Mitochondrial Outer Membrane Permeabilization Research Articles

Alterations in hippocampal mitochondrial dynamics areassociated with neurodegeneration and recognition memory decline in old male mice.

Mitochondrial dynamics is a key process that modulates the ultrastructure, quality and function of mitochondria. It is disrupted in numerous major neurodegenerative disorders including Parkinson's, Alzheimer's and Huntington's disease. Mitochondrial dysfunction has been correlated with the loss of memory. Previous studies suggest the involvement of Vdac1 and Drp1 in outer mitochondrial membrane permeabilization and promotion of mitochondrial fragmentation through Drp1 phosphorylation at S616. However, alterations in mitochondrial dynamics with respect to aging, memory loss and neurodegeneration remain unexplored. Therefore, the present study focuses on the involvement of mitochondrial dynamics in neurodegeneration and recognition memory decline during aging. The recognition memory decline was validated by the novel object recognition test and measurement of hippocampal Arc protein level during aging. The ultrastructure analysis revealed a decline in mitochondrial length and area, while an increase in the number of fragmented, round and disrupted mitochondria in the hippocampus during aging. Disruption was also evident in mitochondrial cristae and membrane with advancing age. The change in mitochondrial morphology was corroborated by an increase in the expression of phospho-Drp1 (S616) and Cyt-c proteins but decline in Mfn2, LC3B, Vdac1, Bcl-XL and Bcl-2 proteins in the hippocampus during aging. Taken together, our findings reveal that an increase in the expression of phospho-Drp1 (S616) and decrease in Mfn2 and LC3B proteins in the hippocampus bring about a reduction in mitochondrial length and area, and rise in mitochondrial fragmentation leading to reduced neuronal cell density, increased neurodegeneration and recognition memory decline in old male mice. Diagram depicts the increase in hippocampal mitochondrial fragmentation during aging of mice. Increased mitochondrial fragmentation causes distorted mitochondrial function such as decrease in ATP/ADP transportation due to decrease in Vdac1 protein level and increase in oxidative damage. These alterations result in hippocampal neurodegeneration and consequently impairment in recognition memory during aging.

Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death.

Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.

The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial-DNA-mediated inflammation

SummaryBAX and BAK are key apoptosis regulators that mediate the decisive step of mitochondrial outer membrane permeabilization. However, the mechanism by which they assemble the apoptotic pore remains obscure. Here, we report that BAX and BAK present distinct oligomerization properties, with BAK organizing into smaller structures with faster kinetics than BAX. BAK recruits and accelerates BAX assembly into oligomers that continue to grow during apoptosis. As a result, BAX and BAK regulate each other as they co-assemble into the same apoptotic pores, which we visualize. The relative availability of BAX and BAK molecules thereby determines the growth rate of the apoptotic pore and the relative kinetics by which mitochondrial contents, most notably mtDNA, are released. This feature of BAX and BAK results in distinct activation kinetics of the cGAS/STING pathway with implications for mtDNA-mediated paracrine inflammatory signaling.

SRT1720 induces SIRT1-independent cell death in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) develops after a long period of human T-cell leukemia virus (HTLV)-1 infection and is associated with host aging in addition to genetic abnormalities in HTLV-1 infected cells. SIRT1 is a histone deacetylase involved in cell cycle and apoptosis. We previously showed the high expression of SIRT1 protein in peripheral blood mononuclear cells from patients with ATL. There have been many reports that SIRT1 inhibitors show tumor-suppressive effects. On the other hand, SIRT1 activator SRT1720 induces the cell death of multiple myeloma and breast cancer cells. However, the effect of SRT1720 on ATL is unknown. This study aimed to evaluate the effect of SRT1720 on cell death in leukemic cell lines in vitro and freshly isolated ATL cells ex vivo and in an ATL in vivo mouse model. SRT1720 reduced cell viability in vitro and ex vivo. Additionally, SRT1720 increased the number of apoptotic cells, as shown by annexin V positive cells, cleaved poly (ADP-ribose) polymerase 1, cleaved caspase-3, and fragmented DNA. SRT1720 also induced mitochondrial outer membrane permeabilization with the generation of mitochondrial reactive oxygen species and autophagy. However, SIRT1 knockdown did not attenuate SRT1720-induced cell death in leukemic cell lines. Finally, SRT1720 treatment decreased the growth of human ATL tumor xenografts in immunodeficient mice. Our study shows that while SRT1720 does not target SIRT1, it induces cell death in ATL cells via apoptosis and autophagy and has antitumor activity.

