Gain Of Function Studies Research Articles

Combination Therapy Blocking TNF Superfamily Members 14 and 15 Reverses Pulmonary Fibrosis

Abstract Currently, anti-inflammatory drugs fail to reduce pulmonary fibrosis in the clinic and there is an urgent unmet need to develop novel anti-fibrotic drugs capable of reversing disease. Our lab has identified two novel mediators of pulmonary fibrosis belonging to the tumor necrosis factor superfamily (TNFSF), LIGHT (TNFSF14) and TL1A (TNFSF15). Here, we show LIGHT and TL1A receptor are both significantly elevated in patient pulmonary fibrosis biopsies as compared to healthy control lungs. Using gain of function studies, we found that LIGHT and TL1A can drive airway remodeling independently of one another and that these TNFSF members synergize to maximize airway inflammation and fibrosis. We show that the combinatorial blockade of LIGHT and TL1A limits TGFb-driven profibrotic effects on fibroblasts in vitro. Moreover, LIGHT and TL1A stimulation of human epithelial cells and fibroblasts reveal distinct fibrotic and inflammatory signatures, including redundant, additive, and synergistic profibrotic activities. Importantly, using antagonistic reagents to neutralize both LIGHT and TL1A signaling in combination post-disease onset, we observe a significant decrease in collagen deposition and smooth muscle hypertrophy in a bleomycin model of pulmonary fibrosis as opposed to respective monotherapies. The implication of this work highlights a therapeutic need of concomitantly targeting LIGHT and TL1A to treat pulmonary fibrosis disorders in humans.

The Gain-of-Function Research in Virology: Concerns, Risks, and Benefits

Gain-of-function (GOF) research is biotechnological research that genetically modifies microbial gene products to enhance their biological function. The basic aim of this research is to elucidate the pathogenesis of infectious diseases, develop new vaccines and therapeutics, improve cancer treatments, and achieve specific goals by adding beneficial trait expression to the genome. Despite these significant potential benefits, GOF research can pose some important and serious biosafety risks. The main group of pathogens of concern in GOF research are respiratory viruses with pandemic potential. While laboratory accidents and ongoing discussions in recent years have raised important awareness that these studies should be carried out in high-safety laboratories, concerns about the results of these studies remain unclear due to the inadequacy or absence of mechanisms to prevent misuse for informal research, despite the strict controls in research funded by international organizations. The most important issue to be discussed is the need to establish regulations that ensure the sustainability of scientific studies and developments that can benefit humanity, as well as strict control or, if necessary, a moratorium on potentially risky research. Debates on the value, safety, ethics, and validity of GOF studies are still ongoing. In this article, it is aimed to examine the status of GOF research in virology, with examples from the current literature, together with benefit and risk perspectives.

Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

Background and AimsEfforts to decode pancreatic ductal adenocarcinoma (PDAC) heterogeneity and the consequent therapeutic selection remains a challenge. We aimed to characterize epigenetically regulated pathways involved in PDAC progression. MethodsGlobal DNA methylation analysis in pancreatic cancer patient tissues and cell lines was performed to identify differentially methylated genes. Targeted bisulfite sequencing and in vitro methylation reporter assays were employed to investigate the direct link between site-specific methylation and transcriptional regulation. A series of in vitro loss- and gain-of function studies, and in vivo xenograft and the KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre) mouse models were used to assess pancreatic cancer cell properties. Gene and protein expression analyses were performed in three different cohorts of pancreatic cancer patients and correlated to clinicopathological parameters. ResultsWe identify Hepatocyte Nuclear Factor 4A (HNF4A) as a novel target of hypermethylation in pancreatic cancer and demonstrate that site-specific proximal promoter methylation drives HNF4A transcriptional repression. Expression analyses in patients, indicate the methylation-associated suppression of HNF4A expression in pancreatic cancer tissues. In vitro and in vivo studies reveal that HNF4A is a novel tumor suppressor in pancreatic cancer, regulating cancer growth and aggressiveness. As evidenced in both the KPC mouse model and human pancreatic cancer tissues, HNF4A expression declines significantly in the early stages of the disease. Most importantly, HNF4 loss correlates with poor overall patient survival. ConclusionsHNF4A silencing, mediated by promoter DNA methylation, drives pancreatic cancer development and aggressiveness leading to poor patient survival.

