Co-existing TP53 and ARID1A mutations promote aggressive endometrial tumorigenesis

TP53 and ARID1A are frequently mutated across cancer but rarely in the same primary tumor. Endometrial cancer has the highest TP53-ARID1A mutual exclusivity rate. However, the functional relationship between TP53 and ARID1A mutations in the endometrium has not been elucidated. We used genetically engineered mice and in vivo genomic approaches to discern both unique and overlapping roles of TP53 and ARID1A in the endometrium. TP53 loss with oncogenic PIK3CAH1047R in the endometrial epithelium results in features of endometrial hyperplasia, adenocarcinoma, and intraepithelial carcinoma. Mutant endometrial epithelial cells were transcriptome profiled and compared to control cells and ARID1A/PIK3CA mutant endometrium. In the context of either TP53 or ARID1A loss, PIK3CA mutant endometrium exhibited inflammatory pathway activation, but other gene expression programs differed based on TP53 or ARID1A status, such as epithelial-to-mesenchymal transition. Gene expression patterns observed in the genetic mouse models are reflective of human tumors with each respective genetic alteration. Consistent with TP53-ARID1A mutual exclusivity, the p53 pathway is activated following ARID1A loss in the endometrial epithelium, where ARID1A normally directly represses p53 pathway genes in vivo, including the stress-inducible transcription factor, ATF3. However, co-existing TP53-ARID1A mutations led to invasive adenocarcinoma associated with mutant ARID1A-driven ATF3 induction, reduced apoptosis, TP63+ squamous differentiation and invasion. These data suggest TP53 and ARID1A mutations drive shared and distinct tumorigenic programs in the endometrium and promote invasive endometrial cancer when existing simultaneously. Hence, TP53 and ARID1A mutations may co-occur in a subset of aggressive or metastatic endometrial cancers, with ARID1A loss promoting squamous differentiation and the acquisition of invasive properties.

The potential role of STAT3 In the APE1/NF-kB regulatory axis in K-rasLSL.G12D/+;Pdx-1-Cre (KC) pancreatic tumor model

APE1/Ref-1 (apurinic/apyrimidinic endonuclease-redox effector factor 1) is a multifunctional protein that has been shown to be overexpressed in multiple types of cancer. The overexpression of APE1/Ref-1 is linked to higher cancer cell survival and increased patient mortality. Furthermore, APE1/Ref-1 is a key regulator of transcription factors (TF) through redox signaling and protein-protein interaction. It is involved in proliferative and inflammatory signaling upregulated in cancer. Transcription factor NF-kB is involved in inflammatory cytokine expression and has been shown to be regulated by Ref-1. My project investigated how Ref-1 regulates NF-kB, specifically Rel-A, in a model using K-rasLSL.G12D/+; Pdx-1-Cre (KC) pancreatic tumor cells (KC3590) derived from genetically engineered mice. Additionally, I explored other TFs within the APE1/Ref-1 signaling pathway, such as STAT3, in this model. My work involved knocking down STAT3 levels within four variations of the KC3590 line. These were the KC3590/ΔNF-kB (parent) and KC3590/ΔNF-kB vector lines (vector) which contain exon deletions within the NF-kB gene rendering it nonfunctional. KC3590/13 and KC3590/15 are cell lines which are KC3590/ΔNF-B cells with functional full-length NF-kB added to the cells. Previous experiments demonstrated that the ΔNF-kB and ΔNF-kB vector lines are resistant to treatment by the specific Ref-1 inhibitors, including APX3330, which inhibit the redox signaling function of Ref-1. Initial data demonstrated that adding back functional NF-kB to the NF-kB deficient cells reestablished sensitivity to APX3330, presumably due to the reintroduction of the Ref-1 target, NF-kB. Knockdown of STAT3 expression in the ΔNF-kB and ΔNF-kB vector lines demonstrated some sensitivity to APX3330, however, in the C13/15 cell lines, no enhanced sensitivity was observed. These data support the hypothesis that NF-kB is the major TF driving the growth of KC pancreatic tumor cells. Subsequent studies will clarify further the role of APE1/Ref-1 regulation in the KC model and the relative importance of APE1/Ref-1’s target TFs.