Methylmercury induces lysosomal membrane permeabilization through JNK-activated Bax lysosomal translocation in neuronal cells

MeHg, an environmental toxicant, is highly toxic to the central nervous system. Recent studies have reported that LMP is an important way in the lysosomal damage. However, the role and molecular mechanism of LMP in MeHg-induced neurotoxicity remain unknown. To study MeHg-induced LMP, we used 10μM MeHg to treat SH-SY5Y cells and 2μM MeHg to treat rat cerebral cortical neurons. Acridine orange (AO) staining and analysis of cathepsin B (CTSB) release were used to determine LMP. We found that MeHg reduced red AO fluorescence and induced CTSB release from lysosomes to the cytoplasm in a time-dependent manner. Moreover, pretreatment with the CTSB inhibitor alleviated cytotoxicity in neuronal cells. These results indicate MeHg induces LMP and subsequent CTSB-dependent cytotoxicity in neuronal cells. Bax is a pore-forming protein, which is involved in mitochondrial outer membrane permeabilization. Intriguingly, we demonstrated that MeHg induced Bax to translocate to lysosomes by using immunofluorescence and Western blot analysis of subcellular fractions. Furthermore, downregulating Bax expression suppressed MeHg-induced LMP. Bax subcellular localization is regulated by protein interaction with the cytoplasmic 14-3-3. Our previous study demonstrated that JNK participated in neurotoxicity through regulating protein interaction. In the current study, we showed that JNK dissociated Bax-14-3-3 complex to facilitate Bax lysosomal translocation. Finally, inhibition of the JNK/Bax pathway could alleviate MeHg-induced cytotoxicity in neuronal cells. The present study implies that inhibiting lysosomal damage (LMP)-related signaling might alleviate MeHg neurotoxicity.

Cytofluorometric assessment of acute cell death responses driven by radiation therapy.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects on malignant cells, largely reflecting the ability of ionizing radiation to cause direct and indirect damage to macromolecules including DNA and lipids. While low-dose RT generally causes limited cytotoxicity in an acute manner (as it imposes insufficient cellular damage to compromise homeostasis, or instead induces the delayed demise of cells that fail to complete mitosis successfully), high RT doses can mediate an acute wave of cell death that begins to manifest shortly (24-72h) after irradiation. Here, we provide two straightforward techniques to assess the acute cytotoxic effects of RT by the flow cytometry-assisted quantification of plasma membrane permeabilization (PMP, a late-stage manifestation of cell death) and either mitochondrial outer membrane permeabilization (MOMP) or phosphatidylserine (PS) externalization (two early-stage signs of cell death) in mouse mammary adenocarcinoma TS/A cells. With minor variations, the same protocols can be straightforwardly adapted to measure acute cell death responses as elicited by RT in a large panel of human and mouse cancer cells lines of different histological derivation.

Mitochondrial Toxicity of Organic Arsenicals.

Arsenic is either notorious toxicant or miracle cure for acute promyelocytic leukemia and several other diseases. It interacts with mitochondria directly or indirectly, by interacting with mitochondrial enzymes, such as respiratory chain complexes and tricarboxylic acid cycle proteins, or affecting mitochondrial homeostasis via ROS or mitochondrial outer membrane permeabilization. Given the ubiquitous presence of mitochondria and indispensable role in cellular metabolism, arsenical-mitochondrial interactions may manifest clinical importance by revealing mechanism of disease curation, preventing severe side effects, and foreseeing potential health issues. Here, we described the interaction between isolated mitochondria and arsenicals.

Myeloid cell leukemia-1: a formidable barrier to anticancer therapeutics and the quest of targeting it.

The antiapoptotic B cell lymphoma-2 (Bcl-2) family members are apical regulators of the intrinsic pathway of apoptosis that orchestrate mitochondrial outer membrane permeabilization (MOMP) through interactions with their proapoptotic counterparts. Overexpression of antiapoptotic Bcl-2 family proteins has been linked to therapy resistance and poor prognosis in diverse cancers. Among the antiapoptotic Bcl-2 family members, predominant overexpression of the prosurvival myeloid cell leukemia-1 (Mcl-1) has been reported in a myriad of hematological malignancies and solid tumors, contributing to therapy resistance and poor outcomes, thus making it a potential druggable target. The unique structure of Mcl-1 and its complex regulatory mechanism makes it an adaptive prosurvival switch that ensures tumor cell survival despite therapeutic intervention. This review focusses on diverse mechanisms adopted by tumor cells to maintain sustained elevated levels of Mcl-1 and how high Mcl-1 levels contribute to resistance in conventional as well as targeted therapies. Moreover, recent developments in the Mcl-1-targeted therapeutics and the underlying challenges and considerations in designing novel Mcl-1 inhibitors are also discussed.