Nuclear Acly protects the liver from ischemia-reperfusion injury.

Hepatic ischemia-reperfusion (IR) injury is the most common complication that occurs in liver surgery and hemorrhagic shock. ATP citrate lyase (Acly) plays a pivotal role in chromatin modification via generating acetyl-CoA for histone acetylation to influence biological processes. We aim to examine the roles of Acly, which is highly expressed in hepatocytes, in liver IR injury. The functions of Acly in hepatic IR injury were examined in the mouse model with a hepatocyte-specific knockout of Acly . The Acly target genes were analyzed by CUT&RUN assay and RNA sequencing. The relationship between the susceptibility of the steatotic liver to IR and Acly was determined by the gain of function studies in mice. Hepatic deficiency of Acly exacerbated liver IR injury. IR induced Acly nuclear translocation in hepatocytes, which spatially fueled nuclear acetyl-CoA. This alteration was associated with enhanced acetylation of H3K9 and subsequent activation of the Foxa2 signaling pathway. Nuclear localization of Acly enabled Foxa2-mediated protective effects after hypoxia-reperfusion in cultured hepatocytes, while cytosolic Acly demonstrated no effect. The presence of steatosis disrupted Acly nuclear translocation. In the steatotic liver, restoration of Acly nuclear localization through overexpression of Rspondin-1 or Rspondin-3 ameliorated the IR-induced injury. Our results indicate that Acly regulates histone modification by means of nuclear AcCoA production in hepatic IR. Disruption of Acly nuclear translocation increases the vulnerability of the steatotic liver to IR. Nuclear Acly thus may serve as a potential therapeutic target for future interventions in hepatic IR injury, particularly in the context of steatosis.

Abstract P3180: Cardiomyocyte Pgc-1α Mediates The Adaptive Response To Endurance Exercise Training In Mice

Exercise has myriad systemic benefits in reducing the incidence from and morbidity of cardio-metabolic diseases. In healthy individuals, aerobic exercise promotes physiologic hypertrophy of the heart, which requires increased mitochondrial numbers and function. In skeletal muscle and heart, a key orchestrator of the adaptation to increased energetic needs is the transcriptional regulator PGC-1α. PGC-1α may mediate cardiac adaptation to physiological stress through its function in mitochondrial biogenesis but also through other mechanisms. The role of cardiac PGC-1α to the systemic training response is poorly understood. We generated PGC-1α cardiomyocyte-specific knockout (cKO) mice to study its role in endurance exercise training. Sedentary cKO mice demonstrated normal cardiac function and no significant differences in acute treadmill exercise tolerance. However, cKO mice demonstrated a lack of ability to augment exercise capacity after 6 weeks of wheel running compared to WT (work achieved 32.9 J in WT [39% increase] vs 23.1 J in cKOs [no change vs baseline], p<0.001, unpaired t-test). This was despite comparable voluntary wheel distance run during training (2.93-3.67 km/day for both over 5 weeks, p=0.58). Unexpectedly, after 6 weeks of training, cKO mice developed a dilated cardiomyopathy at rest and exercise-related dysfunction (contractile reserve [ΔFS stress-rest ] +6.0 in WT vs -6.4% in KO, p<0.05, unpaired t-test). They also demonstrated features of cardiac cachexia as measured by 30% and 21% reductions in inguinal adipose and gastrocnemius mass, respectively (P<0.05, unpaired t-test). Gene expression profiling demonstrated activation of pathological ( Nppa , Nppb , Myh7/Myh6 ) and atrophic pathways ( Gdf11 , Gdf15 , Fstl3 ) in the trained cKOs. In vitro, gain of function studies using adenoviral expression in neonatal rat ventricular myocytes demonstrated that PGC-1α overexpression could abrogate pathologic hypertrophy and atrophy gene expression pathways stimulated by phenylephrine. Our preliminary studies support cardiomyocyte PGC-1α as a key mediator of the adaptive response to exercise training and unveil a key role of this transcriptional activator in protecting from exercise-related cardiac dysfunction in mice.

