TNF Receptor-associated Protein Research Articles

Novel energy optimizer, meldonium, rapidly restores acute hypobaric hypoxia-induced brain injury by targeting phosphoglycerate kinase 1

BackgroundAcute hypobaric hypoxia-induced brain injury has been a challenge in the health management of mountaineers; therefore, new neuroprotective agents are urgently required. Meldonium, a well-known cardioprotective drug, has been reported to have neuroprotective effects. However, the relevant mechanisms have not been elucidated. We hypothesized that meldonium may play a potentially novel role in hypobaric hypoxia cerebral injury.MethodsWe initially evaluated the neuroprotection efficacy of meldonium against acute hypoxia in mice and primary hippocampal neurons. The potential molecular targets of meldonium were screened using drug-target binding Huprot™ microarray chip and mass spectrometry analyses after which they were validated with surface plasmon resonance (SPR), molecular docking, and pull-down assay. The functional effects of such binding were explored through gene knockdown and overexpression.ResultsThe study clearly shows that pretreatment with meldonium rapidly attenuates neuronal pathological damage, cerebral blood flow changes, and mitochondrial damage and its cascade response to oxidative stress injury, thereby improving survival rates in mice brain and primary hippocampal neurons, revealing the remarkable pharmacological efficacy of meldonium in acute high-altitude brain injury. On the one hand, we confirmed that meldonium directly interacts with phosphoglycerate kinase 1 (PGK1) to promote its activity, which improved glycolysis and pyruvate metabolism to promote ATP production. On the other hand, meldonium also ameliorates mitochondrial damage by PGK1 translocating to mitochondria under acute hypoxia to regulate the activity of TNF receptor-associated protein 1 (TRAP1) molecular chaperones.ConclusionThese results further explain the mechanism of meldonium as an energy optimizer and provide a strategy for preventing acute hypobaric hypoxia brain injury at high altitudes.

IFI27 Integrates Succinate and Fatty Acid Oxidation to Promote Adipocyte Thermogenic Adaption.

Mitochondria are the pivot organelles to control metabolism and energy homeostasis. The capacity of mitochondrial metabolic adaptions to cold stress is essential for adipocyte thermogenesis. How brown adipocytes keep mitochondrial fitness upon a challenge of cold-induced oxidative stress has not been well characterized. This manuscript shows that IFI27 plays an important role in cristae morphogenesis, keeping intact succinate dehydrogenase (SDH) function and active fatty acid oxidation to sustain thermogenesis in brown adipocytes. IFI27 protein interaction map identifies SDHB and HADHA as its binding partners. IFI27 physically links SDHB to chaperone TNF receptor associated protein 1 (TRAP1), which shields SDHB from oxidative damage-triggered degradation. Moreover, IFI27 increases hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA) catalytic activity in β-oxidation pathway. The reduced SDH level and fatty acid oxidation in Ifi27-knockout brown fat results in impaired oxygen consumption and defective thermogenesis. Thus, IFI27 is a novel regulator of mitochondrial metabolism and thermogenesis.

CVM-1118 (foslinanib), a 2-phenyl-4-quinolone derivative, promotes apoptosis and inhibits vasculogenic mimicry via targeting TRAP1.

CVM-1118 (foslinanib) is a phosphoric ester compound selected from 2-phenyl-4-quinolone derivatives. The NCI 60 cancer panel screening showed CVM-1125, the major active metabolite of CVM-1118, to exhibit growth inhibitory and cytotoxic effects at nanomolar range. CVM-1118 possesses multiple bioactivities, including inducing cellular apoptosis, cell cycle arrest at G2/M, as well as inhibiting vasculogenic mimicry (VM) formation. The TNF receptor associated protein 1 (TRAP1) was identified as the binding target of CVM-1125 using nematic protein organization technique (NPOT) interactome analysis. Further studies demonstrated CVM-1125 reduced the protein level of TRAP1 and impeded its downstream signaling by reduction of cellular succinate levels and destabilization of HIF-1α. The pharmacogenomic biomarkers associated with CVM-1118 were also examined by Whole Genome CRISPR Knock-Out Screening. Two hits (STK11 and NF2) were confirmed with higher sensitivity to the drug in cell knock-down experiments. Biological assays indicate that the mechanism of action of CVM-1118 is via targeting TRAP1 to induce mitochondrial apoptosis, suppress tumor cell growth, and inhibit vasculogenic mimicry formation. Most importantly, the loss-of-function mutations of STK11 and NF2 are potential biomarkers of CVM-1118 which can be applied in the selection of cancer patients for CVM-1118 treatment. CVM-1118 is currently in its Phase 2a clinical development.

