The Notch signaling pathway plays an important role in cellular proliferation, differentiation, and apoptosis. Unregulated activation of Notch signaling can result in excessive cellular proliferation and cancer. Human T-cell leukemia virus type 1 (HTLV-I) is the etiological agent of adult T-cell leukemia (ATL). The disease has a dismal prognosis and is invariably fatal. In this study, we report a high frequency of constitutively activated Notch in ATL patients. We found activating mutations in Notch in more than 30% of ATL patients. These activating mutations are phenotypically different from those previously reported in T-ALL leukemias and may represent polymorphisms for activated Notch in human cancers. Compared with the exclusive activating frameshift mutations in the proline, glutamic acid, serine, and threonine (PEST) domain in T-ALLs, those in ATLs have, in addition, single-substitution mutations in this domain leading to reduced CDC4/Fbw7-mediated degradation and stabilization of the intracellular cleaved form of Notch1 (ICN1). Finally, we demonstrated that inhibition of Notch signaling by γ-secretase inhibitors reduced tumor cell proliferation and tumor formation in ATL-engrafted mice. These data suggest that activated Notch may be important to ATL pathogenesis and reveal Notch1 as a target for therapeutic intervention in ATL patients.

The Notch signaling pathway is highly conserved from Drosophila to humans and plays an important role in the regulation of cellular proliferation, differentiation and apoptosis.Constitutive activation of Notch signaling has been shown to result in excessive cellular proliferation and a wide range of malignancies, including leukemia, glioblastoma and lung and breast cancers. Notch can also act as a tumor suppressor, and its inactivation has been associated with an increased risk of spontaneous squamous cell carcinoma. This minireview focuses on recent advances related to the mechanisms and roles of activated Notch1, Notch2, Notch3 and Notch4 signaling in human lymphocytic leukemia, myeloid leukemia and B cell lymphoma, as well as their significance, and recent advances in Notch-targeted therapies.

IntroductionHomologous recombination (HR) is a major pathway of doublestrand break (DSB) repair in mammalian cells. 1 Faithful recombination is critical to avoid genetic and genomic aberrations, and involves a complex and orderly assembly of many checkpoints and repair factors. DSBs frequently occur as a result of exposure to irradiation and chemicals. In response to DSBs, activation of ataxia telangiectasia mutated (ATM) initiates a cascade of events, including phosphorylation of histone H2AX (referred to as ␥-H2AX) and downstream effectors such as structural chromosome maintenance 1 (SCM1) and checkpoint kinase 2 (Chk2). 2,3 Chk2 phosphorylates p53, disrupting its interaction with Mdm2 and stabilizing the p53 protein, 4 which pauses the cell cycle so that the cell can attempt to repair its damaged DNA. H2AX phosphorylation plays an important role in both DNA-damage-checkpoint activation and deactivation of the checkpoint signal to allow the cell cycle to resume. HR is very important during DNA replication in the S phase, when DSBs are generated during lagging strand synthesis or when unrepaired lesions cause replication-fork stalling. 5,6 Initiation of HR involves the recruitment of the MRE11/RAD50/NBS1 (MRN) complex to DNA-damaged sites that can be visualized by accumulation of ␥-H2AX as foci. 7 In contrast, DSBs created during the G1 or M phase are preferentially repaired by a nonconservative, nonhomologous-endjoining (NHEJ) pathway. The NHEJ pathway has been shown to be Ku80 and DNA-dependent protein kinase (DNA-PK) dependent. 8 The switch from HR to NHEJ has not been fully elucidated, but can in part be explained by the fact that MRE11-resection activity generates single-stranded DNA for which Ku80 has a poor affinity, allowing for the assembly of the MRN complex and HR repair. Therefore, regulation of DSB access to MRE11 or Ku80 is likely decisive in the fate and type of DNA repair.HTLV-1 is a human retrovirus associated with adult T-cell leukemia/lymphoma, an aggressive disease with a dismal prognosis. 9 Whereas the majority of HTLV-1-infected individuals remain asymptomatic, upwards of 5% of patients ultimately develop adult T-cell leukemia/lymphoma. The molecular mechanisms of HTLV-1 oncogenesis are poorly understood. HTLV-1 disrupts cell-cycle checkpoints, tumor suppressors, and Notch signaling and reactivates telomerase. [10][11][12][13] Unlike animal oncoretroviruses, HTLV-1 does not transduce a protooncogene and does not integrate at specific sites in the human genome, thereby excluding insertional mutagenesis. The end of the HTLV-1 proviral genome encodes for the regulatory proteins p12, p30, and the HTLV-1 bZIP factor (HBZ), which are involved in virus infectivity, immune escape, and establishment of latency. 14-17 HTLV-1 leukemic cells often present numerous genomic alterations, but the genesis and contribution of these chromosomal defects are presently unclear. The viral oncoprotein Tax plays an important role in the initiation of cellular transformation. In addition, several studies have shown...

BackgroundHuman T-cell leukemia virus type I (HTLV-I) has efficiently adapted to its host and establishes a persistent infection characterized by low levels of viral gene expression and slow proliferation of HTLV-I infected cells over decades. We have previously found that HTLV-I p30 is a negative regulator of virus expression.ResultsIn this study we show that p30 targets multiple cell cycle checkpoints resulting in a delayed entry into S phase. We found that p30 binds to cyclin E and CDK2 and prevents the formation of active cyclin E-CDK2 complexes. In turn, this decreases the phosphorylation levels of Rb and prevents the release of E2F and its transcriptional activation of genes required for G1/S transition. Our studies also show that HTLV-II p28 does not bind cyclin E and does not affect cell cycle progression.ConclusionsIn contrast to HTLV-I, the HTLV-II-related retrovirus is not oncogenic in humans. Here we report that the HTLV-I p30 delays cell cycle progression while its homologue, HTLV-II p28, does not, providing evidence for important differences between these two related retrovirus proteins.

BackgroundHTLV-1 is a retrovirus that infects over 20 million people worldwide and is responsible for the hematopoietic malignancy adult T cell leukemia (ATL). We previously demonstrated that Notch is constitutively activated in ATL cells. Activating genetic mutations were found in Notch; however, Notch signaling was also activated in the absence of genetic mutations suggesting the existence of other mechanisms.MethodsWe analyzed the expression of Notch receptor ligands in HTLV-I-transformed cells, ATL patient-derived cell lines, and fresh uncultured ATL samples by RT-PCR, FACS, and immunohistochemistry. We then investigated viral and cellular molecular mechanisms regulating expression of JAG1. Finally, using shRNA knock-down and neutralizing antibodies, we investigated the function of JAG1 in ATL cells.ResultsHere, we report the overexpression of the Notch ligand, JAG1, in freshly uncultured ATL patient samples compared to normal PBMCs. We found that in ATL cells, JAG1 overexpression relies upon the viral protein Tax and cellular miR-124a, STAT3, and NFATc1. Interestingly, our data show that blockade of JAG1 signaling dampens Notch1 downstream signaling and limits cell migration of transformed ATL cells.ConclusionsOur results suggest that targeting JAG1 can block Notch1 activation in HTLV-I-transformed cells and represents a new target for immunotherapy in ATL patients.