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Previous studies showed that Fanconi anemia (FA) murine stem cells have defective reconstitution after bone marrow (BM) transplantation. The mechanism underlying this defect is not known. Here, we report defective homing of FA patient BM progenitors transplanted into mouse models. Using cells from patients carrying mutations in FA complementation group A (FA-A), we show that when transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) recipient mice, FA-A BM cells exhibited impaired homing activity. FA-A cells also showed defects in both cell-cell and cell-matrix adhesion. Complementation of FA-A deficiency by reexpression of FANCA readily restored adhesion of FA-A cells. A significant decrease in the activity of the Rho GTPase Cdc42 was found associated with these defective functions in patient-derived cells, and expression of a constitutively active Cdc42 mutant was able to rescue the adhesion defect of FA-A cells. These results provide the first evidence that FA proteins influence human BM progenitor homing and adhesion via the small GTPase Cdc42-regulated signaling pathway.

The Rho GTPase Cdc42 regulates adhesion, migration, and homing, as well as cell cycle progression, of hematopoietic stem cells, but its role in multilineage blood development remains unclear. We report here that inducible deletion of cdc42 in cdc42-floxed mouse bone marrow by the interferon-responsive, Mx1-Cre–mediated excision led to myeloid and erythroid developmental defects. Cdc42 deletion affected the number of early myeloid progenitors while suppressing erythroid differentiation. Cdc42-deficient mice developed a fatal myeloproliferative disorder manifested by significant leukocytosis with neutrophilia, myeloid hyperproliferation, and myeloid cell infiltration into distal organs. Concurrently, Cdc42 deficiency caused anemia and splenomegaly accompanied with decreased bone marrow erythroid burst-forming units (BFU-Es) and colony-forming units-erythroid (CFU-Es) activities and reduced immature erythroid progenitors, suggesting that Cdc42 deficiency causes a block in the early stage of erythropoiesis. Cdc42 activity is responsive to stimulation by SCF, IL3, SDF-1α, and fibronectin. The increased myelopoiesis and decreased erythropoiesis of the knockout mice are associated with an altered gene transcription program in hematopoietic progenitors, including up-regulation of promyeloid genes such as PU.1, C/EBP1α, and Gfi-1 in the common myeloid progenitors and granulocyte-macrophage progenitors and down-regulation of proerythroid gene such as GATA-2 in the megakaryocyte-erythroid progenitors. Thus, Cdc42 is an essential regulator of the balance between myelopoiesis and erythropoiesis.

Abstract 2019 Rac1, Cdc42 and RhoA, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton and aggregation in platelets. Although they have been implicated in regulation of platelet activation, the unique and redundant roles of each of the Rho GTPase in various signaling cascades and the resulting functional outcomes have yet to be clearly defined. In this study we compared their roles in several aspects of platelet activation by utilizing three small molecule inhibitors, NSC23766, CASIN, and GO4, that specifically suppress endogenous Rac1, Cdc42, and RhoA activities, respectively. These novel pharmacological inhibitors are active in direct binding to their specific GTPase substrates, i.e. NSC23766 to Rac1, CASIN to Cdc42, and G04 to RhoA, and in interfering with the GTP loading exchange reactions of each Rho GTPase catalyzed by respective guanine nucleotide exchange factors at 5–50 uM concentration range. First, effector-domain pull down assays confirmed that treatment of platelets with NSC23766 (30 uM), CASIN (10 uM) or GO4 (30-50 uM) specifically blocked collagen induced Rac1-GTP, Cdc42-GTP, and RhoA-GTP formations, respectively. Incubation of platelets with NSC23766 (30 uM) or CASIN (10 uM) effectively inhibited collagen-induced phosphorylation of the Rac/Cdc42 effector, PAK1. Addition of GO4 (30 uM) to platelets prior to stimulation with thrombin blocked RhoA/ROCK mediated phosphorylation of myosin light chain (MLC). Second, incubation of aspirin treated platelets containing apyrase (3 U/ml) with CASIN (10 uM), but not NSC23766 (30 uM) or GO4 (30 uM), inhibited