In Vivo Metabolic Roles of G Proteins of the Gi Family Studied with Novel Mouse Models

G protein-coupled receptors (GPCRs) are the target of ~30-35% of all FDA-approved drugs. The individual members of the GPCR superfamily couple to one or more functional classes of heterotrimeric G proteins. The physiological outcome of activating a particular GPCR in vivo depends on the pattern of receptor distribution and the type of G proteins activated by the receptor. Based on the structural and functional properties of their α-subunits, heterotrimeric G proteins are subclassified into four major families: Gs, Gi/o, Gq/11, and G12/13. Recent studies with genetically engineered mice have yielded important novel insights into the metabolic roles of Gi/o-type G proteins. For example, recent data indicate that Gi signaling in pancreatic α-cells plays a key role in regulating glucagon release and whole body glucose homeostasis. Receptor-mediated activation of hepatic Gi signaling stimulates hepatic glucose production, suggesting that inhibition of hepatic Gi signaling could prove clinically useful to reduce pathologically elevated blood glucose levels. Activation of adipocyte Gi signaling reduces plasma free fatty acid levels, thus leading to improved insulin sensitivity in obese, glucose-intolerant mice. These new data suggest that Gi-coupled receptors that are enriched in metabolically important cell types represent potential targets for the development of novel drugs useful for the treatment of type 2 diabetes and related metabolic disorders.

HYPERTENSION IN RELATION TO IMMUNE SYSTEM AND WAY OF LIFE ALONG WITH TREATMENT

The objective of the review is to explain the pathophysiology, different causes and various treatments involved in hypertension. This article discusses the disease's pathogenesis, etiology, diagnosis, and immunity. This review looks at the main significant epidemiological and clinical studies on the role of several lifestyle factors in hypertension development. This review examines the numerous mechanisms that cause hypertension in order to discover new treatments. In addition, it covers the many types of hypertension therapy. According to different studies, lifestyle habits may have an impact on blood pressure levels. Moreover, the importance of chronic inflammation in hypertension and its repercussions has been confirmed in genetically engineered mice lacking components of innate and/or adaptive immunity. Immune cell depletion enhances endothelial function, lowers oxidative stress, lowers the vascular tone, and protects against renal interstitial infiltrates, salt retention, and kidney injury. Based on existing literature, there is strong evidence that lifestyle variables can affect blood pressure levels. Then, in hypertensive people, lifestyle changes can help by lowering overall cardiovascular risk and death from any cause. The involvement of immunity as a common thread in the hypertension processes of many organ systems.

TMOD-06. CREATION OF PATIENT-DERIVED LOWER GRADE GLIOMA ORGANOID MODELS FOR PERSONALIZED TREATMENT RESPONSE ASSESSMENT

Creating in vitro models of lower grade glioma represents a major challenge in neuro-oncology research. There are few such models that are tractable and widely used, which has hindered understanding of the biology of these tumors. Recently, substantial progress has been made in generating patient-derived in vitro organoid models of high grade glioma, but modeling lower grade disease remains difficult. Based on our experience creating neurosphere cultures of lower grade astrocytomas from genetically engineered mice, we hypothesized that modifying patient-derived organoid generation protocols to incorporate physiological oxygen levels would allow establishment and propagation of lower grade glioma organoids. In this study, we show that this approach supports efficient organoid model generation from primary glioma specimens across all histological subtypes and tumor grades (WHO Grade I-IV, n = 20). These organoid models retain key characteristics of their respective parental tumors, including IDH mutations and other genetic alterations, metabolite profiles, intratumoral heterogeneity, cellular composition, and cytoarchitectural features. Importantly, lower grade glioma organoids can be cultured for months and reanimated after biobanking. Our high success rate ( >90%) in establishing organoid models from primary lower grade glioma tissue samples further highlighted opportunities for treatment response assessments. To perform longitudinal measurements of therapy-induced changes in glioma organoid viability, we designed a novel, non-invasive imaging assay (termed rapid apex imaging) to determine real-time treatment response in low and high grade gliomas. We evaluated longitudinal responses of glioblastoma and IDH1 R132H-positive Grade II astrocytoma organoids to temozolomide and olaparib with and without radiation treatment. We quantified topological changes in organoid structure by building a bioinformatics tool to translate imaging data into a cellularity metric as a biomarker of organoid response. Our work unveils an effective new method to create in vitro, personalized models of lower grade glioma that supports elucidation of treatment sensitivity profiles.

Genetically engineered mice for combinatorial cardiovascular optobiology

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

Hyaluronic acid nanoparticle-encapsulated microRNA-125b repolarizes tumor-associated macrophages in pancreatic cancer

Aim: To investigate a novel strategy to target tumor-associated macrophages and reprogram them to an antitumor phenotype in pancreatic adenocarcinoma (PDAC). Methods: M2 peptides were conjugated to HA-PEG/HA-PEI polymer to form self-assembled nanoparticles with miR-125b. The efficacy of HA-PEI/PEG-M2peptide nanoparticles in pancreatic tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre genetically engineered mice was evaluated. Results: In vitro M2 macrophage-specific delivery of targeted nanoformulations was demonstrated. Intraperitoneal administration of M2-targeted nanoparticles showed preferential accumulation in the pancreas of KPC-PDAC mice and an above fourfold increase in the M1-to-M2 macrophage ratio compared with transfection with scrambled miR. Conclusion: M2-targeted HA-PEI/PEG nanoparticles with miR-125b can transfect tumor-associated macrophages in pancreatic tissues and may have implications for PDAC immunotherapy.

Elevated murine HB-EGF confers sensitivity to diphtheria toxin in EGFR-mutant lung adenocarcinoma

Conditional ablation of defined cell populations in vivo can be achieved using genetically engineered mice in which the human diphtheria toxin (DT) receptor (DTR) is placed under control of a murine tissue-specific promotor, such that delivery of diphtheria toxin selectively ablates cells expressing the high-affinity human DTR. Cells expressing only the endogenous low-affinity mouse DTR are assumed to be unaffected. Surprisingly, we found that systemic DT administration induced rapid regression of murine EGFR-mutant lung adenocarcinomas in the absence of a transgenic allele containing human DTR. DT enzymatic activity was required for tumor regression, and EGFR-mutant tumor cells were the primary targets of DT toxicity. In FVB mice, EGFR-mutant tumors upregulated expression of HB-EGF, which is the DTR in mice and humans. HB-EGF blockade with CRM197, an enzymatically inactive DT mutant, partially abrogated DT-induced tumor regression. These results suggest that elevated expression of murine HB-EGF (low-affinity DTR) confers sensitivity to DT in EGFR-mutant tumors, demonstrating a biological effect of DT in mice lacking transgenic DTR alleles and highlighting a unique vulnerability of EGFR-mutant lung cancers.

Nf1 and Sh2b3 mutations cooperate in vivo in a mouse model of juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is initiated in early childhood by somatic mutations that activate Ras signaling. While some patients have only a single identifiable oncogenic mutation, others have one or more additional alterations. Such secondary mutations, as a group, are associated with an increased risk of relapse after hematopoietic stem cell transplantation, or transformation to acute myeloid leukemia. These clinical observations suggest a cooperative effect between initiating and secondary mutations. However, the roles of specific genes in the prognosis or clinical presentation of JMML have not been described. In this study, we investigate the impact of secondary SH2B3 mutations in JMML. We find that patients with SH2B3 mutations have adverse outcomes, as well as higher white blood cell counts and hemoglobin F levels in the peripheral blood. We further demonstrate this interaction in genetically engineered mice. Deletion of Sh2b3 cooperates with conditional Nf1 deletion in a dose-dependent fashion. These studies illustrate that haploinsufficiency for Sh2b3 contributes to the severity of myeloproliferative disease and provide an experimental system for testing treatments for a high-risk cohort of JMML patients.

Hippocampal β2-GABAA receptors mediate LTP suppression by etomidate and contribute to long-lasting feedback but not feedforward inhibition of pyramidal neurons

The general anesthetic etomidate, which acts through GABAA receptors, impairs the formation of new memories under anesthesia. This study addresses the molecular and cellular mechanisms by which this occurs. Here, using a new line of genetically engineered mice carrying the GABAAR β2-N265M mutation, we tested the roles of receptors that incorporate GABAA receptor β2 vs. β3 subunits to suppression of long-term potentiation (LTP), a cellular model of learning and memory. We found that brain slices from β2-N265M mice resisted etomidate suppression of LTP, indicating that the β2-GABAARs are an essential target in this model. As these receptors are most heavily expressed by interneurons in the hippocampus, this finding supports a role for interneuron modulation in etomidate control of synaptic plasticity. Nevertheless, β2 subunits are also expressed by pyramidal neurons, so they might also contribute. Therefore, using a previously established line of β3-N265M mice, we also examined the contributions of β2- vs. β3-GABAARs to GABAA,slow dendritic inhibition, because dendritic inhibition is particularly well suited to controlling synaptic plasticity. We also examined their roles in long-lasting suppression of population activity through feedforward and feedback inhibition. We found both β2- and β3-GABAARs contribute to GABAA,slow inhibition, and that both β2- and β3-GABAARs contribute to feedback inhibition, whereas only β3-GABAARs contribute to feedforward inhibition. We conclude that modulation of β2-GABAARs is essential to etomidate suppression of LTP. Furthermore, to the extent that this occurs through GABAARs on pyramidal neurons, it is through modulation of feedback inhibition.