Molecular mechanisms and consequences of mitochondrial permeability transition.

Mitochondrial permeability transition (mPT) is a phenomenon that abruptly causes the flux of low molecular weight solutes (molecular weight up to 1,500) across the generally impermeable inner mitochondrial membrane. The mPT is mediated by the so-called mitochondrial permeability transition pore (mPTP), a supramolecular entity assembled at the interface of the inner and outer mitochondrial membranes. In contrast to mitochondrial outer membrane permeabilization, which mostly activates apoptosis, mPT can trigger different cellular responses, from the physiological regulation of mitophagy to the activation of apoptosis or necrosis. Although there are several molecular candidates for the mPTP, its molecular nature remains contentious. This lack of molecular data was a significant setback that prevented mechanistic insight into the mPTP, pharmacological targeting and the generation of informative animal models. In recent years, experimental evidence has highlighted mitochondrial F1Fo ATP synthase as a participant in mPTP formation, although a molecular model for its transition to the mPTP is still lacking. Recently, the resolution of the F1Fo ATP synthase structure by cryogenic electron microscopy led to a model for mPTP gating. The elusive molecular nature of the mPTP is now being clarified, marking a turning point for understanding mitochondrial biology and its pathophysiological ramifications. This Review provides an up-to-date reference for the understanding of the mammalian mPTP and its cellular functions. We review current insights into the molecular mechanisms of mPT and validated observations - from studies in vivo or in artificial membranes - on mPTP activity and functions. We end with a discussion of the contribution of the mPTP to human disease. Throughout the Review, we highlight the multiple unanswered questions and, when applicable, we also provide alternative interpretations of the recent discoveries.

Bcl-xL and IP3R interaction: Intimate relationship with an uncertain outcome

Bcl-2 family proteins are major apoptosis regulators. They control a key step in apoptosis execution referred to as the mitochondrial outer membrane permeabilization. Several Bcl-2 homologs were also reported to act at the level of the endoplasmic reticulum (ER) where they control intracellular Ca2+ trafficking. There is an increasing body of evidence that, in addition to their conventional role as MOMP regulators, several Bcl-2 family members, including Bcl-xL, are linked to Ca2+ -dependent processes, independent of cell death. Among them Bcl-xL has been proposed to promote IP3R1 channel opening and sustain mitochondrial bioenergetics. A recent article by Rosa and colleagues in Cell Death & Differentiation challenges this model and support the notion that Bcl-xL acts more as a repressor than as a sensitizer of IP3R1 opening. They suggest the existence of intrafamilial competition among the Bcl-2 family of protein with respect to their effect on IP3R Ca2+ permeability, which might be important regarding their respective non-canonical functions. In this regard, the results by Rosa and colleagues open exciting avenues regarding the biological process by which Bcl-xL affects Ca2+ trafficking through IP 3 R channels.

Live-cell visualization of cytochrome c: a tool to explore apoptosis.

Apoptosis dysfunction is associated with several malignancies, including cancer and autoimmune diseases. Apoptosis restoration could be an attractive therapeutic approach to those diseases. Mitochondrial outer membrane permeabilization is regarded as the point of no return in the 'classical' apoptosis triggering pathway. Cytoplasmic release of cytochrome c (cyt c), a mitochondrial electron transporter, is a prominent indicator of such critical step. Therefore, visualizing cyt c efflux in living cells is a convenient approach to address apoptosis triggering and monitor performance of apoptosis restoration strategies. Recent years have been prolific in the development of biosensors to visualize cyt c mitochondrial efflux in living cells, by fluorescence microscopy. These biosensors specifically detect endogenous, untagged cyt c, while showing efficient cellular uptake and reduced cell toxicity. A common aspect is their fluorescence quenching in the absence or presence of bound cyt c, resulting in two main biosensor types: 'turn ON' and 'turn OFF'. In some of these systems, fluorescence intensity of fluorophore-bound aptamers is enhanced upon cyt c binding. In others, cyt c binding to quantum dots quenches their fluorescence. In the present minireview, I describe these biosensors and briefly introduce some hypotheses that could be addressed using these novel tools.