Shedding LIGHT and TL1A on Pulmonary Fibrosis

Abstract Currently, anti-inflammatory drugs fail to reduce pulmonary fibrosis (PF) and tissue remodeling in the clinic. Despite the advancements made in understanding the molecular pathways driving PF, there is still an unmet need to develop innovative and effective anti-fibrotic drugs. Our lab has identified two novel mediators of fibrosis belonging to the tumor necrosis factor superfamily (TNFSF), LIGHT (TNFSF14) and TL1A (TNFSF15). Using a well characterized mouse model of PF driven by bleomycin, we showed that blocking LIGHT or TL1A signaling in isolation, by genetic ablation or antibody-mediated neutralization, significantly reduces PF. In gain of function studies, the in vivo administration of LIGHT or TL1A in isolation recapitulated fibrosis symptoms reminiscent of human disease. Furthermore, our data suggest that LIGHT promotes thymic stromal lymphopoietin (TLSP) expression by epithelial cells, whereas TL1A activates fibroblasts to promote periostin expression. Both TSLP and periostin are drivers of PF. Importantly, we observed an upregulation of LIGHT and DR3 (the receptor for TL1A) in human lung biopsies of patients suffering SSc and IPF. We have found that LIGHT and TL1A can drive fibrosis independently of one another and that the in vivo co-administration of these TNFSF members synergize to maximize inflammation and collagen deposition. Using antagonistic fusion proteins to neutralize LIGHT and TL1A signaling, we will monitor PF in vivo and check whether greater efficacy is achieved when LIGHT and TL1A are targeted concomitantly post-disease onset. The relevance of this work on PF associated with ILDs in humans is tremendous.

KIF14 promotes proliferation, lymphatic metastasis and chemoresistance through G3BP1/YBX1 mediated NF-κB pathway in cholangiocarcinoma.

Cholangiocarcinoma (CCA), a highly lethal and fetal cancer derived from the hepatobiliary system, is featured by aggressive growth and early lymphatic metastasis. Elucidating the underlying mechanism and identifying the effective therapy are critical for advanced CCA patients. In the study, we detected that KIF14 was upregulated in CCA samples, especially in patients with lymph node metastasis and vascular invasion. CCA patients with higher KIF14 were associated with worse overall survival and recurrence-free survival after surgery. Gain-of and loss-of function studies showed that KIF14 enhanced CCA cells proliferation, migration, invasion and lymphatic metastasis whereas its silencing abolished the effects in vivo and in vitro. Mechanistic investigation showed that KIF14 bound to the G3BP1/YBX1 complex and facilitated their interaction, causing increased activity of the NF-κB promoter and activation of NF-κB pathway. Furthermore, increased KIF14 level enhanced chemotherapy-resistance to gemcitabine-based regimen and induced immunosuppressive microenvironment. In addition, KIF14 was direct target of HNF4A and inversely regulated by HNF4A. Together, these findings suggested that KIF14 could be a potential oncogene and a good indicator in predicting prognosis and chemotherapy guidance for CCA patients.

Tendon Cells Root Into (Instead of Attach to) Humeral Bone Head via Fibrocartilage-Enthesis.

