Isoform-specific Roles Research Articles

Endothelial Rho kinase controls blood vessel integrity and angiogenesis.

The Rho kinases 1 and 2 (ROCK1/2) are serine-threonine specific protein kinases that control actin cytoskeleton dynamics. They are expressed in all cells throughout the body, including cardiomyocytes, smooth muscle cells and endothelial cells, and intimately involved in cardiovascular health and disease. Pharmacological ROCK inhibition is beneficial in mouse models of hypertension, atherosclerosis, and neointimal thickening that display overactivated ROCK. However, the consequences of endothelial ROCK signaling deficiency in vivo remain unknown. To address this issue, we analyzed endothelial cell (EC) specific ROCK1 and 2 deletions. We generated Cdh5-CreERT2 driven, tamoxifen inducible loss of function alleles of ROCK1 and ROCK2 and analyzed mouse survival and vascular defects through cellular, biochemical, and molecular biology approaches. We observed that postnatal or adult loss of endothelial ROCK1 and 2 was lethal within a week. Mice succumbed to multi-organ hemorrhage that occurred because of loss of vascular integrity. ECs displayed deficient cytoskeletal actin polymerization that prevented focal adhesion formation and disrupted junctional integrity. Retinal sprouting angiogenesis was also perturbed, as sprouting vessels exhibited lack of polymerized actin and defective lumen formation. In a three-dimensional endothelial sprouting assay, combined knockdown of ROCK1/2 or knockdown or ROCK2 but not ROCK1 led to reduced sprouting, lumenization and cell polarization defects caused by defective actin and altered VE-cadherin dynamics. The isoform specific role of endothelial ROCK2 correlated with ROCK2 substrate specificity for FAK and LIMK. By analyzing single and three allele mutants we show that one intact allele of ROCK2 is sufficient to maintain vascular integrity in vivo . Endothelial ROCK1 and 2 maintain junctional integrity and ensure proper angiogenesis and lumen formation. The presence of one allele of ROCK2 is sufficient to maintain vascular growth and integrity. These data indicate the need of careful consideration for the use of ROCK inhibitors in disease settings.

Animal models of haploinsufficiency revealed the isoform-specific role of GSK-3 in HFD-induced obesity and glucose intolerance.

Glycogen synthase kinase 3 (GSK-3), a serine-threonine kinase with two isoforms (α and β) is implicated in the pathogenesis of type 2 diabetes mellitus (T2D). Recently, we reported the isoform-specific role of GSK-3 in T2D using homozygous GSK-3α/β knockout mice. Although the homozygous inhibition models are idealistic in a preclinical setting, they do not mimic the inhibition seen with pharmacological agents. Hence, in this study, we sought to investigate the dose-response effect of GSK-3α/β inhibition in the pathogenesis of obesity-induced T2D. Specifically, to gain insight into the dose-response effect of GSK-3 isoforms in T2D, we generated tamoxifen-inducible global GSK-3α/β heterozygous mice. GSK-3α/β heterozygous and control mice were fed a high-fat diet (HFD) for 16 wk. At baseline, the body weight and glucose tolerance of GSK-3α heterozygous and controls were comparable. In contrast, at baseline, a modest but significantly higher body weight (higher lean mass) was seen in GSK-3β heterozygous compared with controls. Post-HFD, GSK-3α heterozygous and controls displayed a comparable phenotype. However, GSK-3β heterozygous were significantly protected against obesity-induced glucose intolerance. Interestingly, the improved glucose tolerance in GSK-3β heterozygous animals was dampened with chronic HFD-feeding, likely due to significantly higher fat mass and lower lean mass in the GSK-3β animals. These findings suggest that GSK-3β is the dominant isoform in glucose metabolism. However, to avail the metabolic benefits of GSK-3β inhibition, it is critical to maintain a healthy weight.NEW & NOTEWORTHY The precise isoform-specific role of GSK-3 in obesity-induced glucose intolerance is unclear. To overcome the limitations of pharmacological GSK-3 inhibitors (not isoform-specific) and tissue-specific genetic models, in the present study, we created novel inducible heterozygous mouse models of GSK-3 inhibition that allowed us to delete the gene globally in an isoform-specific and temporal manner to determine the isoform-specific role of GSK-3 in obesity-induced glucose intolerance.

Suppression of neuronal AMPKβ2 isoform impairs recognition memory and synaptic plasticity

AMP-activated protein kinase (AMPK) is an αβγ heterotrimer protein kinase that functions as a molecular sensor to maintain energy homeostasis. Accumulating evidence suggests a role of AMPK signaling in the regulation of synaptic plasticity and cognitive function; however, isoform-specific roles of AMPK in the central nervous system (CNS) remain elusive. Regulation of the AMPK activities has focused on the manipulation of the α or γ subunit. Meanwhile, accumulating evidence indicates that the β subunit is critical for sensing nutrients such as fatty acids and glycogen to control AMPK activity. Here, we generated transgenic mice with conditional suppression of either AMPKβ1 or β2 in neurons and characterized potential isoform-specific roles of AMPKβ in cognitive function and underlying mechanisms. We found that AMPKβ2 (but not β1) suppression resulted in impaired recognition memory, reduced hippocampal synaptic plasticity, and altered structure of hippocampal postsynaptic densities and dendritic spines. Our study implicates a role for the AMPKβ2 isoform in the regulation of synaptic and cognitive function.

