Altered platelet-megakaryocyte endocytosis and trafficking of albumin and fibrinogen in RUNX1 haplodeficiency.

ABSTRACT: Platelet α-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germ line RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMMs), is associated with thrombocytopenia, platelet dysfunction, and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen, and immunoglobulin G (IgG) were decreased in a patient with FPDMM. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen, and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, small interfering RNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared with control cells, with increases in caveolin-1 and flotillin-1 (2 independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes), and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 KD resulted in increased colocalization of albumin with flotillin and fibrinogen with RAB11, suggesting altered trafficking of both proteins. The increased uptake of albumin and fibrinogen, as well as levels of caveolin-1, flotillin-1, LAMP2, and IFITM3, were recapitulated by short hairpin RNA RUNX1 KD in CD34+-derived MK. To our knowledge, these studies provide first evidence that platelet endocytosis of albumin and fibrinogen is impaired in some patients with RUNX1-haplodeficiency and suggest that megakaryocytes have enhanced endocytosis with defective trafficking, leading to loss of these proteins by distinct mechanisms. This study provides new insights into mechanisms governing endocytosis and α-granule deficiencies in RUNX1-haplodeficiency.

Plasma growth factors maintain constitutive translation in platelets to regulate reactivity and thrombotic potential.

ABSTRACT: Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.

Altered Platelet-Megakaryocyte Endocytosis and Trafficking of Albumin and Fibrinogen in RUNX1 Haplodeficiency.

Platelet α-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germline RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMM), is associated with thrombocytopenia, platelet dysfunction and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen and IgG levels were decreased in a FPDMM patient. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, siRNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared to control cells, with increases in caveolin-1 and flotillin-1 (two independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes) and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 knockdown increased colocalization of albumin with flotillin and of fibrinogen with RAB11 suggesting altered trafficking of both. The increased albumin and fibrinogen uptake and levels of caveolin-1, flotillin-1, LAMP2 and IFITM3 were recapitulated by shRNA RUNX1 knockdown in CD34 + -derived MK. These studies provide the first evidence that in RUNX1- haplodeficiency platelet endocytosis of albumin and fibrinogen is impaired and that megakaryocytes have enhanced endocytosis with defective trafficking leading to loss of these proteins by distinct mechanisms. They provide new insights into mechanisms governing endocytosis and α-granule deficiencies in RUNX1- haplodeficiency. Key points: Platelet content and endocytosis of α-granule proteins, albumin, fibrinogen and IgG, are decreased in germline RUNX1 haplodeficiency. In RUNX1 -deficient HEL cells and primary MK endocytosis is enhanced with defective trafficking leading to decreased protein levels.

Defective RAB31-mediated megakaryocytic early endosomal trafficking of VWF, EGFR, and M6PR in RUNX1 deficiency.

Transcription factor RUNX1 is a master regulator of hematopoiesis and megakaryopoiesis. RUNX1 haplodeficiency (RHD) is associated with thrombocytopenia and platelet granule deficiencies and dysfunction. Platelet profiling of our study patient with RHD showed decreased expression of RAB31, a small GTPase whose cell biology in megakaryocytes (MKs)/platelets is unknown. Platelet RAB31 messenger RNA was decreased in the index patient and in 2 additional patients with RHD. Promoter-reporter studies using phorbol 12-myristate 13-acetate-treated megakaryocytic human erythroleukemia cells revealed that RUNX1 regulates RAB31 via binding to its promoter. We investigated RUNX1 and RAB31 roles in endosomal dynamics using immunofluorescence staining for markers of early endosomes (EEs; early endosomal autoantigen 1) and late endosomes (CD63)/multivesicular bodies. Downregulation of RUNX1 or RAB31 (by small interfering RNA or CRISPR/Cas9) showed a striking enlargement of EEs, partially reversed by RAB31 reconstitution. This EE defect was observed in MKs differentiated from a patient-derived induced pluripotent stem cell line (RHD-iMKs). Studies using immunofluorescence staining showed that trafficking of 3 proteins with distinct roles (von Willebrand factor [VWF], a protein trafficked to α-granules; epidermal growth factor receptor; and mannose-6-phosphate) was impaired at the level of EE on downregulation of RAB31 or RUNX1. There was loss of plasma membrane VWF in RUNX1- and RAB31-deficient megakaryocytic human erythroleukemia cells and RHD-iMKs. These studies provide evidence that RAB31 is downregulated in RHD and regulates megakaryocytic vesicle trafficking of 3 major proteins with diverse biological roles. EE defect and impaired vesicle trafficking is a potential mechanism for the α-granule defects observed in RUNX1 deficiency.

