Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of canonical RAS GTPases. Here, we have analyzed the phenotypes of LZTR1 loss-of-function mutants in both fruit flies and mice and have demonstrated biochemical dependency on their RIT1 orthologs. Moreover, we show that LZTR1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of RIT1.

Afadin couples RAS GTPases to the polarity rheostat Scribble

AFDN/Afadin is required for establishment and maintenance of cell-cell contacts and is a unique effector of RAS GTPases. The biological consequences of RAS signalling to AFDN are unknown. Here, we use proximity-based proteomics to generate an interaction map for the long and short isoforms of AFDN, identifying the polarity protein SCRIB/Scribble as the top hit. We reveal that the first PDZ domain of SCRIB and the AFDN FHA domain mediate a direct but non-canonical interaction between these important adhesion and polarity proteins. Further, the dual RA domains of AFDN have broad specificity for RAS and RAP GTPases, and KRAS co-localizes with and promotes AFDN-SCRIB complex formation. Knockout of AFDN or SCRIB in MCF7 epithelial cells disrupts MAPK and PI3K activation and inhibits cell motility in a growth factor-dependent manner. These data have important implications for understanding why cells with activated RAS have reduced cell contacts and polarity defects, and finally begin to characterize AFDN as a RAS effector.

Synthetic Optimization and MAPK Pathway Activation Anticancer Mechanism of Polyisoprenylated Cysteinyl Amide Inhibitors

Abnormalities of the MAPK pathway play vital roles in cancer initiation and progression. RAS GTPases that are key upstream mediators of the pathway are mutated in 30% of human cancers. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed as potential targeted therapies against the RAS-driven cancers. The current study reports on the optimization of the PCAIs and the determination of their mechanisms of action in KRAS-mutant cancer cells. They display ClogP values ranging from 3.01 to 6.35, suppressing the viabilities of KRAS-mutant MDA-MB-231, A549, MIA PaCa-2, and NCI-H1299 cells in 2D and 3D cultures with EC50 values of 2.2 to 6.8, 2.2 to 7.6, 2.3 to 6.5 and 5.0 to 14 µM, respectively. When A549 cells were treated with the PCAIs, NSL-YHJ-2-27, for 48 h, no significant difference was observed in the levels of total or phosphorylated B- and C-Raf proteins. However, at 5 µM, it stimulated the phosphorylation of MEK1/2, ERK1/2, and p90RSK by 84%, 59%, and 160%, respectively, relative to controls. A non-farnesylated analog, NSL-YHJ-2-62, did not elicit similar effects. These data reveal that effects on the RAS-MAPK signaling axis most likely contribute to the anticancer effects of the PCAIs, possibly through the proapoptotic isoforms of p90RSK. The PCAIs may thus have the potential to serve the unmet therapeutic needs of patients with aberrant hyperactive G-protein signaling.

Impaired RAS Proteolysis Drives Clonal Hematopoietic Transformation

Recently, the protein LZTR1 (leucine zipper-like transcriptional regulator 1) was discovered as an adaptor for a cullin 3 complex responsible for ubiquitin-mediated degradation of RAS proteins. While these data provided a novel mechanism for RAS protein regulation, there is considerable controversy as to which RAS paralogs are physiologic substrates of LZTR1. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations in both LZTR1 as well as the RAS GTPase and LZTR1 substrate RIT1 were identified in patients with clonal hematopoietic disorders. However, the effects of these alterations on normal and maliganant hematopoiesis have not been evaluated. Here we utilized a series of genetically engineered murine models for germline and conditional deletion of LZTR1, RIT1, and expression of oncogenic RIT1 mutant which revealed a key role for LZTR1 in the regulation of hematopoietic stem cell (HSC) self-renewal and delineated a series of LZTR1-regulated substrates in hematopoietic cells. Consistent with a role for LZTR1 alterations in the Noonan Syndrome, germline homozygous deletion of Lztr1 was associated with lethality between embryonic day 17.5 and birth. Lztr1-/- fetuses had massive dyserythropoiesis and apoptosis of fetal liver hematopoietic cells. Competitive transplantation of E14.5 Lztr1 null fetal liver or bone marrow from 6-week-old Mx1-cre Lztr1 conditional knockout (cKO) mice resulted in striking increased self-renewal in primary and secondary competitive transplantation assays in vivo (Fig.A-B). Interestingly, recipient animals reconstituted with Lztr1-/- cells developed fatal myeloid and lymphoid leukemias characterized by anemia, thrombocytopenia, and increased myeloid and B-lymphoid cells (Fig.C-D). In order to identify the LZTR1 substrates responsible for effects on HSCs, we evaluated levels of all RAS GTPases in Lztr1 null HSCs. This revealed elevated KRas, NRas, MRas, and Rit1 protein in LZTR1 KO cells (Fig.E), with RIT1 being most prominently elevated. Evaluation of a cohort of 4,113 patients with hematologic malignancies identified 41 patients with somatic RIT1 mutations, the majority of which cluster in the switch II region and escape LZTR1-mediated ubiquitination, resulting in RIT1 protein accumulation (Fig.F-H). Given that the impact of RIT1 mutations on hematopoiesis is unknown, we next compared Lztr1 cKO with conditional expression of one of the most common leukemia-associated RIT1 mutants that escapes LZTR1-mediated ubiquitin (Rit1 M90I). Both Lztr1 cKO and Rit1 M90I conditional expression conferred GM-CSF hypersensitivity to HSCs in vitro, cytokine independent growth to human AML cell lines in vitro, and strong competitive self-renewal in vivo (Fig. I-J). Consistent with RIT1 mutations being found primarily in myeloid neoplasm patients, aged Mx1-cre Rit1M90I/WT mice developed fatal MPN, MDS, and mixed MDS/MPN disorders (Fig.K), which were serially transplantable into sublethally irradiated recipients. Despite convergent effects of LZTR1 and RIT1 on clonal HSC advantage, LZTR1 null cell lines did not solely require RIT1 for HSC advantage as revealed by Lztr1/Rit1 double KO mice. We therefore next carried out a series of experiments in RAS-less cells and whole genome CRISPR screens to delineate factors required for LZTR1 mediated hematopoietic transformation. This revealed that KRAS as well as MRAS and its RAF phosphatase partner SHOC2 were selective dependencies for LZTR1-mediated transformation. These data indicate that multiple RAS GTPases as well as RAF activation are required for LZTR1-mediated transformation (Fig.L). While considerable prior research has evaluated oncogenic alleles of RAS which alter RAS-GTP hydrolysis on hematopoiesis, the role of modulating RAS protein abundance on hematopoiesis is unknown. Here we identify RAS proteolysis as a novel regulator of HSC function, define the spectrum of RIT1 mutations in leukemia, and identify LZTR1 and RIT1 mutations as drivers of leukemogenesis. The discovery of RAS proteolysis as a novel driver of leukemogenesis has important therapeutic implications given efforts to therapeutically degrade RAS family members. Finally, the clinical importance of K/NRAS mutations on resistance to therapies in AML motivates future studies on the potential clinical impact of LZTR1 and RIT1 alterations in myeloid neoplasm patients. Figure 1 Figure 1. Disclosures Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Merck: Consultancy; Foundation Medicine Inc: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

