Abstract #1801: SNS-032 reduced the expression of cyclin D1 and Mcl-1 and inhibited survival in mantle cell lymphoma cell lines

SNS-032 is a potent, selective inhibitor of cyclin-dependent kinases (Cdk) 2, 7 and 9 that inhibits the cell cycle and transcription. Our studies in primary chronic lymphocytic leukemia (CLL) cells demonstrated that SNS-032 inhibited Cdk7 and Cdk9, effectively decreased the phosphorylation of RNA polymerase II (pol II) and inhibited transcription; this was associated with diminished levels of the anti-apoptotic protein Mcl-1 and activation of apoptosis. While indolent CLL cells provided a model to study the action of SNS-032 as an inhibitor of transcription, the present study focuses on evaluating this compound in proliferating cells using four mantle cell lymphoma (MCL) cell lines (Granta 519, Jeko-1, Mino and SP-53). All lines carry the t(11:14)(q13;q32) translocation, that leads to overexpression of cyclin D1, characteristic of MCL. SNS-032 (0.3 to 1 µM) inhibited the phosphorylation of RNA pol II in all 4 cell lines and blocked RNA synthesis. The transcripts and protein levels of short lived protein decreased, including cyclin D1 and Mcl-1. Cell growth was inhibited in a concentration-dependent manner. Phosphorylation of Cdk2 was reduced in Jeko-1, Mino and SP-53 cells, indicating inhibition of Cdk7 activity. However, there was no apparent perturbation in cell cycle distribution following 24 h SNS-032 treatment. Rather, apoptosis was induced rapidly. The strongest effect was seen in JeKo-1 cells, where apoptosis initiated 6 h after addition of SNS-032 (0.3 µM) and was maximal (90% cell death) at 12 h. Since SNS-032 reduced the expression of both Mcl-1 and cyclin D1, siRNA was used to specifically knock down each protein in JeKo-1 cells, and the effect on cell viability was compared by annexin V /PI staining. Mcl-1 and cyclinD1 expression were reduced by their specific siRNA to the average of 9% and 35% measured 24 h after transfection (n=3), respectively. Only Mcl-1 siRNA transfected cells had significant reduction of cell viability, whereas knocking down cyclin D1 did not induce cell death. Cells transfected with both Mcl-1 and cyclin D1 siRNAs did not show additional apoptosis compared to cells transfected with Mcl-1 siRNA alone. Similar results were observed at 48 h. The response to SNS-032 in Granta 519 cells was different from the other cells. Apoptosis was not detected in Granta cells 24 h after SNS-032 incubation (0.3-3 µM). Rather, there was a time- and concentration- dependent inhibition of colony formation, with IC₅₀ of 50 nM after 24 h. SiRNAs of neither Mcl-1 nor cyclinD1 affected Granta cell viability. However, cyclin D1 siRNA, rather than siRNA of Mcl-1, significantly inhibited clonogenic viability of Granta cells. Together, these data indicated that although SNS-032 eliminated both Mcl-1 and cyclin D1, reducing Mcl-1 expression was associated with SNS-032 induced cell death in JeKo-1 cells; eliminating cyclin D1 contributed to the reduced clonogenic survival of Granta cells.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1801.