Large joints are composed of two closely linked cartilages: articular cartilage (AC; rich in type II collagen, a well-studied tissue) and fibrocartilaginous enthesis (FE; rich in type I collagen, common disorder sites of enthesopathy and sporting injuries, although receiving little attention). For many years, both cartilages were thought to be formed by chondrocytes, whereas tendon, which attaches to the humeral bone head, is primarily considered as a completely different connective tissue. In this study, we raised an unconventional hypothesis: tendon cells directly form FE via cell transdifferentiation. To test this hypothesis, we first qualitatively and quantitatively demonstrated distinct differences between AC and FE in cell morphology and cell distribution, mineralization status, extracellular matrix (ECM) contents, and critical ECM protein expression profiles using comprehensive approaches. Next, we traced the cell fate of tendon cells using ScxLin (a tendon specific Cre ScxCreERT2; R26R-tdTomato line) with one-time tamoxifen induction at early (P3) or young adult (P28) stages and harvested mice at different development ages, respectively. Our early tracing data revealed different growth events in tendon and FE: an initial increase but gradual decrease in the ScxLin tendon cells and a continuous expansion in the ScxLin FE cells. The young adult tracing data demonstrated continuous recruitment of ScxLin cells into FE expansion during P28 and P56. A separate tracing line, 3.2 Col 1Lin (a so-called "bone-specific" line), further confirmed the direct contribution of tendon cells for FE cell formation, which occurred in days but FE ECM maturation (including high levels of SOST, a potent Wnt signaling inhibitor) took weeks. Finally, loss of function data using diphtheria toxin fragment A (DTA) in ScxLin cells demonstrated a significant reduction of ScxLin cells in both tendons and FE cells, whereas the gain of function study (by stabilizing β-catenin in ScxLin tendon cells via one-time injection of tamoxifen at P3 and harvesting at P60) displayed great expansion of both ScxLin tendon and FE mass. Together, our studies demonstrated that fibrocartilage is an invaded enthesis likely originating from the tendon via a quick cell transdifferentiation mechanism with a lengthy ECM maturation process. The postnatally formed fibrocartilage roots into existing cartilage and firmly connects tendon and bone instead of acting as a simple attachment site as widely believed. We believe that this study will stimulate more intense exploring in this understudied area, especially for patients with enthesopathy and sporting injuries.

Gain of function studies on predicted host receptors for white spot virus

Three decades after its first outbreak, the shrimp white spot virus (WSV) is still a global cause of concern due to considerable losses and lack of effective control measures. Several candidate host receptor proteins have been identified, but the pathogenesis is not clearly understood, although the key role of the WSV envelope protein VP28 in virus internalization is established. Here, protein-protein docking is applied to evaluate the interaction of VP28 trimeric extracellular region with four host (Penaeus monodon) receptors reported earlier, Rab7 GTPase (PmRab7), glucose transporter 1 (PmGLUT1), C-type lectin (PmCTL) and calreticulin (PmCRT). The stability of predicted complexes evaluated in terms of binding energy per unit buried surface area ranged from −8.46 to −11.82 cal mol−1/Å2, which is not sufficient for functional interaction. Nevertheless, each of these host proteins was tested by a gain-of-function approach by observing their ability to make a fish cell line permissive to the shrimp WSV. Full-length expression constructs of the four receptors were transfected into SSN1 snakehead fish cells that are non-permissive to WSV. Transfected SSN1 cells and WSV permissive insect Sf9 cells were challenged with purified WSV. After 24 h, the presence of receptor transcripts was confirmed in the treated SSN1 cells, and not in the non-transfected SSN1 cells. Further, vp28 transcript was detected in Sf9 cells, but not in any of the treated SSN1 cells, indicating that none of the receptors were singly sufficient to make SSN1 cells permissive to WSV, even though PmRab7 was a strong candidate that alone showed >85% protection in virus neutralization experiments. For the other 3 candidates, previous reports predicted the involvement of co-receptors, which is confirmed here by their inability to act singly.

Orosomucoid 2 maintains hepatic lipid homeostasis through suppression of de novo lipogenesis.