CARD8: A Novel Inflammasome Sensor with Well-Known Anti-Inflammatory and Anti-Apoptotic Activity.

Inflammasomes comprise a group of protein complexes with fundamental roles in the induction of inflammation. Upon sensing stress factors, their assembly induces the activation and release of the pro-inflammatory cytokines interleukin (IL)-1β and -18 and a lytic type of cell death, termed pyroptosis. Recently, CARD8 has joined the group of inflammasome sensors. The carboxy-terminal part of CARD8, consisting of a function-to-find-domain (FIIND) and a caspase activation and recruitment domain (CARD), resembles that of NLR family pyrin domain containing 1 (NLRP1), which is recognized as the main inflammasome sensor in human keratinocytes. The interaction with dipeptidyl peptidases 8 and 9 (DPP8/9) represents an activation checkpoint for both sensors. CARD8 and NLRP1 are activated by viral protease activity targeting their amino-terminal region. However, CARD8 also has some unique features compared to the established inflammasome sensors. Activation of CARD8 occurs independently of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), leading mainly to pyroptosis rather than the activation and secretion of pro-inflammatory cytokines. CARD8 was also shown to have anti-inflammatory and anti-apoptotic activity. It interacts with, and inhibits, several proteins involved in inflammation and cell death, such as the inflammasome sensor NLRP3, CARD-containing proteins caspase-1 and -9, nucleotide-binding oligomerization domain containing 2 (NOD2), or nuclear factor kappa B (NF-κB). Single nucleotide polymorphisms (SNPs) of CARD8, some of them occurring at high frequencies, are associated with various inflammatory diseases. The molecular mechanisms underlying the different pro- and anti-inflammatory activities of CARD8 are incompletely understood. Alternative splicing leads to the generation of multiple CARD8 protein isoforms. Although the functional properties of these isoforms are poorly characterized, there is evidence that suggests isoform-specific roles. The characterization of the functions of these isoforms, together with their cell- and disease-specific expression, might be the key to a better understanding of CARD8's different roles in inflammation and inflammatory diseases.

G272V and P301L Mutations Induce Isoform Specific Tau Mislocalization to Dendritic Spines and Synaptic Dysfunctions in Cellular Models of 3R and 4R Tau Frontotemporal Dementia.

Tau pathologies are detected in the brains of some of the most common neurodegenerative diseases including Alzheimer's disease (AD), Lewy body dementia (LBD), chronic traumatic encephalopathy (CTE), and frontotemporal dementia (FTD). Tau proteins are expressed in six isoforms with either three or four microtubule-binding repeats (3R tau or 4R tau) due to alternative RNA splicing. AD, LBD, and CTE brains contain pathological deposits of both 3R and 4R tau. FTD patients can exhibit either 4R tau pathologies in most cases or 3R tau pathologies less commonly in Pick's disease, which is a subfamily of FTD. Here, we report the isoform-specific roles of tau in FTD. The P301L mutation, linked to familial 4R tau FTD, induces mislocalization of 4R tau to dendritic spines in primary hippocampal cultures that were prepared from neonatal rat pups of both sexes. Contrastingly, the G272V mutation, linked to familial Pick's disease, induces phosphorylation-dependent mislocalization of 3R tau but not 4R tau proteins to dendritic spines. The overexpression of G272V 3R tau but not 4R tau proteins leads to the reduction of dendritic spine density and suppression of mEPSCs in 5-week-old primary rat hippocampal cultures. The decrease in mEPSC amplitude caused by G272V 3R tau is dynamin-dependent whereas that caused by P301L 4R tau is dynamin-independent, indicating that the two tau isoforms activate different signaling pathways responsible for excitatory synaptic dysfunction. Our 3R and 4R tau studies here will shed new light on diverse mechanisms underlying FTD, AD, LBD, and CTE.

Isoform-specific effects of neuronal inhibition of AMPK catalytic subunit on LTD impairments in a mouse model of Alzheimer’s disease

Synaptic dysfunction is highly correlated with cognitive impairments in Alzheimer’s disease (AD), the most common dementia syndrome in the elderly. Long-term potentiation (LTP) and long-term depression (LTD) are two primary forms of synaptic plasticity with opposite direction of synaptic efficiency change. Both LTD and LTD are considered to mediate the cellular process of learning and memory. Substantial studies demonstrate AD-associated deficiency of both LTP and LTD. Meanwhile, the molecular signaling mechanisms underlying impairment of synaptic plasticity, particularly LTD, are poorly understood. By taking advantage of the novel transgenic mouse models recently developed in our lab, here we aimed to investigate the roles of AMP-activated protein kinase (AMPK), a central molecular senor that plays a critical role in maintaining cellular energy homeostasis, in regulation of LTD phenotypes in AD. We found that brain-specific suppression of the AMPKα1 isoform (but not AMPKα2 isoform) was able to alleviate mGluR-LTD deficits in APP/PS1 AD mouse model. Moreover, suppression of either AMPKα isoform failed to alleviate AD-related NMDAR-dependent LTD deficits. Taken together with our recent studies on roles of AMPK signaling in AD pathophysiology, the data indicate isoform-specific roles of AMPK in mediating AD-associated synaptic and cognitive impairments.