Allosteric SHP2 Inhibitor RMC4550 Synergizes with Venetoclax in FLT3 and KIT Mutant Acute Myeloid Leukemia

Mutations in receptor tyrosine kinases (RTK) FLT3 and KIT occur frequently in Acute Myeloid Leukemia (AML) and are associated with high risk of relapse. FLT3 tyrosine kinase inhibitors (TKI) are clinically approved in AML, but resistance is common and involves emerging clones reliant on oncogenic signaling, particularly in the RAS/MAPK pathway. Patients who relapse on FLT3 TKIs have inauspicious prognoses and no specific therapeutic options, highlighting the unmet need for effective strategies to target oncogenic signaling and improve outcomes in relapsed/refractory (R/R) AML.The protein tyrosine phosphatase SHP2 (PTPN11) is a central node in RAS/MAPK activation downstream of various RTKs, including FLT3, acting as a scaffold for adaptor proteins that promote RAS-GTP loading. Novel allosteric inhibitors are being clinically investigated in cancers with signaling activating mutations.Here, we demonstrate that the allosteric SHP2 inhibitor RMC-4550 modulates expression of pro and anti-apoptotics in FLT3 and KIT mutant AML providing rationale for combinatorial targeting of SHP2 and BCL2 as a synergistic approach. We subsequently report the preclinical efficacy of RMC-4550 and the FDA-approved, BCL2 selective inhibitor, Venetoclax combination in both in vitro and in vivo AML models.We evaluated cell viability of multiple AML cell lines treated with RMC-4550. FLT3-ITD (Molm14, MV4-11) and KIT mutant (Kasumi1, SKNO1) lines were sensitive to SHP2 inhibition. RMC-4550 maintained its efficacy in FLT3-ITD Molm14 cells with secondary mutations in FLT3 tyrosine kinase domain (TKD) and in NRAS G12C. RMC-4550 biochemically represses pERK (Figure 1A) and transcriptionally downregulates mRNA expression of DUSP6 and anti-apoptotic BCL2 and MCL1.We functionally evaluated the mitochondrial outer membrane permeabilization (MOMP) in response to SHP2 inhibition using a dynamic iBH3 profiling assay. RMC-4550 increased the overall priming and the dependency on BCL2 in both Molm14 and MV4-11 cell lines (Figure 1A).To investigate the global transcriptomic changes induced by allosteric SHP2 inhibition, we performed total mRNA sequencing on Molm14, MV4-11 and SKNO1 cell lines. GSEA analysis revealed that RMC-4550 significantly upregulated expression of genes repressed by RAS activation, downregulated MYC targets, but also dysregulated genes mediating apoptosis. The most consistently upregulated pro-apoptotic gene was BMF (fold change: 4.39, FDR<0.001). BMF is a BH3-only protein found to be sequestered to motor filaments that, in response to cellular damage signals, is translocated in the cytoplasm and binds pro-survival Bcl2 proteins. The BMF transcript upregulation was confirmed by qPCR and western blot analysis showed a marked overexpression of the BMF protein level upon SHP2 inhibition, particularly in the cytoplasmic subcellular compartment (Figure 1B).We next treated Molm14, MV4-11, Kasumi and SKNO1 lines with incremental doses of RMC-4550 and Venetoclax in an 8x8 combination matrix to assess the synergy of the two compounds using cell viability and apoptosis readouts. The assay showed highly synergistic activity in both FLT3-ITD and KIT lines. Remarkably, we noted a potent synergy in Molm14 cells with concurrent mutation in NRAS G12C (Figure 1C).In a Molm14 cell line xenograft model, we demonstrated that the combination of RMC-4550 (30 mg/kg) and Venetoclax (100 mg/kg) administered orally 5 times a week for 28 days significantly decreased leukemia burden and improved survival (p<0.001) compared to control and single agents (Figure 1D).In a FLT3-ITD AML patient-derived xenograft (PDX) model, the combination of RMC-4550 and Venetoclax markedly decreased %hCD45 in both cardiac blood and spleen of NSGS mice compared to vehicle-treated control (Figure 1E).Supporting a potential therapeutic index for the combination, RMC-4550 and Venetoclax strongly inhibited colony formation in FLT3 AML primary samples compared to samples from healthy volunteers.Collectively, our data suggest that SHP2 inhibition increases the apoptotic dependency on BCL2 through up-regulation of the pro-apoptotic BMF, a mechanistic rationale to synergistically inhibit both targets. We provide preclinical evidence that co-targeting SHP2 and BCL2 is a potential effective therapeutic strategy in RTK-driven AML. [Display omitted] DisclosuresStahlhut: Revolutions Medicine: Current Employment, Current equity holder in publicly-traded company. Smith: Daiichi Sankyo: Consultancy; Amgen: Honoraria; AbbVie: Research Funding; Revolutions Medicine: Research Funding; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Paradoxical implication of BAX/BAK in the persistence of tetraploid cells

