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In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753–65. ©2016 AACR.

Description of additional methods and procedures used in the study.

GO terms for NEK6 upregulated genes Day 5 after castration (filtered for >10 GeneHits).

Legend for Supplemental Figures S1-S6.

Virus-like particles (VLPs) are promising scaffolds for biomaterials as well as diagnostic and therapeutic applications. However, there are some key challenges to be solved, such as the ability to engineer alternate sizes for varied use cases. To this end, we created a library of MS2 VLP variants at two key residues in the coat protein which have been implicated as important to controlling VLP size and geometry. By adapting a method for systematic mutagenesis coupled with size-based selections and high-throughput sequencing as a readout, we developed a quantitative assessment of two residues in MS2 coat protein that govern the size shift in MS2 VLPs. We then applied the strategy to the equivalent residues in Qβ VLPs, an MS2 homolog, and demonstrate that the analogous pair of residues are also able to impact VLP size and shape. These results underscore the power of fitness landscapes in identifying critical features for assembly.

Purpose The purpose of this quality improvement project was to improve consistency of discharge teaching in women who used progesterone-containing hormonal contraceptive medications and received sugammadex during general anesthesia, as there is a risk of unintended pregnancy for 1 week after administration of sugammadex. Design This project used a predesign and postdesign using two separate sample groups of patients and postanesthesia care unit (PACU) nurses. Methods The sample consisted of 31 total women of childbearing age and 59 PACU nurses. Simplification of sugammadex discharge instructions was achieved by incorporating evidence-based recommendations for electronic discharge instructions and nursing education. PACU nurses were educated and surveyed before and after regarding frequency of discharge teaching, clarity, and comprehension of the after-visit summary and knowledge of sugammadex. Patients were called via telephone postoperatively to assess recall of sugammadex discharge teaching. Findings Postoperative patient phone calls identified a small increase in patient recall of discharge instructions from 5 of 14 patients (35.7%) before implementation to 7 of 17 after implementation (41.2%). PACU nurse surveys indicated an increase in self-reported frequency of sugammadex discharge teaching (34.8% vs 64.2%, P = .024) and that new discharge instructions contained more clear, comprehensive information as compared with previous instructions (29.4% vs 75.5%, P = .001). Conclusions This quality improvement project successfully implemented more consistent and comprehensive discharge instructions for women who receive sugammadex intraoperatively. Limitations of the project included a small sample size and short implementation intervals. As a result of switching to uniform discharge instructions, more patients received important discharge teaching from PACU nurses, and the percentage of patients who recalled this information increased.

The purpose of this project was to improve the consistency of verbal and written discharge instructions for women of childbearing age (13-55 years) taking hormonal contraceptives who receive aprepitant perioperatively, to address the need to use a secondary form of birth control for 28 days, as well as to increase the knowledge and confidence of Registered Nurses when providing discharge instructions.This quality improvement project used a pre/post design to evaluate two separate groups of patients and registered nurses.The patient sample consisted of 30 total women of childbearing age who received aprepitant during the perioperative period (15 pre and 15 post). The PACU nurse sample included 15 ambulatory surgery center nurses and 58 main hospital nurses for a combined sample of 73 PACU nurses. The PACU nurses were provided with educational in-service regarding information about aprepitant and its drug interactions. PACU nurses were additionally instructed to provide patient discharge instructions using both a written and verbal format. Patients were called postoperatively before and after both the written after visit summary (AVS) changes and the PACU nurse in-services, PACU nurses were evaluated on their knowledge, confidence, and frequency of discharge teaching before and after their educational in-service. The PACU nurses were surveyed 90 days after the intervention to assess their long-term knowledge retention.There was a significant increase in nurse knowledge about aprepitant from preimplementation to postimplementation (61.39% vs 81.95%, P.001). Nursing knowledge showed a non-significant decline at 90-days postimplementation (81.95% vs 73.68%, P = .096) although remained significantly higher than preimplementation scores (73.68% vs 61.39%, P = .003). There was an overall 33.3% increase in the percentage of patients who were able to recall receiving aprepitant and the need to use a secondary form of birth control when comparing the preintervention group to the postintervention group (26.7% vs 60%, P = .123).The findings suggest that providing a standardized presentation about aprepitant may improve the PACU nurses' ability to verbalize key information about aprepitant, including the need for patients to use a secondary form of birth control. This increase in nursing knowledge and confidence, along with improved written discharge instructions, may have led to improved patient comprehension of aprepitant discharge education. Additionally, there was an increase in the percentage of patients who were able to recall the need to use a secondary form birth control for 28 days, to reduce the likelihood of an unintentional pregnancy.

