Introduction: Chk1 and Chk2 are serine/threonine kinases that are involved in cell cycle control and DNA repair pathways. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1-negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5-2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of baseline genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. Moreover, in all sensitive cell lines Chk1/Chk2 inhibition increased number of γH2AX foci (western blot and immunofluorescence analysis) and phosphorylation of Chk1 (ser317 and ser345) representative of induced DNA damage. Functionally, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15). The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients with 36% of them having IC50 within 100-500 nM. By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells. Noteworthy, in mice transplanted with T-lymphoid leukemia, PF-0477736 increased the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts after 24 hrs to elucidate the mechanisms of action of PF-0477736. Results showed a differential expression (p < 0.05) of genes involved in chromatin modification (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to accumulation of DNA damage and subsequent apoptosis in leukemia cells. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007-2009. PF-0477736 provided by Pfizer.
Inhibition of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) is a novel therapeutic strategy in B- and T-Acute Lymphoblastic Leukemia (ALL).
IACOBUCCI, ILARIA;GHELLI LUSERNA DI RORÀ, ANDREA;Ferrari, Anna;IMBROGNO, ENRICA;Papayannidis, Cristina;VENTURI, CLAUDIA;LONETTI, ANNALISA;GUADAGNUOLO, VIVIANA;CATTINA, FEDERICA;Abbenante, Mariachiara;
2013
Abstract
Introduction: Chk1 and Chk2 are serine/threonine kinases that are involved in cell cycle control and DNA repair pathways. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1-negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5-2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of baseline genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. Moreover, in all sensitive cell lines Chk1/Chk2 inhibition increased number of γH2AX foci (western blot and immunofluorescence analysis) and phosphorylation of Chk1 (ser317 and ser345) representative of induced DNA damage. Functionally, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15). The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients with 36% of them having IC50 within 100-500 nM. By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells. Noteworthy, in mice transplanted with T-lymphoid leukemia, PF-0477736 increased the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts after 24 hrs to elucidate the mechanisms of action of PF-0477736. Results showed a differential expression (p < 0.05) of genes involved in chromatin modification (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to accumulation of DNA damage and subsequent apoptosis in leukemia cells. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007-2009. PF-0477736 provided by Pfizer.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
