Cell Death & Disease

Protein phosphatase-2A (PP2A) is a major source of cellular serine/threonine phosphatase activity. PP2A B-type subunits regulate the intracellular localization and the catalytic activity of PP2A-A/PP2A-C complexes towards individual proteins. There is limited knowledge on how PP2A B-type subunits regulate biologically important functions and if these subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with poor prognosis. Mesenchymal PDAC subtypes are more aggressive and metastasis-prone than epithelial subtypes. We show that mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 and its mRNA Ppp2r3a than epithelial PDAC cells (n=38). Among 17 PP2A B-type subunits, this differential regulation is unique for Ppp2r3a and PR130. The higher levels of PR130 in mesenchymal PDAC cells are linked to their vulnerability to the PP2A inhibitor phendione. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in such cells. These processes occur independently of the major tumor suppressor p53, which is frequently mutated in PDAC cells. Proteomic analyses reveal that phendione upregulates the chaperone heat shock protein HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis. We additionally disclose that phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes mesenchymal PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data illustrate pharmacologically amenable, selective dependencies of mesenchymal PDAC cells on PP2A-PR130 and HSP70. PP2A inhibition triggers a harmful accumulation of protein aggregates in neurons. This undesired mechanism might be exploited to kill mesenchymal tumor cells. O_FIG O_LINKSMALLFIG WIDTH=137 HEIGHT=200 SRC="FIGDIR/small/556106v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@c8a47forg.highwire.dtl.DTLVardef@a7abfforg.highwire.dtl.DTLVardef@d18a3org.highwire.dtl.DTLVardef@1cc9943_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS[tpltrtarr] The PP2A subunit PR130 is a molecular marker of mesenchymal PDAC cells [tpltrtarr]The small molecule PP2A inhibitor phendione selectively kills mesenchymal PDAC cells [tpltrtarr]Phendione decreases PR130 through proteasomes and selectively increases the heat shock protein 70 kDa in mesenchymal PDAC cells [tpltrtarr]HSP70 promotes cell survival upon inhibition of PP2A [tpltrtarr]PP2A-PR130 regulates the accumulation of cytotoxic protein aggregates in mesenchymal PDAC cells

ABSTRACTDouble strand brakes (DSB) accumulate in cellular DNA as a result of deficiencies in homologous recombination repair systems, such as mutations in BRCA genes, or upon antitumoral treatments. In the present study we show that the accumulation of DSB, regardless of its origins, leads to a shift towards oxidative metabolism. We have identified that DSB-induced reactive oxygen species (ROS) promote the activation of NRF2 which downregulates the glycolytic transcription factor HIF-1. HIF-1 inhibition is a key step in this metabolic shift, because leads to the reduction of PDHK1 levels and the consequential activation of pyruvate dehydrogenase, a mitochondrial gatekeeper of cellular metabolism, promoting this metabolic shift. Remarkably, after the induction of DSBs, the tumour is more sensitive to the inhibition of oxidative metabolism since both treatments synergize in vivo, resulting in reduced tumour growth. Therefore, we demonstrate a significant feedback between DSBs induction and cancer cell metabolism that ultimately limits the cells potential for metabolic plasticity, hence sensitizing it to the action of counteracting drugs.

Traditional taxonomies classify ferroptosis and apoptosis as distinct forms of regulated cell death. Here, we challenge this view by defining and validating a previously unrecognized neuronal death entity in traumatic brain injury (TBI), which we term "Ferrapoptosis". This hybrid mode of death is characterized by the coexistence of ferroptotic and apoptotic molecular and ultrastructural features within the same neuron-- ultrastructural alterations that cannot be fully characterized by any known cell death modalities. By integrating single-cell transcriptomics with multi-layered functional assays, we show that Ferrapoptosis dominates in severe injury and the acute phase, and is critically driven by mitochondrial oxidative stress. Over time, its predominance is gradually replaced by death modes in which either ferroptosis or apoptosis alone becomes the major pathway. Genome-wide CRISPR screening further identifies Smg7 as a key regulator that synchronously activates both death programs to drive the hybrid phenotype, whereas genetic or viral inhibition of Smg7 reduces Ferrapoptosis and promotes neurological recovery after TBI in mice. Our work systematically delineates a previously unrecognized form of cell death and its pathogenetic mechanisms, providing experimental evidence to refine cell death classification, and suggesting a conceptual strategy for treating complex diseases such as TBI by targeting shared regulatory nodes. HIGHLIGHTSO_LIDefined and validated a previously unrecognized cell death modality, termed "Ferrapoptosis". C_LIO_LIElucidated the ordered pattern of Ferrapoptosis across key stages of TBI pathogenesis. C_LIO_LIRevealed mitochondrial oxidative stress as the pivotal hub integrating ferroptosis and apoptosis. C_LIO_LIIdentify Smg7 as a core driver of Ferrapoptosis and a promising neuroprotective target. C_LI

