gx-15-070 and Necrosis

Survival in infants younger than 1 year who have acute lymphoblastic leukemia (ALL) is inferior whether MLL is rearranged (R) or germline (G). MLL translocations confer chemotherapy resistance, and infants experience excess complications. We characterized in vitro sensitivity to the pan-antiapoptotic BCL-2 family inhibitor obatoclax mesylate in diagnostic leukemia cells from 54 infants with ALL/bilineal acute leukemia because of the role of prosurvival BCL-2 proteins in resistance, their imbalanced expression in infant ALL, and evidence of obatoclax activity with a favorable toxicity profile in early adult leukemia trials. Overall, half maximal effective concentrations (EC50s) were lower than 176 nM (the maximal plasma concentration [Cmax] with recommended adult dose) in 76% of samples, whether in MLL-AF4, MLL-ENL, or other MLL-R or MLL-G subsets, and regardless of patients' poor prognostic features. However, MLL status and partner genes correlated with EC50. Combined approaches including flow cytometry, Western blot, obatoclax treatment with death pathway inhibition, microarray analyses, and/or electron microscopy indicated a unique killing mechanism involving apoptosis, necroptosis, and autophagy in MLL-AF4 ALL cell lines and primary MLL-R and MLL-G infant ALL cells. This in vitro obatoclax activity and its multiple killing mechanisms across molecular cytogenetic subsets provide a rationale to incorporate a similarly acting compound into combination strategies to combat infant ALL.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Histone-Lysine N-Methyltransferase; Humans; Indoles; Infant; Infant, Newborn; Myeloid-Lymphoid Leukemia Protein; Necrosis; Oncogene Proteins, Fusion; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Pyrroles

Poorly differentiated and anaplastic thyroid carcinomas are very aggressive, almost invariably lethal neoplasms for which no effective treatment exists. These tumors are intrinsically resistant to cell death, even when their driver oncogenic signaling pathways are inhibited.We have undertaken a detailed analysis, in mouse and human thyroid cancer cells, of the mechanism through which Obatoclax, a pan-inhibitor of the anti-apoptotic proteins of the BCL2 family, effectively reduces tumor growth in vitro and in vivo.We demonstrate that Obatoclax does not induce apoptosis, but rather necrosis of thyroid cancer cells, and that non-transformed thyroid cells are significantly less affected by this compound. Surprisingly, we show that Obatoclax rapidly localizes to the lysosomes and induces loss of acidification, block of lysosomal fusion with autophagic vacuoles, and subsequent lysosomal permeabilization. Notably, prior lysosome neutralization using different V-ATPase inhibitors partially protects cancer cells from the toxic effects of Obatoclax. Although inhibition of autophagy does not affect Obatoclax-induced cell death, selective down-regulation of ATG7, but not of ATG5, partially impairs Obatoclax effects, suggesting the existence of autophagy-independent functions for ATG7. Strikingly, Obatoclax killing activity depends only on its accumulation in the lysosomes, and not on its interaction with BCL2 family members.Finally, we show that also other lysosome-targeting compounds, Mefloquine and LLOMe, readily induce necrosis in thyroid cancer cells, and that Mefloquine significantly impairs tumor growth in vivo, highlighting a clear vulnerability of these aggressive, apoptosis-resistant tumors that can be therapeutically exploited.

Topics: Animals; Antineoplastic Agents; Apoptosis; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Cell Proliferation; Enzyme Inhibitors; Humans; Indoles; Lysosomes; Mefloquine; Mice; Mice, Knockout; Necrosis; Proto-Oncogene Proteins c-bcl-2; Pyrroles; RNA Interference; RNA, Small Interfering; Spheroids, Cellular; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Tumor Cells, Cultured

In this study, we explored the antitumor activities of the PARP inhibitor AZD2281 (Olaparib) and the pan-Bcl-2 inhibitor GX15-070 (Obatoclax) in six pancreatic cancer cell lines. While both agents were able to cause growth arrest and limited apoptosis, the combination of the two was able to synergistically cause growth arrest and non-apoptotic cell death. Furthermore, in an in vivo xenograft model, the combination caused substantially increased tumor necrosis compared to either treatment alone. Our results support further investigation of the combination of Bcl-2 and PARP inhibitors for the treatment of pancreatic cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Female; Humans; Indoles; Mice, Inbred BALB C; Mice, Nude; Necrosis; Pancreatic Neoplasms; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Time Factors; Xenograft Model Antitumor Assays

Obatoclax (GX15-070), a small-molecule inhibitor of antiapoptotic Bcl-2 proteins, has been reported to trigger cell death via autophagy. However, the underlying molecular mechanisms have remained elusive. Here, we identify GX15-070-stimulated assembly of the necrosome on autophagosomal membranes as a key event that connects GX15-070-stimulated autophagy to necroptosis. GX15-070 predominately induces a non-apoptotic form of cell death in rhabdomyosarcoma cells, as evident by lack of typical apoptotic features such as DNA fragmentation or caspase activation and by insensitivity to the broad-range caspase inhibitor zVAD.fmk. Instead, GX15-070 triggers massive accumulation of autophagosomes, which are required for GX15-070-induced cell death, as blockade of autophagosome formation by silencing of Atg5 or Atg7 abolishes GX15-070-mediated cell death. Co-immunoprecipitation studies reveal that GX15-070 stimulates the interaction of Atg5, a constituent of autophagosomal membranes, with components of the necrosome such as FADD, RIP1 and RIP3. This GX15-070-induced assembly of the necrosome on autophagosomes occurs in a Atg5-dependent manner, as knockdown of Atg5 abrogates formation of this complex. RIP1 is necessary for GX15-070-induced cell death, as both genetic and pharmacological inhibition of RIP1 by shRNA-mediated knockdown or by the RIP1 inhibitor necrostatin-1 blocks GX15-070-induced cell death. Similarly, RIP3 knockdown rescues GX15-070-mediated cell death and suppression of clonogenic survival. Interestingly, RIP1 or RIP3 silencing has no effect on GX15-070-stimulated autophagosome formation, underlining that RIP1 and RIP3 mediate cell death downstream of autophagy induction. Of note, GX15-070 significantly suppresses tumor growth in a RIP1-dependent manner in the chorioallantoic membrane model in vivo. In conclusion, GX15-070 triggers necroptosis by promoting the assembly of the necrosome on autophagosomes. These findings provide novel insights into the molecular mechanisms of GX15-070-induced non-apoptotic cell death.

Topics: Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Cell Death; Cell Line; Fas-Associated Death Domain Protein; GTPase-Activating Proteins; HEK293 Cells; Humans; Imidazoles; Indoles; Microtubule-Associated Proteins; Necrosis; Phagosomes; Pyrroles; Receptor-Interacting Protein Serine-Threonine Kinases; RNA Interference; RNA, Small Interfering