Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

Cells adapt to endoplasmic reticulum (ER)-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD), however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.Although PP1 activity does not play a major role in regulating the ER stress response in leukemic cells, phosphatase signaling nevertheless significantly limits proteasome inhibitor-mediated ER-stress and apoptosis. Inclusion of specific phosphatase inhibitors might therefore represent an option to improve current proteasome inhibitor-based treatment modalities for hematological cancers

RNA Silencing of Mcl-1 Enhances ABT-737-Mediated Apoptosis in Melanoma: Role for a Caspase-8-Dependent Pathway

BACKGROUND: Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity

Expression of FRA16D/WWOX and FRA3B/FHIT Genes in Hematopoietic Malignancies

The WW domain containing oxidoreductase (WWOX) gene was recently identified as a candidate tumor suppressor gene at a common fragile site, FRA16D. Because the fragile histidine triad (FHIT) gene, a tumor suppressor gene encompassing the most active, common fragile site FRA3B, is frequently deleted in various cancers, we evaluated the expression of WWOX and FHIT in 74 cases of primary hematopoietic neoplasias and 20 leukemia cell lines. Aberration or absence of WWOX transcripts was detected in 51% of the primary cases and 55% of cell lines, and three WWOX nucleotide variants were detected among the leukemia cell lines. FHIT expression was absent or altered in 36% of the primary cases and 15% of cell lines. The occurrence of aberrant FHIT reverse transcription-PCR products correlated significantly with the occurrence of WWOX alterations. Wild-type transcripts of both genes were expressed in normal hematopoiesis along with a small fraction of short transcripts. A DNA blot study showed that WWOX and FHIT genes were deleted in 2 of 18 cases with primary acute leukemias; both genes were not expressed in the 2 cases. Furthermore, treatment of cells with a demethylating or histone acetylating agent in culture resulted in increased expression of WWOX and FHIT mRNA in leukemia cells. Conclusions are that WWOX expression is frequently altered or absent in hematopoietic disorders, often in association with FHIT alterations, and that alterations of these fragile genes may result not only from genomic deletions but also from epigenetic modifications associated with expression of fragility

The First Asian, Single-Center Experience of Blastocyst Preimplantation Genetic Diagnosis with HLA Matching in Thailand for the Prevention of Thalassemia and Subsequent Curative Hematopoietic Stem Cell Transplantation of Twelve Affected Siblings

Background and Methods. In Southeast Asia and particularly in Thailand, β0-thalassemia/hemoglobin E (HbE) disease is a common hereditary hematological disease. In a retrospective analysis, we investigated the outcomes of blastocyst preimplantation genetic diagnosis (PGD) for α- and β-thalassemia exclusion, with human leukocyte antigen (PGD-HLA) matching, from 2007 to 2016 at Superior A.R.T. (Bangkok, Thailand) and subsequent hematopoietic stem cell transplantation (HSCT) treatment of affected siblings. The main outcome measures included the proportion of biopsied cycles with at least one screened embryo suitable for transfer, implantation rate, live birth rate, and curative HSCT post-PGD-HLA. Results. In 221 cycles from 138 patients (104 cycles requiring HLA matching), 90.5% had embryo(s) biopsied for genetic testing. There were 119 embryo transfers for thalassemia (76) and thalassemia-HLA cases (43), respectively, resulting in overall clinical pregnancy rates of 54.6%, implantation rates of 45.7%, and live birth rates of 44.1%. Our dataset included fifteen PGD-HLA live births with successful HSCT in twelve affected siblings, 67% using umbilical cord blood stem cells (UCBSC) as the only SC source. Conclusions. We report favorable thalassemia PGD and PGD-HLA laboratory and clinical outcomes from a single center. The ultimate success in PGD-HLA is of course the cure of a thalassemia-affected sibling by HSCT. Our PGD-HLA HSCT series is the first and largest performed entirely in Asia with twelve successful and two pending cures and predominant UCBSC use