Discovery of Small Molecule Glycolytic Stimulants for Enhanced ApoE Lipidation in Alzheimer's Disease Cell Model.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by pathophysiological deposits of extracellular amyloid beta (Aβ) peptides and intracellular neurofibrillary tangles of tau. The central role of Aβ in AD pathology is well-established, with its increased deposition attributed mainly to its decreased cerebral clearance. Here, it is noteworthy that apolipoprotein E (ApoE), the most significant risk factor for AD, has been shown to play an isoform-specific role in clearing Aβ deposits (ApoE2 > ApoE3 > ApoE4), owing mainly to its lipidation status. In addition to the pathophysiological Aβ deposits, AD is also characterized by abnormal glucose metabolism, which is a distinct event preceding Aβ deposition. The present study established, for the first time, a possible link between these two major AD etiologies, with glucose metabolism directly influencing ApoE lipidation and its secretion by astrocytes expressing human ApoE4. Specifically, glucose dose-dependently activated liver X receptor (LXR), leading to elevated ABCA1 and ABCG1 protein levels and enhanced ApoE lipidation. Moreover, co-treatment with a glycolytic inhibitor significantly inhibited this LXR activation and subsequent ApoE lipidation, further supporting a central role of glucose metabolism in LXR activation leading to enhanced ApoE lipidation, which may help against AD through potential Aβ clearance. Therefore, we hypothesized that pharmacological agents that can target cellular energy metabolism, specifically aerobic glycolysis, may hold significant therapeutic potential against AD. In this context, the present study also led to the discovery of novel, small-molecule stimulants of astrocytic glucose metabolism, leading to significantly enhanced lipidation status of ApoE4 in astrocytic cells. Three such newly discovered compounds (lonidamine, phenformin, and berberine), owing to their promising cellular effect on the glycolysis-ApoE nexus, warrant further investigation in suitable in vivo models of AD.

Abstract 4449: Hepatic steatosis induced by bioactive lipids

Abstract Introduction: HCC is the most common form of liver cancer and is the sixth most common cancer globally. Despite the clinical advancement of immune checkpoint inhibitors, median survival continues to be less than twenty months and most patients develop resistance making it one of the deadliest malignancies. The rate of liver cancer continues to rise, which is coupled to the high prevalence of steatosis and steatohepatitis that accounts for about 25% in the general population. Pathologically, 80% of liver cancer occurs in patients with an underlying liver disease that displays liver steatosis. Thus, it is paramount to better characterize the transition from steatosis to HCC. Methods: Utilizing a Pten deleted mouse model (PtenloxP/loxP; Alb-Cre+) that recapitulates HCC progression, protein and lipid fractions were isolated to explore differentially expressed proteins and their correlations with bioactive lipid metabolism. Global untargeted differential proteomics was done with fractionation using a qExactive. Lipid fractions were analyzed using a Sciex 6500 for targeted lipidomic analysis. In addition, genetic knockout hepatocyte cell lines of each AKT isoform were used to explore the involvement of the PI3K/AKT signal. Further, a phosphoproteomic enrichment was performed to discover downstream signaling targets. Results: The PTEN regulated PI3K/AKT signal is induced in 54% of all liver cancers and represents the dominant signaling pathway regulating liver cancer progression. Untargeted differential proteomic analysis of Pten deleted mice livers revealed significant dysregulation in oxidative stress and eicosanoid metabolism among the top enriched disease and biological functions when the phenotype progressed from steatosis to HCC. Analysis of publicly available liver cancer patient samples from the NCI Proteomic Data Commons further shows strong correlation between PTEN protein abundance and the expression of enzymes involved in eicosanoid metabolism. Our analysis using a bio-active lipid multi reaction monitoring panel of Pten deleted mouse livers further validates significant decreases in resolving eicosanoid levels along with increases in proinflammatory eicosanoid precursors as steatosis progress to HCC. These data suggest a potential hepatic AKT-dependency in regulating the shift towards proinflammatory eicosanoids. Primary isolated hepatocytes from mouse livers lacking either AKT1 or AKT2 were analyzed to elucidate AKT’s regulatory role in hepatic eicosanoid metabolism. Proteomic and lipidomic analysis of these hepatocytes supported a unique AKT isoform specific role in the regulation of eicosanoids via potential isoform specific signaling interactions with MAPK. Conclusion: Eicosanoid metabolism dysregulation plays a key role in the progression from steatosis to HCC and appears to be regulated in an AKT isoform specific manner via interactions with MAPK signaling. Citation Format: Mario Alba, Ielyzaveta Slarve, Brandon Ebright, Yiren Zhou, Whitaker Cohn, Yunyi Jia, Elizabeth Elton, Jared Khan, Aditi Datta, Lina He, Qi Tang, Pranav Pammidimukkala, Taojian Tu, Phillip Nguyen, Jonathan Katz, Julian Whitelegge, Stan Louie, Bangyan Stiles. Hepatic steatosis induced by bioactive lipids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4449.

