G1 Cell Cycle Arrest and Extrinsic Apoptotic Mechanisms Underlying the Anti-Leukemic Activity of CDK7 Inhibitor BS-181.

Shin Young Park,Ki Yun Kim,Young Ho Kim,Do Youn Jun,Su-Kyeong Hwang

doi:10.3390/cancers12123845

Abstract

Simple SummaryChemotherapy resistance in human T-cell acute lymphoblastic leukemia (T-ALL), an aggressive neoplasm, results in poor prognosis despite advances in treatment modalities. Toward the identification of an effective alternative, in the present study, we elucidated the mechanism underlying the antitumor activity of the CDK7 inhibitor BS-181 using malignant cells (Jurkat A3, U937, and HeLa) and normal human peripheral T cells. This is the first report to demonstrate that BS-181 antitumor activity is mainly caused by extrinsic apoptosis induction through cell-surface TRAIL/DR5 levels in human T-ALL Jurkat T cells. Moreover, combined treatment with recombinant TRAIL (rTRAIL) exerted synergistic effects on BS-181 cytotoxicity against malignant cells but not normal human peripheral T cells by augmenting both the extrinsic and intrinsic BCL-2-sensitive apoptosis pathways. Our findings suggest that the combination with rTRAIL may facilitate BS-181 antitumor activity against T-ALL cells while minimizing associated side effects, therefore potentially being applicable to clinical human T-ALL treatment.In vitro antitumor activity of the CDK7 inhibitor BS-181 against human T-ALL Jurkat cells was determined. Treatment of Jurkat clones (JT/Neo) with BS-181 caused cytotoxicity and several apoptotic events, including TRAIL/DR4/DR5 upregulation, c-FLIP down-regulation, BID cleavage, BAK activation, ΔΨm loss, caspase-8/9/3 activation, and PARP cleavage. However, the BCL-2-overexpressing Jurkat clone (JT/BCL-2) abrogated these apoptotic responses. CDK7 catalyzed the activating phosphorylation of CDK1 (Thr161) and CDK2 (Thr160), and CDK-directed retinoblastoma phosphorylation was attenuated in both BS-181-treated Jurkat clones, whereas only JT/BCL-2 cells exhibited G1 cell cycle arrest. The G1-blocker hydroxyurea augmented BS-181-induced apoptosis by enhancing TRAIL/DR4/DR5 upregulation and c-FLIP down-regulation. BS-181-induced FITC–annexin V-positive apoptotic cells were mostly in the sub-G1 and G1 phases. BS-181-induced cytotoxicity and mitochondrial apoptotic events (BAK activation/ΔΨm loss/caspase-9 activation) in Jurkat clones I2.1 (FADD-deficient) and I9.2 (caspase-8-deficient) were significantly lower than in A3 (wild-type). Exogenously added recombinant TRAIL (rTRAIL) markedly synergized BS-181-induced apoptosis in A3 cells but not in normal peripheral T cells. The cotreatment cytotoxicity was significantly reduced by the DR5-blocking antibody but not by the DR4-blocking antibody. These results demonstrated that the BS-181 anti-leukemic activity is attributed to extrinsic TRAIL/DR5-dependent apoptosis preferentially induced in G1-arrested cells, and that BS-181 and rTRAIL in combination may hold promise for T-ALL treatment.

Highlights

Human T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm derived from the malignant transformation of lymphoblasts committed to a T-cell lineage and accounts for approximately20% of all ALL cases [1,2]
A BS-181-induced increase in the number of early and late apoptotic cells was not observed in JT/BCL-2 cells. These results suggested that BS-181 cytotoxicity toward Jurkat T cells was attributed partly to a cytostatic effect exerted by inducing G1 cell cycle arrest and mainly to cell death resulting from BCL-2-sensitive apoptosis induction, which might be preferentially induced in G1 phase cells
Presented, and two additional experiments yielded similar results. These results indicated that BS-181-induced apoptosis in JT/Neo cells was preferentially provoked in G1-arrested cells, consequent to the inhibition of cyclin-dependent kinases (CDKs)-directed Rb phosphorylation required for entry into S phase, and suggested that the G1 phase was more susceptible to BS-181induced TRAIL/death receptor 4 (DR4)/death receptor 5 (DR5) upregulation and c-FLIPL down-regulation, which caused BCL-2-sensitive mitochondrial apoptotic events, such as ΔѰm loss and caspase-9/caspase-3 activation, leading to

Summary

Introduction

Human T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm derived from the malignant transformation of lymphoblasts committed to a T-cell lineage and accounts for approximately20% of all ALL cases [1,2]. To improve the overall survival rates in the chemotherapeutic treatment of T-ALL and other high-risk leukemias, there is a high demand for novel antitumor agents that can minimize drug resistance and side effects [5]. The catalytic activities of CDKs are frequently elevated in conjunction with the uncontrolled proliferation of tumor cells, owing to genetic and epigenetic alterations in CDKs or their regulatory proteins, such as CDK inhibitors and cyclins [9,10,11,12,13]. CDKs have emerged as attractive targets for the development of antitumor agents and chemotherapeutic treatment [8,9,14]. Recent advances in the development of highly specific CDK4/6 inhibitors have led to the approval of palbociclib and ribociclib for breast tumor treatment [15,16,17]. Along with attempts to expand pharmacological target CDKs to non-canonical

Methods

Discussion

Conclusion