Dinaciclib (SCH727965) Is a Novel Cyclin Dependent Kinase Inhibitor That Promotes Selective Apoptosis In CLL Cells and Abrogates the Protective Effects of Microenvironment Cytokines

Abstract 971

The first generation cyclin dependent kinase (CDK) inhibitor flavopiridol has significant clinical activity in refractory CLL including those with del(17p13.1). These successes have prompted development of second generation molecules that lack the protein binding, off-target effects (i.e. GSK3-β inhibition), and complex enterohepatic circulation that exist with flavopiridol. Dinaciclib is a selective inhibitor of CDK 1, 2, 5 and 9 (IC50 of < 5nM) and lacks GSK3-β inhibition. Dinaciclib was selected as a lead CDK inhibitor candidate by an in vivo screen that identified it as having a favorable therapeutic index. Completed phase I studies in solid tumor cancer patients with dinaciclib have confirmed this favorable toxicity profile. We sought to explore the pre-clinical activity of dinaciclib in CLL to support its clinical development. Our studies demonstrate that dinaciclib promotes concentration-dependent apoptosis that is superior to flavopiridol following a clinically relevant 2-hour exposure (74.4% viable with 1μM flavopiridol compared to 26.7% viable with 1μM dinaciclib, p<0.0001). Additionally, dinaciclib, like flavopiridol, lacks cytotoxic effects on normal T-cells under these same conditions. Dinaciclib induced cytotoxicity was independent of IgVH mutational status and fludarabine refractoriness. Surprisingly, CLL cells from patients with del(17p13.1) were significantly more resistant than from patients with normal cytogenetics (68.2% viable versus 19.6%, p=0.0006) to dinaciclib induced cytotoxicity. This is in contrast to flavopiridol (54.1% viable versus 50.0%, p=0.2119) where this has not been seen. This finding is being investigated in the current phase 1 clinical trial of dinaciclib in CLL. Apoptosis induced by dinaciclib is caspase dependent, as co-incubation with the caspase inhibitor z-VAD-fmk rescued dinaciclib induced cytotoxicity. As seen with other CDK inhibitors, dinaciclib potently down-regulated Mcl-1 and Bcl-2 expression at a mRNA and protein level in CLL cells. Given the importance of microenvironment stimuli to CLL survival, we sought to determine how soluble and contact microenvironment effects influence dinaciclib cytotoxicity. T-cells and stromal cells promote CLL cell survival in part through the production of cytokines including CD40 ligand, BAFF, TNF-α, and IL-4. While CD40 ligand, BAFF, TNF-α, and IL-4 all protected CLL cells from spontaneous apoptosis, they did not significantly abrogate dinaciclib-mediated cytotoxicity. Consequently dinaciclib inhibited CD40 ligand induced up-regulation of Mcl-1 protein expression. The mechanism by which this protection is abrogated appears to involve down-regulation of the respective ligand receptor, as expression of each of these receptors was notably reduced at 2 hours in 1μM dinaciclib-treated CLL cells. CLL cells also derive protection from both spontaneous and drug-induced apoptosis by direct co-culture (and contact) with stromal cells. We therefore assessed the cytotoxic effect of dinaciclib on CLL patient cells when cultured in the presence of stromal cells. These studies surprisingly demonstrated loss of adhesion of stromal cells and abrogation of CLL cell protection. To avoid any direct effect of dinaciclib on the stromal cells, we then pre-treated CLL cells with 1μM dinaciclib for 2 hours, followed by washout and incubation on stromal cells. In these experiments, co-culture of CLL cells on stromal cells almost completely abrogated dinaciclib-mediated cytotoxicity. Similar findings were observed with fibronectin-coated plates. Together, these data indicate that stromal protection of CLL cells against dinaciclib is mediated either by cell-cell contact or by cytokines other than those tested. Collectively, these studies support the development of dinaciclib for the treatment of CLL, and also identify distinct microenvironment signals that may modulate response to this novel agent. Additionally, the differential response to del(17p13.1) CLL cells suggest distinct differences between dinaciclib and flavopiridol that are currently under investigation.

This work was supported by the National Cancer Institute Leukemia SPORE P50-CA140158, The Leukemia and Lymphoma Society and The D. Warren Brown Foundation. AJJ is a Paul Calabresi Scholar.