PDGFRβ + cell HIF2α is dispensable for white adipose tissue metabolic remodeling and hepatic lipid accumulation in obese mice

BackgroundObesity is associated with extensive white adipose tissue (WAT) expansion and remodeling. Healthy WAT expansion contributes to the maintenance of energy balance in the liver, thereby ameliorating obesity-related hepatic steatosis. Tissue-resident mesenchymal stromal cell populations, including PDGFRβ + perivascular cells, are increasingly recognized pivotal as determinants of the manner in which WAT expands. However, the full array of regulatory factors controlling WAT stromal cell functions remains to be fully elucidated. Hypoxia-inducible factors (HIFs) are critical regulators in WAT stromal cell populations such as adipocyte precursor cells (APCs). It is revealed that HIF1α activation within PDGFRβ + stromal cells results in the suppression of de novo adipogenesis and the promotion of a pro-fibrogenic cellular program in obese animals. However, the role of HIF2α in PDGFRβ + cells remains undetermined in vivo.MethodsNew genetic models were employed in which HIF1α (encoded by the Hif1a gene) and HIF2α (encoded by the Epas1 gene) are selectively inactivated in PDGFRβ + cells in an inducible manner using tamoxifen (TAM). With these models, both in vitro and in vivo functional analysis of PDGFRβ + cells lacking HIF proteins were performed. Additionally, comprehensive metabolic phenotyping in diet-induced mouse models were performed to investigate the roles of PDGFRβ + cell HIF proteins in WAT remodeling, liver energy balance and systemic metabolism.ResultsUnlike HIF1α inactivation, the new findings in this study suggest that inducible ablation of HIF2α in PDGFRβ + cells does not cause apparent effects on WAT expansion induced by obesogenic diet. The adipogenic ability of PDGFRβ + APCs is not significantly altered by genetic HIF2α ablation. Moreover, no difference of key parameters associated with healthy WAT remodeling such as improvements of WAT insulin sensitivity, reduction in metabolic inflammation, as well as changes in liver fat accumulation or systemic glucose metabolism, is detected in PDGFRβ + cell Epas1-deficient mice.ConclusionThe new findings in this study support that, in contrast to HIF1α, PDGFRβ + cell HIF2α appears dispensable for WAT metabolic remodeling and the resulting effects on liver metabolic homeostasis in diet-induced obesity, underscoring the isoform-specific roles of HIFα proteins in the regulation of adipose tissue biology.

Protein kinase C activity modulates nuclear Lamin A/C dynamics in HeLa cells

The nuclear lamina serves important functions in the nucleus, providing structural support to the nuclear envelope and contributing to chromatin organization. The primary proteins that constitute the lamina are nuclear lamins whose functions are impacted by post-translational modifications, including phosphorylation by protein kinase C (PKC). While PKC-mediated lamin phosphorylation is important for nuclear envelope breakdown during mitosis, less is known about interphase roles for PKC in regulating nuclear structure. Here we show that overexpression of PKC ß, but not PKC α, increases the Lamin A/C mobile fraction in the nuclear envelope in HeLa cells without changing the overall structure of Lamin A/C and Lamin B1 within the nuclear lamina. Conversely, knockdown of PKC ß, but not PKC α, reduces the Lamin A/C mobile fraction. Thus, we demonstrate an isoform-specific role for PKC in regulating interphase Lamin A/C dynamics outside of mitosis.