Histone chaperone SSRP1 is required for apoptosis inhibition and mitochondrial function in HCC via transcriptional promotion of TRAP1.

Epigenetic regulation contributes to human health and disease, especially cancer, but the mechanisms of many epigenetic regulators remain obscure. Most research is focused on gene regulatory processes, such as mRNA translation and DNA damage repair, rather than the effects on biological functions like mitochondrial activity and oxidative phosphorylation. Here, we identified an essential role for the histone chaperone structure-specific recognition protein 1 (SSRP1) in mitochondrial oxidative respiration in hepatocellular carcinoma, and found that SSRP1 suppression led to mitochondrial damage and decreased oxidative respiration. Further, we focused on TNF receptor-associated protein 1 (TRAP1), the only member of the heat shock protein 90 (HSP90) family, which directly interacts with selected respiratory complexes and affects their stability and activity. We confirmed that SSRP1 downregulation caused a decrease in TRAP1 expression at both the mRNA and protein levels. A chromatin immunoprecipitation assay also showed that SSRP1 could deposit in the TRAP1 promoter region, indicating that SSRP1 maintains mitochondrial function and reactive oxygen species levels through TRAP1. Additionally, rescue experiments and animal experiments confirmed the mechanism of SSRP1 and TRAP1 interaction. In summary, we identified a new mechanism that connects mitochondrial respiration and apoptosis, via SSRP1.

O-GlcNAcylation suppresses TRAP1 activity and promotes mitochondrial respiration.

The molecular chaperone TNF-receptor-associated protein-1 (TRAP1) controls mitochondrial respiration through regulation of Krebs cycle and electron transport chain activity. Post-translational modification (PTM) of TRAP1 regulates its activity, thereby controlling global metabolic flux. O-GlcNAcylation is one PTM that is known to impact mitochondrial metabolism, however the major effectors of this regulatory PTM remain inadequately resolved. Here we demonstrate that TRAP1-O-GlcNAcylation decreases TRAP1 ATPase activity, leading to increased mitochondrial metabolism. O-GlcNAcylation of TRAP1 occurs following mitochondrial import and provides critical regulatory feedback, as the impact of O-GlcNAcylation on mitochondrial metabolism shows TRAP1-dependence. Mechanistically, loss of TRAP1-O-GlcNAcylation decreased TRAP1 binding to ATP, and interaction with its client protein succinate dehydrogenase (SDHB). Taken together, TRAP1-O-GlcNAcylation serves to regulate mitochondrial metabolism by the reversible attenuation of TRAP1 chaperone activity.

TRAP1 Chaperones the Metabolic Switch in Cancer.

Mitochondrial function is dependent on molecular chaperones, primarily due to their necessity in the formation of respiratory complexes and clearance of misfolded proteins. Heat shock proteins (Hsps) are a subset of molecular chaperones that function in all subcellular compartments, both constitutively and in response to stress. The Hsp90 chaperone TNF-receptor-associated protein-1 (TRAP1) is primarily localized to the mitochondria and controls both cellular metabolic reprogramming and mitochondrial apoptosis. TRAP1 upregulation facilitates the growth and progression of many cancers by promoting glycolytic metabolism and antagonizing the mitochondrial permeability transition that precedes multiple cell death pathways. TRAP1 attenuation induces apoptosis in cellular models of cancer, identifying TRAP1 as a potential therapeutic target in cancer. Similar to cytosolic Hsp90 proteins, TRAP1 is also subject to post-translational modifications (PTM) that regulate its function and mediate its impact on downstream effectors, or ‘clients’. However, few effectors have been identified to date. Here, we will discuss the consequence of TRAP1 deregulation in cancer and the impact of post-translational modification on the known functions of TRAP1.

TRAP1 regulates the response of colorectal cancer cells to hypoxia and inhibits ribosome biogenesis under conditions of oxygen deprivation.

