Two signaling pathways are activated by antineoplastic therapies that damage DNA and stall replication. In one pathway, double-strand breaks activate ataxia-telangiectasia mutated kinase (ATM) and checkpoint kinase 2 (Chk2), two protein kinases that regulate apoptosis, cell-cycle arrest, and DNA repair. In the second pathway, other types of DNA lesions and replication stress activate the Rad9-Hus1-Rad1 complex and the protein kinases ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (Chk1), leading to changes that block cell-cycle progression, stabilize stalled replication forks, and influence DNA repair. Gemcitabine and cytarabine are two highly active chemotherapeutic agents that disrupt DNA replication. Here, we examine the roles these pathways play in tumor cell survival after treatment with these agents. Cells lacking Rad9, Chk1, or ATR were more sensitive to gemcitabine and cytarabine, consistent with the fact that these agents stall replication forks, and this sensitization was independent of p53 status. Interestingly, ATM depletion sensitized cells to gemcitabine and ionizing radiation but not cytarabine. Together, these results demonstrate that 1) gemcitabine triggers both checkpoint signaling pathways, 2) both pathways contribute to cell survival after gemcitabine-induced replication stress, and 3) although gemcitabine and cytarabine both stall replication forks, ATM plays differential roles in cell survival after treatment with these agents.Gemcitabine (2Ј,2Ј-difluoro 2Ј-deoxycytidine), a pyrimidine-based antimetabolite, is currently licensed for the treatment of pancreatic cancer. Recent clinical studies have also demonstrated extensive activity of this agent against a variety of additional neoplasms, including carcinomas of the ovary, lung, and breast, acute leukemias, and refractory lymphomas (Carmichael, 1998;Nabhan et al., 2001). Because of its widespread use, there is considerable interest in understanding factors that affect sensitivity and resistance to this agent. Earlier studies demonstrated that gemcitabine is taken into cells on concentrative nucleoside transporter 1 and phosphorylated to gemcitabine 5Ј-monophosphate by deoxycytidine kinase (Plunkett et al., 1996). Subsequent addition of 5Ј-phosphates results in the formation of gemcitabine diphosphate and gemcitabine triphosphate, both of which contribute to the antiproliferative effects of gemcitabine (Plunkett et al., 1996). Gemcitabine diphosphate inhibits ribonucleotide reductase, thereby depleting deoxyribonucleotide levels. Gemcitabine triphosphate is a substrate for replicative DNA polymerases and causes chain termination one base pair beyond the site of incorporation.Because gemcitabine inhibits replication, this drug is predicted to activate the S-phase checkpoint, a series of reactions that inhibit DNA synthesis and enhance survival when cells experience replication stress. According to current understanding, the kinases ATR and Chk1 play critical roles in this checkpoint. When replicati...

Adaphostin (NSC 680410), an analog of the tyrphostin AG957, was previously shown to induce Bcr/abl down-regulation followed by loss of clonogenic survival in chronic myelogenous leukemia (CML) cell lines and clinical samples. Adaphostin demonstrated selectivity for CML myeloid progenitors in vitro and remained active in K562 cells selected for imatinib mesylate resistance. In the present study, the mechanism of action of adaphostin was investigated in greater detail in vitro. Initial studies demonstrated that adaphostin induced apoptosis in a variety of Bcr/ abl ؊ cells, including acute myelogenous leukemia (AML) blasts and cell lines as well as chronic lymphocytic leukemia (CLL) samples. Further study demonstrated that adaphostin caused intracellular peroxide production followed by DNA strand breaks and, in cells containing wild-type p53, a typical DNA damage response consisting of p53 phosphorylation and up-regulation. Importantly, the antioxidant N-acetylcysteine (NAC) blunted these events, whereas glutathione depletion with buthionine sulfoximine (BSO) augmented them. Collectively, these results not only outline a mechanism by which adaphostin can damage both myeloid and lymphoid leukemia cells, but also indicate that this novel agent might have a broader spectrum of activity than originally envisioned. IntroductionChronic myelogenous leukemia (CML) is almost universally associated with a t(9;22) chromosomal translocation that juxtaposes the Bcr and Abl genes. 