Roles of Ribosomal S6 Kinases in Acute Leukemia and Normal Hematopoiesis

Ribosomal S6 kinases (RSKs) are serine-threonine kinases that are activated by the Ras/mitogen activated protein kinase signaling pathways and regulate cell proliferation and survival. There are 4 isoforms of RSK (RSK1, 2, 3, 4), and there is evidence that the isoforms mediate tissue-specific cellular functions. We hypothesized that RSK isoforms have distinct roles in hematopoiesisand evaluated RSK as a target for the treatment of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). To investigate isoform expression in hematopoiesis, we sorted cord blood mononuclear cells into hematopoietic progenitor stages and quantified RSK isoform mRNA by RT-qPCR. RSK1 expression remained consistent throughout hematopoiesis, whereas RSK2 expression decreased in the more differentiated populations. RSK3 was elevated in the lymphoid-primed multipotential progenitor and common myeloid progenitor stages and was greatly reduced in the more mature megakaryocyte-erythroid progenitor population. These results suggest that RSK proteins have isoform-specific roles in hematopoiesis. To analyze the importance of each isoform on hematopoiesis, we transduced cord blood CD34+ cells with lentiviral constructs expressing isoform-specific or control shRNAs. After liquid culture for 5 days, the cells were plated in methylcellulose media containing SCF, IL-3, GM-CSF, and EPO to determine the colony-forming potential. RSK1 and RSK2 knockdown decreased the colony-forming unit - erythroid colonies and increased the granulocyte/macrophage colony numbers. RSK3 knockdown increased the burst-forming unit - erythroid colonies and total colony numbers. After liquid culture days 8, 11, and 14, the erythroid and myeloid populations were analyzed by flow cytometry. RSK1 knockdown increased the CD11b+ and CD14+ myeloid populations and decreased the CD71+ erythroid population. RSK3 knockdown increased the erythroid population and decreased the myeloid populations. These data demonstrate that RSK1 and RSK3 have opposing effects on hematopoietic lineage determination. Overexpression of RSK proteins leads to aberrant cell survival and proliferation in many cancers including leukemia. We have previously reported that RSK and phosphorylated RSK are elevated in pediatric AML and are associated with a worse prognosis. There are only two orally bioavailable RSK inhibitors that have moved to phase I clinical trials: TAS0612 and PMD-026. TAS0612 is a multi-kinase inhibitor of RSK, AKT, and S6K. PMD-026 has high specificity to all four RSK isoforms. We evaluated the effects of both compounds in AML and ALL. To determine the effects of the RSK inhibitors on AML, we treated AML cell lines and patient samples with a range of doses of PMD-026, TAS0612, or 0.1% DMSO. After 3 days, we performed CellTiter Glo assays to determine the relative viable cell number and calculate the concentration of drug that inhibits cell growth by 50% (IC50). TAS0612 had an IC50 of 0.096-0.64 µM in AML cell lines Molm13, U937, AML193, and MV4;11. To test the cytotoxicity of TAS0612 on normal hematopoiesis, we plated healthy bone marrow mononuclear cells with TAS0612 in methylcellulose media. Colony formation was not adversely affected by TAS0612 up to 1 µM. PMD-026 was effective in Molm13, Kasumi-1, and MV4;11 with an IC50 of 0.34-0.55 µM and in AML patient samples 3123 and 4192 with an IC50 of 1.5-2.3 µM. PMD-026 did not affect healthy bone marrow colony formation up to 5 µM for a 2.5- to 10-fold therapeutic window. Furthermore, we tested the effects of RSK inhibitors in ALL. While PMD-026 was not effective, we found that B-ALL cell lines (REH, Nalm-6) and T-ALL cell lines (Loucy, Jurkat) were sensitive to RSK inhibition by TAS0612 with an IC50 of 0.056-0.19 µM. Western blot analysis of RSK protein revealed that RSK1, RSK2, and phospho-RSK proteins are elevated in ALL cell lines compared to healthy peripheral blood mononuclear cells. We are investigating the role of RSK and RSK isoforms in ALL. We found that RSK isoform expression is modified during hematopoiesis. Isoform expression influences early-fate decisions to differentiate into erythroid and myeloid lineages. RSK inhibitors PMD-026 and TAS0612 are effective at targeting AML and ALL cells with limited cytotoxicity to healthy cells, making them promising candidates for translation into clinical trials for AML and ALL.

Isoform Specific Roles for the Acute Myeloid Leukemia Stem Cell Antigen CD97

CD97 is an adhesion GPCR protein frequently upregulated in AML and required for LSC self-renewal that is alternatively spliced to form three isoforms that are distinct with respect to their extracellular domain. Our analysis of HSCs from MDS patients with low-risk disease revealed overexpression of CD97 isoform 1 (CD97iso1), while analysis of AML samples from the TCGA dataset revealed preferential expression of CD97iso2 (p<0.0001) (Figure1A). As these data suggested potential isoform specific roles of CD97 in myeloid leukemogenesis, we investigated if the different CD97 isoforms support different functional roles by testing the effect of each isoform on growth, differentiation, and clonogenicity of AML blasts. Consistent with our prior studies demonstrating CD97iso1 promotion of self-renewal in normal CD34+ hematopoietic stem/progenitor cells, over expression of CD97iso1 in AML cell lines increased their serial replating ability, while knockout abrogated their clonogenicity. In stark contrast, overexpression of CD97iso2 or CD97iso3 resulted in decreased clonogenicity upon replating. Overexpression of CD97iso2 conferred the greatest growth advantage to AML cells among the CD97 isoforms. Importantly, knockout of CD97iso2 increased cell differentiation and primed the cells towards apoptosis. Supporting CD97iso2's role in cell growth/survival, daunorubicin treatment increased the expression of CD97iso2. Consistent with CD97's role in increasing migration and invasiveness in solid tumors, we performed trans well migration assays using AML cell lines and observed the highest increase in migration in blasts over expressing CD97iso3 (p<0.01). While CD97 is thought to activate several downstream signaling pathways including the MEK-ERK pathway and PI3K/AKT pathway, it is unknown whether the different CD97 isoforms activate unique downstream signaling pathways, and thus we used an unbiased Bio-ID screen to identify the C-terminal interacting partners of the different CD97 isoforms. We observed preferential enrichment of NLRX1 interactions in CD97iso1 expressing cells, while PI4KA was enriched in CD97iso2 expressing cells. Consistent with NLRX1's role in increasing autophagy, we observed an increase in autophagy upon overexpressing CD97iso1, and consistent with CD97iso2's association with PI4KA, CD97iso2 overexpressing cells showed increased PI4KA and phosphoERK1/2. Finally, confirming non-redundant functional roles of CD97 isoforms in vivo, xenograft studies in NSG mice using AML cell lines revealed that CD97iso2 promoted the most rapid engraftment and shortest survival in recipients (p=0.0001). Given the importance of all the CD97 isoforms in regulating different blast function, we developed antibody-drug conjugates (ADCs) based on human synthetic antibodies that target all isoforms of CD97. ADC against CD97 specifically kills AML cells overexpressing CD97, suggesting the potential use of anti-CD97 ADC for AML therapy. Collectively, these studies demonstrate that CD97 isoforms mediate unique biological roles in vitro and in vivo, and that they likely mediate these functions through differential activation of downstream signaling pathways. We speculate that differential expression of the CD97 isoforms in MDS and AML likely reflects the unique features of MDS HSCs (high levels of CD97iso1 enhancing self-renewal in MDS) versus AML LSCs (CD97iso2 promoting proliferation in AML) (Figure 1B). These studies also highlight the importance of isoform-specific gene expression during myeloid leukemogenesis and suggest that explorations of altered isoform expression in MDS/AML will yield novel insights into the molecular mechanisms that promote disease development and progression.

