IntroductionCheckpoint kinases (ie, Chk1 and Chk2) represent key components of the DNA damage checkpoint machinery, which monitors DNA breaks caused by endogenous/metabolic or environmental genotoxic insults or by replication stress. 1,2 In response to DNA damage, cells activate checkpoint pathways, resulting in cell-cycle arrest, which permits the DNA repair machinery to rectify the damage. Depending on the nature of the DNA lesions and the context in which damage occurs, cells either survive and resume cell-cycle progression through a recovery mechanism when repair is successful or are eliminated by apoptosis if repair fails. Thus, checkpoints provide normal cells with critical surveillance machinery designed to promote genomic integrity and survival. Conversely, checkpoint dysfunction contributes to tumorigenesis by permitting cell proliferation in the face of genomic instability. 3,4 Moreover, checkpoints are activated by numerous chemotherapeutic agents and ionizing radiation. 5 This has prompted the development of anticancer strategies targeting checkpoint machinery. 5,6 Among the diverse checkpoint pathway components, Chk1 represents a particularly attractive target for several reasons, that is, (1) Chk1 is functionally associated with all known checkpoints (eg, the G2-M transition, G1, intra-S, 5 and, most recently, the mitotic spindle checkpoint 7 ); (2) Chk1 is essential for maintenance of genomic integrity, whereas the role of Chk2 is conditional 3 ; and (3) for multiple checkpoints, Chk2 function can be mimicked by Chk1, whereas Chk1 cannot be replaced by a functionally overlapping kinase such as Chk2. 3 Chk1 inhibition (eg, by the Chk1 inhibitor UCN-01) results in abrogation of checkpoints induced by DNA-damaging chemotherapy and radiation, leading to enhanced tumor cell killing. 8,9 Given these findings, a major emphasis has been placed on efforts to combine Chk1 inhibitors (eg, UCN-01 10 or CHIR-124 11 ) with diverse DNA-damaging agents. However, an alternative strategy is based on the concept that transformed cells may be ill-equipped to survive simultaneous interruption of both checkpoint machinery and prosurvival signaling. In this context, our group has reported that exposure of human leukemia and multiple myeloma (MM) cells to UCN-01 induces pronounced activation of MEK1/2 and ERK1/2, 12,13 key components of the Ras/Raf/MEK/ERK cascade that plays a critical role in proliferation and survival of malignant cells. 14 Significantly, disruption of ERK1/2 activation by pharmacologic MEK1/2 inhibitors, 12,13 farnesyltransferase inhibitors (FTIs; eg, L744832) 15,16 or HMG-CoA reductase inhibitors (ie, statins) 17 results in a dramatic increase in apoptosis of hematopoietic malignant cells. Together, these findings suggest that activation of Ras/MEK/ERK signaling cascade may represent a compensatory response to Chk1 inhibitor lethality, and that interruption of this response lowers the death threshold.Although the observation that MEK1/2 inhibitors or FTIs antagonize UCN-01-mediated ERK1/2 activati...

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer͞testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 introncontaining genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.germ line ͉ human ͉ transcript

Interactions between the dual Bcr/Abl and aurora kinase inhibitor MK-0457 and the histone deacetylase inhibitor vorinostat were examined in Bcr/Abl ؉ leukemia cells, including those resistant to imatinib mesylate (IM), particularly those with the T315I mutation. Coadministration of vorinostat dramatically increased MK-0457 lethality in K562 and LAMA84 cells. Notably, the MK-0457/vorinostat regimen was highly active against primary CD34 ؉ chronic myelogenous leukemia ( IntroductionChronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph; 22q), which is responsible for the chimeric fusion oncoprotein Bcr/Abl. The Bcr/Abl kinase is constitutively active and signals downstream to multiple survival pathways, 1 providing CML cells with a survival advantage over their normal counterparts and conferring resistance against cytotoxic agents. 2 The treatment of CML has been revolutionized by the introduction of the kinase inhibitor imatinib mesylate (IM; Gleevec, Novartis, Basel, Switzerland), which is highly active in patients with chronic-phase CML 3 but less active in patients with accelerated or blast-phase disease. 4 However, almost all patients who initially respond eventually develop resistance to this agent. Mechanisms of resistance include bcr/abl gene amplification, increased expression of the Bcr/Abl protein, and most commonly, point mutations in various domains of the Bcr/Abl kinase, including the activation loop, the phosphorylation loop, or the gatekeeper region. 5 This phenomenon stimulated the development of secondgeneration Bcr/Abl kinase inhibitors (eg, dasatinib and nilotinib), which are active against proteins bearing most mutations. 6,7 However, these agents are inactive against cells with gatekeeper region mutations, most notably T315I, 8 prompting the search for newer Bcr/Abl kinase inhibitors active against such mutants.The aurora kinases (A, B, and C) represent a family of serine/threonine kinases involved in the control of mitosis. 9 Deregulation of aurora kinase activity leads to disruption of cell-cycle progression, mitotic abnormalities, and genetic instability. 10 Importantly, aurora kinases are overexpressed and/or activated in a variety of tumor cells, suggesting a role for this family in tumorigenesis. 9,10 MK-0457 is a small-molecule, novel pan-aurora kinase inhibitor 9 with demonstrated activity against wild-type (wt) and mutated Bcr/Abl, [11][12][13] including the T315I mutation, as well as FLT3 and JAK2. MK-0457 delays entry into mitosis, leads to aberrant cytokinesis, induces apoptosis in several human tumor types, and is being evaluated in patients with a variety of malignant diseases. 9 MK-0457 potently inhibits aurora kinases (particularly aurora A and B) in tumor cells, manifested by down-regulation of phosphorylated histone H3 at Ser10. 9 This results in multiple events, including aberrant cell-cycle progression and accumulation of polyploid cells with DNA content of 4N or more, which collectively trigger cell death. 14,15 Very recently, it was repor...

Cancer/testis (CT) genes are normally expressed in germ cells only, yet are reactivated and expressed in some tumors. Of the approximately 40 CT genes or gene families identified to date, 20 are on the X chromosome and are present as multigene families, many with highly conserved members. This indicates that novel CT gene families may be identified by detecting duplicated expressed genes on chromosome X. By searching for transcript clusters that map to multiple locations on the chromosome, followed by in silico analysis of their gene expression profiles, we identified five novel gene families with testis-specific expression and >98% sequence identity among family members. The expression of these genes in normal tissues and various tumor cell lines and specimens was evaluated by qualitative and quantitative RT-PCR, and a novel CT gene family with at least 13 copies was identified on Xq24, designated as CT47. mRNA expression of CT47 was found mainly in the testes, with weak expression in the placenta. Brain tissue was the only positive somatic tissue tested, with an estimated CT47 transcript level 0.09% of that found in testis. Among the tumor specimens tested, CT47 expression was found in approximately 15% of lung cancer and esophageal cancer specimens, but not in colorectal cancer or breast cancer. The putative CT47 protein consists of 288 amino acid residues, with a C-terminus rich in alanine and glutamic acid. The only species other than human in which a gene homologous to CT47 has been detected is the chimpanzee, with the predicted protein showing approximately 80% identity in its carboxy terminal region.