Metabolic rewiring fuels rapid cancer cell proliferation by promoting adjustments in energetic resources, and increasing glucose uptake and its conversion into lactate, even in the presence of oxygen. Furthermore, solid tumors often contain hypoxic areas and can rapidly adapt to low oxygen conditions by activating hypoxia inducible factor (HIF)-1α and several downstream pathways, thus sustaining cell survival and metabolic reprogramming. Since TNF receptor-associated protein 1 (TRAP1) is a HSP90 molecular chaperone upregulated in several human malignancies and is involved in cancer cell adaptation to unfavorable environments and metabolic reprogramming, in the present study, its role was investigated in the adaptive response to hypoxia in human colorectal cancer (CRC) cells and organoids. In the present study, glucose uptake, lactate production and the expression of key metabolic genes were evaluated in TRAP1-silenced CRC cell models under conditions of hypoxia/normoxia. Whole genome gene expression profiling was performed in TRAP1-silenced HCT116 cells exposed to hypoxia to establish the role of TRAP1 in adaptive responses to oxygen deprivation. The results revealed that TRAP1 was involved in regulating hypoxia-induced HIF-1α stabilization and glycolytic metabolism and that glucose transporter 1 expression, glucose uptake and lactate production were partially impaired in TRAP1-silenced CRC cells under hypoxic conditions. At the transcriptional level, the gene expression reprogramming of cancer cells driven by HIF-1α was partially inhibited in TRAP1-silenced CRC cells and organoids exposed to hypoxia. Moreover, Gene Set Enrichment Analysis of TRAP1-silenced HCT116 cells exposed to hypoxia demonstrated that TRAP1 was involved in the regulation of ribosome biogenesis and this occurred with the inhibition of the mTOR pathway. Therefore, as demonstrated herein, TRAP1 is a key factor in maintaining HIF-1α-induced genetic/metabolic program under hypoxic conditions and may represent a promising target for novel metabolic therapies.

Increased Post-Hypoxic Oxidative Stress and Activation of the PERK Branch of the UPR in Trap1-Deficient Drosophila melanogaster Is Abrogated by Metformin

Hypoxia is known to impair mitochondrial and endoplasmic reticulum (ER) homeostasis. Post-hypoxic perturbations of the ER proteostasis result in the accumulation of misfolded/unfolded proteins leading to the activation of the Unfolded Protein Response (UPR). Mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) is reported to preserve mitochondrial membrane potential and to impede reactive oxygen species (ROS) production thereby protecting cells from ER stress as well as oxidative stress. The first-line antidiabetic drug Metformin has been attributed a neuroprotective role after hypoxia. Interestingly, Metformin has been reported to rescue mitochondrial deficits in fibroblasts derived from a patient carrying a homozygous TRAP1 loss-of-function mutation. We sought to investigate a putative link between Metformin, TRAP1, and the UPR after hypoxia. We assessed post-hypoxic/reperfusion longevity, mortality, negative geotaxis, ROS production, metabolic activity, gene expression of antioxidant proteins, and activation of the UPR in Trap1-deficient flies. Following hypoxia, Trap1 deficiency caused higher mortality and greater impairments in negative geotaxis compared to controls. Similarly, post-hypoxic production of ROS and UPR activation was significantly higher in Trap1-deficient compared to control flies. Metformin counteracted the deleterious effects of hypoxia in Trap1-deficient flies but had no protective effect in wild-type flies. We provide evidence that TRAP1 is crucially involved in the post-hypoxic regulation of mitochondrial/ER stress and the activation of the UPR. Metformin appears to rescue Trap1-deficiency after hypoxia mitigating ROS production and downregulating the pro-apoptotic PERK (protein kinase R-like ER kinase) arm of the UPR.

Suppressing TRAP1 sensitizes glioblastoma multiforme cells to temozolomide.

Glioma is a common malignant tumor of the central nervous system, accounting for ~50% of intracranial tumors. The current standard therapy for glioma is surgical resection followed by postoperative adjuvant radiotherapy and temozolomide (TMZ) chemotherapy. However, resistance to TMZ is one of the factors affecting prognosis. It has been reported that TNF receptor-associated protein 1 (TRAP1) is overexpressed in numerous types of tumor and that interfering with its function may abrogate chemotherapy resistance. TRAP1 inhibitor Gamitrinib triphenylphosphonium (G-TPP) and shRNA were used in the present study to suppress the function of this molecule in glioblastoma multiforme (GBM) cell lines. MTT assay was performed to evaluate the combined effect of G-TPP and TMZ treatment. To investigate the underlying mechanism responsible for this combined effect, the mitochondrial unfolded protein response (mtUPR), mitophagy, mitochondrial fusion and reactive oxygen species (ROS) were quantified using western blotting and immunofluorescence techniques. TMZ treatment induced apoptosis in GBM cells by activating the p53 pathway, whilst simultaneously downregulating mitophagy and enhancing mitochondrial fusion. The latter may occur in order to compensate for the defect caused by downregulated mitophagy. Suppressing the function of TRAP1 disturbed this compensatory mechanism by inducing mtUPR, which resulted in a burst of ROS formation and sensitized the GBM cells to the effects of TMZ treatment. Thus, suppressing the function of TRAP1 sensitized GBM cells to TMZ lysis by inducing mtUPR and the subsequent ROS burst. TRAP1 is therefore considered to be a promising target for GBM therapy.