Intrinsic cancer cell phosphoinositide 3-kinase δ regulates fibrosis and vascular development in cholangiocarcinoma.

The class I- phosphatidylinositol-3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform-specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation. Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT-qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ-overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8-based-PI3Kδ overexpression. These PI3Kδ-mediated cell morphogenesis and stroma remodelling were dependent on TGFβ/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression. Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFβ/src/Notch-dependent mechanisms and open new directions for the classification and treatment of CCA patients.

14-3-3 Proteins stabilize actin and vimentin filaments to maintain processes in renal glomerular podocyte.

14-3-3 proteins are a ubiquitously expressed family of adaptor proteins. Despite exhibiting high sequence homology, several 14-3-3 isoforms have isoform-specific binding partners and roles. We reported that 14-3-3β interacts with FKBP12 and synaptopodin to maintain the structure of actin fibers in podocytes. However, the precise localization and differential role of 14-3-3 isoforms in kidneys are unclear. Herein, we showed that 14-3-3β in glomeruli was restricted in podocytes, and 14-3-3σ in glomeruli was expressed in podocytes and mesangial cells. Although 14-3-3β was dominantly co-localized with FKBP12 in the foot processes, a part of 14-3-3β was co-localized with Par3 at the slit diaphragm. 14-3-3β interacted with Par3, and FKBP12 bound to 14-3-3β competitively with Par3. Deletion of 14-3-3β enhanced the interaction of Par3 with Par6 in podocytes. Gene silencing for 14-3-3β altered the structure of actin fibers and process formation. 14-3-3β and synaptopodin expression was decreased in podocyte injury models. In contrast, 14-3-3σ in podocytes was expressed in the primary processes. 14-3-3σ interacted with vimentin but not with the actin-associated proteins FKBP12 and synaptopodin. Gene silencing for 14-3-3σ altered the structure of vimentin fibers and process formation. 14-3-3σ and vimentin expression was increased in the early phase of podocyte injury models but was decreased in the late stage. Together, the localization of 14-3-3β at actin cytoskeleton plays a role in maintaining the foot processes and the Par complex in podocytes. In contrast, 14-3-3σ at vimentin cytoskeleton is essential for maintaining primary processes.

Anthracycline cardiotoxicity is exacerbated by global p38β genetic ablation in a sexually dimorphic manner but unaltered by cardiomyocyte-specific p38α loss.

Severe cardiotoxic effects limit the efficacy of doxorubicin (DOX) as a chemotherapeutic agent. Activation of intracellular stress signaling networks, including p38 mitogen-activated protein kinase (MAPK), has been implicated in DOX-induced cardiotoxicity (DIC). However, the roles of the individual p38 isoforms in DIC remain incompletely elucidated. We recently reported that global p38δ deletion protected female but not male mice from DIC, whereas global p38γ deletion did not significantly modulate it. Here we studied the in vivo roles of p38α and p38β in acute DIC. Male and female mice with cardiomyocyte-specific deletion of p38α or global deletion of p38β and their wild-type counterparts were injected with DOX. Survival and health were tracked for 10 days postinjection. Cardiac function was assessed by echocardiography and electrocardiography and fibrosis by Picrosirius red staining. Expression and activation of signaling proteins and inflammatory markers were measured by Western blot, phosphorylation array, and chemokine/cytokine array. Global p38β deletion significantly aggravated DIC and worsened cardiac electrical and mechanical function deterioration in female mice. Mechanistically, DIC in p38β-null female mice correlated with increased autophagy, sustained hyperactivation of proapoptotic JNK signaling, as well as remodeling of a myocardial inflammatory environment. In contrast, cardiomyocyte-specific deletion of p38α improved survival of DOX30-treated male mice 5 days posttreatment but did not influence cardiac function in DOX-treated male or female mice. Our data highlight the sex- and isoform-specific roles of p38α and p38β MAPKs in DOX-induced cardiac injury and suggest a novel in vivo function of p38β in protecting female mice from DIC.NEW & NOTEWORTHY We show that p38α and p38β have distinct in vivo functions in a murine model of acute DIC. Specifically, although conditional cardiomyocyte-specific p38α deletion exhibited mild cardioprotective effects in male mice, p38β deletion exacerbated the DOX cardiotoxicity in female mice. Our findings caution against employing pyridinyl imidazole inhibitors that target both p38α and p38β isoforms as a cardioprotective strategy against DIC. Such an approach could have undesirable sex-dependent effects, including attenuating p38β-dependent cardioprotection in females.

Paralogous translation factors target distinct mRNAs to differentially regulate tolerance to oxidative stress in yeast.