Pro-apoptotic multi-domain proteins of the BCL2 family such as BAX and BAK are well known for their important role in the induction of mitochondrial outer membrane permeabilization (MOMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. Human or mouse cells lacking both BAX and BAK (due to a double knockout, DKO) are notoriously resistant to MOMP and cell death induction. Here we report the surprising finding that BAX/BAK DKO cells proliferate less than control cells expressing both BAX and BAK (or either BAX or BAK) when they are driven into tetraploidy by transient exposure to the microtubule inhibitor nocodazole. Mechanistically, in contrast to their BAX/BAK-sufficient controls, tetraploid DKO cells activate a senescent program, as indicated by the overexpression of several cyclin-dependent kinase inhibitors and the activation of β-galactosidase. Moreover, DKO cells manifest alterations in ionomycin-mobilizable endoplasmic reticulum (ER) Ca2+ stores and store-operated Ca2+ entry that are affected by tetraploidization. DKO cells manifested reduced expression of endogenous sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (Serca2a) and transfection-enforced reintroduction of Serca2a, or reintroduction of an ER-targeted variant of BAK into DKO cells reestablished the same pattern of Ca2+ fluxes as observed in BAX/BAK-sufficient control cells. Serca2a reexpression and ER-targeted BAK also abolished the tetraploidy-induced senescence of DKO cells, placing ER Ca2+ fluxes downstream of the regulation of senescence by BAX/BAK. In conclusion, it appears that BAX/BAK prevent the induction of a tetraploidization-associated senescence program. Speculatively, this may contribute to the low incidence of cancers in BAX/BAK DKO mice and explain why human cancers rarely lose the expression of both BAX and BAK.

Severe cellular stress activates apoptosis independently of p53 in osteosarcoma

Apoptosis induced by doxorubicin, bortezomib, or paclitaxel, targeting DNA, 26S proteasome, and microtubules respectively, was assessed in two osteosarcoma cells, p53 wild-type U2OS and p53-null MG63 cells. Doxorubicin-induced apoptosis only occurred in U2OS, not in MG63. In contrast, bortezomib and paclitaxel could drive U2OS or MG63 toward apoptosis effectively, suggesting that apoptosis induced by bortezomib or paclitaxel is p53-independent. The expressions of Bcl2 family members such as Bcl2, Bcl-xl, and Puma could be seen in U2OS and MG63 cells with or without doxorubicin, bortezomib, or paclitaxel treatment. In contrast, another member, Bim, only could be observed in U2OS, not in MG63, under the same conditions. Bim knockdown did not affect the doxorubicin-induced apoptosis in U2OS, suggested that a BH3-only protein other than Bim might participate in apoptosis induced by doxorubicin. Using a BH3-mimetic, ABT-263, to inhibit Bcl2 or Bcl-xl produced a limited apoptotic response in U2OS and MG63 cells, suggesting that this BH3-mimetic cannot activate the Bax/Bak pathway efficiently. Significantly, ABT-263 enhanced doxorubicin- and bortezomib-induced apoptosis synergistically in U2OS and MG63 cells. These results implied that the severe cellular stress caused by doxorubicin or bortezomib might be mediated through a dual process to control apoptosis. Respectively, doxorubicin or bortezomib activates a BH3-only protein in one way and corresponding unknown factors in another way to affect mitochondrial outer membrane permeability, resulting in apoptosis. The combination of doxorubicin with ABT-263 could produce synergistic apoptosis in MG63 cells, which lack p53, suggesting that p53 has no role in doxorubicin-induced apoptosis in osteosarcoma. In addition, ABT-263 enhanced paclitaxel to induce moderate levels of apoptosis.

An Autophagy-Disrupting Small Molecule Promotes Cancer Cell Death via Caspase Activation.

A novel autophagy inhibitor, autophazole (Atz), which promoted cancer cell death via caspase activation, is described. This compound was identified from cell-based high-content screening of an imidazole library. The results showed that Atz was internalized into lysosomes of cells where it induced lysosomal membrane permeabilization (LMP). This process generated nonfunctional autolysosomes, thereby inhibiting autophagy. In addition, Atz was found to promote LMP-mediated apoptosis. Specifically, LMP induced by Atz caused release of cathepsins from lysosomes into the cytosol. Cathepsins in the cytosol cleaved Bid to generate tBid, which subsequently activated Bax to induce mitochondrial outer membrane permeabilization (MOMP). This event led to cancer cell death via caspase activation. Overall, the findings suggest that Atz will serve as a new chemical probe in efforts aimed at gaining a better understanding of the autophagic process.

