Targeting Cdc7 Kinase in Multiple Myeloma.

Myeloma remains an incurable cancer and the development of new therapeutic approaches is of fundamental importance. A key feature of progressive myeloma is deregulation of the mechanisms that control progression through the cell cycle, in particular, the increase in the activity of S-phase promoting factors and the loss of cell cycle brakes such as the tumour suppressor protein p53. Moreover, high expression of Mcl-1, a potent anti-apoptotic Bcl2 family member, and defects in the p53 pathway are now known to be instrumental in inhibiting responsiveness to current and emerging myeloma therapeutics. The Cell division cyle 7 (Cdc7) kinase is an essential protein for the initiation of DNA replication and for cell cycle progression. Previous studies have indicated that Cdc7 inhibition can cause p53 independent apoptosis in several cancer models both in vitro and in vivo and currently Cdc7 inhibitors are entering clinical trials as experimental drugs. In this work we study the effects of the prototype Cdc7 protein kinase inhibitor, PHA-767491, in myeloma cells. In addition to targeting Cdc7, PHA-767491 also targets cyclin dependent kinase 9 (CDK9) and can downregulate Mcl-1 in a variety of cancer cell lines. Hypothesis: Given the molecular characteristics of progressive myeloma and the spectrum of activity of PHA-767491, we hypothesized that Cdc7 inhibition could be an effective treatment strategy in myeloma. Methods The Multiple Myeloma Research Consortium Validation-Core cell lines were treated with increasing doses of PHA-767491 as a single agent and in combination with drugs that currently constitute standards of care in myeloma treatment. These lines included lines resistant to conventional anti-myeloma agents (e.g. melphalan, dexamethasone, and doxorubicin) as well as novel anti-MM agents (e.g. immunomodulatory thalidomide derivatives). A number of lines were TP53 mutants. Similarly, CD138+ purified primary cells with clincal resistance to lenalidomide and bortezomib were treated with increasing doses of PHA-767491. Cell viability was measured by Cell TitreGlo 72 hours post treatment allowing IC50 calcuations. Combination indices were calculated as described by Chou et Talalay. Apoptosis was assessed by PI and Annexin V staining using flow cytometry and immunoblotting with relevant antibodies was performed for cell cycle and apoptotic proteins of interest.

We found that PHA-767491 blocks proliferation and induces apoptosis with an IC50 in the low micromolar range in six out of seven MMRC cell lines and in primary cells from patient (s) with clinical resistance to bortezomib and lenalidomide. The averaqe and median IC50 value of MMRC cell lines was 2.25μM and 2μM respectively. TP53 mutation did not alter sensitivity to PHA-767491. Following treatment of myeloma cells with PHA-767491 there was potent inhibition of phosphorylation of the replicative helicase Mcm2 at serine 40/41 (a specific biomarker of Cdc7 activity) and complete downregulation of Mcl-1 by 6 hours, with concomitant caspase 3 cleavage and apoptosis as assessed by accumulation of sub-G1 cells. Annexin V/PI staining confirmed these cells to be apoptotic. When used in combination, PHA-767491 showed synergistic activity with melphalan and doxorubicin (Combination Index IC50 – 0.3, 0.7 respectively), even in the highly doxorubicin-resistant(p170/MDR1 mediated) line RPMI-8226-Dox-40 (Combination IndexIC50 – 0.3).

We suggest that Cdc7 inhibition with PHA-767491 or with small molecules that have similar properties could be an effective strategy in myeloma treatment and merits further evaluation.

Montagnoli:Nerviano Medical Sciences Oncology: Employment.