Karen Ocorr

BACKGROUND: RASopathies constitute a group of rare genetic disorders caused by mutations in genes along the canonical RAS/mitogen-activated protein kinase (MAPK) signaling pathway, affecting cell growth and differentiation. These syndromes, which include Noonan syndrome (NS), are characterized by developmental delays, distinctive facial dysmorphia, and cardiac defects, notably hypertrophic cardiomyopathy (HCM). Despite their prevalence and impact, therapeutic options for RASopathies remain limited. Rigosertib, a novel dual RAS/MAPK and phosphatidylinositol 3-kinase (PI3K)/AKT pathway inhibitor, is currently in clinical trials for treatment of melanoma and recessive dystrophic epidermolysis bullosa. Here, we identify rigosertib as a candidate therapy for RAF1-associated HCM. METHODS: We performed a drug screen of clinically relevant compounds in transgenic Drosophila models of RASopathies to identify candidate therapeutics. Cardiac-targeted Drosophila models expressing RASopathy-associated transgenes were used to evaluate the effects of rigosertib on cardiac hypertrophy and compare its efficacy with the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Therapeutic efficacy was further assessed in a mammalian model using Raf1 ^L613V/+ knock-in mice treated with rigosertib for 6 weeks. Cardiac structure and function were evaluated by echocardiography, histology, and molecular analyses, including assessment of cardiomyocyte size, fetal gene expression, and extracellular signal-regulated kinase/AKT signaling. Additional NS-associated phenotypes, including skeletal growth and craniofacial abnormalities, were also evaluated. RESULTS: Rigosertib was effective across a panel of transgenic Drosophila RASopathy models, suggesting activity against multiple disease variants. In cardiac-targeted fly models, rigosertib reduced cardiac hypertrophy and outperformed trametinib. In Raf1 ^L613V/+ mice, 6 weeks of treatment significantly improved left ventricular chamber dimension, posterior wall thickness, heart mass, and cardiomyocyte size, resulting in reversal of cardiac hypertrophy. Rigosertib also normalized fetal gene expression and inhibited extracellular signal-regulated kinase and AKT signaling in primary cardiomyocytes. In addition to reversing cardiac pathology, rigosertib significantly improved other NS-associated features, including increased bone growth and correction of craniofacial abnormalities. CONCLUSIONS: Together, our findings suggest that rigosertib normalizes and reverses RASopathy-associated HCM and other NS-associated syndromic features, supporting its development as a promising treatment for RAF1-associated HCM and, potentially, other RASopathy-dependent pathologies. This study not only highlights the therapeutic potential of rigosertib but also demonstrates the utility of an integrated approach using Drosophila and mammalian models to elucidate drug effects across complex biological systems.

We previously identified ETS1 as a candidate gene in the rare chromosomal deletion disorder, Jacobsen syndrome (JS, OMIM#147791). Here, we present data from four model systems that define roles for ETS factors in normal heart development and suggest how defects in these genes contribute to congenital heart disease. In frog, knockdown of Ets1 in cardiac mesoderm led to a hypoplastic ventricle with impaired cardiac function and loss of trabecular myocardium. Grafting of wildtype cardiac mesodermal tissue restored the integrity of the ventricular endocardial layer and normal development. In zebrafish, KD of ETS1 and two related ETS factor genes caused severely underdeveloped, poorly functioning ventricles with decreased numbers of endocardial cells that failed to contact the myocardium. Genetic ablation of the endocardium in mice also caused a hypoplastic ventricle with loss of the trabecular myocardium. In Drosophila, loss of the ETS1 ortholog pointed (pnt) affected specification of cardiac precursors and decreased Notch signaling, a pathway previously implicated in some forms of hypoplastic left heart syndrome (HLHS). We also demonstrated genetic interactions between pnt and tinman, the NKX2.5 ortholog, associated with HLHS. Finally, our examination of the heart from a newborn with JS/HLHS showed myofibrillar disarray and myocardial maturation defects. Furthermore, there was a reduction in the coronary vascular endothelium in the LV myocardium from the JS/HLHS patient compared to an age-matched normal heart. Taken together, our studies demonstrate a critical and conserved role of ETS factors for cardiac endothelial function and in ventricular morphogenesis.

