Abstract 4694: The small molecule CHK1/CHK2 inhibitor PF-0477736 (Pfizer) demonstrates single agent activity in diffuse large B-cell lymphoma

Abstract

Abstract Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the DNA damage response pathway. CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, including p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL with dysfunctional p53 axis (harboring p53 mutations and/or CDKN2A loss) have been recently shown to have a dismal outcome. In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a panel of B cell lymphoma cell lines and primary cells, and explore its mechanisms of action. Three Germinal center B cell (GCB) DLBCL derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2 were first screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but with an homozygous deletion of CDKN2A. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity (IC50 10 nM at 24 hrs). The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 25-500 nM induced cell death by apoptosis in a time and dose dependent manner. Of note PF-0477736 100-1000 nM demonstrated activity also in primary DLBCL cells. We found no correlations between baseline levels of CHK1/2 activation and outcome. In the sensitive cell lines inhibition of the downstream target CDC25c ser216 phosphorylation coupled with a marked increase in levels of the DNA damage marker γH2AX was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25 - 250 nM). PF-0477736 at the dose of 50-100 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome, providing the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4694. doi:1538-7445.AM2012-4694