Novel Mechanisms Underlying IMiDs Resistance in Multiple Myeloma: Therapeutic Implications

IntroductionImmunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind to the CRL4CRBN ubiquitin ligase and trigger proteasomal degradation of IKZF1/3 and cytotoxicity of multiple myeloma (MM) cells. Previous studies show that downregulation of CRL4CRBN abrogates sensitivity to IMiDs in MM cell lines. Clinical studies, show that IMiDs achieve better clinical responses in MM patients when combined with proteasome inhibitor bortezomib, which would in principle inhibit proteasomal degradation of IMiDs-targeted proteins. Moreover, some MM cells are resistant to IMiDs treatment despite harboring high CRBN expression levels. Thus, the molecular mechanisms of intrinsic resistance to IMiDs are not yet fully understood. In this study, we found that dual roles of IMiDs in activating CRL4CRBN and a mechanism of action for combination therapy of IMiDs with bortezomib. We also show that non-canonical NF-κB pathway, independent of CRBN, is involved in the IMiDs resistance in MM.Methods and ResultsTo study the molecular mechanisms underlying IMiDs resistance, we first performed genome-wide knockout screening in IMiDs-sensitive MM.1S cells, using a CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 miRNAs. Twenty-eight genes and one miRNA were identified which were associated with resistance to IMiDs. All six sgRNAs targeting CRBN were also identified, consistent with CRBN as the direct target of IMiDs and required for their MM cytotoxicity. Among these validated genes, 7 of 9 components of the COP signalosome (CSN) subunits were found to be required for MM cell sensitivity to IMiDs. Deletion of any of these CSN subunits in MM cells led to a dramatic decrease of CRBN protein level thereby inhibiting IKZF1/3 degradation induced by IMiDs.To investigate whether the CSN regulates CRBN level through the ubiquitin-proteasome pathway, we carried out ubiquitination assays and found that CRBN can be degraded by proteasomes via cullin E3, rather than CRL4, ubiquitin ligase. Moreover, we identified the interaction of CRBN with SCFFbxo7 complex, and demonstrated that CRBN interacted with both CRL4 and SCFFbxo7 with distinct domains. To determine if CRBN is a SCFFbxo7 substrate, we examined the ubiquitination and degradation of CRBN after altering SCFFbxo7 complex. CRBN, when unbound to CRL4, is ubiquitinated by SCFFbxo7, followed by proteasomal degradation. Importantly, deletion of Cul1 or Fbxo7 completely restored the CRBN protein in CSN2 or CSN6 knockout MM cells to levels found in wild type cells, as well as re-sensitized these CSN mutant MM cells to pomalidomide-induced cytotoxicity.Since CRBN protein turnover is controlled by a SCF ubiquitin ligase, we next tested whether proteasome inhibitor, in addition to their inherent anti-MM activities, might similarly enhance IMiDs-induced MM cell cytotoxicity. As expected, sequential treatment with bortezomib and pomalidomide primed MM cells to pomalidomide-induced downregulation of IKZF3 and cell death, associated with synergistic cytotoxicity. We also found that lenalidomide potently inhibited the ubiquitination of CRBN, which was abrogated by loss of lenalidomide binding on CRBN, downregulation of Fbxo7, or Cul1 expression. These data suggest that IMiDs stabilize the CRBN protein both by shifting its assembly with the SCF and CRL4 complexes and by directly blocking its ubiquitination and degradation targeted by SCFFbxo7.From the genome-scale CRISPR-Cas9 knockout screening, we also found that knock-out of TRAF2, a member of the TNF receptor associated factor protein family, conferred resistance of MM cells to IMiDs treatment, without effecting CRBN level and degradation of IKZF1/3 induced by IMiDs. TRAF2 -KO MM cells displayed increased processing of p100 NF-κB2 protein to p52, resulting in hyperactivation of the non-canonical NF-κB pathway. This finding suggests that the non-canonical NF-kB pathway may be involved in the intrinsic resistance of MM to IMiDs.ConclusionsTaken together, our findings therefore identify novel mechanisms of IMiDs resistance, involving the regulation of CRBN and the non-canonical NF-κB pathway, and provide a mechanistic framework to inform combination therapy strategies to overcome IMiDs resistance in MM. DisclosuresZhou:WuXi AppTec Inc.: Employment. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Anderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees.

CD40 intracellular TRAF binding domains modulate B cell activation and experimental autoimmune encephalomyelitis development

Abstract CD40 is a costimulatory surface protein expressed on multiple immune cells, including B cells, where it is required for their full activation. Upon interacting with CD40 ligand, CD40 initiates downstream signaling that culminates in activation of naïve B cells. Previous work from our lab revealed that cell autonomous expression of CD40 on B cells regulates their activity, including germinal center development and antibody class switching. Intracellular (IC) domains of the CD40 protein are crucial for signaling. Several of these domains are known to be associated with TNF receptor associated factor proteins (TRAFs) which facilitate further downstream signaling. These include a TRAF6 binding domain and a TRAF2/3 binding domain. We analyzed the CD40 IC domain requirements for CD40 signaling in B cells and for induction of experimental autoimmune encephalomyelitis (EAE). To do so, multiple mouse strains were generated with elimination of the putative TRAF6 or TRAF2/3 domains by CRISPR technology. Whereas eliminating the putative TRAF6 binding site (Domain 1) had little to no effect on B cell viability and in vitro activation by anti-CD40 antibody, eliminating the putative TRAF2/3 binding site (Domain 2) impaired both these functions. Similarly, generation of high affinity IgG in vivo was also dependent on Domain 2 but not Domain 1. In contrast, both Domain 1 and Domain 2 were required for full development of EAE. Interestingly, mixed BM chimeras revealed that the ameliorating effect of CD40 mutations on EAE was mediated by hematopoietic derived non-B cells. Our results indicate that the CD40 TRAF2/3 binding Domain 2 is critical for CD40 dependent B cell function, while both Domain 1 and Domain 2 expression by other APCs function in EAE induction.

Survival and transcriptomic responses to different Perkinsus marinus exposure methods in an Eastern oyster family

The parasite Perkinsus marinus, which causes Dermo disease, affects Eastern oyster (Crassostrea virginica) survival and is responsible for substantial losses to aquaculture production. Controlled laboratory challenges enable definition and measurement of disease response phenotypes as well as characterization of gene expression patterns during the infection process. The method of parasite exposure and severity of infection likely affect response measures and exploring variation among them can enhance our understanding of this host-parasite interaction. Here we compared response traits and global expression patterns over time for a selectively bred Eastern oyster family exposed to P. marinus via either suspension feeding or direct injection to elucidate mechanisms determining survival. All exposed oysters (n = 56 per group) received a dose of 5 × 106 cultured P. marinus cells g−1 wet tissue weight but oysters in the injected group had higher initial parasite load and significantly reduced survival probability compared to the fed group. Using RNAseq, we identified differentially expressed transcripts between fed and control animals 6 h after exposure and between fed and injected groups at 36 h, 7 d, and 28 d post exposure. Immediately after infection, caspase-8 and transcripts associated with mediating cell damage from reactive oxygen species were upregulated in the fed group compared to unchallenged controls. The comparison between fed and injected groups early in the infection process revealed increased innate immune activity and overexpression of transcripts involved in proinflammatory cytokine pathways, including toll-like receptor 4 and TNF receptor-associated factor 6-like protein, in the injected group. Later in the infection process, the fed group expressed transcripts associated with growth, specifically microtubule-based processes, at a higher level than the injected group. This study demonstrates that dosing method influences initial parasite load, survival, and transcriptomic response to Dermo disease and highlights the importance of considering exposure method when interpreting interactions between P. marinus and the Eastern oyster.

Roles of TNF receptor-associated and Fas-associated death domain proteins in the apoptosis of Eimeria tenella host cells

The present study aimed to investigate the effects of death receptor adapter proteins, namely, TNF receptor-associated death domain (TRADD) and Fas-associated death domain (FADD) proteins, on Eimeria tenella-induced host cell apoptosis. Gene silencing, culture technique for primary chick embryo cecal epithelial cells, enzyme-linked immunosorbent assay, Hoechst–Annexin V/PI apoptosis staining, fluorescence quantitative PCR, and flow cytometry were used to detect the E. tenella host cell apoptotic rate, RIP1 and FADD protein expression levels, and caspase-8 activity of the TRADD siRNA-treated and FADD siRNA-treated groups. Results showed that the apoptotic rate in the TRADD siRNA group was significantly higher than that in the NC siRNA group at 4 h post-infection with E. tenella (P < 0.05). The RIP1 protein expression level in the TRADD siRNA group was significantly lower than that in the NC siRNA group at 4–24 h (P < 0.05). The FADD expression and apoptotic rates in the TRADD siRNA group were significantly lower than those in the NC siRNA group at 24–120 h (P < 0.05). The caspase-8 activity and apoptotic rates in the FADD siRNA group were significantly lower than those in the NC siRNA group (P < 0.05) at 24–120 h. These findings indicated that E. tenella inhibited the host cell apoptosis through the TRADD-RIP1 pathway at the early developmental stage and promoted host cell apoptosis via the TRADD-FADD-caspase-8 apoptotic pathway at the middle and late developmental stages.

TRAF2 Mediates Sensitivity to Immunomodulatory Drugs in the Bone Marrow Microenvironment

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has improved patient outcome in multiple myeloma (MM). Previous studies have shown that IMiDs directly bind cereblon (CRBN), a substrate adaptor of Cullin4 Ring Ligase (CRL4), and activate CRL4CRBN ligase, thereby selectively targeting two B cell transcription factors IKZF1 and IKZF3 for ubiquitylation and proteasomal degradation. We have shown that IMiDs also directly bind and inhibit TP53-regulating kinase activity, resulting in MM cell growth inhibition. Importantly, IMiDs have achieved additive and synergistic anti-MM activity when combined with proteasome inhibitors and monoclonal antibodies in vitro, and are now utilized in combinations to treat newly diagnosed MM. However, development of resistance to IMiDs commonly underlies relapse of disease. To delineate mechanisms of IMiDs resistance, the majority of previous studies have focused on CRBN. Although downregulation or mutations in CRBN can be associated with IMiDs resistance, MM cells can manifest resistance without any CRBN dysfunction, indicating alternative mechanisms of IMiDs resistance. We and others have shown a critical role of the BM microenvironment in MM pathogenesis: secretion of soluble factors from cellular components activates intracellular signaling pathways that promote MM cell migration, proliferation, survival, and drug resistance. To date, however, the mechanisms whether tumor microenvironment mediates IMiDs resistance have not been fully delineated. In our genome-wide CRISPR-Cas9 knockout screening, we have validated that CSN9 signalosome complex regulates sensitivity to IMiDs by modulating CRBN expression. Our screen also identified TRAF2, a member of the TNF receptor associated factor protein family, to regulate IMiDs sensitivity. To confirm modulation of IMiDs sensitivity by TRAF2, we here individually cloned TRAF2 sgRNAs into LentiCRISPRv2 vector, and then re-introduced them into MM cells. As expected, TRAF2-KO MM cells acquired significant resistance to Pom and Len treatment. Importantly, TRAF2 KO showed no effect on CRBN expression; moreover, IMiDs treatment of TRAF2 KO MM cells still triggered IKZF1 and IKZF3 degradation associated with downregulation of IRF4, the main effector of MM cell survival. Importantly, we observed that TRAF2 KO MM cells expressed high levels of p-ERK. We and others have shown that the bone marrow (BM) microenvironment activates ERK signaling pathway and plays a crucial role in drug resistance, and here showed that co-culture of MM cells with BM stromal cells (BMSCs) or culture supernatants (SC-sup) confers resistance to IMiDs, associated with downregulation of TRAF2. These results suggest that IMiDs resistance in the BM microenvironment may be mediated by ERK signaling pathway induced by soluble factors. AZD6244 is a potent and highly selective MEK inhibitor, and we next investigated whether AZD6244 inhibits ERK1/2 activation and overcome IMiDs resistance in TRAF2 KO MM cells. Importantly, addition of AZD6244 overcame resistance to IMiDs in both TRAF2 KO MM cells and SC-sup treated TRAF2 wild-type (WT) cells. In our human MM murine xenograft model, we observed that TRAF2 WT MM cells were sensitive to Pom treatment in vivo, while TRAF2 inducible KD MM cells demonstrated resistance. Importantly, the combination of AZD6244 and Pom significantly reduced growth even of TRAF2 KD MM cells, indicating that MEK inhibitor can inhibit activation of ERK1/2 and overcome IMiDs resistance in vivo. Finally, we confirmed that the number of patient samples with activated ERK pathway was significantly enriched at relapse while on lenalidomide maintenance therapy compared to diagnosis (BIOCARTA ERK pathway analysis 67% vs 39%, respectively, FDR&amp;lt;0.05), implicating ERK activity in clinical resistance to IMiDs. In summary, we have identified and validated TRAF2 as a CRBN-IKZF1/3 axis-independent regulator of BM microenvironment-mediated sensitivity to IMiDs. These studies not only identify a novel mechanism of IMiDs resistance in the tumor microenvironment, but also provide the preclinical rationale for combining inhibitors of MEK/ERK signaling with IMiDs to overcome IMiDs resistance and improve patient outcome. Disclosures Munshi: Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; C4: Current equity holder in private company. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Anderson:Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Honokiol Bis-Dichloroacetate Is a Selective Allosteric Inhibitor of the Mitochondrial Chaperone TRAP1.

Aims: TNF receptor-associated protein 1 (TRAP1), the mitochondrial paralog of the heat shock protein 90 (Hsp90) family of molecular chaperones, is required for neoplastic growth in several tumor cell models, where it inhibits succinate dehydrogenase (SDH) activity, thus favoring bioenergetic rewiring, maintenance of redox homeostasis, and orchestration of a hypoxia-inducible factor 1-alpha (HIF1α)-mediated pseudohypoxic program. Development of selective TRAP1 inhibitors is instrumental for targeted development of antineoplastic drugs, but it has been hampered up to now by the high degree of homology among catalytic pockets of Hsp90 family members. The vegetal derivative honokiol and its lipophilic bis-dichloroacetate ester, honokiol DCA (HDCA), are small-molecule compounds with antineoplastic activity. HDCA leads to oxidative stress and apoptosis in in vivo tumor models and displays an action that is functionally opposed to that of TRAP1, as it induces both SDH and the mitochondrial deacetylase sirtuin-3 (SIRT3), which further enhances SDH activity. We investigated whether HDCA could interact with TRAP1, inhibiting its chaperone function, and the effects of HDCA on tumor cells harboring TRAP1. Results: An allosteric binding site in TRAP1 is able to host HDCA, which inhibits TRAP1 but not Hsp90 ATPase activity. In neoplastic cells, HDCA reverts TRAP1-dependent downregulation of SDH, decreases proliferation rate, increases mitochondrial superoxide levels, and abolishes tumorigenic growth. Innovation: HDCA is a potential lead compound for the generation of antineoplastic approaches based on the allosteric inhibition of TRAP1 chaperone activity. Conclusions: We have identified a selective TRAP1 inhibitor that can be used to better dissect TRAP1 biochemical functions and to tailor novel tumor-targeting strategies.

Development of pyrazolo[3,4-d]pyrimidine-6-amine-based TRAP1 inhibitors that demonstrate in vivo anticancer activity in mouse xenograft models.

TNF Receptor Associated Protein 1 (TRAP1) is a mitochondrial paralog of Hsp90 related to the promotion of tumorigenesis in various cancers via maintaining mitochondrial integrity, reducing the production of reactive oxygen species, and reprogramming cellular metabolism. Consequently, Hsp90 and TRAP1 have been targeted to develop cancer therapeutics. Herein, we report a series of pyrazolo[3,4-d]pyrimidine derivatives that are mitochondria-permeable TRAP1 inhibitors. Structure-based drug design guided the optimization of potency, leading to the identification of compounds 47 and 48 as potent TRAP1 and Hsp90 inhibitors with good metabolic and plasma stability as well as acceptable CYP and hERG inhibition. X-ray co-crystallization studies confirmed both 47 and 48 interact with the ATP binding pocket in the TRAP1 protein. Compounds 47 and 48 demonstrated excellent anticancer efficiency in various cancer cells, with limited toxicity over normal hepatocyte and prostate cells. Mouse PC3 xenograft studies showed 47 and 48 significantly reduced tumor growth.

Improvement of Psoriasis by Alteration of the Gut Environment by Oral Administration of Fucoidan from Cladosiphon Okamuranus.

Psoriasis is a chronic autoimmune inflammatory disease for which there is no cure; it results in skin lesions and has a strong negative impact on patients’ quality of life. Fucoidan from Cladosiphon okamuranus is a dietary seaweed fiber with immunostimulatory effects. The present study reports that the administration of fucoidan provided symptomatic relief of facial itching and altered the gut environment in the TNF receptor-associated factor 3-interacting protein 2 (Traf3ip2) mutant mice (m-Traf3ip2 mice); the Traf3ip2 mutation was responsible for psoriasis in the mouse model used in this study. A fucoidan diet ameliorated symptoms of psoriasis and decreased facial scratching. In fecal microbiota analysis, the fucoidan diet drastically altered the presence of major intestinal opportunistic microbiota. At the same time, the fucoidan diet increased mucin volume in ileum and feces, and IgA contents in cecum. These results suggest that dietary fucoidan may play a significant role in the prevention of dysfunctional immune diseases by improving the intestinal environment and increasing the production of substances that protect the immune system.