A pair of paralogs, Chchd3 and Chchd6, two components of mitochondrial contact site and cristae organizing system (MICOS), have been identified to be candidate pathogenetic genes in congenital heart disease (CHD). Previous research found that knockdown (KD) of the single Chchd3/6 (Chchd3) gene and other MICOS components in Drosophila impaired heart function, likely due to a deficit in mitochondrial organization, ATP production, actomyosin levels, and thus severely diminished contractility. However, the underlying mechanisms of how MICOS deficiency leads to these defects are not clear. Here, we performed genetic manipulations in the Drosophila heart to probe for possible interactions between MICOS-compromised mitochondria and other organelles and processes. We found that moderate reduction in Pink1/parkin-mediated mitophagy synergistically aggravated cardiac Chchd3 KD phenotypes, indicating a major interaction. Further, Chchd3 KD increased the level of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Interestingly, KD of catalase (CAT) also elevated cardiac ROS levels, but surprisingly did not compromise contractility either by itself or in combination with Chchd3 KD to aggravate the cardiac phenotype. However, CAT overexpression (OE) in Chchd3 KD hearts restored contractility, but only partially, even though elevated ROS due to Chchd3 KD was fully normalized. Similarly, counteracting ER stress by overexpressing Xbp1 (or spliced mouse Xbp1) also partially rescued the heart function defects induced by Chchd3 KD. Overall, these data indicate a critical role of mitophagy and ER/oxidative stress in cardiac homeostasis involving Chchd3, which suggests that deficiency of MICOS function contributes to heart dysfunction via multiple stress responsive pathways.

Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD) whose genetic basis remains elusive, likely due to oligogenic complexity. To identify regulators of cardiomyocyte (CM) proliferation relevant to HLHS, we performed a genome-wide siRNA screen in human iPSC-derived CMs, revealing ribosomal protein (RP) genes as the most prominent effectors of CM proliferation. Whole-genome sequencing of 25 HLHS proband–parent trios similarly showed enrichment of rare RP gene variants, including a damaging RPS15A promoter variant shared in a familial CHD case. Cross-species functional analyses demonstrated that perturbation of RP genes impairs cardiac growth: knockdown of RPS15A, RPS17, RPL26L1, RPL39, or RPS15 reduced CM proliferation, caused cardiac malformations in Drosophila , and produced hypoplastic or dysfunctional hearts in zebrafish. Genetic interactions between RP genes and key cardiac transcription factors (TBX5 and NKX2–7) further support their developmental role. Importantly, p53 suppression or Hippo activation partially rescued RP deficiency phenotypes. Together, these findings implicate RP genes as critical regulators of cardiogenesis and candidate contributors to HLHS.

Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD). The pathogenesis of HLHS is poorly understood, and due to the likely oligogenic complexity of the disease, definitive HLHS-causing genes have not yet been identified. Postulating impaired cardiomyocyte proliferation as a likely important contributing mechanism to HLHS pathogenesis, and we conducted a genome-wide siRNA screen to identify genes affecting proliferation of human iPSC-derived cardiomyocytes (hPSC-CMs). This yielded ribosomal protein (RP) genes as the most prominent class of effectors of CM proliferation. In parallel, whole genome sequencing and rare variant filtering of a cohort of 25 HLHS proband-parent trios with poor clinical outcome revealed enrichment of rare variants of RP genes. In addition, in a familial CHD case we identified a rare, predicted-damaging promoter variant affecting RPS15A that was shared between the HLHS proband and a distant relative with CHD. Functional testing with an integrated multi-model system approach reinforced the idea that RP genes are major regulators of cardiac growth and proliferation, thus potentially contributing to the hypoplastic phenotype observed in HLHS patients. Cardiac knockdown (KD) of RP genes with promoter or coding variants (RPS15A, RPS17, RPL26L1, RPL39, RPS15) reduced proliferation in generic hPSC-CMs and caused malformed hearts, heart-loss or even lethality in Drosophila. In zebrafish, diminished rps15a function caused reduced CM numbers, heart looping defects, or weakened contractility, while reduced rps17 or rpl39 function caused reduced ventricular size or systolic atrial dysfunction of the atrium, respectively. Importantly, genetic interactions between RPS15A and core cardiac transcription factors TBX5 in CMs, Drosocross, pannier and tinman in flies, and tbx5 and nkx2-7 (nkx2-5 paralog) in fish, support a specific role for RP genes in heart development. Furthermore, RPS15A KD-induced heart/CM proliferation defects were significantly attenuated by p53 KD in both hPSC-CMs and zebrafish, and by Hippo activation (YAP/yorkie overexpression) in developing fly hearts. Based on these findings, we conclude that RP genes play novel critical roles in cardiogenesis and constitute an emerging class of gene candidates likely involved in HLHS.

