HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity.

Proteasomes degrade intracellular proteins to generate antigenic peptides that are recognized by the adaptive immune system and promote anticancer immunity. However, tumors subvert the antigen presentation machinery to escape immunosurveillance. We hypothesized that proteasome activation could concomitantly increase antigen abundance and diversity in multiple myeloma cells. High-throughput screens revealed that histone deacetylase 6 (HDAC6) inhibitors activated proteasomes to unmask neoantigens and amplify the tumor-specific antigenic landscape. Treatment of patient CD138+ cells with HDAC6 inhibitors significantly promoted the antimyeloma activity of autologous CD8+ T cells. Pharmacologic blockade and genetic ablation of the HDAC6 ubiquitin-binding domain released HR23B, which shuttles ubiquitinylated cargo to proteasomes, while silencing HDAC6 or HR23B in multiple myeloma cells abolished the effect of HDAC6 inhibitors on proteasomes, antigen presentation, and T-cell cytotoxicity. Taken together, our results demonstrate the paradigm-shifting translational impact of proteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets for T cell-directed immunotherapy. SIGNIFICANCE: The elimination of therapy-resistant tumor cells remains a major challenge in the treatment of multiple myeloma. Our study identifies and functionally validates agents that amplify MHC class I-presented antigens and pave the way for the development of proteasome activators as immune adjuvants to enhance immunotherapeutic responses in patients with multiple myeloma.

Piezo1 facilitates optimal T cell activation during tumor challenge.

Functional effector T cells in the tumor microenvironment (TME) are critical for successful anti-tumor responses. T cell anti-tumor function is dependent on their ability to differentiate from a naïve state, infiltrate into the tumor site, and exert cytotoxic functions. The factors dictating whether a particular T cell can successfully undergo these processes during tumor challenge are not yet completely understood. Piezo1 is a mechanosensitive cation channel with high expression on both CD4+ and CD8+ T cells. Previous studies have demonstrated that Piezo1 optimizes T cell activation and restrains the CD4+ regulatory T cell (Treg) pool in vitro and under inflammatory conditions in vivo. However, little is known about the role Piezo1 plays on CD4+ and CD8+ T cells in cancer. We hypothesized that disruption of Piezo1 on T cells impairs anti-tumor immunity in vivo by hindering inflammatory T cell responses. We challenged mice with T cell Piezo1 deletion (P1KO) with tumor models dependent on T cells for immune rejection. P1KO mice had the more aggressive tumors, higher tumor growth rates and were unresponsive to immune-mediated therapeutic interventions. We observed a decreased CD4:CD8 ratio in both the secondary lymphoid organs and TME of P1KO mice that correlated inversely with tumor size. Poor CD4+ helper T cell responses underpinned the immunodeficient phenotype of P1KO mice. Wild type CD8+ T cells are sub-optimally activated in vivo with P1KO CD4+ T cells, taking on a CD25loPD-1hi phenotype. Together, our results suggest that Piezo1 optimizes T cell activation in the context of a tumor response.

Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination.

Peptidylarginine deiminase (PADI) 2 catalyzes the post-translational conversion of peptidyl-arginine to peptidyl-citrulline in a process called citrullination. However, the precise functions of PADI2 in bone formation and homeostasis remain unknown. In this study, our objective was to elucidate the function and regulatory mechanisms of PADI2 in bone formation employing global and osteoblast-specific Padi2 knockout mice. Our findings demonstrate that Padi2 deficiency leads to the loss of bone mass and results in a cleidocranial dysplasia (CCD) phenotype with delayed calvarial ossification and clavicular hypoplasia, due to impaired osteoblast differentiation. Mechanistically, Padi2 depletion significantly reduces RUNX2 levels, as PADI2-dependent stabilization of RUNX2 protected it from ubiquitin-proteasomal degradation. Furthermore, we discovered that PADI2 binds to RUNX2 and citrullinates it, and identified ten PADI2-induced citrullination sites on RUNX2 through high-resolution LC-MS/MS analysis. Among these ten citrullination sites, the R381 mutation in mouse RUNX2 isoform 1 considerably reduces RUNX2 levels, underscoring the critical role of citrullination at this residue in maintaining RUNX2 protein stability. In conclusion, these results indicate that PADI2 plays a distinct role in bone formation and osteoblast differentiation by safeguarding RUNX2 against proteasomal degradation. In addition, we demonstrate that the loss-of-function of PADI2 is associated with CCD, thereby providing a new target for the treatment of bone diseases.