filopodia formation on immobilized fibrinogen or collagen-related peptide (CRP), a GPVI agonist. On the other hand, treatment of platelets with CASIN (10 uM) or GO4 (30 uM), but not with NSC23766 (30 uM), inhibited spreading of platelets on immobilized fibrinogen in the presence of aspirin and apyrase. Third, NSC23766 (3-30 uM), CASIN (3-10 uM), and GO4 (5-50 uM) all inhibited secretion from platelet granules and secretion-dependent aggregation induced by threshold concentration of ADP, collagen, CRP, or thrombin in a concentration-dependent manner. However, while CASIN (10 uM) or GO4 (30 uM) completely blocked collagen or CRP induced aggregation in aspirin treated platelets containing apyrase, NSC23766 (30 uM) showed no effect. Fourth, while pre-incubation of platelets with 5 uM CASIN or 10 uM G04 alone only partially (15%) inhibited CRP induced platelet aggregation in aspirin treated samples, CASIN at 10 uM or a combination of 5 uM CASIN and 5 uM G04 were able to inhibit platelet aggregation by 90%. Fifth, GO4 (30 uM) but not CASIN (10 uM) inhibited thrombin stimulated phosphorylation of p38-MAPK (137%) in aspirin treated platelets in the presence of apyrase. Addition of GO4 (30 uM) or CASIN (10 uM) to aspirin treated platelets containing apyrase inhibited CRP induced phosphorylation of ERK1/2 by 94% and 53% respectively, However, in the absence of aspirin and apyrase GO4 (30 uM), but not CASIN (10 uM), completely inhibited CRP induced phosphorylation of ERK1/2. Finally, although both GO4 (30 uM) and CASIN (10 uM) completely inhibited CRP induced phosphorylation of MLC in aspirin treated platelets containing apyrase, GO4 (30 uM) maximally (94%) while CASIN (10 uM) partially (36%) inhibited phosphorylation of MLC in the absence of aspirin and apyrase. Taken together, these data suggest that: (a) Cdc42 is involved in integrin alphaIIbbeta3 and GPVI mediated filopodia formation, RhoA is involved in regulation of integrin alphaIIbbeta3 induced platelet spreading, whereas Rac1 is critical in secondary mediators (ADP/TXA2) mediated lamellipodia formation; (b) Cdc42 and RhoA regulate platelet aggregation in parallel pathways, possibly by affecting the RhoA/ROCK-MAPK-dependent and -independent phosphorylation of MLC; and (c) the crosstalk among Cdc42, Rac1 and RhoA plays an important role in signaling cascades involved in platelet activation. Disclosures: No relevant conflicts of interest to declare.

Abstract 2997 Poster Board II-975 Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton in platelets. Previously we have shown that Rac1 GTPase is involved in regulation of platelet secretion and aggregation by diverse signaling pathways (J Thromb Haemost 2007; 5: 1747-55). Others have reported that Rac1 is essential for GPVI-, but not G protein-dependent platelet aggregation (Pflugers Arch. 2009; 457:1173-85). Cdc42 was recently reported to be involved in collagen, but not collagen related peptide (CRP), a GPVI specific agonist, induced platelet aggregation (Platelets 2008; 19: 199-210). In this study we have investigated the role of Cdc42 in regulation of platelet function by using complementary approaches of (a) mouse gene targeting of Cdc42, and (b) specific inhibition of Cdc42 activity by a newly identified chemical inhibitor of Cdc42, CASIN (Cdc42 activity-specific inhibitor). Platelets from Cdc42−/− mice exhibited a complete lack of filopodia formation and spreading on collagen coated surfaces. Threshold concentrations of collagen, CRP or thrombin failed to induce shape change or aggregation in platelets from Cdc42−/− mice compared with induction of shape change and maximal aggregation in platelets from Cdc42+/+ mice. Platelets from Cdc42−/− mice, as compared to Cdc42+/+ mice, exhibited a significant inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin from the dense- and alpha-granules respectively. Increasing concentrations of the agonists only partially corrected the defective aggregation and secretion responses in Cdc42−/− platelets. These data provide the genetic evidence that Cdc42 is required for collagen, CRP and thrombin mediated platelet signaling and activation. Treatment of platelets with CASIN, but not a pharmacologically inactive analog, blocked collagen induced activation of Cdc42 without detectably affecting the Rac1 activity. Human platelets