Translation initiation factor 4G (eIF4G) is an integral component of the eIF4F complex which is key to translation initiation for most eukaryotic mRNAs. Many eIF4G isoforms have been described in diverse eukaryotic organisms but we currently have a poor understanding of their functional roles and whether they regulate translation in an mRNA specific manner. The yeast Saccharomyces cerevisiae expresses two eIF4G isoforms, eIF4G1 and eIF4G2, that have previously been considered as functionally redundant with any phenotypic differences arising due to alteration in eIF4G expression levels. Using homogenic strains that express eIF4G1 or eIF4G2 as the sole eIF4G isoforms at comparable expression levels to total eIF4G, we show that eIF4G1 is specifically required to mediate the translational response to oxidative stress. eIF4G1 binds the mRNA cap and remains associated with actively translating ribosomes during oxidative stress conditions and we use quantitative proteomics to show that eIF4G1 promotes oxidative stress-specific proteome changes. eIF4G1, but not eIF4G2, binds the Slf1 LARP protein which appears to mediate the eIF4G1-dependent translational response to oxidative stress. We show similar isoform specific roles for eIF4G in human cells suggesting convergent evolution of multiple eIF4G isoforms offers significant advantages especially where translation must continue under stress conditions.

Generation of a new Adar1p150-/- mouse demonstrates isoform-specific roles in embryonic development and adult homeostasis.

The RNA editing enzyme Adenosine deaminase acting on RNA 1 (ADAR1) is an essential regulator of innate immune activation by both cellular and viral dsRNA. Adenosine-to-Inosine (A-to-I) editing by ADAR1 modifies the sequence and structure of endogenous dsRNA and masks it from the cytoplasmic dsRNA sensor melanoma differentiation-associated protein 5 (MDA5), preventing innate immune activation. Loss of function mutations in ADAR are associated with rare autoinflammatory disorders including Aicardi-Goutieres Syndrome (AGS), defined by a constitutive systemic upregulation of type I interferon (IFN). The murine Adar gene encodes two protein isoforms with distinct functions: ADAR1p110 is constitutively expressed and localizes to the nucleus, whereas ADAR1p150 is primarily cytoplasmic and is inducible by IFN. Recent studies have demonstrated the critical requirement for ADAR1p150 to suppress innate immune activation by self dsRNAs, however, detailed in vivo characterization of the role of ADAR1p150 during development and in adult mice is lacking. We developed a new ADAR1p150-specific knockout mouse mutant based on a single nucleotide deletion that resulted in the loss of the ADARp150 protein without affecting ADAR1p110 expression. The Adar1p150-/- died embryonically at E11.5-E12.5 due to cell death in the fetal liver accompanied by an activated IFN response. Somatic loss of ADAR1p150 in adults was lethal and caused rapid hematopoietic failure, demonstrating an ongoing requirement for ADAR1p150 in vivo. The generation and characterization of this mouse model demonstrates the essential role of ADAR1p150 in vivo and provides a new tool for dissecting the functional differences between ADAR1 isoforms and their physiological contributions.

Stretch regulates alveologenesis and homeostasis via mesenchymal Gαq/11-mediated TGFβ2 activation.

Alveolar development and repair require tight spatiotemporal regulation of numerous signalling pathways that are influenced by chemical and mechanical stimuli. Mesenchymal cells play key roles in numerous developmental processes. Transforming growth factor-β (TGFβ) is essential for alveologenesis and lung repair, and the G protein α subunits Gαq and Gα11 (Gαq/11) transmit mechanical and chemical signals to activate TGFβ in epithelial cells. To understand the role of mesenchymal Gαq/11 in lung development, we generated constitutive (Pdgfrb-Cre+/-;Gnaqfl/fl;Gna11-/-) and inducible (Pdgfrb-Cre/ERT2+/-;Gnaqfl/fl;Gna11-/-) mesenchymal Gαq/11 deleted mice. Mice with constitutive Gαq/11 gene deletion exhibited abnormal alveolar development, with suppressed myofibroblast differentiation, altered mesenchymal cell synthetic function, and reduced lung TGFβ2 deposition, as well as kidney abnormalities. Tamoxifen-induced mesenchymal Gαq/11 gene deletion in adult mice resulted in emphysema associated with reduced TGFβ2 and elastin deposition. Cyclical mechanical stretch-induced TGFβ activation required Gαq/11 signalling and serine protease activity, but was independent of integrins, suggesting an isoform-specific role for TGFβ2 in this model. These data highlight a previously undescribed mechanism of cyclical stretch-induced Gαq/11-dependent TGFβ2 signalling in mesenchymal cells, which is imperative for normal alveologenesis and maintenance of lung homeostasis.

