PHA767491, a Dual Cdc7/CDK9 Inhibitor, with Potential to Target Both Proliferation and Survival in CLL.

Abstract 2366

Poster Board II-343

CLL is characterized by a combination of a block in apoptosis allied to the steady expansion of a small proliferative compartment, which is more pronounced in marrow and nodal tissue than the peripheral blood. Current treatments for CLL are not curative and factors associated with resistance to standard therapy include IgVH mutations and abnormalities of p53. It is thought that persistence of cells within the proliferative nodal compartment contributes to relapse.

PHA-767491 was originally identified as a potent inhibitor of the S phase promoting kinase Cell division cycle 7 (Cdc7), but has also been shown to have inhibitory activity against the cyclin dependent kinase 9 (CDK9). PHA-767491 has been shown to selectively induce apoptosis in cancer cells in a p53 independent fashion (Montagnoli et al Nat Chem Biol. 2008 Jun;4(6):357-65). An orally bioavailable form of PHA767491, NMS-354 is now in phase I clinical trials in solid tumors. Since transcriptional inhibition resulting from CDK9 inhibition could result in depletion of anti-apoptotic proteins with short half-life, such as Mcl-1 and XIAP, both critical for CLL survival, we hypothesized that PHA-767491 and NMS-354 may have considerable activity in CLL, targeting both survival and proliferation pathways and inducing cell death independently of p53 abnormalities. These studies detail the mechanism of action of PHA-767491 on primary CLL cells.

Primary cell samples were isolated from a total of 20 patients with CLL with various stages of disease (8 Binet A, 6 Stage B, and 4 Stage C) and who were either treatment naïve or had received a variety of prior therapies. Patient samples were characterised for cytogenetic abnormalities (11q, 17p and 13q deletion or trisomy 12), IgVH mutation, CD38 expression and B2M levels.

PHA-767491 was equally effective at inducing apoptosis in CLL cells with both favourable and unfavourable prognostic features, including 17p del, 11q del and unmutated IgVH. The concentration of PHA-767491 required to induce cell death in 50% of the CLL cells (IC50) approximated to between 500 nM and 1 mM at 12 hours of treatment. Following treatment with 1 mM significant apoptosis was seen in all samples by 6 hours, and was maximal by 12 hours. Induction of apoptosis (assessed by TMRE, Annexin-V, caspase 3 activation and PARP cleavage) was noted as early as 4 hours. The mechanism of action of PHA-767491 was investigated by western blotting for a variety of apoptotic proteins following incubation with the compound. Rapid downregulation of Mcl-1 was noted within 4 hours of treatment and this was followed later (6-10 hours) by downregulation of XIAP. Levels of Bcl2 and Bcl-XL were unchanged.

We next conducted cell cycle analysis, which showed that the CLL cells were in G0/G1 indicating that the likely mechanism of action in peripheral blood cells ex-vivo is likely to be due to Mcl-1 downregulation rather than Cdc7 inhibition. Subsequent immunohistochemical staining of lymph node biopsies from patients in this study showed evidence of Cdc7 activity in CLL cells (Cdc7, Mcm2 phosphorylation), providing evidence of a proliferative compartment potentially targetable by Cdc7 inhibition. Experiments to explore this aspect are ongoing.

In summary these data suggest that PHA-767491 and related dual Cdc7/CDK9 inhibitors may have a role in treatment of high risk CLL and that further investigation is warranted.