pre-incubated with CASIN (10 micro-M) exhibited a complete lack of filopodia formation and spreading on collagen coated surfaces. Further, treatment of platelets with CASIN (1-10 micro-M) inhibited: (a) aggregation induced by collagen, CRP, thrombin, ADP or U46619; (b) release of P-selectin and secretion of ATP induced by U46619; and (c) collagen induced phosphorylation of Akt. Addition of CASIN to platelets also blocked collagen or CRP induced aggregation in aspirinated platelets in the presence of apyrase. In other experiments, addition of CASIN to citrated platelet-rich plasma inhibited thrombin induced clot retraction. Significantly, removal of CASIN from the platelet samples by washing reversed inhibition of aggregation as well as clot retraction, reflecting a reversible suppression of Cdc42 activity by CASIN. Administration of CASIN into C57Bl/6 mice inhibited ex vivo platelet aggregation induced by collagen or ADP as well as significantly prolonged tail bleeding times. These data suggest that: (a) Cdc42 plays an essential, non-redundant role in platelet filopodia formation, spreading, secretion, aggregation and clot retraction; (b) Cdc42 is involved in GPVI, non-GPVI- and G protein-dependent signaling in platelets; (c) the pharmacologic inhibitor CASIN is capable of specifically and reversibly inhibiting Cdc42 activity in platelets, mimicking Cdc42 genetic knockout in mice. Altogether, our studies strongly implicate Cdc42 as a novel anti-platelet target, and present evidence that the Cdc42 specific small molecule inhibitor, CASIN, may have therapeutic potential. Disclosures: No relevant conflicts of interest to declare.

Fanconi anemia (FA) is a devastating blood disease associated with bone marrow (BM) failure. Currently the effective treatment for FA is BM transplantation. We and others showed that FA murine hematopoietic stem and progenitor cells (HSC/Ps) have defective hematopoietic reconstitution after BM transplantation. In addition, several clinical gene therapy trials in FA patients have failed to show sustained engraftment of FA HSC/Ps complemented with a functional FA gene. The mechanism underlying this engraftment defect remains unknown. To determine whether the poor engraftment of FA HSC/Ps observed in mouse knockout models and clinical gene therapy trials in FA patients results from an intrinsic defect of the FA HSC/Ps or an impaired microenvironment in the FA bone marrow, we have assessed the self-renewal ability of HSC/Ps and hematopoietic supportive capacity of BM stromal cells from FA-A patients. First, we performed quantitative cobblestone area-forming cell (CAFC) assays using MS-5 stromal layer. CAFCs were enumerated at 2 and 5 weeks of cocultures between BM mononuclear cells from a normal donor and three FA-A patients. Early-appearing CAFCs (week 2) represent transient repopulating cells equivalent to progenitor colony-forming units (CFUs), whereas late-appearing CAFCs (week 5) are representative of long-term repopulating stem cells. The frequency of FA-A CAFCs was 15–20 folds decreased compared with that of the normal donor at both week 2 and week 5. Second, we carried out long-term culture-initiating cell (LTC-IC) assays using BM mononuclear cells and BM-derived stromals from normal donors or FA-A patients. Compared to coculture with normal stromal cells, the recovery of colony forming cells (CFCs) that grew on FA-A stromal layer after 5–6 weeks incubation was significantly decreased for either normal or FA-A HSC/Ps (41.4% ± 3.1 CFC in normal HSC/Ps - normal stroma compared to 14.3% ± 1.6 CFC in normal HSC/Ps – FA-A stroma and 2.4 ± 0.6 CFC in FA-A HSC/Ps- FA-A stroma). Finally, we conducted CFC and cell-cell adhesion assays using BM-derived stromal cells from normal donors and FA-A patients. We observed that FA-A BM stroma failed to support HSC/P cells from either normal donors or FA-A patients. Specifically, severe adhesion defect was found in adhesion of normal and FA-A HSC/Ps with FA-A stromal layer (5.5% ± 0.6 CFC adhesion in normal HSC/Ps - normal stroma compared to 2.4% ± 0.5 CFC adhesion in normal HSC/Ps – FA-A stroma and 1.0 ± 0.7 CFC adhesion in FA-A HSC/Ps- FA-A stroma). These results suggest that both a cell-autonomous defect of FA HSC/Ps and an impaired FA BM microenvironment may contribute to the abnormal hematopoiesis in FA.