Ribosomopathies are a class of disorders caused by defects in the structure or function of the ribosome and characterized by tissue-specific abnormalities. Diamond Blackfan anemia (DBA) arises from different mutations, predominantly in genes encoding ribosomal proteins (RPs). Apart from the anemia, skeletal defects are among the most common anomalies observed in patients with DBA, but they are virtually restricted to radial ray and other upper limb defects. What leads to these site-specific skeletal defects in DBA remains a mystery. Using a novel mouse model for RP haploinsufficiency, we observed specific, differential defects of the limbs. Using complementary in vitro and in vivo approaches, we demonstrate that reduced WNT signaling and subsequent increased β-catenin degradation in concert with increased expression of p53 contribute to mesenchymal lineage failure. We observed differential defects in the proliferation and differentiation of mesenchymal stem cells (MSCs) from the forelimb versus the hind limbs of the RP haploinsufficient mice that persisted after birth and were partially rescued by allelic reduction of Trp53. These defects are associated with a global decrease in protein translation in RP haploinsufficient MSCs, with the effect more pronounced in cells isolated from the forelimbs. Together these results demonstrate translational differences inherent to the MSC, explaining the site-specific skeletal defects observed in DBA.

Studies of the severely pancytopenic scat mouse model first demonstrated the crucial role of RASA3, a dual RAS and RAP GTPase activating protein (GAP), in hematopoiesis. RASA3 is required for survival in utero; germline deletion is lethal at E12.5–13.5 due to severe hemorrhage. Here, conditional deletion in hematopoietic stem and progenitor cells (HSPCs) using Vav-iCre recapitulates the null phenotype demonstrating that RASA3 is required at the stem and progenitor level to maintain blood vessel development and integrity and effective blood production. In adults, bone marrow blood cell production and spleen stress erythropoiesis are suppressed significantly upon induction of RASA3 deficiency, leading to pancytopenia and death within two weeks. Notably, RASA3 missense mutations in two mouse models, scat (G125V) and hlb381 (H794L), show dramatically different hematopoietic consequences specific to both genetic background and molecular variant. The mutation effect is mediated at least in part by differential effects on RAS and RAP activation. In addition, we show that the role of RASA3 is conserved during human terminal erythropoiesis, highlighting a potential function for the RASA3-RAS axis in disordered erythropoiesis in humans. Finally, global transcriptomic studies in scat suggest potential targets to ameliorate disease progression., Author summary Hematopoiesis is the process by which blood cells are formed. An individual must have a normal complement of red blood cells to prevent anemia, platelets to control bleeding, and white blood cells to maintain immune functions. All blood cells are derived from hematopoietic stem cells that differentiate into progenitor cells that then develop into mature circulating cells. We studied several mouse strains carrying different mutations in the gene encoding RASA3 and human CD34+ cells, which can be induced to produce blood cells in culture. We show that RASA3 is required at the earliest stages of blood formation, the stem and progenitor cells, and that the complement of genes other than RASA3, or the genetic background, profoundly alters the overall effect on blood formation. Further, the molecular nature of the mutation in RASA3 also has a profound and independent effect on overall blood formation. One mutant mouse strain, designated scat, suffers cyclic anemia characterized by severe anemic crisis episodes interspersed with remissions where the anemia significantly improves. Comparison of scat crisis and remission hematopoietic stem and progenitor cells reveals striking differences in gene expression. Analyses of these expression differences provide clues to processes that potentially drive improvement of anemia in scat and provide new avenues to pursue in future studies to identify novel therapeutics for anemia.