Extrinsic apoptosis relies on TNF-family receptor activation by immune cells or receptor-activating biologics. Here, we monitored cell cycle progression at minutes resolution to relate apoptosis kinetics and cell-to-cell heterogeneities in death decisions to cell cycle phases. Interestingly, we found that cells in S phase delay TRAIL receptor-induced death in favour for mitosis, thereby passing on an apoptosis-primed state to their offspring. This translates into two distinct fates, apoptosis execution post mitosis or cell survival from inefficient apoptosis. Transmitotic resistance is linked to Mcl-1 upregulation from mid S phase onwards, which allows cells to pass through mitosis with activated caspase-8, and with cells escaping apoptosis after mitosis sustaining sublethal DNA damage. Antagonizing Mcl-1 by BH3-mimetics suppresses cell cycle-dependent delays in apoptosis, prevents apoptosis-resistant progression through mitosis and averts unwanted survival from apoptosis induction. Cell cycle progression therefore modulates signal transduction during extrinsic apoptosis, with Mcl-1 governing decision making between death, proliferation and survival from inefficient apoptosis induction. Cell cycle progression thus is a crucial process from which cell-to-cell heterogeneities in fates and treatment outcomes emerge in isogenic cell populations during extrinsic apoptosis signalling.

Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localization and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localization is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we showed that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs demonstrated a reduced expression of the Unfolded Protein Response (UPR) markers upon ER stress and displayed reduced inositol trisphosphate receptor (IP3R)-mediated ER calcium release. Collectively, our data show that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the ER, apart from its cliche regulation of apoptosis.

Understanding how the malignant cells respond to chemotherapy is essential to prevent the development of resistance and to improve the efficiency of anti-cancer drugs. Recently, we established that, by intrinsic and paracrine mechanisms, taxol treatment in breast tumor cells increases NOXA a pro-apoptotic protein functioning as an endogenous inhibitor of survival protein MCL-1, thereby enhancing cytotoxic load on the compensatory survival protein BCL-xL. We herein sought to define the contribution of NOXA/MCL-1 to the modality of cell death secretome composition upon anti-mitotic treatment associated with a BCL-xL antagonist. We observed that genetic inactivation of NOXA (enforcing MCL-1 pro-survival activity) in cancer cells not only delays their death when exposed to taxol in combination with the BCL-xL antagonist A1331852, but also alters its morphological characteristics with the apparition of features evoking pyroptosis. We identified the Caspase3-GSDME axis as regulating pyroptotic-like features suggesting that NOXA may act as a negative regulator of this cell death process (and MCL-1 as a positive regulator for it). Furthermore, comparative analysis of secretomes from the NOXA proficient or deficient cancer cells treated by taxol reveals variations in inflammatory cytokine production including those of IL-1{beta} and IL-18. Thus, our results show that anti-mitotic treatments are able to induce death by apoptosis and/or pyroptosis depending on BCL-2 family balance in breast cancer cells. Furthermore, NOXA/MCL-1 ratio appears to control the communication between these two types of cell death and their associated extracellular inflammatory signals in coordination with the pore-forming gasdermin GSDME.

