Summary Acute myeloid leukemia (AML) is an aggressive cancer with very poor outcomes. To identify additional drivers of leukemogenesis, we analyzed sequence data from 1,727 unique individual AML patients, which revealed mutations in ubiquitin ligase family genes in 11.2% of adult AML samples with mutual exclusivity. The Skp1/Cul1/Fbox (SCF) E3 ubiquitin ligase complex gene FBXO11 was the most significantly downregulated gene of the SCF complex in AML. FBXO11 catalyzes K63-linked ubiquitination of a novel target, LONP1, which promotes entry into mitochondria, thereby enhancing mitochondrial respiration. Reduced mitochondrial respiration secondary to FBXO11 depletion imparts myeloid-biased stem cell properties in primary CD34 + hematopoietic stem progenitor cells (HSPC). In a human xenograft model, depletion of FBXO11 cooperated with AML1-ETO and mutant KRAS G12D to generate serially transplantable AML enriched for primitive cells. Our findings suggest that reduced FBXO11 primes HSPC for myeloid-biased self-renewal through attenuation of LONP1-mediated regulation of mitochondrial respiration.
ABSTRACT Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear. Here, we identify EHD2 and EHBP1 as novel regulators of Notch activation in endothelial cells through controlling endocytosis of Delta-like ligand 4 (Dll4). Knockout of EHBP1 and EHD2 in zebrafish produced a significant increase in ectopic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. In vitro , EHBP1 and EHD2 localized to plasma membrane-bound Dll4 and actin independently of clathrin. Disruption of caveolin endocytosis resulted in EHBP1 and EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization in endothelial cells. Overall, we demonstrate that EHBP1 and EHD2 regulate Dll4 endocytosis by anchoring caveolar endocytic pits to the actin cytoskeleton.
ABSTRACT In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35’s primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization, which is required to setup proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent regulator of actin architecture during blood vessel development.
Abstract In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35’s primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence, apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization through limiting Rac1 and RhoA activity, which is required to set up proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent brake of actin remodeling during blood vessel development.
Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear about how this system is regulated. Our objective was to determine the involvement of Epsin 15 Homology Domain Containing 2 (EHD2) in delta-like ligand 4 (Dll4) endocytosis during Notch activation.
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