Association of Early Hysterectomy With Risk of Cardiovascular Disease in Korean Women.

Importance: Women who undergo surgical hysterectomy before natural menopause may have an earlier increase in hematocrit and storage iron levels than those who continue menstruation, thereby increasing the risk of cardiovascular disease (CVD) at ages younger than usually seen. Examining this issue may provide important implications for women's cardiovascular health to both physicians and patients. Objective: To evaluate the association of hysterectomy with the risk of incident CVD among women before age 50 years. Design, Setting, and Participants: In this Korean population-based cohort study, 135 575 women aged 40 to 49 years were evaluated from January 1, 2011, to December 31, 2014. After propensity score matching in covariates including age, socioeconomic status, region, Charlson Comorbidity Index, hypertension, diabetes, dyslipidemia, menopause, menopausal hormone therapy, and adnexal surgery before inclusion, 55 539 pairs were included in the hysterectomy and nonhysterectomy groups. Participants were followed up until December 31, 2020. Data analysis was conducted from December 20, 2021, to February 17, 2022. Main Outcomes and Measures: The primary outcome was an incidental CVD, a composite of myocardial infarction, coronary artery revascularization, and stroke. The individual components of the primary outcome were also evaluated. Results: A total of 55 539 pairs were included; median age in the combined groups was 45 (IQR, 42-47) years. During median follow-up periods in the hysterectomy group of 7.9 (IQR, 6.8-8.9) years and nonhysterectomy group of 7.9 (IQR, 6.8-8.8) years, the incidence of CVD was 115 per 100 000 person-years for the hysterectomy group and 96 per 100 000 person-years for the nonhysterectomy group. After adjusting for confounding factors, the hysterectomy group had an increased risk of CVD compared with the nonhysterectomy group (hazard ratio [HR], 1.25; 95% CI, 1.09-1.44). The incidences of myocardial infarction and coronary artery revascularization were comparable between the groups, whereas the risk of stroke was significantly higher in the hysterectomy group (HR, 1.31; 95% CI, 1.12-1.53). Even after excluding women who underwent oophorectomy, the hysterectomy group had higher risks of CVD (HR, 1.24; 95% CI, 1.06-1.44). Conclusions and Relevance: The findings of this cohort study suggest early menopause owing to hysterectomy was associated with increased risks for a composite of CVD, particularly stroke.

The synthetic oleanane triterpenoid CDDO-2P-Im binds GRP78/BiP to induce unfolded protein response-mediated apoptosis in myeloma.

Synthetic oleanane triterpenoids (SOTs) are small molecules with broad anticancer properties. A recently developed SOT, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4(-pyridin-2-yl)-1H-imidazole (CDDO-2P-Im or '2P-Im'), exhibits enhanced activity and improved pharmacokinetics over CDDO-Im, a previous generation SOT. However, the mechanisms leading to these properties are not defined. Here, we show the synergy of 2P-Im and the proteasome inhibitor ixazomib in human multiple myeloma (MM) cells and 2P-Im activity in a murine model of plasmacytoma. RNA sequencing and quantitative reverse transcription PCR revealed the upregulation of the unfolded protein response (UPR) in MM cells upon 2P-lm treatment, implicating the activation of the UPR as a key step in 2P-Im-induced apoptosis. Supporting this hypothesis, the deletion of genes encoding either protein kinase R-like endoplasmic reticulum kinase (PERK) or DNA damage-inducible transcript 3 protein (DDIT3; also known as CHOP) impaired the MM response to 2P-Im, as did treatment with ISRIB, integrated stress response inhibitor, which inhibits UPR signaling downstream of PERK. Finally, both drug affinity responsive target stability and thermal shift assays demonstrated direct binding of 2P-Im to endoplasmic reticulum chaperone BiP (GRP78/BiP), a stress-inducible key signaling molecule of the UPR. These data reveal GRP78/BiP as a novel target of SOTs, and specifically of 2P-Im, and suggest the potential broader utility of this class of small molecules as modulators of the UPR.

Oral TGF-ßR1 inhibitor Vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity.