PTK2, a non-receptor tyrosine kinase, plays a critical role in regulating essential cellular functions, including cell survival, by reducing p53 levels and activating the PI3K/AKT pathway. However, the mechanism underlying PTK2 stabilization during stress remains unclear. In this study, we identified Bclaf1, a multifunctional protein known to stabilize partners, as a PTK2 interactor. Using advanced microscopy techniques we identified nuclear Bclaf1 biomolecular condensates containing PTK2 in cardiomyocytes under oxidative stress. While diffuse PTK2 in the nucleus was susceptible to ubiquitination, PTK2 sequestered in the Bclaf1 condensates was protected from the ubiquitin-proteasome system (UPS). The K926 residue was identified as a ubiquitination site on PTK2, and subsequent proteasome inhibition experiments confirmed the role of the UPS in PTK2 homeostasis. Furthermore, disrupting Bclaf1 biomolecular condensates lead to PTK2 degradation, subsequently increasing p53 levels and activating apoptosis. Our findings support the role of Bclaf1 in the formation of pro-survival nuclear condensates that sequester and stabilize PTK2, promoting cardiomyocyte survival during oxidative stress. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/636487v3_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@15377a9org.highwire.dtl.DTLVardef@d5325forg.highwire.dtl.DTLVardef@de0ac9org.highwire.dtl.DTLVardef@196325d_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefMoretto et al. demonstrated that Bclaf1 undergo liquid-liquid phase separation (LLPS) during doxorubicin (Dox) induced oxidative stress to generate membraneless organelles where PTK2 is sequestered and protected from the ubiquitin-proteasome system (UPS). The maintenance of PTK2 stability may favor the p53 and Mdm2 interaction, which results in p53 ubiquitination and degradation, promoting cell survival. Loss of Bclaf1 disrupts PTK2 stabilization, leading to the ubiquitination and degradation of this kinase and downstream upregulation of p53, increasing cell death. HighlightsO_LIBclaf1 undergoes LLPS to stabilize protein partners during stress. C_LIO_LIPTK2 inside the Bclaf1 biomolecular condensates is protected from the UPS to promote cardiomyocyte survival during dox-induced oxidative stress. C_LIO_LIThe PTK2 ubiquitination site, lysine 926, was identified and the action of the UPS on PTK2 proteostasis was confirmed. C_LIO_LIBclaf1 knockdown results in overall protein ubiquitination and in PTK2 ubiquitination and degradation, resulting in increased levels of p53 and PUMA. C_LI

Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy eliminating cancers effectively and safely is the current clinical choice. Since aggressive tumors have inherent or acquired resistance to multidisciplinary therapies targeting apoptosis, tumor-specific induction of another cell death modality is a promising avenue to meet the goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can induce cell death in OS and OC via a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) because of the characteristic unipolar mitochondrial perinuclear aggregation. APAM had potent antitumor activity both in vitro and in vivo. APAM caused apoptosis, necrotic cell death, and autophagy. APAM contained hydrogen peroxide and increased mitochondrial ROS (mROS), while the antioxidant N-acetylcysteine (NAC) prevented cell death. MPMC occurred following mitochondrial fragmentation coinciding with nuclear damages. MPMC was accompanied by the tubulin network remodeling and mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC and the microtubule inhibitor, Nocodazole. Increased Cardiolipin (CL) oxidation was also seen, and NAC and the peroxy radical scavenger Ferrostatin-1 prevented it. In contrast, in fibroblasts, APAM induced minimal cell death, mROS generation, mLPO accumulation, CL oxidation, and MPMC. These results suggest that MPMC is a tumor-specific cause of cell death via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC might serve as a promising target for exerting tumor-specific cytotoxicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/466636v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1118e2corg.highwire.dtl.DTLVardef@18cf99corg.highwire.dtl.DTLVardef@57a7edorg.highwire.dtl.DTLVardef@d1f447_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Lysosomes are known to contribute to the development of drug resistance through a variety of mechanisms that include the sequestration of drugs within their compartments and the activation of adaptive stress pathways. Although targeting POL I (RNA polymerase I) exhibits anticancer effects, little attention has been paid to the contribution of lysosomes to the efficacy and resistance of RNA POL I inhibitors. In this study, we investigated this aspect in the context of two potent POL I inhibitors, CX-3543 (Quarfloxin) and CX-5461 (Pidnarulex). Unexpectedly, CX-3543 was discovered to be sequestered in the lysosomal compartment. This resulted in the permeabilization of lysosomal membranes (LMP) and the subsequent activation of cellular stress adaptation pathways, including the transcription factor (TFEB) and autophagy. Disruption of TFEB or autophagy increased cell sensitivity to CX-3543, highlighting the cytoprotective role of these processes against cell death induced by this compound. Moreover, targeting lysosomal membranes using chloroquine derivatives or blue light excitation induced substantial LMP, resulting in the liberation of CX-3543 from lysosomes. This effect amplified both the inhibition of DNA-to-RNA transcription and cell death induced by CX-3543. Similar effects were observed when chloroquine derivatives were combined with CX-5461. Furthermore, combining CX-3543 with the chloroquine derivative DC661 reduced the growth of fibrosarcoma established in immunocompetent mice more efficiently than either agent alone. Altogether, our results uncover an unanticipated lysosome-related mechanism that contributes to the resistance of cancer cells to POL I transcription inhibitors, as well as a strategy to combat this resistance.

