Gain Of Function Research Articles

The association of back pain with physical inactivity and hypothyroidism in pregnant women.

During pregnancy, structural and functional changes usually occur in the body, which has various consequences, including lower back pain (LBP) and hypothyroidism. One of the risk factors for these problems is physical inactivity. This study aimed to investigate the association of back pain and physical inactivity, weight gain, and hypothyroidism in pregnant women. In this cohort study, 420 pregnant women (26.333 ± 5.820 years old) were included. At first, participants answered this question: "Do you have any plans for pregnancy in the next month?" If the answer was yes, further evaluations were performed. The physical activity and pain intensity were measured by the International Physical Activity Questionnaire Short Form (IPAQ-S) and Visual Analogue Scale. Serum TSH was measured by automated chemiluminescence and commercial kits. Measurements were conducted before, the first, second, and third trimester of pregnancy. Women reporting LBP were less engaged in physical activities and weight gained in the second and third trimesters of pregnancy was significantly higher than pregnant women without LBP (p< 0.05). TSH level and weight gained in pregnant women with low physical activity level was significantly higher than pregnant women with moderate and high physical activity (p< 0.05) (without significant difference in TSH and BMI). The physical inactivity (before: OR: 1.11 95% CI: 0.89 to 1.22; first trimester: OR: 1.09 95% CI: 1.02 to 1.59; second trimester: OR: 0.92 95% CI: 0.87 to 1.31; third trimester: OR: 1.12 95% CI: 1.02 to 1.39), TSH (OR: 0.85 95% CI: 0.57 to 1.29), and weight gain (second trimester: OR: 0.87 95% CI: 0.92 to 1.59; third trimester: OR: 1.44 95% CI: 1.02 to 1.98; p< 0.05) did predict increased pain intensity. Using health-oriented approaches to increase physical activity and normalize thyroid function and weight gain during pregnancy can have beneficial effects on LBP.

Mis-localization of endogenous TDP-43 leads to ALS-like early-stage metabolic dysfunction and progressive motor deficits

BackgroundThe key pathological signature of ALS/ FTLD is the mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm. However, TDP-43 gain of function in the cytoplasm is still poorly understood since TDP-43 animal models recapitulating mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm are missing.MethodsCRISPR/Cas9 technology was used to generate a zebrafish line (called CytoTDP), that mis-locates endogenous TDP-43 from the nucleus to the cytoplasm. Phenotypic characterization of motor neurons and the neuromuscular junction was performed by immunostaining, microglia were immunohistochemically localized by whole-mount tissue clearing and muscle ultrastructure was analyzed by scanning electron microscopy. Behavior was investigated by video tracking and quantitative analysis of swimming parameters. RNA sequencing was used to identify mis-regulated pathways with validation by molecular analysis.ResultsCytoTDP fish have early larval phenotypes resembling clinical features of ALS such as progressive motor defects, neurodegeneration and muscle atrophy. Taking advantage of zebrafish’s embryonic development that solely relys on yolk usage until 5 days post fertilization, we demonstrated that microglia proliferation and activation in the hypothalamus is independent from food intake. By comparing CytoTDP to a previously generated TDP-43 knockout line, transcriptomic analyses revealed that mis-localization of endogenous TDP-43, rather than TDP-43 nuclear loss of function, leads to early onset metabolic dysfunction.ConclusionsThe new TDP-43 model mimics the ALS/FTLD hallmark of progressive motor dysfunction. Our results suggest that functional deficits of the hypothalamus, the metabolic regulatory center, might be the primary cause of weight loss in ALS patients. Cytoplasmic gain of function of endogenous TDP-43 leads to metabolic dysfunction in vivo that are reminiscent of early ALS clinical non-motor metabolic alterations. Thus, the CytoTDP zebrafish model offers a unique opportunity to identify mis-regulated targets for therapeutic intervention early in disease progression.

Postfilter for Dual Channel Speech Enhancement Using Coherence and Statistical Model-Based Noise Estimation.

A multichannel speech enhancement system usually consists of spatial filters such as adaptive beamformers followed by postfilters, which suppress remaining noise. Accurate estimation of the power spectral density (PSD) of the residual noise is crucial for successful noise reduction in the postfilters. In this paper, we propose a postfilter utilizing proposed a posteriori speech presence probability (SPP) and noise PSD estimators, which are based on both the coherence and the statistical models. We model the coherence-based a posteriori SPP as a simple function of the magnitude of coherence between two microphone signals and combine it with a single-channel SPP based on statistical models. The coherence-based estimator for the PSD of the noise remaining in the beamformer output in the presence of speech is derived using the pseudo-coherence considering the effect of the beamformers, which is used to construct the coherence-based noise PSD estimator. Then, the final noise PSD estimator is obtained by combining the coherence-based and statistical model-based noise PSD estimators with the proposed SPP. The spectral gain function is also modified, incorporating the proposed SPP. Experimental results demonstrate that the proposed method led to more accurate noise PSD estimation and perceptual evaluation of speech quality scores in various diffuse noise environments, and did not degrade the speech quality under the presence of directional interference, although the proposed method utilizes the coherence information.

Hypoxia inducible factor 2α promotes tolerogenic macrophage development during cardiac transplantation through transcriptional regulation of colony stimulating factor 1 receptor

Solid organ transplantation mobilizes myeloid cells, including monocytes and macrophages, which are central protagonists of allograft rejection. However, myeloid cells can also be functionally reprogrammed by perioperative costimulatory blockade to promote a state of transplantation tolerance. Transplantation tolerance holds promise to reduce complications from chronic immunosuppression and promote long-term survival in transplant recipients. We sought to identify different mediators of transplantation tolerance by performing single-cell RNA sequencing of acute rejecting or tolerized cardiac allografts. This led to the unbiased identification of the transcription factor, hypoxia inducible factor (HIF)-2α, in a subset of tolerogenic monocytes. Using flow cytometric analyses and mice with conditional loss or gain of function, we uncovered that myeloid cell expression of HIF-2α was required for costimulatory blockade-induced transplantation tolerance. While HIF-2α was dispensable for mobilization of tolerogenic monocytes, which were sourced in part from the spleen, it promoted the expression of colony stimulating factor 1 receptor (CSF1R). CSF1R mediates monocyte differentiation into tolerogenic macrophages and was found to be a direct transcriptional target of HIF-2α in splenic monocytes. Administration of the HIF stabilizer, roxadustat, within micelles to target myeloid cells, increased HIF-2α in splenic monocytes, which was associated with increased CSF1R expression and enhanced cardiac allograft survival. These data support further exploration of HIF-2α activation in myeloid cells as a therapeutic strategy for transplantation tolerance.

Elucidating the Role of Human ALAS2 C-terminal Mutations Resulting in Loss of Function and Disease.

The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme in vitro, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.

Hyper-methylation and DNMT3A mediated LTC4S downregulation promoted lung adenocarcinoma tumorigenesis via mTORC1 signaling pathway

BackgroundLung adenocarcinoma is a malignancy characterized by high mortality rates and unfavorable prognosis. However, the role of Leukotriene C4 Synthase (LTC4S) in lung cancer remains uninvestigated. MethodsThe expression and prognostic value of LTC4S in LUAD were analyzed using the GEPIA online database. Subsequently, the function of LTC4S in lung cancer cells was examined through gain-of function experiments, using assays to evaluate tumor malignant behavior. Subcutaneous xenograft experiments in vivo was used for investigating the functions of LTC4S. Then, tumor hallmark pathways were analyzed by GSEA. Western blot assay was used to validate the impact of LTC4S on mTORC1 pathway. Finally, the correlation of mRNA and methylation of LTC4S were analyzed by cBioPortal. qRT-PCR, ChIP-qPCR and ChIP-Atlas were used to verify the regulation factors of LTC4S low expression in LUAD cells. ResultsLTC4S presented significant decreased expression and favorable prognostic significance in LUAD. LTC4S was correlated with clinical stages in LUAD, which showed decreased expression gradually and significantly along with TNM stages. LTC4S-co-expressed genes were closely related to Ras signaling pathway, and MAPK signaling pathway. Overexpression of LTC4S inhibited cancer malignant phenotype and tumor growth in vitro and vivo. GSEA analysis and Western blot assay suggested low expression of LTC4S activated mTORC1 signaling pathway in LUAD. Moreover, the DNA methylation level of LTC4S in LUAD tissue was markedly elevated compared to normal tissue. The hypermethylation of the LTC4S promoter by DNMT3A leads to the decreased expression of LTC4S in LUAD. ConclusionsIn conclusion, low expression of LTC4S serves as an unfavorable prognostic marker and the critical function of LTC4S in controlling the progression of LUAD. This highlights the promise for exploring the clinical benefits of manipulating LTC4S in LUAD targeted therapies.

COVID-19 and silent hypoxemia in a minimal closed-loop model of the respiratory rhythm generator

Silent hypoxemia, or “happy hypoxia,” is a puzzling phenomenon in which patients who have contracted COVID-19 exhibit very low oxygen saturation (SaO2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\ ext {SaO}_2$$\\end{document} < 80%) but do not experience discomfort in breathing. The mechanism by which this blunted response to hypoxia occurs is unknown. We have previously shown that a computational model of the respiratory neural network (Diekman et al. in J Neurophysiol 118(4):2194–2215, 2017) can be used to test hypotheses focused on changes in chemosensory inputs to the central pattern generator (CPG). We hypothesize that altered chemosensory function at the level of the carotid bodies and/or the nucleus tractus solitarii are responsible for the blunted response to hypoxia. Here, we use our model to explore this hypothesis by altering the properties of the gain function representing oxygen sensing inputs to the CPG. We then vary other parameters in the model and show that oxygen carrying capacity is the most salient factor for producing silent hypoxemia. We call for clinicians to measure hematocrit as a clinical index of altered physiology in response to COVID-19 infection.

Thyroid disfunction and obesity - what comes first? A complex relationship between hypothyroidism and weight gain

Introduction: Obesity and mild thyroid failure are common diseases, and the complex relationship between thyroid function and excess weight gain is still being researched. This study analyzes and summarizes the current scientific reports on the intricate correlation between thyroid dysfunction and obesity. Material and methods: We have gathered the available materials and scientific reports, analyzing and summarizing them in a single study. Aim of study: We aimed to summarize the studies conducted so far by analyzing the available scientific reports to answer the question of ‘what comes first’ and to understand the correlation between thyroid dysfunction and excessive weight gain. Discussion: Thyroid dysfunction in the form of hypothyroidism is associated with metabolic abnormalities, which may play a crucial role in the development of metabolic syndrome. On the other hand, excess level of leptin associated with excess adipose tissue is a potential cause of disruptions in the hypothalamic-pituitary-thyroid axis, manifested as elevated TSH levels. Therefore, it is worth considering certain aspects regarding the inclusion of hormone replacement therapy in obese patients presenting laboratory features of subclinical hypothyroidism. Conclusion. Obesity-induced thyroid dysfunction is typically reversible with weight loss and hormone replacement therapy should be considered for obese patients with subclinical hypothyroidism only if thyroid antibodies are present to confirm an autoimmune thyroid disorder.

Targeting metabolic reprogramming to overcome drug resistance in advanced bladder cancer: insights from gemcitabine- and cisplatin-resistant models.

Gemcitabine plus cisplatin (GC) combination chemotherapy is the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease. However, most cases develop resistance to this therapy. We investigated whether drug resistance could be targeted through metabolic reprogramming therapies. Metabolomics analyses in our lab's gemcitabine- and cisplatin-resistant cell lines revealed increased phosphoglycerate dehydrogenase (PHGDH) expression in gemcitabine-resistant cells compared with parental cells. Isocitrate dehydrogenase 2 (IDH2) gain of function stabilized hypoxia-inducible factor1α (HIF1α) expression, stimulating aerobic glycolysis. In gemcitabine-resistant cells, elevated fumaric acid suppressed prolyl hydroxylase domain-containing protein 2/Egl nine homolog 1 (PHD2) and stabilized HIF1α expression. PHGDH downregulation or inhibition in gemcitabine-resistant BC cells inhibited their proliferation, migration, and invasion. Cisplatin-resistant cells showed elevated fatty acid metabolism, upregulating fatty acid synthase (FASN) downstream of tyrosine kinase. Using the fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor erdafitinib, we inhibited malonyl-CoA production, which is crucial for fatty acid synthesis, and thereby suppressed upregulated HIF1α expression. Combination treatment with NCT503 and erdafitinib synergistically suppressed tumor cell proliferation and induced apoptosis in vitro and in vivo. Understanding these mechanisms could enable innovative BC therapeutic strategies to be developed.

Staphylococcal peroxidase inhibitor (SPIN): Investigation of the inhibitory N-terminal domain via a stabilizing disulfide insertion

Staphylococcus aureus secretes an array of small proteins that inhibit key enzyme-catalyzed reactions necessary for proper function of the human innate immune system. Among these, the Staphylococcal Peroxidase Inhibitor, SPIN, blocks the activity of myeloperoxidase (MPO) and thereby disrupts the HOCl-generating system of neutrophils. Previous studies on S. aureus SPIN have shown that it relies on a C-terminal α-helical bundle domain to mediate initial binding to MPO, but requires a disordered N-terminal region to fold into a β-hairpin conformation to inhibit MPO activity. To further investigate the structure/function relationship of SPIN, we introduced two cysteine residues into its N-terminal region to trap SPIN in its MPO-bound conformation and characterized the modified protein, which we refer to here as SPIN-CYS. Although control experiments confirmed the presence of the disulfide bond in SPIN-CYS, solution structure determination revealed that the N-terminal region of SPIN-CYS adopted a physically constrained series of lariat-like structures rather than a well-defined β-hairpin. Nevertheless, SPIN-CYS exhibited a gain in inhibitory potency against human MPO when compared to wild-type SPIN. This gain of function persisted even in the presence of deleterious mutations within the C-terminal α-helical bundle domain. Surface plasmon resonance studies showed that the gain in potency arose through an increase in apparent affinity of SPIN-CYS for MPO, which was driven primarily by an increased association rate with MPO when compared to wild-type SPIN. Together, this work provides new information on the coupled binding and folding events required to manifest biological activity of this unusual MPO inhibitor.

Development of SYK NanoBRET Cellular Target Engagement Assays for Gain-of-Function Variants.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y).

Identification and Properties of TRPV4 Mutant Channels Present in Polycystic Kidney Disease Patients.

Polycystic kidney disease (PKD), a disease characterized by the enlargement of the kidney through cystic growth is the fourth leading cause of end-stage kidney disease world-wide. Transient receptor potential Vanilloid 4 (TRPV4), a calcium-permeable TRP, channel participates in kidney cell physiology and since TRPV4 forms complexes with another channel whose malfunction is associated to PKD, TRPP2 (or PKD2), we sought to determine whether patients with PKD, exhibit previously unknown mutations in TRPV4. Here, we report the presence of mutations in the TRPV4 gene in patients diagnosed with PKD and determine that they produce gain-of-function (GOF). Mutations in the sequence of the TRPV4 gene have been associated to a broad spectrum of neuropathies and skeletal dysplasias but not PKD, and their biophysical effects on channel function have not been elucidated. We identified and examined the functional behavior of a novel E6K mutant and of the previously known S94L and A217S mutant TRVP4 channels. The A217S mutation has been associated to mixed neuropathy and/or skeletal dysplasia phenotypes, however, the PKD carriers of these variants had not been diagnosed with these reported clinical manifestations. The presence of certain mutations in TRPV4 may influence the progression and severity of PKD through GOF mechanisms. PKD patients carrying TRVP4 mutations are putatively more likely to require dialysis or renal transplant as compared to those without these mutations.

Event-triggered prescribed-time control for a class of uncertain nonlinear systems using finite time-varying gain

This paper addresses the event-triggered prescribed-time control problem for a class of high-order nonlinear systems based on finite time-varying gain. Due to the existence of unknown gain functions and system uncertainties, the resultant control with prescribed-time convergence performance becomes nontrivial. The problem becomes even more complex due to the use of event-based communication instead of continuous communication. To tackle the aforementioned challenges, this paper proposes an event-triggered prescribed-time stabilization approach with the following key steps. Firstly, we establish a new prescribed-time stability lemma to overcome the technical difficulty arising from the prescribed-time controller design, and stability analysis. Secondly, we give the controller design procedure upon using the backstepping technique. Thirdly, we redesign the event-triggering threshold condition based on time-varying functions, which allows the controller terminal jitter to be mitigated and the controller to be implemented straightforwardly in practice. Furthermore, the proposed control scheme avoids Zeno behavior. The numerical simulation confirms the effectiveness of the proposed control scheme.

Abstract A020: TP53 mutation and prediction of platinum response in BRCA-mutated ovarian cancer: A prospective case-series analysis

Abstract Background and aim of the study: Currently, platinum sensitivity (PS) is a prerequisite for first-line PARP inhibitors (PARPi) in locally advanced and relapsed high grade serous ovarian cancer (HGS-OC). BRCA mutations are recognized as predictive of PS and, therefore, of response to PARPi . Notably, platinum and PARPi cytotoxic action is mainly related by their ability of inducing p53-mediated apoptosis. Therefore, the integrity of p53 machinery is crucial for platinum-related activity whereas the presence of p53 mutation is a fairly frequent event in ovarian cancer, particularly in HGS-OC and in BRCA mutated ones. Patients and methods: We prospectively analyzed 208 women with primary ovarian cancer undergoing surgery at the Department of Gynecologic Oncology, ARNAS G. Brotzu, Cagliari, Italy, between 2019 and 2023. Somatic NGS analysis was performed to detect BRCA and HRD mutations. TP53 mutations were classified according to according to hotspot, structural (missense or nonsense) and functional classification as “gain of function” (GOF) or “loss of function” (LOF), based on the IARC TP53 database. Comparative testing with Fisher's exact test was used to examine TP53 mutation distribution and associations with clinicopathologic factors and PS. The BRCA mutation status was further used to stratify the analysis. Results: Globally, we included 127 adult HGS-OC pts. 84.2% had stage III-IV disease. TP53 mutation was found in 83.4 % of the entire cohort. Somatic BRCA mutations were found in 28.3% pts. Overall, HGS-OC with somatic BRCA mutations had higher TP53 mutation frequency (88.8%) when compared to BRCA WT (81.3%, p=0.1510). Employing the structural classification scheme, most cancers harbored a missense TP53 mutation (76.5%). LOF TP53 mutations were found in 59.4% while GOF in 31.2%. No significant disparity was observed in the distribution of specific TP53 mutations within each classification scheme between cases with BRCA mutations and those without. As for BRCA mutated pts, TP53 WT were all PS. Among those p53 mutated, GOF mutations were associated with PS in 7 pts and platinum resistance in 3 pts; LOF mutations were associated with PS in 7 pts and platinum resistance in 12 pts. The difference in distribution of PS between functional categories of p53 mutations was significant (p=0.0291). As for BRCA WT pts, TP53 WT were all PS. Among TP53 mutated, GOF mutations were associated with PS in 14 pts and platinum resistance in 10 pts; viceversa, LOF mutations were associated with PS in 19 pts and platinum resistance in 25 pts, even if these findings were not statistically significant (p=0.2357). Of relevance, in 5 cases where LOF mutations of p53 was associated with null expression of HIC p53, patients were refractory to platinum-based chemotherapy. Conclusions: Even if preliminary, our data show that HGS-OC harboring p53 null mutations are the poorest prognostic subgroup, especially in terms of PS. Further studies are needed to confirm our findings and the role of TP53 mutation as a biomarker of inherent or acquired platinum resistance. Citation Format: Eleonora Lai, Manuela Neri, Elisabetta Sanna, Sonia Nemolato, Fabio Bardanzellu, Mario Scartozzi, Sabrina Giglio, Antonio Macciò, Clelia Madeddu. TP53 mutation and prediction of platinum response in BRCA-mutated ovarian cancer: A prospective case-series analysis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A020.

Abstract B008: Utilizing pathway incompatibility for synthetic lethality: A therapeutic strategy for B-cell lymphoma

Abstract Previous pan-cancer analyses revealed co-occurring mutations exploitable for combination therapies and mutually exclusive mutations assumed to reflect functional redundancy (Mina M et al. 2017; Sanchez-Vega F et al. 2018). However, we recently highlighted an alternative scenario when mutual exclusivity reflects the incapability of two pathways to function together, a phenomenon we term “pathway incompatibility” (Chan LN et al. 2020). We found that STAT5-and ERK-activating genetic lesions were not only mutually exclusive in B-ALL cases, but also reciprocally inhibited each other at the signal transduction level, inducing cell death instead of promoting leukemogenesis. Notably, we demonstrated, for the first time, that pharmacological activation of STAT5 in ERK-driven B-ALL cells and ERK in STAT5-driven B-ALL cells represents a tractable synthetic lethality (Chan LN et al. 2020). We now take the important next step of identifying potential candidates for synthetic lethal genetic interactions resulting from pathway incompatibility in B-cell malignancies more broadly. Diffuse large B-cell lymphoma (DLBCL) is a rapidly growing blood cancer affecting older adults and constituting about 25% of all lymphoma cases in the US. Around 40% of patients develop relapsed or refractory DLBCL (Pfreundschuh M et al. 2006; Ngu H et al. 2022). Thus, there is an unmet need to develop new therapeutic strategies to improve clinical outcomes. Studying 1684 DLBCL cases from published datasets, we found that about 30% of DLBCL cases carry loss-of-function (LOF) CBP or gain-of-function (GOF) MYD88 genetic lesions. CBP encodes a histone acetyltransferase and, together with p300, functions as a transcriptional coactivator and tumor suppressor. Conversely, GOF MYD88 mutations drive lymphomagenesis by regulating kinases and transcription factors. Our analysis revealed that LOF CBP and GOF MYD88 genetic lesions co-occurred less frequently than expected by chance (odds ratio: 0.57, P=0.0), indicating their mutual exclusivity. Experiments with human DLBCL cell lines expressing wild-type MYD88 and CBP showed that CBP/p300 inhibitors did not impact cell viability. However, cells expressing a GOF MYD88 mutant or stimulated with a toll-like receptor (TLR) agonist to activate MYD88 became sensitized to CBP/p300 inhibitors, resulting in cell death. This suggests that LOF CBP and GOF MYD88 are incompatible for cell survival, with their co-occurrence resulting in synthetic lethality. We then tested a synthetic lethality-based therapeutic concept based on CBP-MYD88 incompatibility in DLBCL. We found that MYD88-mutated DLBCL cell lines were more sensitive to pharmacological inhibition of CBP/p300, while CBP-deficient DLBCL cell lines showed increased sensitivity to TLR agonist treatment. In summary, we identified mutual exclusivity between LOF CBP and GOF MYD88 lesions in DLBCL, where their co-occurrence induces cell death. Moreover, we demonstrated the therapeutic potential of synthetic lethality resulting from co-introduction of LOF CBP and GOF MYD88 in DLBCL. Citation Format: Mia Knupke, Lai Chan. Utilizing pathway incompatibility for synthetic lethality: A therapeutic strategy for B-cell lymphoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B008.

An Integrative Transcriptome Subtraction Strategy to Identify Human lncRNAs That Specifically Play a Role in Activation of Human Hepatic Stellate Cells.

Fibrotic liver features excessive deposition of extracellular matrix (ECM), primarily produced from "activated" hepatic stellate cells (HSCs). While targeting human HSCs (hHSCs) in fibrosis therapeutics shows promise, the overall understanding of hHSC activation remains limited, in part because it is very challenging to define the role of human long non-coding RNAs (lncRNAs) in hHSC activation. To address this challenge, we identified another cell type that acts via a diverse gene network to promote fibrogenesis. Then, we identified the lncRNAs that were differentially regulated in activated hHSCs and the other profibrotic cell. Next, we conducted concurrent analysis to identify those lncRNAs that were specifically involved in fibrogenesis. We tested and confirmed that transdifferentiation of vascular smooth muscle cells (VSMCs) represents such a process. By overlapping TGFβ-regulated lncRNAs in multiple sets of hHSCs and VSMCs, we identified a highly selected list of lncRNA candidates that could specifically play a role in hHSC activation. We experimentally characterized one human lncRNA, named CARMN, which was significantly regulated by TGFβ in all conditions above. CARMN knockdown significantly reduced the expression levels of a panel of marker genes for hHSC activation, as well as the levels of ECM deposition and hHSC migration. Conversely, gain of function of CARMN using CRISPR activation (CRISPR-a) yielded the completely opposite effects. Taken together, our work addresses a bottleneck in identifying human lncRNAs that specifically play a role in hHSC activation and provides a framework to effectively select human lncRNAs with significant pathophysiological role.

Effects of protein-enriched nutritional support on skeletal muscle mass and rehabilitative outcomes in brain tumor patients: a randomized controlled trial

Patients with brain tumors require extensive and prolonged rehabilitation efforts as they suffer from lesion-induced motor weakness as well as treatment-related side effects, often leading to a significant decline in function. Protein supplements have shown positive effects on promoting muscle strength and physical performance in various tumor etiologies. However, reports on their effects specifically in brain tumor patients remain scarce. This study aims to investigate the feasibility and efficacy of protein supplements in enhancing rehabilitative outcomes via muscle strengthening and functional gain in brain tumor patients with neurological demise. Sixty brain tumor patients were randomly assigned to either a protein supplement or a control group, receiving either protein supplements or a placebo for 6 weeks, in conjunction with conventional rehabilitation therapy. Assessments before and after the intervention included laboratory tests, anthropometric measures using bioimpedance analysis, and functional assessments, which included the MMSE, the modified Barthel Index, the Beck Depression Inventory, the Brief Fatigue Inventory, the Timed Up and Go test, the 6-min walk test, the isokinetic quadriceps muscle strength test, and the handgrip power. After the intervention, the levels of serum hemoglobin, protein, albumin, and C-reactive protein were improved in both groups, however, the change was significant only in the protein group. The muscle strength was enhanced in both groups, however, the significant increase in pinch grasp power was only noted in the protein group (P < 0.05). The distance on 6MWT was also significantly extended at follow-up in the protein group (P < 0.01). In the subgroup analysis according to nutritional status, the moderate malnutrition group showed greater augmentation of muscle mass than those with adequate nutrition (P < 0.05). Interestingly, the amelioration of malnutrition was observed only the in protein group. This study using protein supplements to promote the rehabilitative potential of brain tumor patients revealed a significant effect on improving hemodynamic nutritional indices, muscle power reimbursement, and functional improvement, especially in malnourished patients. The safety and feasibility of protein supplements in brain tumor patients were affirmative in this study. Further studies with more patients may help confirm the secondary functional gain resulting from increased muscle power.Trial registration: This study was retrospectively registered in the Clinical Research Information Service, CRIS no. KCT0009113 on Jan 12, 2024.

Gene replacement therapy for spinal muscular atrophy: safety and preliminary efficacy in a Brazilian cohort.

Spinal muscular atrophy (SMA) is a motor neuron disease associated with progressive muscle weakness, ventilatory failure, and reduced survival. Onasemnogene abeparvovec is the first gene replacement therapy (GT) approved to treat this condition. An observational retrospective study was conducted to assess adverse events and efficacy of GT in SMA patients. Forty-one patients with SMA (58.5% females and 80.1% SMA type 1) were included. The mean age at GT dosing was 18 (±6.4) months. Thirty-six patients (87.8%) were under previous treatment with nusinersen, and 10 (24.4%) continued nusinersen after GT. Mean CHOP-INTEND increased 13 points after 6 months and this finding did not differ between groups according to nusinersen maintenance after GT (p = 0.949). Among SMA type 1 patients, 14 (46.6%) reached the ability to sit alone. Liver transaminases elevation at least two times higher than the upper limit of normal value occurred in 29 (70.7%) patients. Thrombocytopenia occurred in 13 (31.7%) patients, and one presented thrombotic microangiopathy. Older age (>2 years) was associated with more prolonged use of corticosteroids (p = 0.021). GT is effective in SMA patients, combined nusinersen after GT did not appear to add gain in motor function and older age is associated with prolonged corticosteroid use.

Disturbance in cerebral blood microcirculation and hypoxic-ischemic microenvironment are associated with the development of brain metastasis.

Brain metastases (BM) constitute an increasing challenge in oncology due to their impact on neurological function, limited treatment options, and poor prognosis. BM occur through extravasation of circulating tumor cells across the blood-brain barrier. However, the extravasation processes are still poorly understood. We here propose a brain colonization process which mimics infarction-like microenvironmental reactions, that is dependent on Angiopoietin (Ang-2) and vascular endothelial growth factor (VEGF). In this study, intracardiac BM models were used, and cerebral blood microcirculation was monitored by 2-photon microscopy through a cranial window. BM formation was observed using cranial magnetic resonance, bioluminescent imaging, and post-mortem autopsy. Ang-2/VEGF targeting strategies and Ang-2 gain-of-function (GOF) mice were employed to interfere with BM formation. In addition, vascular and stromal factors as well as clinical outcome were analyzed in BM patients. Blood vessel occlusions by cancer cells were detected, accompanied by significant disturbances of cerebral blood microcirculation, and focal stroke-like histological signs. Cerebral endothelial cells showed an elevated Ang-2 expression both in mouse and human BM. Ang-2 GOF resulted in an increased BM burden. Combined anti-Ang-2/anti-VEGF therapy led to a decrease in brain metastasis size and number. Ang-2 expression in tumor vessels of established human brain metastases negatively correlated with survival. Our observations revealed a relationship between disturbance of cerebral blood microcirculation and brain metastasis formation. This suggests that vessel occlusion by tumor cells facilitates brain metastatic extravasation and seeding, while combined inhibition of microenvironmental effects of Ang-2 and VEGF prevent the outgrowth of macrometastases.

Hif3α Plays Key Roles in the Progression of Alzheimer's Disease Caused by Circadian Rhythm Disruption through Regulating the m6A/KDM3A/TGF-β1 Axis.

Disrupted circadian rhythms are associated with the onset of chronic diseases and impairments, including cancer, diabetes, and hypertension. However, whether circadian disruptions accelerate the progression of Alzheimer's disease and the respective pathway remains unclear. In this study, we constructed animal models using male C57BL/6N and APP/PS1 mice. Irregular illumination during sleeping hours was administered to the mice in our intervention groups to consistently disrupt their circadian rhythms. The impact of the intervention was evaluated through body weight tracking, cerebral index determination, histopathological staining, and biochemical marker analysis. Transcriptomic sequencing identified critical genes, with the data subsequently validated using RNA m6A detection and site analysis. The evaluations revealed that circadian disruptions impaired normal weight gain, liver and kidney functions, neuronal cells, and overall brain function. Transcriptomic sequencing data revealed a trend of elevating expression of Hif3α mRNA in the intervention groups. Further analysis of specific gene sites revealed that m6A methylation of the Hif3α gene at m6A site 3632 primarily drove the observed variations in HIF3A protein expression in our model. Furthermore, the expression of proteins in PC12 cells, N2a cells, and mice brains validated that an increase in HIF3A expression decreased KDM3A and TGF-β1 protein expression. Our study reveals a hitherto unknown pathway through which the disruption of circadian rhythms, by triggering m6A methylation at m6A site 3632 in the Hif3α gene, leads to the initiation and acceleration of AD. These findings provide valuable insights and guidelines for treating AD patients and enhancing caregiving by professionals.