Abstract 1772: Inhibition of DNA repair by the small molecule Chk1/Chk2 inhibitor PF-0477736 (Pfizer) in B-acute lymphoblastic leukemia (ALL)
Authors: Iacobucci Ilaria , Cattina Federica , Pomella Silvia , Brighenti Elisa , Papayannidis Cristina , Derenzini Enrico , Lonetti Annalisa , Ferrari Anna , Falzacappa Maria Vittoria Verga , Guadagnuolo Viviana , Aluigi Michela , Ottaviani Emanuela , Abbenante Maria Chiara , Soverini Simona , Russo Domenico , Pelicci Pier Giuseppe , Baccarani Michele , Martinelli Giovanni 
Published: August 2012

Abstract
Checkpoint kinase 1 (Chk1) and 2 (Chk2) are serine/threonine kinases involved in the DNA damage response and in the regulation of cell cycle progression at S-G2 phase. Based on the potential utility of DNA checkpoint inhibition in enhancing tumor cell death, we investigated the preclinical activity of PF-0477736 (Pfizer), a potent and selective Chk1/2 inhibitor, in B-ALL and we determined potential biomarkers of functional inhibition. BCR-ABL1-positive (BV-173, SUPB-15) and negative cell lines (NALM6, NALM19, REH) were incubated with increasing concentrations of PF-0477736 (0.005-2 μM) for 24, 48 and 72 hours. PF-0477736 inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1-1.5 µM, with BV-173 being the most sensitive, while NALM6 the most resistant (WST-1 assay, Roche). Consistent with the viability results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in all cell lines. Western Blot analysis showed that PF-0477736 decreases the inhibitory phosphorylation of Cdc25c Ser216 which is inactivated by Chk1 to prevent mitotic entry, and increases phosphorylation of γH2AX Ser139, a marker of DNA damage. The efficacy of PF-0477736 was thereafter confirmed in primary blast cells from 11 B-ALL patients. Based on the viability results, three groups of patients were identified: very good responders, 46% (IC50 ranged from 0.1-0.5 µM at 24 hours); good responders, 36% (IC50 ranged from 0.6-1 µM at 24 hours); poor responders, 18% (IC50 &gt; 1 µM at 24 hours). Since multiple studies reported a higher activity of PF-0477736 against p53-defective cancer cells, we performed a mutational screening of all coding exons of p53. All cell lines and primary leukemia blasts lacked mutations, demonstrating that in B-ALL the sensitivity to PF-0477736 is independent of p53 status. Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated cell lines and their untreated counterparts. Consistent with a specific Chk1-mechanism of action, treatment resulted in differential expression (p &lt; 0.05) of genes involved in apoptosis and cell cycle (e.g. CEBPB, CUL1, Histone H1-H2A, 2B family clusters) and DNA damage (DDIT3, GADD34 and GADD45a), suggesting that PF-0477736 contributes to a checkpoint abrogation and to an activation of DNA damage response in B-ALL cells. In conclusion, for the first time we demonstrated the efficacy of PF-0477736 in vitro models of B- ALL, suggesting that single-agent Chk1/2 inhibition may be further evaluated in clinical trials. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, PIO program, Programma Ricerca Regione-Università 2007-2009. PF-0477736 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 1772. doi:1538-7445.AM2012-1772