Abstract B029: Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma

Abstract While KRAS is among the most frequently mutated oncogenes, our understanding of the mechanisms of KRAS-driven oncogenesis remains limited. A significant remaining gap is a lack of understanding of tissue-specific effectors of Ras activation and the role of specific KRAS mutations in determining downstream vulnerabilities. Previously, we used a combination of proteomics and CRISPR/Cas9 screens in human lung adenocarcinoma (LUAD) cells to identify a KRAS-specific vulnerability induced by the combined loss of RHOA and the long isoform of RAP1GDS1, suggesting a potentially novel approach for targeting KRAS-driven cancer. Here we use biochemical, proteomic, and genetic approaches to dissect the isoform-specific roles of RAP1GDS1 to elucidate its synthetic lethal interaction with RHOA in KRAS-driven LUAD. We performed AP/MS in KRAS-mutant A549 cells using both the long (RAP1GDS1-607) and the short (RAP1GDS1-558) isoforms of RAP1GDS1 as bait to identify overlapping and isoform-specific RAP1GDS1 interactors. We enriched this analysis with orthologous datasets from DepMap, published protein-protein interaction (PPI) data and RAP1GDS1-specific prenylome. This comprehensive analysis identified a cluster of RAB GTPases, proteins involved in vesicular transport and lysosomal function, as specific interactors of the long RAP1GDS1 isoform. We subsequently used the proximity ligation assay (PLA) to confirm the direct interaction between RAP1GDS1-607 and two of the strongest interactors - RAB7A and RAB22A – according to the PPI data. We also depleted either or both RAP1GDS1 isoforms in cells expressing GFP-tagged RAB7A, RAB22A or KRAS and used live cell imaging to determine how loss of RAP1GDS1 changes their subcellular localization. We found that depletion of both RAP1GDS1 isoforms and, to a lesser extent, single loss of the long RAP1GDS1 isoform, significantly decreased KRAS localization at the plasma membrane. We also used PLA to confirm that RAP1GDS1-607 has a stronger interaction with KRAS compared to the short isoform. Moreover, we demonstrated that loss of the long RAP1GDS1 isoform significantly decreased the membrane localization levels of GTP-bound KRAS, compared to the control or RAP1GDS1-558 knock-down. Finally, to identify the molecular mechanism of R1G1/RhoA synthetic lethality with KRAS, we performed a Genome Wide-CRISPR screen in R1G1-607 and RhoA knock-out cells. We identified the strongest hits and designed a focused CRISPR library of approximately 400 genes to screen a larger panel of KRAS-mutant LUAD cell lines (H23, A549 and H358). The aim is to identify other genes that are synthetic lethal in combination with RhoA or R1G1-607 loss and genes that could mediate the lethal effect of their combined loss. Citation Format: Marta Roman Moreno, Kaja Kostyrko, Kari Herrington, Michael Bassik, Peter Jackson, Eric Alejandro Sweet-Cordero. Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B029.

Cardiac Na/K-ATPase alpha1 isoform-specific role in hypertrophic response: role of metabolic and oxidative signaling

Cardiac Na/K-ATPase (NKA) inhibition by cardiotonic steroids (CTS) modulates intracellular Na + and Ca 2+ , leading to increased force of contraction. In contrast, hypertrophy of cardiomyocytes (CM) is triggered by CTS through stimulation of reactive oxygen species (ROS) and fetal gene re-expression independently of changes in intracellular Na + or Ca 2+ . Based on recent evidence that NKAα1 serves an isoform-specific function in the regulation of oxidative metabolism and ROS generation, we used a genetic approach in the mouse and in human cardiac cells (AC16) to test the hypothesis that NKAα1 is required for cardiac hypertrophy. Cardio α1 -/- mice, a conditional KO of NKAα1 in the heart, were viable without overt abnormality of cardiac structure or function. NKAα1 was undetectable in heart lysates or isolated CM of adult cardio α1 -/- mice. Despite a compensatory upregulation of NKAα2, Na/K-ATPase activity in crude heart homogenates and NKA currents in CM were reduced by about 60%. Decreased ROS (assessed by total protein carbonylation) and reduced fatty acid oxidation (FAO) and oxidative phosphorylation pathways (revealed by RNA-seq analysis) were noted at baseline. RT-qPCR revealed downregulated genes involved with FAO and the electron transport chain (such as, Acadl and Ndufa9, about 20% decrease, n=6) in cardio α1 -/- hearts. The activity of the electron transport chain complex I was decreased in cardio α1 -/- hearts (0.3354 vs 0.2732 mOD/min/mg of protein, p<0.05). When 3-month-old male mice were challenged with angiotensin II (1.5 mg.kg -1 .day -1 for 1 week, n=6-15), cardio α1 -/- mice did not develop cardiac hypertrophy, as assessed by heart weight to tibia length ratio, but displayed exacerbated cardiac fibrosis, revealed by Trichrome staining, consistent with the postulated role of CM NKAα1 isoform-specific function in cardiac hypertrophy in vivo. In vitro, CRISPR-Cas9-mediated KO of NKAα1 in AC16 cells led to a drastic downregulation of NKAα1 compared to WT AC16 cells. In contrast to WT AC16 cells, NKAα1 KO cells did not respond to the α-adrenergic agonist phenylephrine (50 μM for 24h) with the expected increase in cell area (2243 vs 3520 μm 2 for WT, p<0.05; 2454 vs 2563 μm 2 for KO, p>0.05). These data suggest that NKAα1 isoform-specific function is required for cardiac hypertrophy. Mechanistically, our data support an isoform-specific regulatory function of NKAα1 in normal FAO, electron transport chain activity, and ROS generation, which are critical for CM hypertrophy. Supported by NIH Grant R15 HL145666 and American Heart Association Grant 22POST917776 (Marco T. Pessoa) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.