Osteosarcoma (OS) is an aggressive malignant bone cancer, with refractory and metastatic disease remaining a significant challenge. Transforming growth factor-ß1 (TGF-ß) is a potent immune suppressive cytokine in OS and the TGF-ß is increased in the sera of OS patients and this increase is associated with high-grade OS and lung metastases. Therefore, blocking TGF-ß1 signaling may be a novel therapy for OS treatment. Here we show that blocking TGF-ß1 signaling using TGF-ßR1 inhibitor, Vactosertib, significantly inhibited OS proliferation in vitro and in vivo. Notably, Vactosertib inhibits c-Myc expression in the OS cells. Vactosertib increased immune effectors (IFNγ+CD8+ cells and NK cells) and inhibited immune suppressors (M2-like TAM, MDSC) in the OS tumor microenvironment. Our results suggest that inhibition of TGF-ß1 signaling is an effective therapeutic strategy against OS through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response.

Isolation and identification of extracellular matrix proteins from oil-based CASPERized mouse brains for matrisomal analysis.

The extracellular matrix (ECM) components present within all tissues and organs help to maintain the cytoskeletal architecture and tissue morphology. Although the ECM plays a role in cellular events and signaling pathways, it has not been well studied due its insolubility and complexity. Brain tissue has a higher cell density and weaker mechanical strength than other tissues in the body. When removing cells using a general decellularization method to produce scaffolds and obtain ECM proteins, various problems must be considered because tissues are easily damaged. To retain the brain shape and ECM components, we performed decellularization in combination with polymerization. We immersed mouse brains in oil for polymerization and decellularization via O-CASPER (Oil-based Clinically and Experimentally Applicable Acellular Tissue Scaffold Production for Tissue Engineering and Regenerative Medicine) and then isolated ECM components using sequential matrisome preparation reagents (SMPRs), namely, RIPA, PNGase F, and concanavalin A. Adult mouse brains were preserved with our decellularization method. Western blot and LC-MS/MS analyses revealed that ECM components, including collagen and laminin, were isolated efficiently from decellularized mouse brains using SMPRs. Our method will be useful to obtain matrisomal data and perform functional studies using adult mouse brains and other tissues.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair.

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

Regulation of BRCA1 stability through the tandem UBX domains of isoleucyl-tRNA synthetase 1.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.

Pyruvate dehydrogenase kinase 4 promotes ubiquitin-proteasome system-dependent muscle atrophy.

BACKGROUND: Muscle atrophy, leading to muscular dysfunction and weakness, is an adverse outcome of sustained period of glucocorticoids usage. However, the molecular mechanism underlying this detrimental condition is currently unclear. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in skeletal muscle and has been implicated in the pathogenesis of several diseases. The current study was designed to investigated and delineate the role of PDK4 in the context of muscle atrophy, which could be identified as a potential therapeutic avenue to protect against dexamethasone-induced muscle wasting. METHODS: The dexamethasone-induced muscle atrophy in C2C12 myotubes was evaluated at the molecular level by expression of key genes and proteins involved in myogenesis, using immunoblotting and qPCR analyses. Muscle dysfunction was studied in vivo in wild-type and PDK4 knockout mice treated with dexamethasone (25 mg/kg body weight, i.p., 10 days). Body weight, grip strength, muscle weight and muscle histology were assessed. The expression of myogenesis markers were analysed using qPCR, immunoblotting and immunoprecipitation. The study was extended to in vitro human skeletal muscle atrophy analysis. RESULTS: Knockdown of PDK4 was found to prevent glucocorticoid-induced muscle atrophy and dysfunction in C2C12 myotubes, which was indicated by induction of myogenin (0.3271 ± 0.102 vs 2.163 ± 0.192, ****P < 0.0001) and myosin heavy chain (0.3901 ± 0.047 vs. 0.7222 ± 0.082, **P < 0.01) protein levels and reduction of muscle atrophy F-box (10.77 ± 2.674 vs. 1.518 ± 0.172, **P < 0.01) expression. In dexamethasone-induced muscle atrophy model, mice with genetic ablation of PDK4 revealed increased muscle strength (162.1 ± 22.75 vs. 200.1 ± 37.09 g, ***P < 0.001) and muscle fibres (54.20 ± 11.85% vs. 84.07 ± 28.41%, ****P < 0.0001). To explore the mechanism, we performed coimmunoprecipitation and liquid chromatography-mass spectrometry analysis and found that myogenin is novel substrate of PDK4. PDK4 phosphorylates myogenin at S43/T57 amino acid residues, which facilitates the recruitment of muscle atrophy F-box to myogenin and leads to its subsequent ubiquitination and degradation. Finally, overexpression of non-phosphorylatable myogenin mutant using intramuscular injection prevented dexamethasone-induced muscle atrophy and preserved muscle fibres. CONCLUSIONS: We have demonstrated that PDK4 mediates dexamethasone-induced skeletal muscle atrophy. Mechanistically, PDK4 phosphorylates and degrades myogenin via recruitment of E3 ubiquitin ligase, muscle atrophy F-box. Rescue of muscle regeneration by genetic ablation of PDK4 or overexpression of non-phosphorylatable myogenin mutant indicates PDK4 as an amenable therapeutic target in muscle atrophy.

Smad4-deficient T cells promote colitis-associated colon cancer via an IFN-γ-dependent suppression of 15-hydroxyprostaglandin dehydrogenase.

Immune cells and the cytokines they produce are important mediators of the transition from colitis to colon cancer, but the mechanisms mediating this disease progression are poorly understood. Interferon gamma (IFN-γ) is known to contribute to the pathogenesis of colitis through immune modulatory mechanisms, and through direct effects on endothelial and epithelial homeostasis. Here we explore whether IFN-γ influences tumor progression by expanding the effector memory T cells (TEM) population and restricting the expression of tumor suppressors in a preclinical model of spontaneous colitis-associated colorectal cancer (CAC). We show that IFN-γ expression is significantly increased both in the T cells and the colonic mucosal epithelia of mice with a T cell-restricted deletion of the TGF-ß intermediate, SMAD4 (Smad4TKO). The increase of IFN-γ expression correlates with the onset of spontaneous CAC in Smad4TKO mice by 6 months of age. This phenotype is greatly ameliorated by the introduction of a germline deletion of IFN-γ in Smad4TKO mice (Smad4TKO/IFN-γKO, DKO). DKO mice had a significantly reduced incidence and progression of CAC, and a decrease in the number of mucosal CD4+ TEM cells, when compared to those of Smad4TKO mice. Similarly, the colon epithelia of DKO mice exhibited a non-oncogenic signature with a decrease in the expression of iNOS and p-STAT1, and a restoration of the tumor suppressor gene, 15-hydroxyprostaglandin dehydrogenase (15-PGDH). In vitro, treatment of human colon cancer cells with IFN-γ decreased the expression of 15-PGDH. Our data suggest that Smad4-deficient T cells promote CAC through mechanisms that include an IFN-γ-dependent suppression of the tumor suppressor 15-PGDH.

Noncanonical PDK4 action alters mitochondrial dynamics to affect the cellular respiratory status.

Dynamic regulation of mitochondrial morphology provides cells with the flexibility required to adapt and respond to electron transport chain (ETC) toxins and mitochondrial DNA-linked disease mutations, yet the mechanisms underpinning the regulation of mitochondrial dynamics machinery by these stimuli is poorly understood. Here, we show that pyruvate dehydrogenase kinase 4 (PDK4) is genetically required for cells to undergo rapid mitochondrial fragmentation when challenged with ETC toxins. Moreover, PDK4 overexpression was sufficient to promote mitochondrial fission even in the absence of mitochondrial stress. Importantly, we observed that the PDK4-mediated regulation of mitochondrial fission was independent of its canonical function, i.e., inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Phosphoproteomic screen for PDK4 substrates, followed by nonphosphorylatable and phosphomimetic mutations of the PDK4 site revealed cytoplasmic GTPase, Septin 2 (SEPT2), as the key effector molecule that acts as a receptor for DRP1 in the outer mitochondrial membrane to promote mitochondrial fission. Conversely, inhibition of the PDK4-SEPT2 axis could restore the balance in mitochondrial dynamics and reinvigorates cellular respiration in mitochondrial fusion factor, mitofusin 2-deficient cells. Furthermore, PDK4-mediated mitochondrial reshaping limits mitochondrial bioenergetics and supports cancer cell growth. Our results identify the PDK4-SEPT2-DRP1 axis as a regulator of mitochondrial function at the interface between cellular bioenergetics and mitochondrial dynamics.

Overexpression of VEGF in the MOPC 315 Plasmacytoma Induces Tumor Immunity in Mice.

Vascular endothelial growth factor (VEGF) has important effects on hematopoietic and immune cells. A link between VEGF expression, tumor progression, and metastasis has been established in various solid tumors; however, the impact of VEGF expression by hematopoietic neoplasias remains unclear. Here, we investigated the role of VEGF in plasma cell neoplasia. Overexpression of VEGF in MOPC 315 tumor cells (MOPCSVm) had no effect on their growth in vitro. However, constitutive ectopic expression of VEGF dramatically reduced tumorigenicity of MOPC 315 when implanted subcutaneously into BALB/c mice. Mice implanted with MOPCSVm effectively rejected tumor grafts and showed strong cytotoxic T lymphocyte (CTL) activity against parental MOPC 315 cells. MOPCSVm implants were not rejected in nude mice, suggesting the process is T-cell-dependent. Adoptive transfer of splenocytes from recipients inoculated with MOPCSVm cells conferred immunity to naïve BALB/c mice, and mice surviving inoculation with MOPCSVm rejected the parental MOPC 315 tumor cells following a second inoculation. Immunohistochemical analysis showed that MOPCSVm induced a massive infiltration of CD3+ cells and MHC class II+ cells in vivo. In addition, exogenous VEGF induced the expression of CCR3 in T cells in vitro. Together, these data are the first to demonstrate that overexpression of VEGF in plasmacytoma inhibits tumor growth and enhances T-cell-mediated antitumor immune response.

Reciprocal interactions among Cobll1, PACSIN2, and SH3BP1 regulate drug resistance in chronic myeloid leukemia.

Cobll1 affects blast crisis (BC) progression and tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML). PACSIN2, a novel Cobll1 binding protein, activates TKI-induced apoptosis in K562 cells, and this activation is suppressed by Cobll1 through the interaction between PACSIN2 and Cobll1. PACSIN2 also binds and inhibits SH3BP1 which activates the downstream Rac1 pathway and induces TKI resistance. PACSIN2 competitively interacts with Cobll1 or SH3BP1 with a higher affinity for Cobll1. Cobll1 preferentially binds to PACSIN2, releasing SH3BP1 to promote the SH3BP1/Rac1 pathway and suppress TKI-mediated apoptosis and eventually leading to TKI resistance. Similar interactions among Cobll1, PACSIN2, and SH3BP1 control hematopoiesis during vertebrate embryogenesis. Clinical analysis showed that most patients with CML have Cobll1 and SH3BP1 expression at the BC phase and BC patients with Cobll1 and SH3BP1 expression showed severe progression with a higher blast percentage than those without any Cobll1, PACSIN2, or SH3BP1 expression. Our study details the molecular mechanism of the Cobll1/PACSIN2/SH3BP1 pathway in regulating drug resistance and BC progression in CML.

Mitoquinone Inactivates Mitochondrial Chaperone TRAP1 by Blocking the Client Binding Site.

Heat shock protein 90 (Hsp90) family proteins are molecular chaperones that modulate the functions of various substrate proteins (clients) implicated in pro-tumorigenic pathways. In this study, the mitochondria-targeted antioxidant mitoquinone (MitoQ) was identified as a potent inhibitor of mitochondrial Hsp90, known as a tumor necrosis factor receptor-associated protein 1 (TRAP1). Structural analyses revealed an asymmetric bipartite interaction between MitoQ and the previously unrecognized drug binding sites located in the middle domain of TRAP1, believed to be a client binding region. MitoQ effectively competed with TRAP1 clients, and MitoQ treatment facilitated the identification of 103 TRAP1-interacting mitochondrial proteins in cancer cells. MitoQ and its redox-crippled SB-U014/SB-U015 exhibited more potent anticancer activity in vitro and in vivo than previously reported mitochondria-targeted TRAP1 inhibitors. The findings indicate that targeting the client binding site of Hsp90 family proteins offers a novel strategy for the development of potent anticancer drugs.

AGL9: A Novel Hepatoprotective Peptide from the Larvae of Edible Insects Alleviates Obesity-Induced Hepatic Inflammation by Regulating AMPK/Nrf2 Signaling.