Bone morphogenetic proteins (BMP) induce apoptosis in myeloma cells and the mechanism behind this could point to new therapeutic targets. Here, we did a whole genome CRISPR/Cas9 knockout screen using the INA-6 myeloma cell line. Apoptosis was induced with BMP9 and the relative amounts of sgRNAs in treated versus control cells were determined with next-generation sequencing. We identified key positive control genes and a substantial number of novel genes that could be involved in BMP-induced apoptosis. One of the overrepresented genes was the known BMP target gene ID3. We found that ID3 was potently induced by BMP9 treatment and that depletion of ID3 protected cells from c-MYC downregulation and apoptosis. ID3 is known to heterodimerize with basic helix-loop-helix (bHLH) TCF transcription factors. In the screen, TCF3, TCF4, and TCF12 were among genes that potentially protected cells from apoptosis. Knockdown of TCF3, and to some extent TCF12, led to lower basal c-MYC levels and lower cell viability, and this was more pronounced after BMP9-treatment. Our results suggest that ID3 plays an important role in regulating the survival of myeloma cells, at least in part by forming heterodimers with TCF3 and thus preventing expression of the c-MYC oncogene.

Breast cancer (BC) is the most common type of cancer among women worldwide and underlies relapse, disease progression and metastasis. Resistance to chemotherapy and programmed cell death (PCD), including apoptosis, strongly affects therapy success and remains a major challenge. Representative and translational models to understand, manipulate and cultivate advanced BC and to model PCD resistance are therefore urgently required. Smac mimetics are promising compounds to circumvent apoptosis resistance and are able to induce caspase-independent necroptosis, a lytic and inflammatory mode of PCD. Here, we apply primary, patient-derived human mammary organoids (hMOs) to investigate alternative forms of PCD to overcome apoptosis resistance. Using time lapse brightfield with immunofluorescent confocal microscopy, biochemistry and gene expression analysis, we demonstrate that Smac mimetics induce apoptosis in primary hMOs. By mimicking apoptosis resistance via caspase inhibition, hMOs undergo necroptosis, associated with expression and secretion of inflammatory mediators. Inhibition of linear ubiquitination by the LUBAC inhibitor HOIPIN-8 prevents necroptosis, as well as the expression and release of inflammatory mediators in hMOs. Our findings demonstrate that primary hMOs are effective models to model, study and manipulate PCD responses and inflammation in in primary BC organoids and open new therapeutic screening options for chemotherapy-resistant BC.

