Abstract 2499: Single agent activity of checkpoint kinase inhibitor PF-477736, in a MYC-driven lymphoma model
Authors: Petranel T. Ferrão, Carleen Cullinane, Jeanette Raleigh, Sreesha Srinivasa, Wendy Levin, Ricky W. Johnstone, Grant A. McArthur
Journal: Cancer Research (2010)

Abstract CHK1 and CHK2 are protein kinases that function as effectors of cell cycle checkpoint arrest following DNA damage. Small molecule inhibitors with specificity for CHK1 such as PF-477736 (Blasina et al, 2008 Mol Cancer Ther 7:2394) and others with specificity for CHK1 and CHK2, are currently in phase 1 clinical evaluation in combination with chemotherapeutic agents known to induce DNA damage. We therefore hypothesized that inhibitors of CHK1/CHK2 may have single agent activity in tumours with oncogene-induced DNA damage. Cell lines derived from Eμ-MYC driven murine lymphomas display intrinsic DNA damage detected by γH2AX foci staining. Within 2 hours of 500nM PF-477736 treatment, a dramatic increase in the intensity of γH2AX staining and proportion of cells with γH2AX staining, was observed in all Eμ-MYC lines. An increase in mitotic cells detected by expression of phospho-Histone3, was also observed in all Eμ-MYC lines with a high proportion displaying features of aberrant mitosis, demonstrating inhibition of the S and/or G2/M checkpoints resulting in premature entry to mitosis. Early apoptosis was detected in 6 separate Eμ-MYC lines expressing functional p53, following treatment in vitro (&amp;gt;50% Annexin V positive cells 4 hrs after 500nM PF-477736). Subsequent cell death assessed as the proportion of PI positive cells, was observed in all cell lines expressing functional p53 (IC50 200-500nM at 16 hrs). Enforced expression of anti-apoptotic protein Bcl-2 by retroviral transduction abrogated cell death at up to 3μM PF-477736. Two separate Eμ-MYC lines expressing spontaneously arising mutant forms of p53, and three separate Eμ-MYC lines derived on a p53 null background, were relatively insensitive to PF-477736 (IC50&amp;gt;1μM at 16hrs), indicating that functional p53 is necessary to mediate apoptosis/death following accumulation of DNA damage due to CHK1 inhibition. Treatment with AZD7762 a CHK1/2 inhibitor, also resulted in death of Eμ-MYC lines in vitro, (IC50 200nM-2μM at 16 hrs), however the variation in sensitivity could not be attributed to p53 status. Treatment of C57Bl/6 mice transplanted with an Eμ-MYC line expressing functional p53, resulted in a dramatic increase in the proportion of apoptotic/dead cells in the established lymphomas within 6 hours following 20mg/kg PF-477736 i.p. A significant decrease in the wbc of 5 mice, from an average 48.2×106 wbc/ml pre-treatment to 31.8×106 at 16 hours post PF-477736 (P=0.0053), compared to vehicle treated mice (51.1×106 pre-treatment and 60.4×106 post-treatment), together with a lower average spleen weight of the PF-477736 treated mice (0.52g) compared to vehicle treated mice (0.68g), was observed. We propose that checkpoint kinase inhibitors will be beneficial as therapeutic agents able to specifically exploit tumours containing intrinsic DNA damage that are reliant on checkpoints for maintenance of genomic integrity, such as in “MYC-driven cancers”. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2499.