Positive transcription elongation factor b (P-TEFb) is a therapeutic target in human multiple myeloma.

Yu Zhang,Yun Dai,Robert Z Orlowski,Steven Grant,Liang Zhou,Yun Leng

doi:10.18632/oncotarget.19761

Abstract

The role of the positive RNA Pol II regulator, P-TEFb (positive transcription elongation factor b), in maintenance of the anti-apoptotic protein Mcl-1 and bortezomib (btz) resistance was investigated in human multiple myeloma (MM) cells. Mcl-1 was up-regulated in all MM lines tested, including bortezomib-resistant lines, human MM xenograft mouse models, and primary CD138+ MM cells. Mcl-1 over-expression significantly reduced bortezomib lethality, indicating a functional role for Mcl-1 in bortezomib resistance. MM cell lines, primary MM specimens, and murine xenografts exhibited constitutive P-TEFb activation, manifested by high CTD (carboxy-terminal domain) S2 phosphorylation, associated with a) P-TEFb subunit up-regulation i.e., CDK9 (42 and 55 kDa isoforms) and cyclin T1; and b) marked CDK9 (42 kDa) T186 phosphorylation. In marked contrast, normal hematopoietic cells failed to exhibit up-regulation of p-CTD, CDK9, cyclin T1, or Mcl-1. CDK9 or cyclin T1 shRNA knock-down dramatically inhibited CTD S2 phosphorylation and down-regulated Mcl-1. Moreover, CRISPR-Cas CDK9 knock-out triggered apoptosis in MM cells and dramatically diminished cell growth. Pan-CDK e.g., dinaciclib or alvocidib and selective CDK9 inhibitors (CDK9i) recapitulated the effects of genetic P-TEFb disruption. CDK9 shRNA or CDK9 inhibitors significantly potentiated the susceptibility of MM cells, including bortezomib-resistant cells, to proteasome inhibitors. Analogously, CDK9 or cyclin T1 knock-down or CDK9 inhibitors markedly increased BH3-mimetic lethality in bortezomib-resistant cells. Finally, pan-CDK inhibition reduced human drug-naïve or bortezomib-resistant CD138+ cells and restored bone marrow architecture in vivo. Collectively, these findings implicate constitutive P-TEFb activation in high Mcl-1 maintenance in MM, and validate targeting the P-TEFb complex to circumvent bortezomib-resistance.

Highlights

Multiple myeloma (MM) is an accumulative disease of mature plasma cells, and despite recent advances in treatment, including the introduction of effective new drugs such as proteasome inhibitors and immunomodulatory agents, it remains largely incurable
Exposure of U266 or bortezomib-resistant PS-R cells to CDK9i resulted in sharp reductions in S2 carboxyterminal domain (CTD) and S5 CTD phosphorylation (Figure 3D). This was accompanied by down-regulation of phosphorylated RNA Pol IIo and diminished expression of Mcl-1. These findings argue that both cyclin T1 and CDK9 play significant functional roles in maintaining Mcl-1 expression in bortezomib-resistant or -sensitive cells, and that the consequences of genetic disruption of the P-TEFb apparatus can be recapitulated by pharmacological CDK9 inhibitors, including in bortezomib-resistant MM cells
Parallel results were observed in bortezomibsensitive U266 or -resistant (PS-R) cells (Figure 4B) as well as RPMI8226/LR5 cells (Figure 4C) or bortezomib-resistant RPMI8226/VR cells [22]. These findings indicate that clinically relevant CDK9 inhibitors block RNA Pol II CTD phosphorylation in MM cells at early intervals, resulting in Mcl-1 down-regulation and cell death, and that analogous events occur in bortezomib-resistant cells

Summary

Introduction

Multiple myeloma (MM) is an accumulative disease of mature plasma cells, and despite recent advances in treatment, including the introduction of effective new drugs such as proteasome inhibitors and immunomodulatory agents, it remains largely incurable. MM is characterized by dysregulation of members of the Bcl-2 family of pro- and anti-apoptotic proteins. The multidomain anti-apoptotic protein Mcl-1 (myeloid cell leukemia-1) is frequently over-expressed in MM [1]. Proteasome inhibitors such as bortezomib, by blocking Mcl-1 degradation, induce Mcl-1 accumulation, which may contribute to resistance to such agents [6, 7]. These considerations provide a strong rationale for targeting Mcl-1 in MM, in the setting of proteasome inhibitor resistance

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