Non-alcoholic fatty liver disease (NAFLD) is caused by imbalance in lipid metabolism. In this study, we show that the hepatokine orosomucoid (ORM) 2 is a key regulator of de novo lipogenesis in the liver. Hepatic and plasma ORM2 levels are markedly decreased in obese murine models and patients with NAFLD. Through multiple loss- and gain-of function studies, we demonstrate that ORM2 is essential to maintain hepatic and systemic lipid homeostasis. At the mechanistic level, ORM2 binds to inositol 1, 4, 5-trisphosphate receptor type 2 to activate AMP-activated protein kinase signaling, thereby inhibiting sterol regulatory element binding protein 1c-mediated lipogenic gene program. Notably, intraperitoneal injections of recombinant ORM2 protein or stabilized ORM2-FC fusion protein markedly improved liver steatosis, steatohepatitis and atherosclerosis in preclinical mouse models, without adverse effects on body weight or food intake. Thus, these findings suggest that ORM2 may serve as a potential target for therapeutic intervention in NAFLD, non-alcoholic steatohepatitis and related lipid disorders.

SPARC, a Novel Regulator of Vascular Cell Function in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH can be caused by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMCs) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVECs). On reexposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, the authors aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature. After microarray analysis, the authors assessed the role of SPARC (secreted protein acidic and rich in cysteine) in PH using lung tissue from idiopathic pulmonary arterial hypertension (IPAH) patients, as well as from chronically hypoxic mice. In vitro studies were conducted in primary human PASMCs and PMVECs. In vivo function of SPARC was proven in chronic hypoxia-induced PH in mice by using an adeno-associated virus-mediated Sparc knockdown approach. C57BL/6J mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent reexposure to normoxia for different time points. Microarray analysis of the pulmonary vascular compartment after laser microdissection identified Sparc as one of the genes downregulated at all reoxygenation time points investigated. Intriguingly, SPARC was vice versa upregulated in lungs during development of hypoxia-induced PH in mice as well as in IPAH, although SPARC plasma levels were not elevated in PH. TGF-β1 (transforming growth factor β1) or HIF2A (hypoxia-inducible factor 2A) signaling pathways induced SPARC expression in human PASMCs. In loss of function studies, SPARC silencing enhanced apoptosis and reduced proliferation. In gain of function studies, elevated SPARC levels induced PASMCs, but not PMVECs, proliferation. Coculture and conditioned medium experiments revealed that PMVECs-secreted SPARC acts as a paracrine factor triggering PASMCs proliferation. Contrary to the authors' expectations, in vivo congenital Sparc knockout mice were not protected from hypoxia-induced PH, most probably because of counter-regulatory proproliferative signaling. However, adeno-associated virus-mediated Sparc knockdown in adult mice significantly improved hemodynamic and cardiac function in PH mice. This study provides evidence for the involvement of SPARC in the pathogenesis of human PH and chronic hypoxia-induced PH in mice, most likely by affecting vascular cell function.

DNA-Binding domain mutations confer severe outcome at an early age among STAT1 gain-of-function patients.

STAT1gain-of-function (GOF) is an immune dysregulatory disorder with poorly studied genotype-phenotype correlation, impeding prognostication and early intervention. Given previous mechanistic studies, as well as anecdotal clinical reports, we sought to systematically determine whether DNA-binding domain (DBD) mutations in STAT1 result in a different phenotype than mutations in other gene domains. Negative prognostic features previously identified by the International STAT1 GOF Study Group (invasive infections, intracranial aneurysms, and malignancy), as well as other clinical features and mortality, were compared within a cohort of 30 patients with STAT1 GOF diagnosed at our center, consisting of 9 patients with DBD mutations and 21 patients with non-DBD mutations. We subsequently re-analyzed mortality data from a large, previously-published 274-patient cohort by the International STAT1 GOF Study Group. While no differences were noted with respect to malignancy or symptomatic aneurysms, invasive /opportunistic infections were substantially more common among DBD patients, as were sinopulmonary infections, bronchiectasis, enteropathy, endocrinopathies, lymphoproliferative manifestations, and recurrent fevers/HLH. DBD patients also had a lower probability of survival and younger age of mortality compared with non-DBD patients. Our re-evaluation of the published data from the International STAT1 GOF Study Group revealed a similar finding of earlier mortality among patients harboring DBD mutations. We report that STAT1 GOF patients with DBD mutations may be regarded as a unique subgroup, impacted more by early-onset profound combined immunodeficiency and with earlier mortality. These findings may impact clinical decision making with respect to early intervention, and in particular hematopoietic stem cell transplant considerations, in such patients.