Platelet microRNAs inhibit primary tumor growth via broad modulation of tumor cell mRNA expression in ectopic pancreatic cancer in mice.

We investigated the contributions of platelet microRNAs (miRNAs) to the rate of growth and regulation of gene expression in primary ectopic tumors using mouse models. We previously identified an inhibitory role for platelets in solid tumor growth, mediated by tumor infiltration of platelet microvesicles (microparticles) which are enriched in platelet-derived miRNAs. To investigate the specific roles of platelet miRNAs in tumor growth models, we implanted pancreatic ductal adenocarcinoma cells as a bolus into mice with megakaryocyte-/platelet-specific depletion of mature miRNAs. We observed an ~50% increase in the rate of growth of ectopic primary tumors in these mice compared to controls including at early stages, associated with reduced apoptosis in the tumors, in particular in tumor cells associated with platelet microvesicles-which were depleted of platelet-enriched miRNAs-demonstrating a specific role for platelet miRNAs in modulation of primary tumor growth. Differential expression RNA sequencing of tumor cells isolated from advanced primary tumors revealed a broad cohort of mRNAs modulated in the tumor cells as a function of host platelet miRNAs. Altered genes comprised 548 up-regulated transcripts and 43 down-regulated transcripts, mostly mRNAs altogether spanning a variety of growth signaling pathways-notably pathways related to epithelial-mesenchymal transition-in tumor cells from platelet miRNA-deleted mice compared with those from control mice. Tumors in platelet miRNA-depleted mice showed more sarcomatoid growth and more advanced tumor grade, indicating roles for host platelet miRNAs in tumor plasticity. We further validated increased protein expression of selected genes associated with increased cognate mRNAs in the tumors due to platelet miRNA depletion in the host animals, providing proof of principle of widespread effects of platelet miRNAs on tumor cell functional gene expression in primary tumors in vivo. Together, these data demonstrate that platelet-derived miRNAs modulate solid tumor growth in vivo by broad-spectrum restructuring of the tumor cell transcriptome.

Platelets and extracellular vesicles and their cross talk with cancer.

Platelets play significant and varied roles in cancer progression, as detailed throughout this review series, via direct interactions with cancer cells and by long-range indirect interactions mediated by platelet releasates. Microvesicles (MVs; also referred to as microparticles) released from activated platelets have emerged as major contributors to the platelet-cancer nexus. Interactions of platelet-derived MVs (PMVs) with cancer cells can promote disease progression through multiple mechanisms, but PMVs also harbor antitumor functions. This complex relationship derives from PMVs' binding to both cancer cells and nontransformed cells in the tumor microenvironment and transferring platelet-derived contents to the target cell, each of which can have stimulatory or modulatory effects. MVs are extracellular vesicles of heterogeneous size, ranging from 100 nm to 1 µm in diameter, shed by living cells during the outward budding of the plasma membrane, entrapping local cytosolic contents in an apparently stochastic manner. Hence, PMVs are encapsulated by a lipid bilayer harboring surface proteins and lipids mirroring the platelet exterior, with internal components including platelet-derived mature messenger RNAs, pre-mRNAs, microRNAs, and other noncoding RNAs, proteins, second messengers, and mitochondria. Each of these elements engages in established and putative PMV functions in cancer. In addition, PMVs contribute to cancer comorbidities because of their roles in coagulation and thrombosis and via interactions with inflammatory cells. However, separating the effects of PMVs from those of platelets in cancer contexts continues to be a major hurdle. This review summarizes our emerging understanding of the complex roles of PMVs in the development and progression of cancer and cancer comorbidities.

High-efficiency unassisted transfection of platelets with naked double-stranded miRNAs modulates signal-activated translation and platelet function.