Inherited bone marrow failure syndromes (IBMFS) are a heterogenous group of disorders characterized by dysregulated hematopoiesis across various lineages, predisposition to malignancy, and diverse syndromic features. The scat (severe combined anemia and thrombocytopenia) mouse model has been characterized as a unique model of IBMFS. Scat carries an autosomal recessive missense mutation in the Rasa3 gene that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein, and its loss of function in scat results in increased erythroid Ras activity, increased reactive oxygen species, and altered cell cycle progression, all culminating in delayed terminal erythroid differentiation. However, the precise mechanism of RASA3 regulation of erythroid differentiation remains undefined, and elucidation of this mechanism is crucial to identifying new therapeutic targets in inherited anemia. Considering the role of RASA3 as regulator of Ras signaling and cell cycle progression, and the importance of these processes to erythroid differentiation, we sought to first characterize the coordination of Ras signaling pathways and cell cycle progression in normal murine erythropoiesis, then observe how loss of RASA3 function alters this regulatory axis. We observed that wild type (WT) erythroblasts demonstrate population-specific influence of ERK and PI3K/AKT signaling in regulating cell cycle progression. Inhibition of both pathways with increasing doses of U0126 and LY294002, respectively, induced accumulation in G0/G1 from the proerythroblast stage until the late basophilic/polychromatic stage (U0126 vehicle vs. 1uM p= 0.0023; LY294002 vehicle vs 1uM p=0.0389). At these later stages, ERK and PI3K/AKT inhibition led to a decrease in G0/G1 percentages, suggesting a stage-specific switch in signaling mediated cell cycle regulation, with PI3K inhibition demonstrating more potent and consistent effects (U0126 vehicle vs 1uM p=0.0406, 0.0481; LY294002 vehicle vs 1uM p=0.0003, 0.0197, 0.0086, n=3). These patterns suggest that ERK and AKT may facilitate cell cycle progression past the G0/G1 checkpoint in early erythropoiesis while inducing cell cycle exit or accumulation in G0/G1 in late erythropoiesis. In scat, we previously characterized increased active Ras in erythroid cells and a delay in terminal erythroid differentiation with accumulation at the polychromatic stage. We therefore next sought to examine the potential mechanistic contribution of altered PI3K/AKT signaling and cell cycle progression to the differentiation delay seen in scat. Phospho-flow analyses demonstrate that scat bone marrow-derived basophilic and polychromatic erythroblasts have increased AKT activation compared to WT (p=0.0402, p=0.0559, respectively; n=4), with similar trends evident in scat spleen basophilic erythroblasts (p=0.064; n=4). These results are consistent with increased Ras activation in scat. Ex vivo EdU/PI analyses revealed that scat bone marrow-derived polychromatic erythroblasts demonstrate G0/G1 accumulation (p=0.0466) and decreased progression to S-phase (0.0414; n=6), also with similar trends in scat spleen basophilic erythroblasts (p=0.004; n=6). These results correlate with the observed differentiation delay in scat and indicate that RASA3 regulates stage-specific signaling and cell cycle progression during erythropoiesis. To study if cell cycle dysregulation in scat begins at an earlier stage of erythroid differentiation, we analyzed murine hematopoietic and erythroid progenitors as Ter119-, cKit+ cells expressing increasing levels of CD71 and found that both CD71lo and CD71med bone marrow-derived scat progenitors present with G0/G1 accumulation (p=0.0015, p=0.0073, respectively) and decreased progression to S-phase (p=0.0014, p=0.0241; n=7). This suggests a dynamic relationship between RASA3, Ras signaling, and cell cycle progression throughout early and late erythroid differentiation. Together, these findings support the role of RASA3 as a regulator of the signaling networks governing erythropoiesis and reveal a new targetable axis in a model of inherited bone marrow failure. Disclosures No relevant conflicts of interest to declare.

Anemic Nan mice carry a mutation (E339D) in the second zinc finger of erythroid transcription factor KLF1. Nan-KLF1 fails to bind a subset of normal KLF1 targets and ectopically binds a large set of genes not normally engaged by KLF1, resulting in a corrupted fetal liver transcriptome. Here, we performed RNAseq using flow cytometric-sorted spleen erythroid precursors from adult Nan and WT littermates rendered anemic by phlebotomy to identify global transcriptome changes specific to the Nan Klf1 mutation as opposed to anemia generally. Mutant Nan-KLF1 leads to extensive and progressive transcriptome corruption in adult spleen erythroid precursors such that stress erythropoiesis is severely compromised. Terminal erythroid differentiation is defective in the bone marrow as well. Principle component analysis reveals two major patterns of differential gene expression predicting that defects in basic cellular processes including translation, cell cycle, and DNA repair could contribute to disordered erythropoiesis and anemia in Nan. Significant erythroid precursor stage specific changes were identified in some of these processes in Nan. Remarkably, however, despite expression changes in large numbers of associated genes, most basic cellular processes were intact in Nan indicating that developing red cells display significant physiological resiliency and establish new homeostatic set points in vivo.

