MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network.

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.

A trifluorinated thiazoline scaffold leading to pro-apoptotic agents targeting prohibitins.

A new class of small molecules, with an unprecedented trifluorothiazoline scaffold, were synthesized and their pro-apoptotic activity was evaluated. With an EC50 in the low micromolar range, these compounds proved to be potent inducers of apoptosis in a broad spectrum of tumor cell lines, regardless of the functional status of p53. Fast structure-activity relationship studies allowed the preparation of the strongest apoptosis-inducing candidate. Using a high performance affinity purification approach, we identified prohibitins 1 and 2, key proteins involved in the maintenance of cell viability, as the targets for these compounds.

AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts.

5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1 (-/-) /α2 (-/-) MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.

Cell death through the ages: The ICDS 25th Anniversary Meeting.

In June of 2019, the International Cell Death Society (ICDS) held its 25th anniversary meeting in New York City at the Icahn School of Medicine at Mount Sinai organized by Drs. Richard A. Lockshin (St. John's University, USA), Zahra Zakeri (Queens College, USA), and Jerry Edward Chipuk (Icahn School of Medicine at Mount Sinai, USA). The three-day event, entitled 'Cell death through the ages: The ICDS 25th anniversary meeting', hosted ninety-one delegates including thirty-four speakers and twenty-two poster presentations. Additionally, the organizers gave special recognition to the twenty-one previous ICDS Lifetime Achievement awardees-those who have significantly contributed to the field of cell death and the growth of the organization. Here, we provide a summary of the meeting and highlight trending research in the fields of cell death, autophagy, immunology, and their impact on health and disease.

MDM2 and mitochondrial function: One complex intersection.

Decades of research reveal that MDM2 participates in cellular processes ranging from macro-molecular metabolism to cancer signaling mechanisms. Two recent studies uncovered a new role for MDM2 in mitochondrial bioenergetics. Through the negative regulation of NDUFS1 (NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1) and MT-ND6 (NADH dehydrogenase 6), MDM2 decreases the function and efficiency of Complex I (CI). These observations propose several important questions: (1) Where does MDM2 affect CI activity? (2) What are the cellular consequences of MDM2-mediated regulation of CI? (3) What are the physiological implications of these interactions? Here, we will address these questions and position these observations within the MDM2 literature.

Regulation of tumor-stroma interactions by the unfolded protein response.

The unfolded protein response (UPR) is a conserved adaptive pathway that helps cells cope with the protein misfolding burden within the endoplasmic reticulum (ER). Imbalance between protein folding demand and capacity in the ER leads to a situation called ER stress that is often observed in highly proliferative and secretory tumor cells. As such, activation of the UPR signaling has emerged as a key adaptive mechanism promoting cancer progression. It is becoming widely acknowledged that, in addition to its intrinsic effect on tumor biology, the UPR can also regulate tumor microenvironment. In this review, we discuss how the UPR coordinates the crosstalk between tumor and stromal cells, such as endothelial cells, normal parenchymal cells, and immune cells. In addition, we further describe the involvement of ER stress signaling in the response to current treatments as well as its impact on antitumor immunity mainly driven by immunogenic cell death. Finally, in this context, we discuss the relevance of targeting ER stress/UPR signaling as a potential anticancer approach.

Caspase 1/11 Deficiency or Pharmacological Inhibition Mitigates Psoriasis-Like Phenotype in Mice.

Inflammatory caspases, activated within the inflammasome, are responsible for the maturation and secretion of IL-1ß/IL-18. Although their expression in psoriasis was shown several years ago, little is known about the role of inflammatory caspases in the context of psoriasis. Here, we confirmed that caspases 1, 4, and 5 are activated in lesional skin from psoriasis patients. We showed in three psoriasis-like models that inflammatory caspases are activated, and accordingly, caspase 1/11 invalidation or pharmacological inhibition by Ac-YVAD-CMK (i.e., Ac-Tyr-Val-Ala-Asp-chloromethylketone) injection induced a decrease in ear thickness, erythema, scaling, inflammatory cytokine expression, and immune cell infiltration in mice. We observed that keratinocytes were primed to secrete IL-1ß when cultured in conditions mimicking psoriasis. Generation of chimeric mice by bone marrow transplantation was carried out to decipher the respective contribution of keratinocytes and/or immune cells in the activation of inflammatory caspases during psoriasis-like inflammatory response. Our data showed that the presence of caspase 1/11 in the immune system is sufficient for a fully inflammatory response, whereas the absence of caspase 1/11 in keratinocytes/fibroblasts had no impact. In summary, our study indicates that inflammatory caspases activated in immune cells are implicated in psoriasis pathogenesis.