Gasdermins mediate cellular release of mitochondrial DNA during pyroptosis and apoptosis.

Pyroptosis and intrinsic apoptosis are two forms of regulated cell death driven by active caspases where plasma membrane permeabilization is induced by gasdermin pores. Caspase-1 induces gasdermin D pore formation during pyroptosis, whereas caspase-3 promotes gasdermin E pore formation during apoptosis. These two types of cell death are accompanied by mitochondrial outer membrane permeabilization due to BAK/BAX pore formation in the external membrane of mitochondria, and to some extent, this complex also affects the inner mitochondrial membrane facilitating mitochondrial DNA relocalization from the matrix to the cytosol. However, the detailed mechanism responsible for this process has not been investigated. Herein, we reported that gasdermin processing is required to induce mitochondrial DNA release from cells during pyroptosis and apoptosis. Gasdermin targeted at the plasma membrane promotes a fast mitochondrial collapse along with the initial accumulation of mitochondrial DNA in the cytosol and then facilitates the DNA's release from the cell when the plasma membrane ruptures. These findings demonstrate that gasdermin action has a critical effect on the plasma membrane and facilitates the release of mitochondrial DNA as a damage-associated molecular pattern.

Abstract 1937: Quinacrine-induced autophagy in ovarian cancer triggers cathepsin-L mediated lysosomal/mitochondrial membrane permeabilization and cell death

Abstract Background: Lysosomes are acidic organelles that contain numerous cathepsins and acid hydrolases. Lysosomal membrane permeabilization (LMP) and the presence of cathepsins in the cytosol triggers intrinsic apoptotic pathway through cathepsin L (CTSL) mediated degradation of critical proteins. This process is mediated by the proteolytic activation of the pro-apoptotic molecule Bid, resulting in mitochondrial outer membrane permeability (MOMP), cyt c release and then, cell death. Based on our previous study that quinacrine (QC) induces autophagy in ovarian cancer, the current study is aimed to investigate the potential role of cathepsins in QC induced LMP and MOMP in ovarian cancer. Methods: MagicRed and Mitotracker Red staining was used to assess the QC induced CTSL activation and LMP. To identify the role of specific cathepsin in QC mediated cytotoxicity, different specific cathepsin inhibitors pepstatin A, CA-074Me, Z-FY(tBU)-DMK were used and cell death was assessed by clonogenic assay, PARP cleavage and annexin v-PI staining. Using ATG5 and/or CTSL knockdown cells and pharmacological inhibition with 3-MA/chloroquine and/or CTSL overexpression clones, QC-induced flux in CTSL activation was determined using several different approaches including evaluating autophagy flux using tandem dual labeled RFP/GFP LC3BII by immunoflourescence analysis. Recombinant CTSL was used to validate identify p62 as novel substrate of CTSL. QC-induced autophagy and CTSL activation were validated by western blot analysis in HeyA8MDR xenograft tumors. Results: QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, MOMP, and mitochondrial cytochrome c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas ectopic expression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL suggesting that CTSL plays a role in QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockdown cells or with autophagy inhibitor chloroquine. Importantly we show upregulation of CTSL in QC treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome c, tBid, cleaved PARP, and caspase3. Conclusion: Taken together, the data suggests that QC-induced autophagy and CTSL upregulation promotes a positive feedback loop leading to excessive autophagic flux, LMP, MOMP to promote QC-induced cell death in ovarian cancer cells. Citation Format: Prabhu Thirusangu, Christopher L. Pathoulas, Upasana Ray, Yinan xiao, Julie Staub, Ashwani Khurana, Vijayalakshmi Shridhar. Quinacrine-induced autophagy in ovarian cancer triggers cathepsin-L mediated lysosomal/mitochondrial membrane permeabilization and cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1937.