RASA3 is a Ras GTPase activating protein that plays a critical role in blood formation. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a missense mutation in Rasa3. Homozygotes present with a phenotype characteristic of bone marrow failure that is accompanied by alternating episodes of crisis and remission. The mechanism leading to impaired erythropoiesis and peripheral cell destruction as evidenced by membrane fragmentation in scat is unclear, although we previously reported that the mislocalization of RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the disease. In this study, we further characterized the bone marrow failure in scat and found that RASA3 plays a central role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels during terminal erythroid differentiation, without inducing apoptosis of the precursors. In scat mice undergoing crises, there is a consistent pattern of an increased proportion of cells in the G0/G1 phase at the basophilic and polychromatophilic stages of erythroid differentiation, suggesting that RASA3 is involved in the G1 checkpoint. However, this increase in G1 is transient, and either resolves or becomes indiscernible by the orthochromatic stage. In addition, while ROS levels are normal early in erythropoiesis, there is accumulation of superoxide levels at the reticulocyte stage (DHE increased 40% in scat; p = 0.02) even though mitochondria, a potential source for ROS, are eliminated normally. Surprisingly, apoptosis is significantly decreased in the scat bone marrow at the proerythroblastic (15.3%; p = 0.004), polychromatophilic (8.5%; p = 0.01), and orthochromatic (4.2%; p = 0.02) stages. Together, these data indicate that ROS accumulation at the reticulocyte stage, without apoptosis, contributes to the membrane fragmentation observed in scat. Finally, the cell cycle defect and increased levels of ROS suggest that scat is a model of bone marrow failure with characteristics of aplastic anemia.

The Ras GTPase-activating protein, RASA3, plays a critical non-redundant role in vertebrate erythropoiesis. The autosomal recessive scat mouse model carries a missense (G125V) mutation in Rasa3 that results in severe anemia, thrombocytopenia and leukopenia with markedly reduced survival. Terminal erythroid differentiation is dysregulated in scat bone marrow (BM) and spleen with delayed cell cycle progression, decreased apoptosis, and increased reactive oxygen species. To further study the role of RASA3 in hematopoiesis, we created a conditional Rasa3 allele. Germline null mice die at E12.5-13.5 of severe hemorrhage, pallor and reduced fetal liver size. Specific ablation of Rasa3 in hematopoietic stem and progenitor cells (HSPCs) and endothelial cells (ECs) driven by Vav-Cre and Tek-Cre recapitulates the null phenotype, highlighting the specific requirement of RASA3 in hematopoietic and vascular compartments. To study RASA3 deficiency specifically in terminal erythropoiesis, we utilized EpoR-Cre. Surprisingly, EpoR-Cre driven deletion does not produce an abnormal erythroid phenotype, and normal levels of RASA3 protein are seen on western blots of Ter119+ spleen and BM cells despite efficient deletion of the floxed Rasa3 allele, suggesting that RASA3 produced early in HSPCs is sufficient to maintain normal terminal erythroid differentiation. Indeed, deletion of Rasa3 in the entire hematopoietic system at 8 weeks of age via inducible Mx1-Cre results in death within 2 weeks of induction with profound anemia, thrombocytopenia, and leukopenia, recapitulating the scat phenotype. While HSC numbers are normal in the RASA3 depleted BM of this model, the frequency and absolute numbers of all progenitors are significantly decreased, and spleen stress erythropoiesis is significantly blunted. Altered Ras activity is associated with delayed erythropoiesis. ERK signaling, a key downstream Ras effector, is hyperactivated 3-fold by western blotting in scat BM CD45-depleted cells vs. wild type (WT). To extend RASA3 studies to human cells, we utilized cord blood CD34+ cells in a three-phase erythroid differentiation culture system. Similar to the mouse models, shRNA knockdown of RASA3 leads to delayed erythroid differentiation at culture day 11 based on glycophorin A, Band 3 and a4-integrin expression levels. Dysregulated cell cycle progression with accumulation in the S-phase is also evident at day 12 of culture. Unlike the mouse models, however, ERK activation was decreased (67-72%) in serum-starved sorted proerythroblasts and later day 11 populations of RASA3 knockdown cells treated with erythropoietin (Epo), stem cell factor (SCF), or both. To elucidate additional molecular mechanisms downstream of RASA3, we performed RNAseq on total spleen and BM, as well as sorted stem and myeloid progenitors (SMPs) of scat mice and WT littermate controls. Principle component analysis confirms that genotype is the primary contributing factor to differential gene expression. In preliminary analyses, MAPK signaling, including negative regulators of ERK, phosphatidyl-inositol signaling, cell cycle regulation, and transcriptional regulation emerge from pathways and GO term analyses of differentially expressed genes. In conclusion, we show that (1) absence of RASA3 results in embryonic lethality with evidence of severe HSPC/EC defects; (2) RASA3 is required for hematopoiesis starting at the HPC stage or earlier; (3) missense mutations (scat) or loss of RASA3 in adults cause pancytopenia and premature death; (4) mechanistically, aberrant ERK signaling and cell cycle defects contribute to the RASA3 deficient erythroid phenotype, while RNAseq studies provide further potential mechanistic targets. Disclosures No relevant conflicts of interest to declare.