In this study, we investigated the anti-obesity properties of the novel peptide Ala-Gly-Leu-Gln-Phe-Pro-Val-Gly-Arg (AGL9), isolated from the enzymatic hydrolysate of Allomyrinadichotoma larvae. To investigate the preventive effects of AGL9 against hepatic steatosis and its possible mechanisms of action, we established an nonalcoholic fatty liver disease (NAFLD) model by feeding C57BL/6 mice a high-fat diet. NAFLD mice were administered 100 mg/kg AGL9 and 60 mg/kg orlistat via gavage (10 mL/kg) for 5 weeks, followed by the collection of blood and liver tissues. We found that AGL9 normalized the levels of serum alanine aminotransferase, aspartate aminotransferase, triglyceride, total cholesterol, high-density lipoprotein, very low-density lipoprotein (LDL)/LDL, adiponectin, and leptin in these mice. Additionally, AGL9 activated the protein-level expression of 5' AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation and the transcript-level expression of sterol regulatory element-binding protein-1c, fatty acid synthase, superoxide dismutase, glutathione peroxidase, glucocorticoid receptor, nuclear respiratory factor 2, tumor necrosis factor-α, interleukin-1ß, interleukin-6, and monocyte chemoattractant protein-1 in hepatocytes. These results showed that AGL9 exhibited hepatoprotective effects by attenuating lipid deposition, oxidative stress, and inflammation via inhibition of AMPK/Nrf2 signaling, thereby reducing the production of hepatic proinflammatory mediators and indicating AGL9 as a potential therapeutic strategy for NAFLD.

Novel therapies emerging in oncology to target the TGF-ß pathway.

The TGF-ß signaling pathway governs key cellular processes under physiologic conditions and is deregulated in many pathologies, including cancer. TGF-ß is a multifunctional cytokine that acts in a cell- and context-dependent manner as a tumor promoter or tumor suppressor. As a tumor promoter, the TGF-ß pathway enhances cell proliferation, migratory invasion, metastatic spread within the tumor microenvironment and suppresses immunosurveillance. Collectively, the pleiotropic nature of TGF-ß signaling contributes to drug resistance, tumor escape and undermines clinical response to therapy. Based upon a wealth of preclinical studies, the TGF-ß pathway has been pharmacologically targeted using small molecule inhibitors, TGF-ß-directed chimeric monoclonal antibodies, ligand traps, antisense oligonucleotides and vaccines that have been now evaluated in clinical trials. Here, we have assessed the safety and efficacy of TGF-ß pathway antagonists from multiple drug classes that have been evaluated in completed and ongoing trials. We highlight Vactosertib, a highly potent small molecule TGF-ß type 1 receptor kinase inhibitor that is well-tolerated with an acceptable safety profile that has shown efficacy against multiple types of cancer. The TGF-ß ligand traps Bintrafusp alfa (a bifunctional conjugate that binds TGF-ß and PD-L1), AVID200 (a computationally designed trap of TGF-ß receptor ectodomains fused to an Fc domain) and Luspatercept (a recombinant fusion that links the activin receptor IIb to IgG) offer new ways to fight difficult-to-treat cancers. While TGF-ß pathway antagonists are rapidly emerging as highly promising, safe and effective anticancer agents, significant challenges remain. Minimizing the unintentional inhibition of tumor-suppressing activity and inflammatory effects with the desired restraint on tumor-promoting activities has impeded the clinical development of TGF-ß pathway antagonists. A better understanding of the mechanistic details of the TGF-ß pathway should lead to more effective TGF-ß antagonists and uncover biomarkers that better stratify patient selection, improve patient responses and further the clinical development of TGF-ß antagonists.

PDK2 Deficiency Prevents Ovariectomy-Induced Bone Loss in Mice by Regulating the RANKL-NFATc1 Pathway During Osteoclastogenesis.

