Responses in mantle cell lymphoma cells to SNS-032 depend on the biological context of each cell line.

SNS-032 is a potent inhibitor of cyclin-dependent kinases (Cdk) 2, 7, and 9 that regulate the cell cycle and transcription. Our studies in indolent primary chronic lymphocytic leukemia cells showed that SNS-032 inhibited transcription, diminished the antiapoptotic protein Mcl-1, and induced apoptosis. The present study focuses on evaluating this compound in four proliferating mantle cell lymphoma lines (Jeko-1, Granta 519, Mino, and SP-53). Consistent with its action against Cdk9 and Cdk7, SNS-032 inhibited the phosphorylation of RNA pol II in all four lines and blocked RNA synthesis. The transcripts and protein levels of short-lived proteins decreased, including cyclin D1 and Mcl-1. Cell growth was inhibited in a concentration-dependent manner in all lines. Apoptosis was induced in JeKo-1, Mino, and SP-53 cells without disrupting cell cycle distribution. However, apoptosis was limited in Granta cells; rather, there was a significant reduction of clonogenic survival. Small interfering RNA was used to specifically knock down Mcl-1 and cyclin D1 in JeKo-1 and Granta cells. Knocking down Mcl-1 induced significant apoptosis in Jeko-1 cells but not Granta cells. Reducing cyclin D1, rather than Mcl-1, was associated with loss of clonogenic survival in Granta cells. Thus, these results indicated that mantle cell lymphoma cell lines have distinct mechanisms sustaining their survival, and the mechanism of action of SNS-032 is dependent on the biological context of an individual line.

Mechanism of action of SNS-032, a novel cyclin-dependent kinase inhibitor, in chronic lymphocytic leukemia.

Inhibitors of cyclin-dependent kinases (Cdks) have been reported to have activities in chronic lymphocytic leukemia cells by inhibiting Cdk7 and Cdk9, which control transcription. Here we studied the novel Cdk inhibitor SNS-032, which exhibits potent and selective inhibitory activity against Cdk2, Cdk7, and Cdk9. We hypothesized that transient inhibition of transcription by SNS-032 would decrease antiapoptotic proteins, resulting in cell death. SNS-032 effectively killed chronic lymphocytic leukemia cells in vitro regardless of prognostic indicators and treatment history. This was associated with inhibition of phosphorylation of RNA polymerase II and inhibition of RNA synthesis. Consistent with the intrinsic turnover rates of their transcripts and proteins, antiapoptotic proteins, such as Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP), were rapidly reduced on exposure to SNS-032, whereas Bcl-2 protein was not affected. The initial decrease of Mcl-1 protein was the result of transcriptional inhibition rather than cleavage by caspase. Compared with flavopiridol and roscovitine, SNS-032 was more potent, both in inhibition of RNA synthesis and at induction of apoptosis. SNS-032 activity was readily reversible; removal of SNS-032 reactivated RNA polymerase II, which led to resynthesis of Mcl-1 and cell survival. Thus, these data support the clinical development of SNS-032 in diseases that require short-lived oncoproteins for survival.

Molecular basis for G2 arrest induced by 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine and consequences of checkpoint abrogation.

2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by beta-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G2. The purpose of this investigation was to define the molecular basis for G2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G1. Cell death was first evident in hematologic cell lines after G1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G2. Some breaks were manifested as chromosomal aberrations when the G2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies.