JAG1 overexpression contributes to Notch1 signaling and the migration of HTLV-1-transformed ATL cells.

BACKGROUND: HTLV-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. METHODS: We 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. RESULTS: Here, 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. CONCLUSIONS: Our results suggest that targeting JAG1 can block Notch1 activation in HTLV-I-transformed cells and represents a new target for immunotherapy in ATL patients.

Small PARP inhibitor PJ-34 induces cell cycle arrest and apoptosis of adult T-cell leukemia cells.

BACKGROUND: HTLV-I is associated with the development of an aggressive form of lymphocytic leukemia known as adult T-cell leukemia/lymphoma (ATLL). A major obstacle for effective treatment of ATLL resides in the genetic diversity of tumor cells and their ability to acquire resistance to chemotherapy regimens. As a result, most patients relapse and current therapeutic approaches still have limited long-term survival benefits. Hence, the development of novel approaches is greatly needed. METHODS: In this study, we found that a small molecule inhibitor of poly (ADP-ribose) polymerase (PARP), PJ-34, is very effective in activating S/G2M cell cycle checkpoints, resulting in permanent cell cycle arrest and reactivation of p53 transcription functions and caspase-3-dependent apoptosis of HTLV-I-transformed and patient-derived ATLL tumor cells. We also found that HTLV-I-transformed MT-2 cells are resistant to PJ-34 therapy associated with reduced cleaved caspase-3 activation and increased expression of RelA/p65. CONCLUSION: Since PJ-34 has been tested in clinical trials for the treatment of solid tumors, our results suggest that some ATLL patients may be good candidates to benefit from PJ-34 therapy.

Tax impairs DNA replication forks and increases DNA breaks in specific oncogenic genome regions.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-I) is a human retrovirus associated with adult T-cell leukemia (ATL), an aggressive CD4 T-cell proliferative disease with dismal prognosis. The long latency preceding the development of the disease and the low incidence suggests that the virus itself is not sufficient for transformation and that genetic defects are required to create a permissive environment for leukemia. In fact, ATL cells are characterized by profound genetic modifications including structural and numerical chromosome alterations. RESULTS: In this study we used molecular combing techniques to study the effect of the oncoprotein Tax on DNA replication. We found that replication forks have difficulties replicating complex DNA, fork progression is slower, and they pause or stall more frequently in the presence of Tax expression. Our results also show that Tax-associated replication defects are partially compensated by an increase in the firing of back-up origins. Consistent with these effects of Tax on DNA replication, an increase in double strand DNA breaks (DDSB) was seen in Tax expressing cells. Tax-mediated increases in DDSBs were associated with the ability of Tax to activate NF-kB and to stimulate intracellular nitric oxide production. We also demonstrated a reduced expression of human translesion synthesis (TLS) DNA polymerases Pol-H and Pol-K in HTLV-I-transformed T cells and ATL cells. This was associated with an increase in DNA breaks induced by Tax at specific genome regions, such as the c-Myc and the Bcl-2 major breakpoints. Consistent with the notion that the non-homologous end joining (NHEJ) pathway is hyperactive in HTLV-I-transformed cells, we found that inhibition of the NHEJ pathway induces significant killing of HTLV-I transformed cells and patient-derived leukemic ATL cells. CONCLUSION: Our results suggest that, replication problems increase genetic instability in HTLV-I-transformed cells. As a result, abuse of NHEJ and a defective homologous repair (HR) DNA repair pathway can be targeted as a new therapeutic approach for the treatment of adult T-cell leukemia.