Estrogen deficiency leads to osteoporosis as a result of an imbalance in bone remodeling due to greater bone resorption. Estrogen deficiency increases the osteoclastic resorption of bone, and many of the FDA-approved therapies for osteoporosis are antiresorptive drugs that mainly act by reducing osteoclast activity. The mitochondrial enzyme pyruvate dehydrogenase kinase (PDK) is a critical regulator of aerobic glycolysis that exerts its effects by phosphorylating the pyruvate dehydrogenase complex (PDC), which is responsible for oxidative phosphorylation. In the present study, we found that during osteoclast differentiation, PDK2 expression increased more than that of the other PDK isoenzymes. Bone loss was delayed and the number of osteoclasts was lower in ovariectomized (OVX) Pdk2-/- mice than in OVX wild-type mice. The differentiation of osteoclasts was suppressed in Pdk2-/- bone marrow-derived monocyte/macrophage lineage cells, which was associated with lower phosphorylation of cAMP response element-binding protein (CREB) and c-FOS, and a consequent reduction in NFATc1 transcription. Administration of AZD7545, a specific inhibitor of PDK2, prevented the OVX-induced bone loss and reduced the phosphorylation of CREB and c-FOS, and the protein expression of NFATc1, in osteoclasts. Collectively, these results indicate that the inhibition of PDK2 prevents osteoporosis in estrogen-deficient mice by reducing aberrant osteoclast activation, probably via inhibition of the RANKL-CREB-cFOS-NFATc1 pathway. These findings imply that PDK2 inhibitors might be repurposed for the therapy of estrogen deficiency-induced osteoporosis. © 2020 American Society for Bone and Mineral Research (ASBMR).

Loss of p27Kip1 leads to expansion of CD4+ effector memory T cells and accelerates colitis-associated colon cancer in mice with a T cell lineage restricted deletion of Smad4.

The cyclin-dependent kinase inhibitor p27Kip1 is a tumor suppressor whose intrinsic activity in cancer cells correlates with tumor aggressiveness, invasiveness, and impaired tumor cell differentiation. Here we explore whether p27Kip1 indirectly influences tumor progression by restricting expansion and survival of effector memory T cell (TEM) populations in a preclinical model of spontaneous colitis-associated colorectal cancer (CAC). We show mRNA and protein expression of p27Kip1 to be significantly decreased in the colons of mice with a T cell-restricted deletion of the TGF-ß intermediate, SMAD4 (Smad4TKO). Loss of p27Kip1 expression in T cells correlates with the onset of spontaneous CAC in Smad4TKO mice by 8 months of age. This phenotype is greatly accelerated by the introduction of a germline deletion of CDKN1b (the gene encoding p27Kip1) in Smad4TKO mice (Smad4TKO/p27Kip1-/-, DKO). DKO mice display colon carcinoma by 3 months of age and increased mortality compared to Smad4TKO. Importantly, the phenotype in DKO mice is associated with a significant increase in the frequency of effector CD4 T cells expressing abundant IFN-γ and with a concomitant decrease in Foxp3+ regulatory T cells, both in the intestinal mucosa and in the periphery. In addition, induction of inflammatory mediators (IFN-γ, TNF-γ, IL-6, IL-1ß, iNOS) and activation of Stat1, Stat3, and IκB is also observed in the colon as early as 1-2 months of age. Our data suggest that genomic alterations known to influence p27Kip1 abundance in gastrointestinal cancers may indirectly promote epithelial malignancy by augmenting the production of inflammatory mediators from a spontaneously expanding pool of TEM cells.

Common plasma protein marker LCAT in aggressive human breast cancer and canine mammary tumor.

Breast cancer is one of the most frequently diagnosed cancers. Although biomarkers are continuously being discovered, few specific markers, rather than classification markers, representing the aggressiveness and invasiveness of breast cancer are known. In this study, we used samples from canine mammary tumors in a comparative approach. We subjected 36 fractions of both canine normal and mammary tumor plasmas to highperformance quantitative proteomics analysis. Among the identified proteins, LCAT was selectively expressed in mixed tumor samples. With further MRM and Western blot validation, we discovered that the LCAT protein is an indicator of aggressive mammary tumors, an advanced stage of cancer, possibly highly metastatic. Interestingly, we also found that LCAT is overexpressed in high-grade and lymph-node-positive breast cancer in silico data. We also demonstrated that LCAT is highly expressed in the sera of advanced-stage human breast cancers within the same classification. In conclusion, we identified a possible common plasma protein biomarker, LCAT, that is highly expressed in aggressive human breast cancer and canine mammary tumor. [BMB Reports 2020; 53(12): 664-669].