Molecular and cellular evidence for the impact of a hypertrophic cardiomyopathy-associated RAF1 variant on the structure and function of contractile machinery in bioartificial cardiac tissues.

Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.

Inhibition of the RacGEF VAV3 by the small molecule IODVA1 impedes RAC signaling and overcomes resistance to tyrosine kinase inhibition in acute lymphoblastic leukemia.

Aberrant RHO guanine nucleotide exchange factor (RhoGEF) activation is chief mechanism driving abnormal activation of their GTPase targets in transformation and tumorigenesis. Consequently, a small-molecule inhibitor of RhoGEF can make an anti-cancer drug. We used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like acute lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation-dependent RacGEF, as the target of the small molecule IODVA1. We show that through binding to VAV3, IODVA1 inhibits RAC activation and signaling and increases pro-apoptotic activity in BCR-ABL1-transformed cells. Consistent with this mechanism of action, cellular and animal models of BCR-ABL1-induced leukemia in Vav3-null background do not respond to IODVA1. By durably decreasing in vivo RAC signaling, IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal. Importantly, IODVA1 suppresses the leukemic burden in the treatment refractory pediatric Ph+ and TKI-resistant Ph+ B-ALL patient-derived xenograft models better than standard-of-care dasatinib or ponatinib and provides a more durable response after treatment withdrawal. Pediatric leukemia samples with diverse genetic lesions show high sensitivity to IODVA1 ex vivo and this sensitivity is VAV3 dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapy.

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.

Structural snapshots of RAF kinase interactions.

RAF (rapidly accelerated fibrosarcoma) Ser/Thr kinases (ARAF, BRAF, and CRAF) link the RAS (rat sarcoma) protein family with the MAPK (mitogen-activated protein kinase) pathway and control cell growth, differentiation, development, aging, and tumorigenesis. Their activity is specifically modulated by protein-protein interactions, post-translational modifications, and conformational changes in specific spatiotemporal patterns via various upstream regulators, including the kinases, phosphatase, GTPases, and scaffold and modulator proteins. Dephosphorylation of Ser-259 (CRAF numbering) and dissociation of 14-3-3 release the RAF regulatory domains RAS-binding domain and cysteine-rich domain for interaction with RAS-GTP and membrane lipids. This, in turn, results in RAF phosphorylation at Ser-621 and 14-3-3 reassociation, followed by its dimerization and ultimately substrate binding and phosphorylation. This review focuses on structural understanding of how distinct binding partners trigger a cascade of molecular events that induces RAF kinase activation.

Rho inhibition by lovastatin affects apoptosis and DSB repair of primary human lung cells in vitro and lung tissue in vivo following fractionated irradiation.

Thoracic radiotherapy causes damage of normal lung tissue, which limits the cumulative radiation dose and, hence, confines the anticancer efficacy of radiotherapy and impacts the quality of life of tumor patients. Ras-homologous (Rho) small GTPases regulate multiple stress responses and cell death. Therefore, we investigated whether pharmacological targeting of Rho signaling by the HMG-CoA-reductase inhibitor lovastatin influences ionizing radiation (IR)-induced toxicity in primary human lung fibroblasts, lung epithelial and lung microvascular endothelial cells in vitro and subchronic mouse lung tissue damage following hypo-fractionated irradiation (4x4 Gy). The statin improved the repair of radiation-induced DNA double-strand breaks (DSBs) in all cell types and, moreover, protected lung endothelial cells from IR-induced caspase-dependent apoptosis, likely involving p53-regulated mechanisms. Under the in vivo situation, treatment with lovastatin or the Rac1-specific small molecule inhibitor EHT1864 attenuated the IR-induced increase in breathing frequency and reduced the percentage of γH2AX and 53BP1-positive cells. This indicates that inhibition of Rac1 signaling lowers IR-induced residual DNA damage by promoting DNA repair. Moreover, lovastatin and EHT1864 protected lung tissue from IR-triggered apoptosis and mitigated the IR-stimulated increase in regenerative proliferation. Our data document beneficial anti-apoptotic and genoprotective effects of pharmacological targeting of Rho signaling following hypo-fractionated irradiation of lung cells in vitro and in vivo. Rac1-targeting drugs might be particular useful for supportive care in radiation oncology and, moreover, applicable to improve the anticancer efficacy of radiotherapy by widening the therapeutic window of thoracic radiation exposure.