We sought novel approaches to improve transfection efficiencies of microRNAs (miRNAs) in platelets, and to apply these approaches to investigate the roles of miRNAs in regulating signal-activated protein translation and functional effects. We found that ex vivo human platelets support gymnosis---internalization of ectopic miRNAs following co-incubation in the absence of conventional transfection reagents or schemes---and subsequently incorporate transfected miRNA into ARGONAUTE2 (AGO2)-based RNA-induced silencing complexes (RISC). Thrombin/fibrinogen stimulation activated translation of miR-223-3p target SEPTIN2, which was suppressed by miR-223-3p transfection in an AGO2/RISC-dependent manner. Thrombin/fibrinogen-induced exosome and microvesicle generation was inhibited by miR-223-3p transfection, and this effect was reversed with a RISC inhibitor. Platelet gymnosis of naked miRNAs appeared to be mediated in part by endocytic pathways including clathrin-dependent and fluid-phase endocytosis and caveolae. These results demonstrate the ability of ex vivo platelets to internalize ectopic miRNAs by unassisted transfection, and utilize them to modulate signal-activated translation and platelet function. Our results identify new roles for miR-223-3p in extracellular vesicle generation in stimulated platelets. High-efficiency gymnotic transfection of miRNAs in ex vivo platelets may be a broadly useful tool for exploring molecular genetic regulation of platelet function.

GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6-/- platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5'-diphosphate stimulation in GRK6-/- platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation.

Concepts and advances in cancer therapeutic vulnerabilities in RAS membrane targeting.

For decades oncogenic RAS proteins were considered undruggable due to a lack of accessible binding pockets on the protein surfaces. Seminal early research in RAS biology uncovered the basic paradigm of post-translational isoprenylation of RAS polypeptides, typically with covalent attachment of a farnesyl group, leading to isoprenyl-mediated RAS anchorage at the plasma membrane and signal initiation at those sites. However, the failure of farnesyltransferase inhibitors to translate to the clinic stymied anti-RAS therapy development. Over the past ten years, a more complete picture has emerged of RAS protein maturation, intracellular trafficking, and location, positioning and retention in subdomains at the plasma membrane, with a corresponding expansion in our understanding of how these properties of RAS contribute to signal outputs. Each of these aspects of RAS regulation presents a potential vulnerability in RAS function that may be exploited for therapeutic targeting, and inhibitors have been identified or developed that interfere with RAS for nearly all of them. This review will summarize current understanding of RAS membrane targeting with a focus on highlighting development and outcomes of inhibitors at each step.

Defective RAB1B-related megakaryocytic ER-to-Golgi transport in RUNX1 haplodeficiency: impact on von Willebrand factor.

Patients with RUNX1 haplodeficiency have thrombocytopenia, platelet dysfunction, and deficiencies of α-granules and dense granules. Platelet expression profiling of a patient with a heterozygous RUNX1 mutation (c.969-323G>T) revealed decreased RAB1B, which encodes a small G protein. RAB GTPases regulate vesicle trafficking, and RAB1B is implicated in endoplasmic reticulum (ER)-to-Golgi transport in nonhematopoietic cells, but its role in megakaryocytes (MK) is unknown. We addressed the hypothesis that RAB1B is a transcriptional target of RUNX1 and that RAB1B regulates ER-to-Golgi transport in MK cells. Chromatin immunoprecipitation studies and electrophoretic mobility shift assay using phorbol 12-myristate 13-acetate (PMA)-treated human erythroleukemia cells revealed RUNX1 binding to RAB1B promoter region RUNX1 consensus sites, and their mutation reduced the promoter activity. RAB1B promoter activity and protein expression were inhibited by RUNX1 siRNA and enhanced by RUNX1 overexpression. These indicate that RAB1B is a direct RUNX1 target, providing a mechanism for decreased RAB1B in patient platelets. Vesicle trafficking from ER to Golgi in PMA-treated human erythroleukemia cells was impaired along with Golgi disruption on siRNA downregulation of RUNX1 or RAB1B. The effects of RUNX1 knockdown were reversed by RAB1B reconstitution. Trafficking of von Willebrand factor (vWF), an α-granule MK synthesized protein, was impaired with RUNX1 or RAB1B downregulation and reconstituted by ectopic RAB1B expression. Platelet vWF was decreased in patients with RUNX1 mutations. Thus, ER-to-Golgi transport, an early critical step in protein trafficking to granules, is impaired in megakaryocytic cells on RUNX1 downregulation, secondary to decreased RAB1B expression. Impaired RAB1B mediated ER-to-Golgi transport contributes to platelet α-granule defects in RUNX1 haplodeficiency.

Platelet Microparticles and miRNA Transfer in Cancer Progression: Many Targets, Modes of Action, and Effects Across Cancer Stages.