Pharmacological data point to RAC1 as promising target for protection against anthracycline-induced cardiotoxicity, yet supporting genetic evidence is limited. Moreover, the relevance of RAC1 for cross-organ injury and cross-agent-induced normal tissue toxicity is unknown. Here, we employed a Mx1-Cre-based mouse model that enables an inducible Rac1 knockout across different organs in order to comparatively analyze the influence of RAC1 on doxorubicin (DOX) and cisplatin (CisPt)-induced acute stress responses of the heart, kidney and liver. Following DOX treatment, the extend of DNA-double strand break (DSB) formation and the percentage of apoptotic cells were reduced in all three organs (i.e. heart, liver, kidney) of RAC1 deficient (Rac1-/-) animals as compared to the wildtype control (Rac1+/+). By contrast, in the absence of RAC1, CisPt-induced DNA damage was reduced only in the liver and the frequency of CisPt-stimulated apoptotic cell death remained unaffected by the Rac1 status in all organs. These findings demonstrate a strikingly organ- and agent-specific relevance of RAC1-regulated mechanisms for normal tissue damage evoked by genotoxic anticancer therapeutics. Accordingly, protein levels of phosphorylated DNA damage response (DDR)-related factors were also reduced in the kidney and liver of DOX treated Rac1-/- animals, but not in the heart. Yet, DOX-triggered mRNA expression of surrogate markers related to inflammation, fibrosis and senescence was preferentially reduced in the heart and kidney of Rac1 deficient mice. Taken together, RAC1 plays a so far unknown distinct role in the pathophysiology of anticancer drug-induced normal tissue damage by influencing DNA damage formation, activation of DDR, apoptosis induction as well as inflammation-, fibrosis- and senescence-associated responses in a pronounced agent- and organ-specific manner. Targeting of RAC1 appears particularly effective in the context of anthracycline-based therapeutic regimen for conferring broad organoprotection to both the heart and detoxifying organs. Translational PerspectiveThis study provides the first genetic evidence identifying RAC1 as a critical mediator of anthracycline-induced cardiac, hepatic and renal injury. We demonstrate that genetic deletion of Rac1 alleviates chemotherapy-induced DNA damage, apoptosis, and stress responses related to senescence, fibrosis, and inflammation in an agent- and organ-specific manner. These findings highlight RAC1 as a putatively clinically relevant target for the development of organoprotective pharmacological strategies aiming to improve the tolerability of anthracycline- and cisplatin-based therapeutic regimen and, accordingly, enhancing the quality of life and prognosis of tumor patients.

The tumor suppressor p73 is a member of the p53 family and transcriptionally activates multiple p53-targets involved in cell cycle regulation and apoptosis. In addition to pro- apoptotic signaling, outcomes of p73 activation include cell survival signals. Thus, p73 activity and targets may provide insight in cell fate outcomes between cell survival and apoptosis following cellular stress. We report that cellular FLICE inhibitory protein (c- FLIP), a master antiapoptotic factor, is a transcriptional target of p73. The activation of p73 ( and {beta} isoforms) transcriptionally upregulates c-FLIP-L/S expression in cancer cells. The cell fate decision following p73 activation is determined by the adjustment of the balance of outcomes of p73 activation between p73-induced pro-apoptotic signaling and c-FLIP-L/S expression in cancer cells. p73 primes extrinsic apoptosis via an autocrine death ligand-DR5 axis, and the priming appears to be titrated at the level of c-FLIP-L/S. The p73-upregulation of c-FLIP-L/S increases the threshold of extrinsic apoptosis. Cells with poor priming levels convert to cell cycle arrest and survival. Depletion of c-FLIP-L/S increases the p73-priming levels towards extrinsic apoptosis and sensitizes cancer cells to p73-primed extrinsic apoptosis. We further identified a small-molecule CB-7587351 ("switcher compound") that alters p73 activation outcomes through c-FLIP-L/S protein degradation. Therapeutic activation of p73 can restore p53- signaling in mutant p53-expressing cancer cells effectively bypassing the p53 deficiency in cancer cells. Our discovery of p73 transcriptional upregulation of c-FLIP provides a promising strategy for depleting c-FLIP to improve antitumor efficacy of p73-targeting cancer therapy for p53-mutant tumors.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer. Loss of von Hippel-Lindau (VHL) and the consequent activation of hypoxia-inducible factor- (HIF, especially HIF2) plays an essential role in ccRCC initiation and progression. The approved HIF2 inhibitor belzutifan faces the challenge of resistance, presenting an opportunity of co-targeting HIF2 and another vulnerability. This study elucidates the synthetic essentiality of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) in VHL-deficient ccRCC, uncovering a novel reciprocal regulation between HIF2 and TRAIL. TRAIL was identified as a direct transcriptional target of HIF2 and paradoxically found to be crucial for cell proliferation, primarily by activating the p38 MAPK pathway and facilitating G1/S phase transition. Depletion of endogenous TRAIL or inhibition of HIF2 with belzutifan sensitizes ccRCC cells to recombinant TRAIL, presenting a promising avenue for combination therapy to overcome both TRAIL resistance and belzutifan resistance in treating ccRCC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/621197v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1d603b9org.highwire.dtl.DTLVardef@18e6dc7org.highwire.dtl.DTLVardef@1c953cdorg.highwire.dtl.DTLVardef@9cd144_HPS_FORMAT_FIGEXP M_FIG C_FIG

