induced autophagy that protects the cells from death. Since class III PI3K isoform PIK3C3/Vps34 participates in autophagy initiation and progression, we predicted that a PI3Kδ and Vps34 dual inhibitor might improve the anti-proliferative activity observed for PI3Kδ-targeted inhibitors. We discovered a highly potent ATP-competitive PI3Kδ/Vps34 dual inhibitor, PI3KD/V-IN-01, which displayed 10-1500 fold selectivity over other PI3K isoforms and did not inhibit any other kinases in the kinome. In cells, PI3KD/V-IN-01 showed 30-300 fold selectivity between PI3Kδ and other class I PI3K isoforms. PI3KD/V-IN-01 exhibited better anti-proliferative activity against AML, CLL and Burkitt lymphoma cell lines than known selective PI3Kδ and Vps34 inhibitors. Interestingly, we observed FLT3-ITD AML cells are more sensitive to PI3KD/V-IN-01 than the FLT3 wt expressing cells. In AML cell inoculated xenograft mouse model, PI3KD/V-IN-01 exhibited dose-dependent anti-tumor growth efficacies. These results suggest that dual inhibition of PI3Kδ and Vps34 might be a useful approach to improve the PI3Kδ inhibitor's anti-tumor efficacy.

Highlights

PI3Kδ belongs to the Class I PI3K family, which consists of PI3Kα, β and γ, and is predominantly expressed in leukocytes.[1]
A focused medicinal chemistry approach based on an aminothiazole scaffold led to a highly potent PI3Kδ/Vps34 dual inhibitor, PI3KD/V-IN-01. In the ADP-GloTM biochemical assay with purified PI3K isoform proteins, PI3KD/V-IN-01 displayed an IC50 of 6 nM against PI3Kδ and 19 nM against VPS34. (Figure 1B) Among other PI3Ks, it exhibited an IC50 of 64 nM against Class I PI3K PI3Kα, 111 nM against PI3Kβ, 119 nM against PI3Kγ
The activity-based in vitro IP kinase assay demonstrated only modest inhibition of mTORC1 kinase by PI3KD/V-IN-01 (EC50 of 1700 nM), which is much less potent than activity displayed against PI3Kδ and vps34 kinases. (Supplemental Figure 3) In HeLa cells, PI3KD/VIN-01 effectively prevented LC3BII accumulation in the presence of EBSS (Earle’s balanced salt solution) and HCQ with an EC50 of 413 nM. (Figure 1E) In addition, an immunofluorescence experiment showed that PI3KD/V-IN-01 increased LC3B puncta in HeLa cells in a dose-dependent way, which was similar to the Vps34 specific inhibitor Vps34-IN-1, but not for the PI3Kδ inhibitor CAL-101 or pan-PI3K inhibitor GDC0941. (Figure 1F and Supplemental Figure 2) This is not surprising as HeLa cells express Vps34 but not PI3Kδ

Summary

Introduction

PI3Kδ belongs to the Class I PI3K family, which consists of PI3Kα, β and γ, and is predominantly expressed in leukocytes.[1]. [4] The seminal discovery of the PI3Kδ selective inhibitor, CAL-101 (Idelalisib), and its successful clinical application toward CLL and Indolent NHL, have validated PI3Kδ as a drug discovery target.[5] both preclinical and clinical tests have shown that inhibition of PI3Kδ alone only exerted limited cytotoxicity against the transformed cells directly, but rather inhibited cell survival by interfering with the microenvironment, such as by blocking the cytokines TNF-α, IL-6, etc to prevent the leukemic cells from circulating back to the lymph nodes and bone marrow for the further proliferation.[6] In order to improve clinical efficacy, various combination therapies have been suggested such as those involving in chemotherapy, and other signaling pathway inhibitors including MEK, BRAF, MYC, PARP, BCL-2 and autophagy.[7] Among them, the autophagy signaling pathway has attracted special attention since inhibition of the PI3K/AKT/mTOR signaling pathway is known to induce autophagy, which could provide a prosurvival mechanism for the cancer cells to escape apoptosis. Simultaneous inhibition of PI3Kδ and autophagy might enhance the clinical efficacy of PI3Kδ inhibitors

Methods

Results

Conclusion