CD56 Has a Critical Role in Regulating Multiple Myeloma Cell Growth and Response to Therapies

Multiple myeloma (MM) is a disease derived from genetically abnormal clonal plasma cells. MM cells aberrantly express several surface antigens compared with normal plasma cells. Among others, CD56/NCAM1 is present at variable levels in approximately 70% of MM patients. Very little is known about its role in MM; however, CD56 positivity in MM correlates with greater osteolytic burden and lower frequency of good prognostic features, such as the presence of t(11;14).We first analyzed 569 patients with MM diagnosed between 1/1/2005 and 12/31/2014 at the Ohio State University Wexner Medical Center, stratifying them based on the percentage of CD56-expressing clonal MM cells. The mean percentage of CD56-expressing clonal MM was 26.5%, with range from 0 to 100%; the Mean Fluorescent Intensity values varied, with a quarter of patients expressing CD56 at high intensity. We then evaluated patient outcomes based on the percentage of CD56-expressing clonal MM cells. We noticed that MM patients with more than 30 or 50 percent of CD56-expressing MM clonal cells have inferior clinical outcomes than patients with less than 30 or 50 percent of CD56-expressing MM clonal cells, with median overall survival of 9.61 versus 7.64 years (log-rank p-value: 0.004) or 9.30 versus 6.47 years (log-rank p-value: 0.0009), respectively.We then demonstrated by conventional and real-time PCR analyses that the predominately expressed CD56 isoform in MM has signaling potential with a transmembrane portion and cytosolic tail. Therefore, we evaluated the functional role of CD56 in MM. By gain-of function studies in U266 and MM.1S MM cell lines, we showed that overexpression of CD56 promotes MM growth and viability; the opposite effect occurred with CD56 silencing in H929, OPM-2, and RPMI-8226 MM cell lines, which leads to reduced MM growth and increased apoptotic cell death. Overexpression of CD56 resulted in the phosphorylation and hence activation of ribosomal protein S6 kinase A3 (RSK2) and of the transcription factor, cAMP responsive element binding protein 1 (CREB1). This induced CREB1 binding to DNA consensus CRE elements, and promoted transcription of CREB1 targets, the anti-apoptotic genes BCL2 and MCL1. CD56 silencing in H929 and OPM-2 MM cell lines had opposite effects, with reduction of phospho-RSK2, phospho-CREB1, MCL1, and BCL2 levels.We then used shRNAs targeting RSK2 and CREB1 or specific inhibitors (BI-D1870 that is a RSK2 inhibitor, and 666-15 that is a CREB1 inhibitor) at 0.1-1 microM concentration. We evaluated viability by MTT assay or Zombie dye staining on CD138 positive MM cells and apoptosis by Annexin V-PI staining. We demonstrated that CD56 positive MM cell lines (H929, OPM-2, and RPMI-8226) or patients with high CD56 expression (>30% of CD56-expressing clonal MM cells) are more sensitive to RSK2/CREB1 inhibition compared with CD56 negative MM cell lines (U266, L363, and MM.1S) or patients with low CD56 expression (<30% of CD56-expressing clonal MM cells). Using similar strategies, we also proved that CREB1 is essential to CD56-protumoral phenotype, since CREB1 inhibition reduces cellular growth and viability in CD56 overexpressing U266 cells. RSK2 and CREB1 inhibition mimic CD56 silencing with decrease of BCL2 and MCL1 mRNA and protein levels. Furthermore, we observed that CD56 signaling by CREB1 activation decreases CRBN expression, reducing responses to lenalidomide. Conversely, CREB1/RSK2 blockade rescued CRBN levels in CD56 positive MM cells and increased lenalidomide response. These results support the hypothesis that targeting CREB1 is hence a so far unexplored but potentially effective synthetic lethal strategy for CD56-expressing MM patients.In conclusion, our study defines an effective threshold for therapeutic intervention in CD56-expressing MM patients. Moreover, our data pioneer the use of CREB1/RSK2 inhibition in CD56-expressing MM cells, either as single agents or in combination with lenalidomide, suggesting that CD56 can be a prognostic and predictive factor of response in MM. DisclosuresNo relevant conflicts of interest to declare.