1,2 Because the resulting kinase, p210 Bcr/abl , is found exclusively in malignant hematopoietic cells, there has been considerable interest in identifying inhibitors of this enzyme. Several classes of p210 Bcr/abl inhibitors have been identified, including the 2-phenylaminopyrimidine derivative imatinib mesylate, 3 the pyrido [2,3-d]pyrimidine derivative PD180970, 4 and the tyrphostin AG957. 5 Clinical studies performed to date have established that imatinib mesylate is highly active in chronic-phase CML. 6,7 In contrast, patients with CML in blast crisis or t(9;22) ϩ acute lymphocytic leukemia (ALL) tend to have shorter remissions. 8,9 At the time of relapse, cells from the latter patients exhibit imatinib mesylate resistance in vitro. 10 Although the molecular basis of imatinib mesylate resistance remains somewhat controversial, 11,12 there is considerable interest in overcoming this resistance. Potential strategies include combining imatinib mesylate with conventional cytotoxic agents 13 or arsenic trioxide 9 as well as using Bcr/abl kinase inhibitors that are unaffected by alterations that cause imatinib mesylate resistance. 14,15 The tyrphostin AG957 was originally identified as an inhibitor of p210 Bcr/abl kinase that is not competitive with adenosine triphosphate (ATP). 5 Subsequent studies demonstrated that adaphostin, the adamatyl ester analog of AG957, has a longer serum half-life and displays greater antiproliferative effects against Bcr/abl ϩ leukemia cells in the National Cancer Institute (NCI) hollow fiber assay. Recent comparison of th...

The mechanism of cytotoxicity of farnesyltransferase inhibitors is incompletely understood and seems to vary depending on the cell type. To identify potential determinants of sensitivity or resistance for study in the accompanying clinical trial ( IntroductionFarnesyltransferase inhibitors (FTIs) are currently undergoing extensive clinical testing in various hematologic malignancies. [1][2][3] These agents inhibit farnesyltransferase, an enzyme that transfers the 15-carbon farnesyl group from farnesyl pyrophosphate to a variety of polypeptide acceptors, including the chaperone heat shock protein 40/HDJ-2; the nuclear intermediate filament proteins prelamin A and lamin B; the centromere protein CENP E; and small GTP-binding proteins of the Ras, Rho, and Rheb families. 4,5 Collectively, inhibition of farnesylation of these polypeptides leads to diminished cell proliferation. In addition, FTIs induce cell death in some model systems under certain conditions. These cytotoxic effects have been attributed to FTI-induced inhibition of prosurvival signaling by Akt,6,7 signal transducers and activators of transcription, [8][9][10] mitogen-activated protein kinases (MAPKs), 9,[11][12][13] or the Rheb target mammalian target of rapamycin. 14 Recent work has especially emphasized the role of Rheb inhibition as a mechanism of FTI-induced antilymphoma effects in murine lymphomas and leukemia. 15 Alternatively, it has been suggested that FTIs induce apoptosis by causing up-regulation of the proapoptotic Bcl-2 family members Bax, 16 Bak, 17 or Puma. 18 Although FTIs were initially developed based on the premise that inhibition of farnesylation would abrogate signaling by mutant Ras proteins, 19 these agents have demonstrated little efficacy in solid tumors. [20][21][22] In contrast, tantalizing activity was observed in several hematologic malignancies. [1][2][3] In particular, the orally bioavailable nonpeptidimimetic FTI tipifarnib 23 demonstrated activity in adults with acute leukemia. The initial phase 1 trial not only established a maximum tolerated dose in patients with relapsed and refractory acute leukemias but also determined that tipifarnib levels in bone marrow were 1.6-8 nmol/mg of tissue at this dose, demonstrated FT inhibition in leukemia cells in situ, and provided evidence of activity in relapsed AML. 24 Subsequent phase 2 and phase 3 studies have demonstrated response rates of 11%-23% in elderly patients with previously untreated poor risk acute myeloid leukemia (AML). 25,26 In an effort to select the subset of AML patients most likely to respond, Raponi et al empirically identified a 2-transcript signature, characterized by a high ratio of mRNA encoding the Ras guanine nucleotide exchange factor RasGRP1 27 relative to mRNA encoding the repair protein aprataxin, that had a 92% negative predictive value and a 28% positive predictive value in 2 single-agent phase 2 tipifarnib AML trials. 28 Based on these results, gene signature-guided trials of tipifarnib in acute leukemia are being initiated.Tipifarnib also has...