Abstract 1947: Brought to light: the homotypic interaction pattern of Bcl-2 effector transmembrane domains in a bimolecular luciferase complementation assay

Abstract The intrinsic apoptosis signaling pathway comprises the intimate interaction network of the Bcl-2 protein family, in which both pro- and antiapoptotic members pushing cell fate to either survival or death. As a critical event, oligomerization of pore-forming effector proteins BAX, BAK and BOK drive mitochondrial outer membrane permeabilization (MOMP), which results in Cytochrome C release and ultimately apoptotic cell death. Thus far, investigations focus on protein-protein interactions mediated by the BH3-domain and hydrophobic groove. These interactions are targeted by BH3 mimetics that represent highly efficient anti-cancer drugs. In contrast, the C-terminal α-9 helices, known as transmembrane domains (TMD), tend to be neglected as a potential secondary interaction site with other Bcl-2 proteins. However, TMDs strongly affect subcellular localization and there is increasing evidence that TMDs crucially influence Bcl-2 protein interactions and therefore apoptosis signaling. Although TMDs are widely abundant in pro- and anti-apoptotic Bcl-2 proteins, their contribution to the complex Bcl-2 interaction network remains largely in the dark. Aiming to shed more light on the interactions of TMD among effector Bcl-2 proteins, we generated a bimolecular complementation assay in which effector TMD peptides are coupled to a bimolecular split-luciferase system. In addition, vectors encode for simultaneous expression of fluorophores to allow control of transgene expression. Human cell lines were co-transfected with combinations of TMD expression constructs yielding luminescence-based interaction data with a fluorescence-based normalization. Interestingly, we found an almost exclusively homotypic TMD interaction among the TMDs of BAX, BAK and BOK. Moreover, confocal microscopy with modified fluorophore-coupled TMD peptides revealed that TMD sequences were sufficient to locate BAX TMD and BAK TMD peptides effectively to mitochondria, while BOK TMD peptides were mainly localized to the endoplasmic reticulum. Strikingly, exchanging TMD sequences in full-length effector proteins profoundly modulated cell death induction. These results strongly suggest a central functional relevance of the α-9-helices in promoting homotypic interactions of effectors BAX, BAK and BOK during oligomerization and pore formation. In contrast, putative heterotypic interaction among effector proteins is not reflected by the newly generated TMD interaction assay. Thus, effector TMDs are a vital factor for specific protein localization and cell death induction displaying specific functionality in each effector protein. Our findings underline the importance of further investigating the TMD region as a potential oncogenic feature influencing interaction of Bcl-2 proteins and therefore as a potential target for therapeutic intervention. Citation Format: Tobias B. Beigl, Sandra Weller, Benjamin Schäfer, Kathrin Böpple, Lara-Sophie Rieder, Hans-Georg Kopp, Markus Rehm, Walter E. Aulitzky, Frank Essmann. Brought to light: the homotypic interaction pattern of Bcl-2 effector transmembrane domains in a bimolecular luciferase complementation assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1947.

Abstract CT224: Phase Ib study of the safety and efficacy of Gemtuzumab Ozogamicin (GO) and Venetoclax in patients with relapsed or refractory CD33+ acute myeloid leukemia: Big Ten Cancer Research Consortium BTCRC-AML17-113