SMYD2-mediated TRAF2 methylation promotes the NF-κB signaling pathways in inflammatory diseases.

BackgroundThe methylation of lysine residues has been involved in the multiple biological and diseases processes. Recently, some particular non‐histone proteins have been elucidated to be methylated by SMYD2, a SET and MYND domain protein with lysine methyltransferase activity.MethodsSMYD2 was evaluated in synovial tissue and cells derived from rheumatoid arthritis patients. We confirmed TRAF2 could be methylated by SMYD2 using Mass spectrometry, pull‐down, immunoprecipitation, methyltransferase assay, ubiquitination assay, luciferase reporter assays, and western blot analyses. Using loss‐ and gain‐of function studies, we explored the biological functions of SMYD2 in vitro and in vivo. Using acute and chronic inflammation with different mice models to determine the impact of SMYD2.ResultsHere, we first time confirmed that the cytoplasmic protein TRAF2 as the kernel node for NF‐κB signaling pathway could be methylated by SMYD2. SMYD2‐mediated TRAF2 methylation contributed to the durative sensitization of NF‐κB signaling transduction through restraining its own proteolysis and enhancing the activity. In addition, we found knocking down of SMYD2 has different degrees of mitigation in acute and chronic inflammation mice models. Furthermore, as the lysine‐specific demethylase, LSD1 could resist methylation on TRAF2 induced by SMYD2.ConclusionsOur data uncovered an unprecedented cytoplasmic protein network that employed methylation of TRAF2 for the maintenance of NF‐κB activation during inflammatory diseases.

Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma

Multiple myeloma (MM) is a heterogeneous disease characterized by significant genomic instability. Recently, a causal role for the AID/APOBEC deaminases in inducing somatic mutations in myeloma has been reported. We have identified APOBEC/AID as a prominent mutational signature at diagnosis with further increase at relapse in MM. In this study, we identified upregulation of several members of APOBEC3 family (A3A, A3B, A3C, and A3G) with A3G, as one of the most expressed APOBECs. We investigated the role of APOBEC3G in MM and observed that A3G expression and APOBEC deaminase activity is elevated in myeloma cell lines and patient samples. Loss-of and gain-of function studies demonstrated that APOBEC3G significantly contributes to increase in DNA damage (abasic sites and DNA breaks) in MM cells. Evaluation of the impact on genome stability, using SNP arrays and whole genome sequencing, indicated that elevated APOBEC3G contributes to ongoing acquisition of both the copy number and mutational changes in MM cells over time. Elevated APOBEC3G also contributed to increased homologous recombination activity, a mechanism that can utilize increased DNA breaks to mediate genomic rearrangements in cancer cells. These data identify APOBEC3G as a novel gene impacting genomic evolution and underlying mechanisms in MM.

MASTL regulates EGFR signaling to impact pancreatic cancer progression.

Pancreatic cancer (PC) remains a major cause of cancer-related deaths primarily due to its inherent potential of therapy resistance. Checkpoint inhibitors have emerged as promising anti-cancer agents when used in combination with conventional anti-cancer therapies. Recent studies have highlighted a critical role of the Greatwall kinase (microtubule-associated serine/threonine-protein kinase-like (MASTL)) in promoting oncogenic malignancy and resistance to anti-cancer therapies; however, its role in PC remains unknown. Based on a comprehensive investigation involving PC patient samples, murine models of PC progression (Kras;PdxCre-KC and Kras;p53;PdxCre-KPC), and loss and gain of function studies, we report a previously undescribed critical role of MASTL in promoting cancer malignancy and therapy resistance. Mechanistically, MASTL promotes PC by modulating the epidermal growth factor receptor protein stability and, thereupon, kinase signaling. We further demonstrate that combinatorial therapy targeting MASTL promotes the efficacy of the cell-killing effects of Gemcitabine using both genetic and pharmacological inhibitions. Taken together, this study identifies a key role of MASTL in promoting PC progression and its utility as a novel target in promoting sensitivity to the anti-PC therapies.