GAPDH Overexpression in the T Cell Lineage Promotes Angioimmunoblastic T Cell Lymphoma through an NF-κB-Dependent Mechanism.

GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.

GAPDH Expression Predicts the Response to R-CHOP, the Tumor Metabolic Status, and the Response of DLBCL Patients to Metabolic Inhibitors.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease treated with anti-CD20-based immuno-chemotherapy (R-CHOP). We identified that low levels of GAPDH predict a poor response to R-CHOP treatment. Importantly, we demonstrated that GAPDHlow lymphomas use OxPhos metabolism and rely on mTORC1 signaling and glutaminolysis. Consistently, disruptors of OxPhos metabolism (phenformin) or glutaminolysis (L-asparaginase) induce cytotoxic responses in GAPDHlow B cells and improve GAPDHlow B cell-lymphoma-bearing mice survival, while they are low or not efficient on GAPDHhigh B cell lymphomas. Ultimately, we selected four GAPDHlow DLBCL patients, who were refractory to all anti-CD20-based therapies, and targeted DLBCL metabolism using L-asparaginase (K), mTOR inhibitor (T), and metformin (M) (called KTM therapy). Three out of the four patients presented a complete response upon one cycle of KTM. These findings establish that the GAPDH expression level predicts DLBCL patients' response to R-CHOP treatment and their sensitivity to specific metabolic inhibitors.

Reshaping the Immune Tumor Microenvironment Through IRE1 Signaling.

The ability of a tumor cell to cope with environmental and intracellular stress depends on its capacity to activate appropriate adaptive pathways. As such, the endoplasmic reticulum (ER) adjusts the adaptive capacity of tumor cells by engaging the unfolded protein response (UPR). The UPR maintains the functionality of the secretory pathway, thereby allowing tumor cells to shape their microenvironment, thus likely determining the nature of the tumor immune response. Consequently, this makes the UPR very relevant in the context of cancer therapeutics. We focus here on inositol-requiring enzyme 1α (IRE1) and compile novel molecular mechanisms demonstrating that tumoral UPR controls the tumor microenvironment (TME) and the immune response, therefore opening potential novel therapeutic avenues.

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance.

Dietary restriction (DR) was shown to impact on tumor growth with very variable effects depending on the cancer type. However, how DR limits cancer progression remains largely unknown. Here, we demonstrate that feeding mice a low-protein (Low PROT) isocaloric diet but not a low-carbohydrate (Low CHO) diet reduced tumor growth in three independent mouse cancer models. Surprisingly, this effect relies on anticancer immunosurveillance, as depleting CD8+ T cells, antigen-presenting cells (APCs), or using immunodeficient mice prevented the beneficial effect of the diet. Mechanistically, we established that a Low PROT diet induces the unfolded protein response (UPR) in tumor cells through the activation of IRE1α and RIG1 signaling, thereby resulting in cytokine production and mounting an efficient anticancer immune response. Collectively, our data suggest that a Low PROT diet induces an IRE1α-dependent UPR in cancer cells, enhancing a CD8-mediated T cell response against tumors.

Parkin-Independent Mitophagy Controls Chemotherapeutic Response in Cancer Cells.

Mitophagy is an evolutionarily conserved process that selectively targets impaired mitochondria for degradation. Defects in mitophagy are often associated with diverse pathologies, including cancer. Because the main known regulators of mitophagy are frequently inactivated in cancer cells, the mechanisms that regulate mitophagy in cancer cells are not fully understood. Here, we identified an E3 ubiquitin ligase (ARIH1/HHARI) that triggers mitophagy in cancer cells in a PINK1-dependent manner. We found that ARIH1/HHARI polyubiquitinates damaged mitochondria, leading to their removal via autophagy. Importantly, ARIH1 is widely expressed in cancer cells, notably in breast and lung adenocarcinomas; ARIH1 expression protects against chemotherapy-induced death. These data challenge the view that the main regulators of mitophagy are tumor suppressors, arguing instead that ARIH1-mediated mitophagy promotes therapeutic resistance.