HSPA1A, HSPA1L and TRAP1 heat shock genes may be associated with prognosis in ovarian epithelial cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with the presence of chemoresistance contributing to the poor prognosis. Heat Shock Proteins (HSPs) genes are activated in response to pathophysiological stress and serve a role in a variety of stages in carcinogenesis, acting primarily as anti-apoptotic agents and in chemotherapy resistance in a variety of tumor types. The current study evaluated the HSP gene expression profile in women with ovarian cancer (OC) and their correlation with clinical and pathological aspects of patients with OC. A total of 51 patients included in the current study were divided into four groups: Primary Epithelial Ovarian Cancer (EOC; n=14), metastatic EOC (n=11), ovarian serous cystadenoma (n=7) and no evidence of ovarian malignancy or control groups (n=19). RNA extraction and reverse transcription-quantitative (RT-q) PCR was then performed on the samples obtained. RT-qPCR was performed to compare TNF receptor associated protein 1 (TRAP1), heat shock protein family (HSP) HSPB1, HSPD1, HSPA1A and HSPA1L expression in primary and metastatic EOCs. TRAP1, HSPB1, HSPD1, HSPA1A and HSPA1L gene expression did not differ among groups. HSPA1A, HSPA1L and TRAP1 were revealed to be underexpressed in the primary and metastatic EOC groups, with HSPA1L exhibiting the lowest expression. TRAP1 expression was higher in tumors at stages I/II compared with those at stages III/IV. No correlation was exhibited between HSP expression and age, menarche, menopause, parity, period after menopause initiation, cytoreduction, CA-125 or overall and disease-free survival. HSPA1A was negatively correlated with the risk of mortality from OC. The results indicated that the downregulation of HSPA1A, HSPA1L and TRAP1 could be associated with the clinical prognostic features of women with EOC.

Selection of Reference Genes for Normalization of Gene Expression Data in Blood of Machado-Joseph Disease/Spinocerebellar Ataxia Type 3 (MJD/SCA3) Subjects.

Alongside with the emergent clinical trials for Machado-Joseph disease/Spinocerebellar ataxia type 3 (MJD/SCA3) comes the need to identify molecular biomarkers of disease that can be tracked throughout the trial. MJD is an autosomal dominant neurodegenerative disorder caused by expansion of a CAG repeat in the coding region of the ATXN3 gene. Previous findings indicate the potential of transcriptional alterations in blood of MJD patients as biomarkers of disease. Accurate quantification of gene expression levels by quantitative real-time PCR (qPCR) depends on data normalization, usually performed using reference genes. Because the expression level of routinely used housekeeping genes may vary in multiple biological and experimental conditions, reference gene controls should be validated in each specific experimental design. Here, we aimed to evaluate the expression behavior of five housekeeping genes previously reported as stably expressed in peripheral blood of patients from several disorders-peptidylprolyl isomerase B (PPIB), TNF receptor associated protein 1 (TRAP1), beta-2-microglobulin (B2M), 2,4-dienoyl-CoA reductase 1 (DECR1), and folylpolyglutamate synthase (FPGS). Expression levels of these five genes were assessed by qPCR in blood from MJD subjects (preataxic and patients) and matched controls. While all housekeeping genes, here studied, were stably expressed in our sets of samples, TRAP1 showed to be the most stable gene by NormFinder and BestKeeper. We, therefore, conclude that any of these genes could be used as reference gene in future qPCR studies using blood samples from MJD subjects.

Association of TRAP1 with infliximab-induced mucosal healing in Crohn's disease.

Anti-tumor necrosis factor (TNF) agents, such as infliximab (IFX), have been increasingly used to induce and maintain disease remission in patients with Crohn's disease (CD). Despite a considerable non-response rate, little is known about the genetic predictors of response to anti-TNF therapy in CD. Our aim in this study was to investigate the genetic factors associated with response to anti-TNF therapy in patients with CD. We performed a two-stage genome-wide association study (GWAS) to identify loci influencing the response to IFX among Korean patients with CD, comprising 42 good responders with mucosal healing and 70 non-responders. The achievement of mucosal healing was assessed by endoscopy and imaging. The functional significance of TRAP1 (TNF receptor associated protein 1) was examined using dextran sodium sulfate-induced colitis model in TRAP1 transgenic mice. The GWAS identified rs2158962, an intronic single nucleotide polymorphism (SNP) of TRAP1, significantly associated with mucosal healing (odds ratio=4.94; Pcombined =1.35×10-7 ). In the dextran sodium sulfate-induced acute colitis, TRAP1 transgenic mice showed a better response to IFX than the wild-type mice. The TRAP1 gene is associated with mucosal healing in CD patients following IFX therapy. Identifying the genetic predictors of mucosal healing to anti-TNF therapy can prevent patients from exposure to ineffective therapies.