Introduction. Chk1 and Chk2 are serine/threonine kinases that play a critical role in determining cellular responses to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. We explored the cellular effects of single-agent inhibition of Chk1/2 by PF-0477736 and its potential use as a therapeutic strategy for the treatment of B-ALL. Methods. Cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Results. 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 M) for 24, 48 and 72 hours. 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. All cell lines were TP53 wild-type, CDKN2A deleted. 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. Functionally, PF-0477736 decreased the inhibitory phosphorylation of Cdc25c Ser216 which is inactivated by Chk1 to prevent mitotic entry and increased the number of H2AX foci, a markers of DNA damage, that culminated in a proportion of cells developing intense staining for H2AX together with nuclear morphological characteristics of apoptosis as demonstrated by immunofluorescence analysis. The efficacy of PF-0477736 was thereafter confirmed in primary blasts from 11 B-ALL patients. Based on the viability results, three groups of patients were identified: very good responders, 46% (IC50: 0.1-0.5 μM at 24 hours); good responders, 36% (IC50: 0.6-1 μM at 24 hours); poor responders, 18% (IC50 > 1 μM at 24 hours). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis was performed on treated cell lines and their untreated counterparts (DMSO 0.1%) after 24 hours. Consistent with a specific Chk1- mechanism of action, treatment resulted in differential expression (p < 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 accumulation of DNA damage and subsequent apoptosis in B-ALL cells. Conclusions. 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-Ravenna, FIRB2006, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer.

SINGLE-AGENT INHIBITION OF CHECKPOINT KINASE 1 (CHK1) AND 2 (CHK2) BY PF-0477736 (PFIZER) AS A NEW PROMISING THERAPY IN B-ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

IACOBUCCI, ILARIA;GHELLI LUSERNA DI RORÀ, ANDREA;DERENZINI, ENRICO;Imbrogno E;PAPAYANNIDIS, CRISTINA;LONETTI, ANNALISA;SOVERINI, SIMONA;MARTINELLI, GIOVANNI
2012

Abstract

Introduction. Chk1 and Chk2 are serine/threonine kinases that play a critical role in determining cellular responses to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. We explored the cellular effects of single-agent inhibition of Chk1/2 by PF-0477736 and its potential use as a therapeutic strategy for the treatment of B-ALL. Methods. Cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Results. 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 M) for 24, 48 and 72 hours. 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. All cell lines were TP53 wild-type, CDKN2A deleted. 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. Functionally, PF-0477736 decreased the inhibitory phosphorylation of Cdc25c Ser216 which is inactivated by Chk1 to prevent mitotic entry and increased the number of H2AX foci, a markers of DNA damage, that culminated in a proportion of cells developing intense staining for H2AX together with nuclear morphological characteristics of apoptosis as demonstrated by immunofluorescence analysis. The efficacy of PF-0477736 was thereafter confirmed in primary blasts from 11 B-ALL patients. Based on the viability results, three groups of patients were identified: very good responders, 46% (IC50: 0.1-0.5 μM at 24 hours); good responders, 36% (IC50: 0.6-1 μM at 24 hours); poor responders, 18% (IC50 > 1 μM at 24 hours). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis was performed on treated cell lines and their untreated counterparts (DMSO 0.1%) after 24 hours. Consistent with a specific Chk1- mechanism of action, treatment resulted in differential expression (p < 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 accumulation of DNA damage and subsequent apoptosis in B-ALL cells. Conclusions. 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-Ravenna, FIRB2006, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer.
2012
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Iacobucci I; Ghelli A; Derenzini E; Imbrogno E; Ferrari A; Cattina F; Pomella S; Papayannidis C; Venturi C; Guadagnuolo V; Lonetti A; Verga Falzacappa MV; Ottaviani E; Abbenante M; Soverini S; Russo D; Pellicci PG; Baccarani M; Martinelli G
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/154800
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