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Purpose:Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it.Experimental Design:We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions of BIM gene harbored by three commonly used MCL cell lines (JEKO-1, MINO, and Z138) were not found by array comparative genomic hybridization using a validation set of 24 primary MCL samples.Results:We demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics.Conclusions:Our data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

Supplemental Figure 1. Next-generation sequencing of PDXs and primary MCL cells Supplemental Figure 2. IHC analysis of primary MCL samples and murine xenografts Supplemental Figure 3. Array comparative genomic hybridization of 24 primary MCL samples Supplemental Figure 4. Sensitivity of HBL2 and MAVER-1 cells resistant to venetoclax to BCL-XL inhibitors WEHI-539 and A1155463 Supplemental Table 1. Complete list of protein coding variants Supplemental Table 2. FISH analyses of the established murine PDXs of MCL Supplemental Table 3. Baseline characteristics of patients Supplemental Table 4. IHC analysis of MCL samples and xenografts Supplemental Table 5. Tumor and spleen weights at the end of in vivo experiments

Mammalian genes were long thought to be constrained within somatic cells in most cell types. This concept was challenged recently when cellular organelles including mitochondria were shown to move between mammalian cells in culture via cytoplasmic bridges. Recent research in animals indicates transfer of mitochondria in cancer and during lung injury in vivo, with considerable functional consequences. Since these pioneering discoveries, many studies have confirmed horizontal mitochondrial transfer (HMT) in vivo, and its functional characteristics and consequences have been described. Additional support for this phenomenon has come from phylogenetic studies. Apparently, mitochondrial trafficking between cells occurs more frequently than previously thought and contributes to diverse processes including bioenergetic crosstalk and homeostasis, disease treatment and recovery, and development of resistance to cancer therapy. Here we highlight current knowledge of HMT between cells, focusing primarily on in vivo systems, and contend that this process is not only (patho)physiologically relevant, but also can be exploited for the design of novel therapeutic approaches.

Mitochondrial Complex II (CII) activity supports multiple types of respiration and connects metabolic state with succinate signaling. It is therefore unsurprising that CII activity has been linked to biological phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, metabolic shifts in cancer, immune activation, and ischemia reperfusion injury. In each of these processes, CII activity may be regulated at the level of assembly, with the disassembled SDHA subunit stored as metastable species with one or more assembly factors. However, both the exact molecular nature of these species and the mechanism of release of SDHA from these metastable species is unclear. In this work, we identified that transfer of SDHA into CII proceeds through at least three metastable intermediates, we assigned the sequence of appearance of these intermediates, and we determined structures for two of these. A complex between SDHA and one of its assembly factors, succinate dehydrogenase assembly factor 2 (SDHAF2), is likely the main alternative species that accumulates in cells. A second assembly factor, succinate dehydrogenase assembly factor 4 (SDHAF4), must displace SDHAF2 for assembly to proceed. Changes in intrinsic disorder in both SDHAF2 and SDHAF4 are required to release SDHA from the metastable complexes. Evaluation of unrelated biological complexes suggests that changes in disorder may guide post-folding biogenesis during both self-assembly and assisted assembly.

Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor–like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on humanoolemmaand played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes.

Background Pheochromocytoma and paraganglioma (PPGL) are neuroendocrine tumors with frequent mutations in genes linked to the tricarboxylic acid cycle. However, no pathogenic variant has been found to date in succinyl-CoA ligase (SUCL), an enzyme that provides substrate for succinate dehydrogenase (SDH; mitochondrial complex II [CII]), a known tumor suppressor in PPGL. Methods A cohort of 352 patients with apparently sporadic PPGL underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health, including the SUCLG2 subunit of SUCL. Gene deletion, succinate levels, and protein levels were assessed in tumors where possible. To confirm the possible mechanism, we used a progenitor cell line, hPheo1, derived from a human pheochromocytoma, and ablated and re-expressed SUCLG2. Results We describe 8 germline variants in the guanosine triphosphate–binding domain of SUCLG2 in 15 patients (15 of 352, 4.3%) with apparently sporadic PPGL. Analysis of SUCLG2-mutated tumors and SUCLG2-deficient hPheo1 cells revealed absence of SUCLG2 protein, decrease in the level of the SDHB subunit of SDH, and faulty assembly of the complex II, resulting in aberrant respiration and elevated succinate accumulation. Conclusions Our study suggests SUCLG2 as a novel candidate gene in the genetic landscape of PPGL. Large-scale sequencing may uncover additional cases harboring SUCLG2 variants and provide more detailed information about their prevalence and penetrance.