Background: RASopathies constitute a group of rare genetic disorders caused by mutations in genes that reside along the canonical Ras/MAPK signaling pathway, affecting cell growth and differentiation. These syndromes, which include conditions like Noonan syndrome (NS), are characterized by developmental delays, distinctive facial dysmorphia, and a variety of cardiac defects, notably hypertrophic cardiomyopathy (HCM). Despite their prevalence and impact, therapeutic options for RASopathies remain limited. Rigosertib, a novel dual Ras/MAPK and PI3K/AKT pathway inhibitor, is currently in clinical trials for treatment of melanoma and recessive dystrophic epidermolysis bullosa. Here, we identify rigosertib as a candidate therapy for RAF1-associated HCM. Methods and Results: Our Drosophila screen of clinically relevant drugs and compounds identified rigosertib as broadly effective across a panel of transgenic RASopathy fly transgenic models, indicating that rigosertib may be effective against multiple disease isoforms. Analysis of a Drosophila model targeting a RAF1 ^L613V transgene to the heart found that rigosertib reduced aspects of cardiac hypertrophy. Rigosertib treatment prevented or regressed cellular hypertrophy in human induced pluripotent stem cell-(iPSC-) derived cardiomyocytes homozygous for the NS-associated RAF1 ^S257L allele. We extended these findings to a mammalian model, using Raf1 ^L613V/+ KI mice to explore the therapeutic implications of rigosertib on RAF1-driven HCM. Longitudinal six-week treatment with rigosertib in these mice resulted in significant improvement in left ventricular chamber dimension and posterior wall thickness, total heart mass, size of individual cardiomyocytes (CMs), as well as reversal of cardiac hypertrophy. Rigosertib treatment also led to normalized fetal gene expression and inhibition of ERK and AKT pathway activities in primary CMs isolated from Raf1 ^L613V/+ mice. Cardiac function, as assessed by echocardiography, showed significant improvement in ejection fraction and fractional shortening, with molecular studies confirming downregulation of hypertrophic markers and signaling pathways. Together with the Drosophila data, these mammalian results support the potential and use for rigosertib to reverse pathological hypertrophy in NS through targeted pathway inhibition in patients. Moreover, in addition to its effects in the heart, rigosertib treatment in mice also significantly improved other NS-associated syndromic features, including increasing bone growth and correcting craniofacial abnormalities. Conclusions: Taken together, our findings suggest rigosertib effectively normalizes and reverses RASopathy-associated HCM as well as other NS-associated syndromic features, supporting its potential for development as a promising treatment for RAF1-associated HCM and, potentially, other RASopathies-dependent pathologies. This study not only highlights the therapeutic potential of rigosertib but also demonstrates the utility of an integrated approach using Drosophila, iPSC and mammalian models to elucidate drug effects across complex biological systems.

Cover legend: The cover image is based on the Research Article 3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex by Zer Vue et al., https://doi.org/10.1111/acel.14009

During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block‐face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase‐quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3, Chchd6, and Mitofilin. We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age‐related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age‐related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue‐dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.

Obesity and type 2 diabetes are at epidemic levels and a significant proportion of these patients are diagnosed with left ventricular hypertrophy. CREB R egulated T ranscription C o-activator ( CRTC ) is a key regulator of metabolism in mammalian hepatocytes, where it is activated by calcineurin (CaN) to increase expression of gluconeogenic genes. CaN is known its role in pathological cardiac hypertrophy, however, a role for CRTC in the heart has not been identified. In Drosophila , CRTC null mutants have little body fat and exhibit severe cardiac restriction, myofibrillar disorganization, cardiac fibrosis and tachycardia, all hallmarks of heart disease. Cardiac-specific knockdown of CRTC , or its coactivator CREBb , mimicked the reduced body fat and heart defects of CRTC null mutants. Comparative gene expression in CRTC loss- or gain-of-function fly hearts revealed contra-regulation of genes involved in glucose, fatty acid, and amino acid metabolism, suggesting that CRTC also acts as a metabolic switch in the heart. Among the contra-regulated genes with conserved CREB binding sites, we identified the fly ortholog of Sarcalumenin, which is a Ca ²⁺ -binding protein in the sarcoplasmic reticulum. Cardiac knockdown recapitulated the loss of CRTC cardiac restriction and fibrotic phenotypes, suggesting it is a downstream effector of CRTC we named thinman ( tmn ). Importantly, cardiac overexpression of either CaN or CRTC in flies caused hypertrophy that was reversed in a CRTC mutant background, suggesting CRTC mediates hypertrophy downstream of CaN, perhaps as an alternative to NFAT. CRTC novel role in the heart is likely conserved in vertebrates as knockdown in zebrafish also caused cardiac restriction, as in fl ies. These data suggest that CRTC is involved in myocardial cell maintenance and that CaN-CRTC- Sarcalumenin/ tmn signaling represents a novel and conserved pathway underlying cardiac hypertrophy.