An unmet challenge in managing breast cancer is treatment failure due to resistance to apoptosis-inducing chemotherapies. Thus, it is important to identify novel non-apoptotic therapeutic agents. Several non-apoptotic programmed cell death pathways utilize specific cellular signaling events to trigger lytic and pro-inflammatory cell death. PANoptosis, which encompasses pyroptosis, apoptosis and necroptosis, is of paramount importance in the regulation of cell death and immune responses. Our study illustrates that ophiobolin A (OpA) is an anti-cancer agent that triggers lytic cell death in breast cancer cells, including triple-negative breast cancer (TNBC), via a mechanism dependent on RIPK1. This study reveals that OpA induces typical pyroptosis-like characteristics, including cellular swelling, plasma membrane rupture, GSDMD cleavage and release of cytokines in breast cancer cells. The involvement of caspase 3, RIPK1, and GSDMD suggests that PANoptosis is activated upon OpA treatment in breast cancer. The induction of pro-inflammatory cell death suggests potential applications for OpA in cancer treatment.

Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). We previously demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a Bcl-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). These findings implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic Bcl-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we observed that BAPTAi alone induced apoptosis in lymphoma cell models that were highly sensitive to S63845, an Mcl-1 antagonist. BAPTAi provoked a rapid decline in Mcl-1 protein levels by inhibiting mTORC1-driven MCL-1 translation. Overexpression of nondegradable Mcl-1 rescued BAPTAi-induced cell death. We further examined how BAPTAi diminished mTORC1 activity and found that BAPTAi impaired glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, an up to now unkown effect of BAPTAi. All aforementioned effects of BAPTAi were also elicited by a BAPTAi analog with low affinity for Ca2+. Thus, our work reveals PFKFB3 inhibition as an unappreciated Ca2+-independent mechanism by which BAPTAi impairs cellular metabolism and ultimately the survival of Mcl-1-dependent cancer cells. Our work has two important implications. First, direct inhibition of PFKFB3 emerged as a key regulator of mTORC1 activity and a promising target in the treatment of Mcl-1-dependent cancers. Second, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

PINK1, a mitochondria targeted Serine/Threonine kinase, regulates ATP production by phosphorylating the Complex I subunit NdufA10. However, when in the presence of depolarized mitochondria, PINK1 phosphorylates ubiquitin and Parkin triggering mitochondria clearance. Mutations in PINK1 have been linked to early-onset recessive familial forms of Parkinsons disease (PD). Deficits in Complex I enzymatic activity and an increase in oxidative damage have been identified in multiple brain regions of PD patients. Unravelling how PINK1 activity regulates mitochondria fate is pivotal. In the present study we characterized how human PD-related PINK1 mutants affect major PINK1 functions. Using molecular dynamics, we gain mechanistic insight into how specific mutations alter the tertiary structure and stability of PINK1s ATP-binding pocket, leading to an increased rigidity and stability. More importantly, we report a structural explanation for the enhanced kinase function of the PINK1-G411S mutant.