Purpose: Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it. Experimental Design: We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions of BIM gene harbored by three commonly used MCL cell lines (JEKO-1, MINO, and Z138) were not found by array comparative genomic hybridization using a validation set of 24 primary MCL samples. Results: We demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics. Conclusions: Our data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

Mitochondria are organelles present in most eukaryotic cells, where they play major and multifaceted roles. The classical notion of the main mitochondrial function as the powerhouse of the cell per se has been complemented by recent discoveries pointing to mitochondria as organelles affecting a number of other auxiliary processes. They go beyond the classical energy provision via acting as a relay point of many catabolic and anabolic processes, to signaling pathways critically affecting cell growth by their implication in de novo pyrimidine synthesis. These additional roles further underscore the importance of mitochondrial homeostasis in various tissues, where its deregulation promotes a number of pathologies. While it has long been known that mitochondria can move within a cell to sites where they are needed, recent research has uncovered that mitochondria can also move between cells. While this intriguing field of research is only emerging, it is clear that mobilization of mitochondria requires a complex apparatus that critically involves mitochondrial proteins of the Miro family, whose role goes beyond the mitochondrial transfer, as will be covered in this review.

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors derived from neural crest cells that are frequently linked to mutations including those in Krebs cycle enzymes, particularly succinate dehydrogenase (SDH). Succinyl-CoA ligase (SUCL) catalyzes reversible conversion of succinyl-CoA to succinate providing the substrate for SDH. While mitochondrial diseases were documented for the mutations in SUCL subunits G1 and A2, the association of GDP/GTP-specific subunit SUCLG2 mutations with specific pathologies including cancer have not been reported. In our study, 352 patients with apparently sporadic PPGLs underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health. Additionally, human pheochromocytoma (hPheo1) cells were used for gene manipulation to produce SUCLG2 knock-out (KO). Tumor tissues and hPheo1 SUCLG2 KO cells were used for further analysis focusing on mechanism of germline variants effect on mitochondrial functions. We detected eight germline SUCLG2 mutations in 15 patients which represents 4.3% of the cohort. Germline variants together with LOH led to decreased levels of SDH subunit B resulting in aberrant respiration and accumulation of succinate, well recognized oncometabolite. Manipulation of SUCLG2 in hPheo1 cells confirmed decrease in SDHB leading to faulty assembly of mitochondrial complex II and alteration of its respiration and activity. In summary, our study identified an association between SUCLG2 and PPGL. Larger scale sequencing and uncovering additional cases bearing SUCLG2 variants will further clarify the relationship between SUCLG2 and SDHx, particularly SDHB, as well as their role in disease etiology.

Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments., Intracellular trafficking of organelles is driven by kinesin-1 stepping along microtubules, but crowding conditions impede kinesin-1 motility. Here authors demonstrate that TRAK1, an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces.

Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it.We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions ofWe demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethalityOur data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that can trigger apoptosis in many types of human cancer cells via engagement of its two pro-apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). TRAIL can also activate several other signaling pathways such as activation of stress kinases, canonical NF-κB signaling and necroptosis. Though both receptors are ubiquitously expressed, their relative participation in TRAIL-induced signaling is still largely unknown. To analyze TRAIL receptor-specific signaling, we prepared Strep-tagged, trimerized variants of recombinant human TRAIL with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, we examined the contribution of individual pro-apoptotic receptors to TRAIL-induced signaling pathways. We found that in TRAIL-resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeds comparably via both DR4- and DR5-activated signaling. TRAIL-induced apoptosis, enhanced by the inhibitor of the Bcl-2 family ABT-737, or by the translation inhibitor homoharringtonine, proceeded in both cell lines predominantly via the DR5 receptor. ShRNA-mediated downregulation of DR4 or DR5 receptors in HT-29 cells also pointed to a stronger contribution of DR5 in TRAIL-induced apoptosis. In contrast to apoptosis, necroptotic signaling was activated similarly by both DR4- or DR5-specific ligands. Activation of auxiliary signaling pathways involving NF-κB or stress kinases proceeded under apoptotic conditions mainly in a DR5-dependent manner, while these signaling pathways were during necroptosis similarly activated by either of these ligands. Our study provides the first systematic insight into DR4-/DR5-specific signaling in colorectal and pancreatic cancer cells.

Contains fulltext : 202265.pdf (Publisher’s version ) (Closed access) Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis.