Platelet-derived microparticles (PMPs) have long been known to increase in circulation in the presence of cancer, and have been considered to be cancer promoting by multiple mechanisms including shrouding of circulating tumor cells allowing immune evasion, inducing a procoagulant state associated with increased risk for venous thromboembolic events in cancer patients, and supporting metastatic dissemination by establishment of niches for anchorage of circulating tumor cells. These modes of PMP-enhanced progression of late stage cancer are generally based on the adhesive and procoagulant surfaces of PMPs. However, it is now clear that PMPs can also act as intercellular signaling vesicles, by fusion with target cells and transfer of a broad array of platelet-derived molecular contents including growth factors, angiogenic modulators, second messengers, lipids, and nucleic acids. It is also now well established that PMPs are major repositories of microRNAs (miRNAs). In recent years, new roles of PMPs in cancer have begun emerging, primarily reflecting their ability to transfer miRNA contents and modulate gene expression in target cells, allowing PMPs to affect cancer development at many stages. PMPs have been shown to interact with and transfer miRNAs to various blood vascular cells including endothelium, macrophages and neutrophils. As each of these contributes to cancer progression, PMP-mediated miRNA transfer can affect immune response, NETosis, tumor angiogenesis, and likely other cancer-associated processes. Recently, PMP miRNA transfer was found to suppress primary tumor growth, via PMP infiltration in solid tumors, anchorage to tumor cells and direct miRNA transfer, resulting in tumor cell gene suppression and inhibition of tumor growth. This mini-review will summarize current knowledge of PMP-miRNA interactions with cancer-associated cells and effects in cancer progression, and will indicate new research directions for understanding platelet-cancer interactions.

Transcription Factor RUNX1 Regulates Platelet PCTP (Phosphatidylcholine Transfer Protein): Implications for Cardiovascular Events: Differential Effects of RUNX1 Variants.

BACKGROUND: PCTP (phosphatidylcholine transfer protein) regulates the intermembrane transfer of phosphatidylcholine. Higher platelet PCTP expression is associated with increased platelet responses on activation of protease-activated receptor 4 thrombin receptors noted in black subjects compared with white subjects. Little is known about the regulation of platelet PCTP. Haplodeficiency of RUNX1, a major hematopoietic transcription factor, is associated with thrombocytopenia and impaired platelet responses on activation. Platelet expression profiling of a patient with a RUNX1 loss-of-function mutation revealed a 10-fold downregulation of the PCTP gene compared with healthy controls. METHODS: We pursued the hypothesis that PCTP is regulated by RUNX1 and that PCTP expression is correlated with cardiovascular events. We studied RUNX1 binding to the PCTP promoter using DNA-protein binding studies and human erythroleukemia cells and promoter activity using luciferase reporter studies. We assessed the relationship between RUNX1 and PCTP in peripheral blood RNA and PCTP and death or myocardial infarction in 2 separate patient cohorts (587 total patients) with cardiovascular disease. RESULTS: Platelet PCTP protein in the patient was reduced by ≈50%. DNA-protein binding studies showed RUNX1 binding to consensus sites in ≈1 kB of PCTP promoter. PCTP expression was increased with RUNX1 overexpression and reduced with RUNX1 knockdown in human erythroleukemia cells, indicating that PCTP is regulated by RUNX1. Studies in 2 cohorts of patients showed that RUNX1 expression in blood correlated with PCTP gene expression; PCTP expression was higher in black compared with white subjects and was associated with future death/myocardial infarction after adjustment for age, sex, and race (odds ratio, 2.05; 95% confidence interval 1.6-2.7; P<0.0001). RUNX1 expression is known to initiate at 2 alternative promoters, a distal P1 and a proximal P2 promoter. In patient cohorts, there were differential effects of RUNX1 isoforms on PCTP expression with a negative correlation in blood between RUNX1 expressed from the P1 promoter and PCTP expression. CONCLUSIONS: PCTP is a direct transcriptional target of RUNX1. PCTP expression is associated with death/myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events.

Platelet microparticles infiltrating solid tumors transfer miRNAs that suppress tumor growth.

Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 was a major species in this transfer. PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by PMPs. Conversely, Par4-deleted mice, which had reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial mt-Nd2, and Snora75, a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability.

Regulation of Ras signaling and function by plasma membrane microdomains.