Mitochondrial permeabilization engages NF-κB-dependent anti-tumour activity under caspase deficiency.

Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the downregulation of inhibitor of apoptosis proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies.

Low carbohydrate diet prevents Mcl-1-mediated resistance to BH3-mimetics.

Overexpression of Mcl-1 is implicated in resistance of several cancers to chemotherapeutic treatment, therefore identifying a safe way to decrease its expression in tumor cells represents a central goal. We investigated if a modulation of the diet could impact on Mcl-1 expression using a Myc-driven lymphoma model. We established that a partial reduction of caloric intake by 25% represents an efficient way to decrease Mcl-1 expression in tumor cells. Furthermore, using isocaloric custom diets, we observed that carbohydrates (CHO) are the main regulators of Mcl-1 expression within the food. Indeed, feeding lymphoma-bearing mice with a diet having 25% less carbohydrates was sufficient to decrease Mcl-1 expression by 50% in lymphoma cells. We showed that a low CHO diet resulted in AMPK activation and mTOR inhibition leading to eukaryotic elongation factor 2 (eEF2) inhibition, blocking protein translation elongation. Strikingly, a low CHO diet was sufficient to sensitize Myc-driven lymphoma-bearing mice to ABT-737-induced cell death in vivo. Thus reducing carbohydrate intake may represent a safe way to decrease Mcl-1 expression and to sensitize tumor cells to anti-cancer therapeutics.

Epigenetic profile in chronic lymphocytic leukemia using methylation-specific multiplex ligation-dependent probe amplification.

AIM: To analyze the methylation status of 35 tumor suppressor genes using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in chronic lymphocytic leukemia (CLL). MATERIALS & METHODS: The DNA of 37 samples from patients with CLL, six healthy donors, and Jurkat and Ramos cell lines was analyzed by MS-MLPA. RESULTS: Our results confirm that hypermethylation is a common and not randomly distributed event in CLL, and some genes, such as WT1, CDH13, IGSF4/TSLC1, GATA5, DAPK1 and RARB, are hypermethylated in more than 25% of the analyzed samples. Importantly, MS-MLPA also detected hypermethylation of some genes not reported previously in CLL, and their methylation status was confirmed by bisulfite sequencing. CONCLUSION: These results indicate that MS-MLPA is a useful technique for the detection of methylation in CLL samples. Selecting CLL-specific methylation targets in order to generate a CLL-specific MS-MLPA probe set could enhance its usefulness as a tool in studies of risk stratification and guiding the best therapeutic decision.

Glycogen synthase kinase-3ß is involved in ligand-dependent activation of transcription and cellular localization of the glucocorticoid receptor.

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in different cell types and therefore are widely used to treat a variety of diseases including autoimmune disorders and cancer. This effect is mediated by the GC receptor (GR), a ligand-activated transcription factor that translocates into the nucleus where it modulates transcription of target genes in a promoter-specific manner. Glycogen synthase kinase-3 (GSK3) regulates GR response by genomic and nongenomic mechanisms, although the specific role of each isoform is not well defined. We used GSK3 pharmacological inhibitors and isoform-specific small interfering RNA to evaluate the role of GSK3 in the genomic regulation induced by GC. GSK3 inhibition resulted in the reduction of GC-induced mRNA expression of GC-induced genes such as BIM, HIAP1, and GILZ. Knockdown of GSK3ß but not GSK3α reduced endogenous GILZ induction in response to dexamethasone and GR-dependent reporter gene activity. Chromatin immunoprecipitation experiments revealed that GSK3 inhibition impaired the dexamethasone-mediated binding of GR and RNA polymerase II to endogenous GILZ promoter. These results indicate that GSK3ß is important for GR transactivation activity and that GSK3ß inhibition suppresses GC-stimulated gene expression. Furthermore, we show that genomic regulation by the GR is independent of known GSK3ß phosphorylation sites. We propose that GC-dependent transcriptional activation requires functional GSK3ß signaling and that altered GSK3ß activity influences cell response to GC.

Analysis of apoptosis regulatory genes altered by histone deacetylase inhibitors in chronic lymphocytic leukemia cells.

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.