Although improvements in knowledge of genetic basis of ALL have been made and many new drugs are now available, the prognosis for adult patients with ALL is still a challenge. Current therapies based on chemotherapy regimens or on tailored targets do not completely eradicate the leukemia clone and relapse is a predictable event. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), an ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Chk1 and Chk2 are serine/threonine kinases which following activation by a broad spectrum of damaged DNA coordinate the DNA damage response. By quantitative PCR, higher transcript levels of Chk1 were found in 8 leukemia cell lines and 54 newly diagnosed ALL cases compared to normal bone marrow mononuclear cells (p value < 0.001). Immunohistochemistry of formalin-fixed paraffin-embedded tissue samples collected from 60 ALL patients showed a diffuse positivity for Chk1, Chk2 and the phosphorylated forms of Chk1 (Ser345) in 51/54 (96%), 55/57 (94%) and 45/56 (80%) of the cases respectively, whereas 15/55 (27%) of ALLs stained for phosphorylated form of Chk2 (Thr68). Interestingly, in our ALL series, genomic damage was suggested by the nuclear labeling for γ-H2A.X molecule in 40/59 (68%) of samples. In B-/T-ALL cell lines (BV-173, SUP-B15, REH, NALM-6, NALM-19, MOLT-4, RPMI8402 and CCRF-CEM) inhibition of Chk1 resulted in a dose and time-dependent cytotoxicity (IC50 from 57.4 nM to 1423.0 at 24 hours), induction of apoptosis and increased DNA damage as demonstrated by increased levels of phosphorylated Chk1 (Ser317 and Ser345) and H2A.X (Ser139). Moreover, PF-00477736 efficiently triggered the Chk1-Cdc25-Cdc2 pathway with a decreased phosphorylation of Cdc25c (Ser216) and Cdc2 (Tyr15). Gene expression profiling analysis shed light on molecular mechanisms driven by PF-00477736 revealing a deregulation of genes involved in DNA damage checkpoint, cell cycle control and apoptosis. Among these, Gadd45a and Plk3 were confirmed by qPCR analysis up-regulated, whereas Cdk4 and Chk2 resulted down-regulated between treated and untreated samples. The efficacy of PF-00477736 as a single agent was confirmed ex-vivo in primary leukemic blasts separated from 14 ALL patients but not in in primary cultures of normal bone marrow mononuclear cells. Furthermore, in mice transplanted with T-lymphoid leukemia PF-0477736 increased the survival of treated mice compared with mice treated with vehicle (p = 0.0016). In conclusion, in vitro, ex-vivo and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia and they provide a strong rationale for its future clinical investigation.

In vitro and in vivo single-agent efficacy of checkpoint kinase inhibition in acute lymphoblastic leukemia

Ilaria Iacobucci;Andrea Ghelli Luserna Di Rorà;Claudio Agostinelli;Enrico Derenzini;Anna Ferrari;Cristina Papayannidis;Annalisa Lonetti;Simona Righi;Enrica Imbrogno;Claudia Venturi;Viviana Guadagnuolo;Federica Cattina;Emanuela Ottaviani;Maria Chiara Abbenante;Domenico Russo;Pier Luigi Zinzani;Stefano Pileri;Giovanni Martinelli
2014

Abstract

Although improvements in knowledge of genetic basis of ALL have been made and many new drugs are now available, the prognosis for adult patients with ALL is still a challenge. Current therapies based on chemotherapy regimens or on tailored targets do not completely eradicate the leukemia clone and relapse is a predictable event. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), an ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Chk1 and Chk2 are serine/threonine kinases which following activation by a broad spectrum of damaged DNA coordinate the DNA damage response. By quantitative PCR, higher transcript levels of Chk1 were found in 8 leukemia cell lines and 54 newly diagnosed ALL cases compared to normal bone marrow mononuclear cells (p value < 0.001). Immunohistochemistry of formalin-fixed paraffin-embedded tissue samples collected from 60 ALL patients showed a diffuse positivity for Chk1, Chk2 and the phosphorylated forms of Chk1 (Ser345) in 51/54 (96%), 55/57 (94%) and 45/56 (80%) of the cases respectively, whereas 15/55 (27%) of ALLs stained for phosphorylated form of Chk2 (Thr68). Interestingly, in our ALL series, genomic damage was suggested by the nuclear labeling for γ-H2A.X molecule in 40/59 (68%) of samples. In B-/T-ALL cell lines (BV-173, SUP-B15, REH, NALM-6, NALM-19, MOLT-4, RPMI8402 and CCRF-CEM) inhibition of Chk1 resulted in a dose and time-dependent cytotoxicity (IC50 from 57.4 nM to 1423.0 at 24 hours), induction of apoptosis and increased DNA damage as demonstrated by increased levels of phosphorylated Chk1 (Ser317 and Ser345) and H2A.X (Ser139). Moreover, PF-00477736 efficiently triggered the Chk1-Cdc25-Cdc2 pathway with a decreased phosphorylation of Cdc25c (Ser216) and Cdc2 (Tyr15). Gene expression profiling analysis shed light on molecular mechanisms driven by PF-00477736 revealing a deregulation of genes involved in DNA damage checkpoint, cell cycle control and apoptosis. Among these, Gadd45a and Plk3 were confirmed by qPCR analysis up-regulated, whereas Cdk4 and Chk2 resulted down-regulated between treated and untreated samples. The efficacy of PF-00477736 as a single agent was confirmed ex-vivo in primary leukemic blasts separated from 14 ALL patients but not in in primary cultures of normal bone marrow mononuclear cells. Furthermore, in mice transplanted with T-lymphoid leukemia PF-0477736 increased the survival of treated mice compared with mice treated with vehicle (p = 0.0016). In conclusion, in vitro, ex-vivo and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia and they provide a strong rationale for its future clinical investigation.
Ilaria Iacobucci, Andrea Ghelli Luserna Di Rorà, Maria Vittoria Verga Falzacappa, Claudio Agostinelli, Enrico Derenzini, Anna Ferrari, Cristina Papayannidis, Annalisa Lonetti, Simona Righi, Enrica Imbrogno, Claudia Venturi, Viviana Guadagnuolo, Federica Cattina, Emanuela Ottaviani, Maria Chiara Abbenante, Domenico Russo, Pier Luigi Zinzani, Stefano Pileri, Pier Giuseppe Pelicci, Giovanni Martinelli
File in questo prodotto:
Eventuali allegati, non sono esposti

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/628657
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? 0
social impact