Together H-, N- and KRAS mutations are major contributors to ~30% of all human cancers. Thus, Ras inhibition remains an important anti-cancer strategy. The molecular mechanisms of isotypic Ras oncogenesis are still not completely understood. Monopharmacological therapeutics have not been successful in the clinic. These disappointing outcomes have led to attempts to target elements downstream of Ras, mainly targeting either the Phosphatidylinositol 3-Kinase (PI3K) or Mitogen-Activated Protein Kinase (MAPK) pathways. While several such approaches are moderately effective, recent efforts have focused on preclinical evaluation of combination therapies to improve efficacies. This review will detail current understanding of the contributions of plasma membrane microdomain targeting of Ras to mitogenic and tumorigenic signaling and tumor progression. Moreover, this review will outline novel approaches to target Ras in cancers, including targeting schemes for new drug development, as well as putative re-purposing of drugs in current use to take advantage of blunting Ras signaling by interfering with Ras plasma membrane microdomain targeting and retention.

Inhibition of Galectin-1 Sensitizes HRAS-driven Tumor Growth to Rapamycin Treatment.

The goal of this study was to develop combinatorial application of two drugs currently either in active use as anticancer agents (rapamycin) or in clinical trials (OTX008) as a novel strategy to inhibit Harvey RAS (HRAS)-driven tumor progression. HRAS anchored to the plasma membrane shuttles from the lipid ordered (Lo) domain to the lipid ordered/lipid disordered border upon activation, and retention of HRAS at these sites requires galectin-1. We recently showed that genetically enforced Lo sequestration of HRAS inhibited mitogen-activated protein kinase (MAPK) signaling, but not phoshatidylinositol 3-kinase (PI3K) activation. Here we show that inhibition of galectin-1 with OTX008 sequestered HRAS in the Lo domain, blocked HRAS-mediated MAPK signaling, and attenuated HRAS-driven tumor progression in mice. HRAS-driven tumor growth was also attenuated by treatment with mammalian target of rapamycin (mTOR) inhibitor rapamycin, and this effect was further enhanced in tumors driven by Lo-sequestered HRAS. These drugs also revealed bidirectional cross-talk in HRAS pathways. Moreover, dual pathway inhibition with OTX008 and rapamycin resulted in nearly complete ablation of HRAS-driven tumor growth. These findings indicate that membrane microdomain sequestration of HRAS with galectin-1 inhibition, coupled with mTOR inhibition, may support a novel therapeutic approach to treat HRAS-mutant cancer.

Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets.

Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (ß3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband ß3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/ß3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband ß3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband ß3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.

RLIP76 regulates Arf6-dependent cell spreading and migration by linking ARNO with activated R-Ras at recycling endosomes.

R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(Δ1-180) or RLIP76(Δ180-192). Spreading and migration required RLIP76(1-180), and RLIP76(Δ1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(Δ1-180). Similarly, RLIP76(Δ1-180) or RLIP76(Δ180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.

The RLIP76 N-terminus binds ARNO to regulate PI 3-kinase, Arf6 and Rac signaling, cell spreading and migration.

RLIP76 is a multifunctional protein involved in tumor growth and angiogenesis, and a promising therapeutic target in many cancers. RLIP76 harbors docking sites for many proteins, and we have found that it interacts with ARNO, a guanine nucleotide exchange factor for Arf6, and that RLIP76 regulates activation of Rac1 via Arf6, and regulates cell spreading and migration in an ARNO and Arf6-dependent manner. Here we show that ARNO interacts with the RLIP76 N-terminal domain, and this domain was required for RLIP76-dependent cell spreading and migration. We identified two sites in the RLIP76 N-terminus with differential effects on ARNO binding and downstream signaling: Ser29/Ser30 and Ser62. Ser29/30 mutation to Alanine inhibited ARNO interaction and was sufficient to block RLIP76-dependent cell spreading and migration, as well as RLIP76-dependent Arf6 activation. In contrast, RLIP76(S62A) interacted with ARNO and supported Arf6 activation. However, both sets of mutations blocked Rac1 activation. RLIP76-mediated Rac and Arf6 activation required PI3K activity. S29/30A mutations inhibited RLIP76-dependent PI3K activation, but S62A mutation did not. Together these results show that ARNO interaction with the RLIP76 N-terminus regulates cell spreading and motility via PI3K and Arf6, independent of RLIP76 control of Rac.