METTL3 Promotes Esophageal Squamous Cell Carcinoma Metastasis Through Enhancing GLS2 Expression.

Recent studies have identified pleiotropic roles of methyltransferase-like 3 (METTL3) in tumor progression. However, the roles of METTL3 in esophageal squamous cell carcinoma (ESCC) are still unclear. Here, we investigated the function and mechanism of METTL3 in ESCC tumorigenesis. We reported that higher METTL3 expression was found in ESCC tissues and was markedly associated with depth of invasion and poor prognosis. Loss- and gain-of function studies showed that METTL3 promoted the migration and invasion of ESCC cells in vitro. Integrated methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-Seq) analysis first demonstrated that glutaminase 2 (GLS2) was regulated by METTL3 via m6A modification. Our findings identified METTL3/GLS2 signaling as a potential therapeutic target in antimetastatic strategies against ESCC.

Activated Protein Phosphatase 2A Disrupts Nutrient Sensing Balance Between Mechanistic Target of Rapamycin Complex 1 and Adenosine Monophosphate-Activated Protein Kinase, Causing Sarcopenia in Alcohol-Associated Liver Disease.

Despite the high clinical significance of sarcopenia in alcohol-associated cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol-induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine monophosphate-activated protein kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown). Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from patients with alcohol-associated cirrhosis were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue adenosine triphosphate, indicating a "pseudofed" state. We identified protein phosphatase (PP) 2A as a key mediator of ethanol-induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A to PP2A, resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3-kinase-γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol-induced molecular and phenotypic perturbations in wild-type mice were observed in PI3Kγ-/- mice even at baseline. Importantly, overexpressing kinase-active PI3Kγ but not the kinase-dead mutant reversed ethanol-induced molecular perturbations. Our study describes the mechanistic underpinnings for ethanol-mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ-PP2A axis.

CRISPR Interference (CRISPRi) and CRISPR Activation (CRISPRa) to Explore the Oncogenic lncRNA Network.

The human genome contains thousands of long noncoding RNAs (lncRNAs), even outnumbering protein-coding genes. These molecules can play a pivotal role in the development and progression of human disease, including cancer, and are susceptible to therapeutic intervention. Evidence of biologic function, however, is still missing for the vast majority of them. Both loss-of-function (LOF) and gain-of-function (GOF) studies are therefore necessary to advance our understanding of lncRNA networks and programs driving tumorigenesis. Here, we describe a protocol to perform lncRNA's LOF or GOF studies in multiple myeloma (MM) cells, using CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) technologies, respectively. These approaches have many advantages, including applicability to large-scale genetic screens in mammalian cells and possible reversibility of modulating effects; moreover, CRISPRa offers the unique opportunity to enhance lncRNA expression at the site of transcription, with relevant biologic implications.

P.870Relationship between type of antipsychotic treatment and psychopathology in acute schizophrenia and schizoaffective patients

Much of what we know today about the molecular and cellular mechanisms underlying the development of the somatosensory cortex and patterning of the thalamocortical afferents comes from studies on the phenotypes of mice, in which a variety of genes have been altered using targeted mutations or other experimental means. Coupled with the recent advances in imaging of axons and dendrites in the cerebral cortex of living mice and rats, genetic loss of function and gain of function studies are likely to uncover many mysteries of the cortical development in general, and differentiation and patterning of the somatosensory cortex. This chapter provides an overview of the recent advances in mechanistic explanations of how the somatosensory cortex develops and the patterning of the thalamocortical axons which provide the primary sensory inputs from the sensory periphery via the dorsal column lemniscal and trigeminal pathways. Since the vast majority of the developmental studies use the mouse as a model, this species is the focus of the chapter.