Abstract Background:AML is predominantly a disease of the elderly, yet outcomes remain dismal, particularly for relapsed/refractory (R/R) AML patients (pts). Gemtuzumab Ozogamicin (GO) is a monoclonal antibody targeting CD33–commonly expressed on AML blasts, and, critically, AML stem cells (LSC)–linked to the cytotoxin calicheamicin. GO resistance mechanisms include (i) decreased/aberrant blast CD33 expression, (ii) p-glycoprotein export of calicheamicin, and (iii) apoptosis resistance due to deficient activation of mitochondrial outer membrane permeabilization, a process highly dependent on BCL-2 expression. GO-induced apoptosis depends on the pro-apoptotic proteins Bax and Bak and is inhibited by overexpression of the anti-apoptotic proteins BCL-2 or BCL-XL. Venetoclax (VEN) is a BH3 mimetic, binding BCL-2, dislodging its binding to Bak/Bax, and thus facilitating apoptosis. LSC overexpress BCL-2, however VEN monotherapy is not effective in AML, as resistance develops rapidly. Hypothesis: VEN targeting of BCL-2 proteins that protect LSC from GO-induced apoptosis will synergistically increase GO efficacy. Correlative studies include pre-treatment AML blast BH3 profiling, CD33 expression (including sequencing for isoforms), and BCL-2, BCL-XL, and MCL-1 protein levels; MRD measurement at post-therapy time points using digital drop PCR technology; and quality of life assessments (EORTC QLQ-C30, FACT-Fatigue) Methods:This is a single arm, open-label, multi-center (BTCRC), dose-escalation phase Ib study of combination of VEN and GO in R/R AML pts (18-75y), using a 3+3 design. Major eligibility: ECOG 0-2, adequate organ function, CD33+ in ≥ 20% AML blasts, ≤ 3 lines of prior therapy, and no prior use of GO or VEN, previous VOD, BMT within 2 months, CNS disease, or history of HIV. Induction: 3-day VEN ramp-up to the target dose of 200 (cohort i), 400 (ii), or 600 (iii) mg daily x 28 d, with GO 3mg/m2 infused days 1, 4, and 7. If CR/CRi achieved, pts proceed to BMT if applicable, otherwise, if in CR/CRi (provided ANC &amp;gt; 1000, plts &amp;gt; 100K) or PR (regardless of counts), they are consolidated with VEN at the prescribed dose x 28d and GO 3mg/m2 on days 1 and 4 (Cycle 2). If BMT not applicable, and pt remaining in CR/CRi or PR (as above), then proceed to VEN alone as Maintenance in cycles 3+ until progression or toxicity. The primary endpoint is MTD of VEN with GO. Secondary endpoints include ORR, anti-leukemic activity, characterization of AEs, and estimates of RFS, EFS, and OS. Progress: This study is currently open to its second dosing cohort and has enrolled 5 pts to date. No dose-limiting toxicities have been encountered. However, the COVID-19 pandemic has had a negative impact on enrollment, which is expected to improve as vaccinations expand. ClinicalTrials.gov NCT04070768. Citation Format: Saad Arain, Pritesh Patel, Karen Sweiss, Brian Parkin, Heiko Konig, Gregory Calip, Betul Gok Yavuz, John Quigley. Phase Ib study of the safety and efficacy of Gemtuzumab Ozogamicin (GO) and Venetoclax in patients with relapsed or refractory CD33+ acute myeloid leukemia: Big Ten Cancer Research Consortium BTCRC-AML17-113 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT224.

Abstract 1946: Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression

Abstract 50 Years after the generation of the most studied human breast cancer cell line, MCF-7, many research results fundamentally changed patient outcomes for the better. The mortality of breast cancer in women has decreased significantly, especially in the last 30 years. But the total number of deaths is rising again in the last ten years. Considering that breast cancer is the most common cancer in women and the second most common cancer overall, with incidences of 24,2% and 11,6%, respectively, total numbers of new cases are in the millions every year. First line drugs for the treatment of breast cancer often target microtubules (e.g. Vinblastine and Paclitaxel) or induce DNA damage (e.g. Carboplatin and Cisplatin (CisPt)). In addition to these classic therapeutic drugs new compounds that specifically target cancer-driving molecular alterations have been developed. Among these is the group of BH3-mimetics. The efficacy of several BH3-mimetics is investigated in numerous clinical trials while the BCL-2 specific inhibitor ABT-199 (Venetoclax) is already approved for clinical application in chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML) and small lymphocytic lymphoma (SLL). BH3-mimetics target the apoptosis machinery by interacting with anti-apoptotic Bcl-2 proteins, mimicking the function of pro-apoptotic BH3-only proteins. In turn, the pro-apoptotic potential of pore-forming BCL-2 effectors BAX, BAK and BOK is released inducing mitochondrial outer membrane permeabilization and cell death. Recently we reported synergistic cell death induction by ABT-199 in combination with the proteasome inhibitor Bortezomib (BOZ) in soft tissue sarcoma (STS) cells. ABT-199 and BOZ induce accumulation of BOK and the BH3-only protein NOXA (Muenchow et al. 2020). Here, we combined ABT-199, CisPt and Nutlin-3 in breast cancer cell lines that differ in expression of the estrogen receptor (ER) and p53 activity. Similar to the results in STS cells synergistic induction of apoptotic cell death was observed by FACS analysis of the mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin-V). Western blot analysis revealed that ABT-199 induced accumulation of NOXA and MCL-1, irrespective of the ER status or p53 mutation. Simultaneous treatment with ABT-199 and CisPt or Nutlin-3 further increased expression of NOXA and MCL-1. Interestingly, qRT-PCR revealed enhanced expression of NOXA and MCL-1 mRNA in ABT-199 treated cells indicating transcriptional regulation. However, transcriptional induction of NOXA was also detected independent of p53 activity. Thus, combined therapeutic approaches using CisPt and ABT-199 should be effective irrespective of ER and p53 expression. Nevertheless, in p53 proficient tumors Nutlins might increase therapeutic efficacy. Citation Format: Benjamin Schaefer, Sandra Weller, Tobias Beigl, Kathrin Boepple, Reiner Hoppe, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1946.