Hematopoietic stem/progenitor cells (HSPCs) are maintained by strictly regulated signals in the bone marrow microenvironment. One challenge in understanding the complex mode of HSPC regulation is to link intracellular signal components with extracellular stimuli. R-Ras is a member of the Ras family small GTPases. Previous mouse genetic studies suggest that R-Ras mRNA is primarily expressed in endothelial cells and R-Ras is involved in vascular angiogenesis. In clonal cell lines, although dominant mutant overexpression studies suggest a possible role of R-Ras in regulating cell adhesion and spreading, proliferation and/or differentiation in a cell-type dependent manner, it remains controversial whether R-Ras activity may promote or inhibit cell adhesion and migration. Here, in a mouse knockout model, we have examined the role of R-Ras in HSPC regulation by a combined in vivo and in vitro approach. Firstly, we found that R-Ras is expressed in the Lin− low density bone marrow cells of wild-type mice, and R-Ras activity in the cells is downregulated by cytokines and chemokines such as SCF and SDF-1a (∼ 20% and 40% of unstimulated control, respectively). Secondly, R-Ras deficiency did not significantly affect peripheral blood CBC, nor alter the frequency or distribution of long-term and short-term hematopoietic stem cells (defined by IL7Ra−Lin−Sca-1+c-Kit+CD34− and IL7Ra−Lin−Sca-1+c-Kit+CD34+ genotypes, respectively) in the bone marrow, peripheral blood and spleen. Competitive repopulation experiments using the wild-type and R-Ras−/− bone marrow cells at 1:1 ratio in lethally irradiated recipient mice showed no significant difference of blood cells of the two genotypes in the recipients up to 6 months post-transplantation. R-Ras−/− bone marrow cells did not show a detectable difference in colony forming unit activities assayed in the presence of various combinations of SCF, TPO, EPO, IL3, G-CSF and serum, compared with the matching wild-type cells. Thirdly, upon challenge with G-CSF, a HSPC mobilizing agent, R-Ras−/− mice demonstrated a markedly enhanced ability to mobilize HSPCs from bone marrow to peripheral blood as revealed by genotypic and colony-forming unit analyses (WT: 150 vs. KO: 320 per 200uL blood, p=0.018), and R-Ras−/− HSPCs exhibit significantly decreased homing activity (WT: 4.3% vs. KO: 2.8%, p

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and predisposition to malignancy. One of the potential therapeutic options for patients with FA is collection of autologous multipotent hematopoietic progenitors prior to the development of severe pancytopenia for autologous transplantation and gene therapy. However, poor engraftment of FA hematopoietic cells represents a major obstacle for effective transplantation. Our current study attempted to investigate the mechanism underlying defective engraftment of FA bone marrow (BM) cells. Using BM cells from patients carrying mutations in the FA complementation group A (FA-A), we demonstrate that SDF (Stromal cell-derived factor)-1alpha – and integrin-mediated migration and adhesion, respectively, is defective in FA primary BM cells compared to those from normal donors (more than 2-fold decrease in both migration and adhesion compared to normal BM cells). Complementation of the FA-A defect by retrovirus gene transfer of FANCA gene almost completely restores the ability of the BM cells to migrate towards the chemokine SDF-1alpha. Similar results are obtained with primary fibroblast cells derived from a FA-A patient, which show 3-fold and 35% decrease in adhesion and migration, respectively, compared to FANCA-corrected cells. Furthermore, when transplanted into immunodeficient Nod/scid recipient mice, the FA-A BM cells exhibited significantly impaired homing function whereas normal cells were efficiently homed in the bone marrow. A significant decrease in the activity of the Rho GTPase Cdc42 in FA-A cells is found associated with the patient cell defective functions. Taken together, these data suggest that the FA proteins play a role in the regulation of cell adhesion and migration in addition to maintaining genomic stability, influencing homing and engraftment, possibly via the small GTPase signaling pathway. These findings may have implications in development of strategies for restoring engraftment function of FA hematopoietic cells.