EGFR-RAS-MAPK signaling is confined to the plasma membrane and associated endorecycling protrusions

The subcellular localization of RAS GTPases defines the operational compartment of the EGFR-ERK1/2 signaling pathway within cells. Hence, we used live-cell imaging to demonstrate that endogenous KRAS and NRAS tagged with mNeonGreen are predominantly localized to the plasma membrane. NRAS was also present in the Golgi apparatus and a tubular, plasma-membrane derived endorecycling compartment, enriched in recycling endosome markers (TERC). In EGF-stimulated cells, there was essentially no colocalization of either mNeonGreen-KRAS or mNeonGreen-NRAS with endosomal EGFR, which, by contrast, remained associated with endogenous Grb2-mNeonGreen, a receptor adaptor upstream of RAS. ERK1/2 activity was diminished by blocking cell surface EGFR with cetuximab, even after most ligand-bound, Grb2-associated EGFRs were internalized. Endogenous mCherry-tagged RAF1, an effector of RAS, was recruited to the plasma membrane, with subsequent accumulation in mNG-NRAS–containing TERCs. We propose that a small pool of surface EGFRs sustain signaling within the RAS-ERK1/2 pathway and that RAS activation persists in TERCs, whereas endosomal EGFR does not significantly contribute to ERK1/2 activity.

Ras Family of Small GTPases in CRC: New Perspectives for Overcoming Drug Resistance

Colorectal cancer remains among the cancers with the highest incidence, prevalence, and mortality worldwide. Although the development of targeted therapies against the EGFR and VEGFR membrane receptors has considerably improved survival in these patients, the appearance of resistance means that their success is still limited. Overactivation of several members of the Ras-GTPase family is one of the main actors in both tumour progression and the lack of response to cytotoxic and targeted therapies. This fact has led many resources to be devoted over the last decades to the development of targeted therapies against these proteins. However, they have not been as successful as expected in their move to the clinic so far. In this review, we will analyse the role of these Ras-GTPases in the emergence and development of colorectal cancer and their relationship with resistance to targeted therapies, as well as the status and new advances in the design of targeted therapies against these proteins and their possible clinical implications.

Dissecting the Involvement of Ras GTPases in Kidney Fibrosis

Many different regulatory mechanisms of renal fibrosis are known to date, and those related to transforming growth factor-β1 (TGF-β1)-induced signaling have been studied in greater depth. However, in recent years, other signaling pathways have been identified, which contribute to the regulation of these pathological processes. Several studies by our team and others have revealed the involvement of small Ras GTPases in the regulation of the cellular processes that occur in renal fibrosis, such as the activation and proliferation of myofibroblasts or the accumulation of extracellular matrix (ECM) proteins. Intracellular signaling mediated by TGF-β1 and Ras GTPases are closely related, and this interaction also occurs during the development of renal fibrosis. In this review, we update the available in vitro and in vivo knowledge on the role of Ras and its main effectors, such as Erk and Akt, in the cellular mechanisms that occur during the regulation of kidney fibrosis (ECM synthesis, accumulation and activation of myofibroblasts, apoptosis and survival of tubular epithelial cells), as well as the therapeutic strategies for targeting the Ras pathway to intervene on the development of renal fibrosis.

The Small GTPases in Fungal Signaling Conservation and Function

Monomeric GTPases, which belong to the Ras superfamily, are small proteins involved in many biological processes. They are fine-tuned regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several families have been identified in organisms from different kingdoms. Overall, the most studied families are Ras, Rho, Rab, Ran, Arf, and Miro. Recently, a new family named Big Ras GTPases was reported. As a general rule, the proteins of all families have five characteristic motifs (G1–G5), and some specific features for each family have been described. Here, we present an exhaustive analysis of these small GTPase families in fungi, using 56 different genomes belonging to different phyla. For this purpose, we used distinct approaches such as phylogenetics and sequences analysis. The main functions described for monomeric GTPases in fungi include morphogenesis, secondary metabolism, vesicle trafficking, and virulence, which are discussed here. Their participation during fungus–plant interactions is reviewed as well.