The canonical Wnt signaling pathway plays an important role in embryogenesis, and the establishment of neurogenic niches. It is involved in proliferation and differentiation of neural progenitors, since elevated Wnt/β-catenin signaling promotes differentiation of neural stem/progenitor cells (NS/PCs1) towards neuroblasts. Nevertheless, it remains elusive how the differentiation program of neural progenitors is influenced by the Wnt signaling output. Using transgenic mouse models, we found that in vitro activation of Wnt signaling resulted in higher expression of β-catenin protein and Wnt/β-catenin target genes, while Wnt signaling inhibition resulted in the reverse effect. Within differentiated cells, we identified three electrophysiologically and immunocytochemically distinct cell types, whose incidence was markedly affected by the Wnt signaling output. Activation of the pathway suppressed gliogenesis, and promoted differentiation of NS/PCs towards a neuronal phenotype, while its inhibition led to suppressed neurogenesis and increased counts of cells of glial phenotype. Moreover, Wnt signaling hyperactivation resulted in an increased incidence of cells expressing outwardly rectifying K+ currents, together with inwardly rectifying Na+ currents, a typical current pattern of immature neurons, while blocking the pathway led to the opposite effect. Taken together, our data indicate that the Wnt signaling pathway orchestrates neonatal NS/PCs differentiation towards cells with neuronal characteristics, which might be important for nervous tissue regeneration during central nervous system disorders. Furthermore, the transgenic mouse strains used in this study may serve as a convenient tool to manipulate β-catenin-dependent signaling in neural progenitors in the neonatal brain.

Introduction Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2 and good sensitivity to a small molecule BCL2 inhibitor venetoclax. In the present study we analyzed molecular mechanisms of venetoclax resistance in MCL cells, and tested strategies to overcome it based on concurrent targeting of BCL2 a MCL1. Methods Cell death was determined by flow cytometry using Annexin-V/PI staining. Establishment of MCL cell clones with knock-down or transgenic overexpression of MCL1, BIM and NOXA, western blotting, immunohistochemistry of formalin-fixed paraffin-embedded tissue sections, and immunoprecipitation experiments were carried out as previously described (Klanova et al, Clin Cancer Res, 2016). All PDXs were derived in our laboratory from patients with relapsed MCL. All PDX were confirmed by NGS to keep majority of somatic mutations with the primary MCL cells from which they were derived. Samples were sequenced using SureSelectXT Human All Exon V6+UTR (Agilent Technologies, Santa Clara, CA) on the NextSeq 500 (Illumina, San Diego, CA) instrument according to manufacturer's protocols. Experimental therapies were implemented using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Therapy was initiated when all mice developed palpable subcutaneous tumors (= day 1, D1). Venetoclax (VTX) and S63845 were from MedchemExpress, carfilzomib (CFZ) was from Charles University General hospital pharmacy. Carfilzomib (4 mg / kg) was administered intravenously (IV) on days 1 and 6. Venetoclax (40 mg / kg) was given by oral gavage on days 1, 2, 3, 6 and 7. S63845 (25 mg / kg) was administered IV on days 1, 2, 3, 6 and 7. Tumor volumes were calculated using the following formula: π / 6 × tumor length × width × height. Results By transgenic overexpression or shRNA-mediated knock-down we confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. We demonstrated that both BIM and NOXA are differentially expressed between MCL cell lines on one side, and primary MCL cells and patient-derived xenograft (PDX) cells on the other side. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, biallelic deletions of BIM harbored by three commonly used MCL cell lines (JEKO-1, MINO and Z138), and previously reported to be present in approx. 30% of MCL patients, were not found in primary MCL cells. As a consequence, vast majority of the in vitro data was implemented on venetoclax-sensitive cell lines HBL2 and MAVER-1, whose patterns of expression of BCL2, MCL1, BIM and NOXA are similar to primary MCL cells. We demonstrated that MCL1, another key anti-apoptotic protein, plays an essential role in mediating resistance to venetoclax. First, MCL1 functions as a buffer for BIM released from BCL2 upon binding of venetoclax thereby preventing activation of BAX and induction of apoptosis. Second, marked upregulation of MCL1 protein was associated with acquired resistance to venetoclax in two most sensitive MCL cell lines HBL2 and MAVER-1. Based on the in vitro data we proposed two experimental treatment strategies that co-targeted MCL1 (along with inhibition of BCL2 with venetoclax): a direct blockage with a highly specific small molecule MCL1 inhibitor S63845, and an indirect blockage achieved by proteasome inhibitor carfilzomib that upregulates the proapoptotic protein NOXA that specifically binds and blocks MCL1. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo inducing the longest "remissions" of MCL bearing mice (i.e. temporary disappearance of subcutaneous MCL tumors) using a panel of four different PDXs derived from patients with relapsed / refractory MCL with complex karyotype changes (Figure 1). The combination of carfilzomib and venetoclax was far less effective, and at the same time more toxic suggesting functional blockage of MCL1 induced by overexpressed NOXA is either incomplete or insufficient. Conclusions Our data strongly support investigation of venetoclax in combination with S63845 as an innovative proapoptotic treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds. Figure 1 Figure 1. Disclosures Trněný: Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Gilead: Honoraria; Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Abbvie: Honoraria, Research Funding; F. Hoffman-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Sandoz: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board.