Atypically expressed transglutaminase 2 (TG2) has been identified as a poor prognostic factor in a variety of cancers. In this study, we evaluated the contribution of TG2 to the prolonged cell survival of differentiated acute promyelocytic leukaemia (APL) cells in response to the standard treatment with combined retinoic acid (ATRA) and arsenic trioxide (ATO). We report that one advantage of ATRA + ATO treatment compared to ATRA alone diminishes the amount of activated and non-activated CD11b/CD18 and CD11c/CD18 cell surface integrin receptors. These changes suppress ATRA-induced TG2 docking on the cytosolic part of CD18 {beta}2-integrin subunits and reduce cell survival. In addition, TG2 overexpresses and hyperactivates the phosphatidylinositol-3-kinase (PI3K), phospho-AKT S473, and phospho-mTOR S2481 signalling axis. mTORC2 acts as a functional switch between cell survival and death by promoting the full activation of AKT. We show that TG2 presumably triggers the formation of a signalosome platform, hyperactivates downstream mTORC2-AKT signalling, which in turn phosphorylates and inhibits the activity of FOXO3, a key pro-apoptotic transcription factor. In contrast, the absence of TG2 restores basic phospho-mTOR S2481, phospho-AKT S473, PI3K, and PTEN expression and activity, thereby sensitising APL cells to ATO-induced cell death. We conclude, that atypically expressed TG2 may serve as a hub, facilitating signal transduction via signalosome formation by the CD18 subunit with both PI3K hyperactivation and PTEN inactivation through the PI3K PTEN cycle in ATRA-treated APL cells.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure and highly resistant to chemotherapy, potentially due to upregulated expression of the pro-survival proteins, BCL2/BCL-XL/MCL-1. Using clinically-relevant models of MPM we show that patient-derived primary MPM cell lines and ex-vivo 3D tumour explants are highly resistant to apoptosis induced by the BCL2/BCL-XL inhibitor, ABT-737. Importantly, we discover that 2-deoxyglucose (2DG), a glycolytic inhibitor, can sensitize MPM cells to ABT-737 and show this correlates with loss of the pro-survival protein, MCL-1. siRNA knockdown of MCL-1 (MCL-1 KD) combined with ABT-737 induced BAX/BAK-dependent, but BIM/PUMA-independent apoptosis, mimicking 2DG/ABT-737 treatment. MCL-1 KD/ABT-737 induced mitochondrial cytochrome c release and caspase-independent inhibition of mitochondrial respiration. Moreover, we observed a hitherto unreported caspase-dependent cleavage of glycolytic enzymes and subsequent inhibition of glycolysis. 2DG inhibited ERK/STAT3 activity, decreased MCL-1 mRNA and protein levels, with concurrent activation of AKT, which limited loss of MCL-1 protein. However, co-treatment with a specific AKT inhibitor, AZD5363, and 2DG/ABT-737 potently induced cell death and inhibited clonogenic cell survival, while in MPM 3D tumour explants MCL-1 protein expression decreased significantly following 2DG or 2DG/AZD5363 treatment. Notably, a similar synergy was observed in MPM cell lines and MPM 3D tumour explants using ABT-737 in combination with the recently developed MCL-1 inhibitor, S63845. Importantly, our study provides a mechanistic explanation for the chemoresistance of MPM and highlights how this can be overcome by a combination of metabolic reprogramming and/or simultaneous targeting of MCL-1 and BCL-2/BCL-XL using BH3-mimetics.

Oxidative stress is a key factor causing mitochondrial dysfunction and retinal ganglion cell (RGC) death in glaucomatous neurodegeneration. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway is involved in mitochondrial protection, promoting RGC survival. Soluble adenylyl cyclase (sAC) is one of the key regulators of the cAMP/PKA signaling pathway. However, the precise molecular mechanisms underlying the sAC-mediated signaling pathway and mitochondrial protection in RGCs that counter oxidative stress are not well characterized. Here, we demonstrate that sAC plays a critical role in protecting RGC mitochondria from oxidative stress. Using mouse models of oxidative stress, we found that activating sAC protected RGCs, blocked AMP-activated protein kinase activation, inhibited glial activation, and improved visual function. Moreover, we found that this is the result of preserving mitochondrial dynamics (fusion and fission), promoting mitochondrial bioenergetics and biogenesis, and preventing metabolic stress and apoptotic cell death in a paraquat oxidative stress model. Notably, sAC activation ameliorated mitochondrial dysfunction in RGCs by enhancing mitochondrial biogenesis, preserving mitochondrial structure, and increasing ATP production in oxidatively stressed RGCs. These findings suggest that activating sAC enhances the mitochondrial structure and function in RGCs to counter oxidative stress, consequently promoting RGC protection. We propose that modulation of the sAC-mediated signaling pathway has therapeutic potential acting on RGC mitochondria for treating glaucoma and other retinal diseases.