In an effort to improve pedestrian safety, several states in the United States changed their pedestrian laws by changing the requirement that drivers yield to pedestrians in crosswalks to a requirement that drivers stop for pedestrians in crosswalks. This study examined whether this change had an effect on pedestrian safety in the United States, with its focus on low-speed roads. To examine the association between changes in pedestrian laws and changes in pedestrian-involved fatal crashes, three approaches were employed: before-after analysis, time-series analysis, and cross-sectional analysis. Pedestrian-involved fatal traffic crashes on low-speed roads were extracted from the U.S. national fatal crash database, the Fatality Analysis Reporting System (FARS), from 1980 through 2005. This study found no statistically significant reduction in pedestrian-involved fatal crashes attributable to changes in the laws, yet this finding is not definitive because of study limitations such as the omission of relevant exposure data.

RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a G125V mutation in Rasa3 that leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Our previous studies demonstrated that this mutation in Rasa3 causes defects in several aspects of erythropoiesis, including a significant delay of erythroid differentiation at the polychromatophilic stage, decreased hemoglobinization, defects in cell cycle progression past the G1 checkpoint, and increased reactive oxygen species (ROS) during terminal erythroid differentiation as well as in scat peripheral blood reticulocytes and red blood cells. We previously reported that the mislocalization of mutated RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the erythropoietic defect in scat, and the observed cell cycle arrest and increased ROS likely also contribute to this unique disease phenotype. Our current efforts are focused on further elucidation of the mechanism and specific disruptions in Ras signaling that lead to anemia, membrane fragmentation, and the cyclic phenotype in scat. Interestingly, we report here that apoptosis is not increased during scat crisis, and that mitochondria, a potential source of ROS, are normally eliminated at the reticulocyte stage. The dramatic nature of remission, with complete normalization of all hematologic parameters, led us to hypothesize that a secreted factor may be mediating the cyclic phenotype of scat. Differences in the cytokine profile of the serum of scat mice compared to wild type suggest that, indeed, one or several secreted factor(s) may be influencing the occurrence of bone marrow failure. Levels of galectin-1, a known mediator of cell-cell interactions and intracellular signaling in the hematopoietic niche, are consistently decreased in scat serum according to a multispot anti-cytokine antibody array (23,326.5 ± 21,439.7 integrated density in scat vs. 31,019.6± 20,110.7 in controls, p Strengthening the notion that RASA3 has a critical conserved role in vertebrate terminal erythropoiesis, the characteristics of bone marrow failure seen in scat have been reproduced in human CD34+ cells using siRNA and shRNA knockdowns of Rasa3 . Similar to the changes seen in scat, cells with decreased RASA3 demonstrated delayed terminal erythroid differentiation and defective hemoglobinization. Finally, analysis of Ras expression and functional pull-down studies in human CD34+ cells revealed that, while K-Ras is the major active isoform expressed during terminal erythroid differentiation, H-Ras is also active during human erythropoiesis. Future studies with CD34+ Rasa3 knockdown cells will explore the influence of RASA3 on human K- and H-Ras signaling in erythropoiesis. Taken together, our studies further characterize the vital role of RASA3 in hemoglobinization, cell cycle progression, and cell survival during terminal erythroid differentiation, as well as identify novel targets for investigation of unknown mechanisms (e.g., dysregulated cytokine secretion) of bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.