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