Abstract 3735: A novel ALDH1A selective inhibitor induces necroptosis in ovarian cancer stem-like cells

Abstract

Background: The high relapse rate in ovarian cancer patients is highly consistent with a cancer stem cell (CSC) model in which rare, inherently chemoresistant CSC are capable of proliferating and differentiating to cause disease. We recently demonstrated ovarian CSC (OvCSC) can be defined by Aldehyde Dehydrogenase (ALDH) enzymatic activity. ALDH expression is increased in chemotherapy resistant OvCSC and ALDH KO can reverse chemotherapy resistance. While ALDH inhibitors exist, they are not isoform specific. We therefore identified and screened numerous putative ALDH inhibitors, based on molecular homology to the known ALDH inhibitor DEAB, and assessed their impact on OvCSC. Methods and Results: While most ALDH inhibitors, including DEAB, had no impact on CSC, we identified a novel compound, 673A which specifically depletes OvCSC in vitro. Supporting functional depletion of OvCSC, treatment of primary tumor ascites resulted in 4-20 fold decrease tumor sphere formation. ALDH inhibition is highly synergistic with cisplatin both in vitro and in vivo as assessed by cell growth curves and tumor growth. Pre-treatment of tumor cells with 673 significantly reduced tumor in tumor initiation and growth rates. Unlike non-CSC depleting ALDH inhibitors, 673A is a pan-ALDH1A isoform specific inhibitor with IC50<230 nM for ALDH1A1, 1A2, and 1A3. OvCSC Caspase independent cell death was accompanied by negative Annexin V stain and HMB1 translocation to cytoplasm. IF demonstrated nuclear swelling and loss of DNA in a manner consistent with karyolysis and programmed cell necrosis. TEM of treated cells demonstrated clear cellular, nuclear, and organelle swelling and severe vacuolation of cytoplasm consistent with necrosis. Consistent with the induction of programmed cell necrosis, 673A treatment resulted in increased intracellular calcium. Furthermore, the intracellular calcium chelator BAPTA was able to rescue ALDH inhibition-induced necrosis. As RIP1 and RIP3 kinase activity is known to be a major mediator of necroptosis, we next assessed the impact of the RIP1 kinase inhibitor, Nec-1, and down-regulation of RIP1 and RIP3 by siRNA. Inhibition or depletion of RIP Kinase 1 did not prevent 673A mediated necroptosis of OvCSC. 673A initiates necroptotic cell death through translocation of RIPK3 downstream target, MLKL, to membrane and activation of mitochondria fission protein DRP1 and its translocation from cytoplasm to mitochondria. Conclusions: We have identified a novel ALDH inhibitor, 673A, which selective inhibits three ALDH1A isoforms. 673A mediated ALDH1A isoform inhibition triggers programmed cell necrosis of OvCSC. Our data suggest that pan ALDH1A1 targeted therapy this may be a powerful new therapeutic approach to target OvCSC and bypass their resistance to apoptosis inducing drugs. Future studies will elucidate the importance of mitochondria metabolic pathways (Glycolysis, OXPHOS, and TCA cycle) in ALDH inhibition-induced necroptosis. Citation Format: Ilana Chefetz-Menaker, Kun Yang, Ron Buckanovich. A novel ALDH1A selective inhibitor induces necroptosis in ovarian cancer stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3735.

Personalized Medicine–Based Approach to Model Patterns of Chemoresistance and Tumor Recurrence Using Ovarian Cancer Stem Cell Spheroids

“…Indeed, in line with previously published research, the use of cisplatin along with the ALDH targeting compound 673A resulted in a marked loss of ALDH + OvCSC progeny within spheroids(31,42). Our platform was able to discern the sensitivity of the three different patient samples to the synergistic cisplatin/673A and cisplatin/ruxolitinib combination therapies, and this correlated with their clinical history for platinum response, as well as, the amounts of OvCSC populations contained within the primary and spheroid samples.…”

“…We further demonstrate the predictive potency of spheroid-based therapeutic assays compared to those conducted in vivo in murine xenograft models established from OvCSC spheroids. We perform drug sensitivity assays in the context of OvCSCs, by using the conventional chemotherapeutic drug, cisplatin, and combining it with a novel ALDH targeting compound, in development, compound 673A(31), as well as, a prominent JAK2 inhibitor, ruxolitinib(32). Platinum based agents are the core chemotherapeutic for treatment of ovarian cancer(33,34).…”

Ovarian Cancers: Genetic Abnormalities, Tumor Heterogeneity and Progression, Clonal Evolution and Cancer Stem Cells

“…Recently, compound 673A, an ALDH inhibitor, was preliminary tested on ovarian cancer cells, showing that it induces necroptosis of ovarian cancer stem-like cells [ 221 ]. This compound was tested also in combination with cisplatin on tumor spheroids isolated from primary tumors: spheroids resistant to these two drugs have the tendency to the loss of CD133 expression and a decrease of ALDH activity [ 222 ].…”

“…One study was carried out using a cell population CD44 + MyD88 + enriched in epithelial ovarian cancer stem cells: this cell population exhibits multiple properties of ovarian cancer stem cells, including chemoresistance and the property to differentiate into multiple cell types [ 231 ]. These cells are able to generate a differentiated cell progeny, CD44 − MyD88 − which have lost the self-renewal capacity, are more sensitive to chemotherapy and are differentiated [ 221 ]. CD44 + MyD88 + epithelial ovarian stem cells grow as adherent cells, but when grown at high cell density are able to generate some spheroids, containing epithelial ovarian cancer cells that have undergone an epithelial-mesenchymal transition (as evidenced by the acquisition of mesenchymal markers and of TWIST-1, SLUG and VIMENTIN, mesenchymal markers associated with the process of EMT); the spheroid mesenchymal cells are able to revert to an epithelial phenotype when switched into appropriate cell culture conditions (i.e., they undergo MET) [ 231 ].…”