In Vitro and in Vivo Single-Agent Efficacy of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) Inhibitor PF-0477736 (Pfizer) in B- and T-Acute Lymphoblastic Leukemia (ALL)

Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. 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.

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).

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), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic 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. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, 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), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). 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 leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (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 checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression.

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.

Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.