Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Alantolactone is a natural product that potently inhibits YAP1/TAZ through promotion of reactive oxygen species accumulation.

Yes-associated protein 1 (YAP1) and its paralogue PDZ-binding motif (TAZ) play pivotal roles in cell proliferation, migration, and invasion, and abnormal activation of these TEAD transcriptional coactivators is found in diverse cancers in humans and mice. Targeting YAP1/TAZ signaling is thus a promising therapeutic avenue but, to date, few selective YAP1/TAZ inhibitors have been effective against cancer cells either in vitro or in vivo. We screened chemical libraries for potent YAP1/TAZ inhibitors using a highly sensitive luciferase reporter system to monitor YAP1/TAZ-TEAD transcriptional activity in cells. Among 29 049 low-molecular-weight compounds screened, we obtained nine hits, and the four of these that were the most effective shared a core structure with the natural product alantolactone (ALT). We also tested 16 other structural derivatives of ALT and found that natural ALT was the most efficient at increasing ROS-induced LATS kinase activities and thus YAP1/TAZ phosphorylation. Phosphorylated YAP1/TAZ proteins were subject to nuclear exclusion and proteosomic degradation such that the growth of ALT-treated tumor cells was inhibited both in vitro and in vivo. Our data show for the first time that ALT can be used to target the ROS-YAP pathway driving tumor cell growth and so could be a potent anticancer drug.

Factors Associated with Difficulty in Returning to School in Students After Traumatic Brain Injury.

Traumatic brain injury (TBI) often causes behavioral problems and difficulties with school work, but the specific factors associated with difficulty in returning to school after TBI still remain unclear. The purpose of this study was to investigate factors associated with difficulty in returning to school within 1 year of injury in students with traumatic brain injury. This study is a secondary analysis of existing data sets. We recruited patients aged 16 years in the United States with a primary rehabilitation diagnosis of TBI registered in the Traumatic Brain Injury Model Systems National Database. We compared variables between the students who returned to school and those who did not return to school. In addition, subgroup analyses were performed focused on traumatic brain injury severity. We excluded those were received <10 years of schooling, and 309 eligible students were identified for the analysis. Of these, 246 (80%) did not return to school within 1 year of injury. There were fewer cases of severe TBI in the group of students who returned to school than in the group who did not return to school (29% vs 44%, P = 0.03). The duration of rehabilitation was significantly longer in the group who returned to school than in the group who did not return to school (mean days 40 vs 29, P = 0.001), and a subgroup analysis showed in the severe traumatic brain injury group (mean days 46 vs 29; P = 0.02) and the non-severe traumatic brain injury group (mean days 37 vs 26; P = 0.02) similar results. Insufficient amount of rehabilitation was associated with difficulty in returning to school in students after TBI, regardless of the severity of the injury.

PI(3,4)P2 plays critical roles in the regulation of focal adhesion dynamics of MDA-MB-231 breast cancer cells.

Phosphoinositides play pivotal roles in the regulation of cancer cell phenotypes. Among them, phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2 ) localizes to the invadopodia, and positively regulates tumor cell invasion. In this study, we examined the effect of PI(3,4)P2 on focal adhesion dynamics in MDA-MB-231 basal breast cancer cells. Knockdown of SHIP2, a phosphatidylinositol 3,4,5-trisphosphatase (PIP3 ) 5-phosphatase that generates PI(3,4)P2 , in MDA-MB-231 breast cancer cells, induced the development of focal adhesions and cell spreading, leading to the suppression of invasion. In contrast, knockdown of PTEN, a 3-phosphatase that de-phosphorylates PIP3 and PI(3,4)P2 , induced cell shrinkage and increased cell invasion. Interestingly, additional knockdown of SHIP2 rescued these phenotypes. Overexpression of the TAPP1 PH domain, which binds to PI(3,4)P2 , and knockdown of Lpd, a downstream effector of PI(3,4)P2 , resulted in similar phenotypes to those induced by SHIP2 knockdown. Taken together, our results suggest that inhibition of PI(3,4)P2 generation and/or downstream signaling could be useful for inhibiting breast cancer metastasis.

Management strategies in Lynch syndrome and familial adenomatous polyposis: a national healthcare survey in Japan.

Lynch syndrome (LS) and familial adenomatous polyposis (FAP) are major sources of hereditary colorectal cancer (CRC) and are associated with other malignancies. There is some heterogeneity in management strategies in Japan. We undertook a survey of management of hereditary CRC in hospitals that are members of the Japan Society of Colorectal Cancer Research. One hundred and ninety departments responded, of which 127 were from designated cancer care hospitals (DCCHs) according to the Japanese government. There were 25 488 operations for CRC in these departments in 2015. The DCCHs performed better with regard to usage of Japan Society of Colorectal Cancer Research guidelines, referring new CRC patients for LS screening, and having in-house genetic counselors and knowledge of treatment for LS. There were 174 patients diagnosed with LS and 602 undergoing follow-up in 2011-2015, which is fewer than the number expected from CRC operations in 2015. These numbers were not affected by whether the institution was a DCCH. Universal screening for LS was carried out in 8% of the departments. In contrast, 541 patients were diagnosed with FAP and 273 received preventive proctocolectomy/colectomy in 2011-2015. The DCCH departments undertook more surgery than non-DCCH departments, although most of the management, including surgical procedures and use of non-steroidal anti-inflammatory drugs, was similar. Management of desmoid tumor in the abdominal cavity differed according to the number of patients treated. In conclusion, there was heterogeneity in management of LS but not FAP. Most patients with LS may be overlooked and universal screening for LS is not common in Japan.

Regulation of CD44 expression and focal adhesion by Golgi phosphatidylinositol 4-phosphate in breast cancer.

CD44, a transmembrane receptor, is expressed in the standard or variant form and plays a critical role in tumor progression and metastasis. This protein regulates cell adhesion and migration in breast cancer cells. We previously reported that phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi regulates cell migration and invasion in breast cancer cell lines. In this study, we showed that an increase in PI(4)P levels at the Golgi by knockdown of PI(4)P phosphatase SAC1 increased the expression of standard CD44, variant CD44, and ezrin/radixin phosphorylation and enhanced the formation of focal adhesions mediated by CD44 and ezrin/radixin in MCF7 and SK-BR-3 cells. In contrast, knockdown of PI 4-kinase IIIß in highly invasive MDA-MB-231 cells decreased these factors. These results suggest that SAC1 expression and PI(4)P at the Golgi are important in tumor progression and metastasis and are potential prognostic markers of breast cancers.

SETBP1 is dispensable for normal and malignant hematopoiesis.

SETBP1 is a potential epigenetic regulator whose hotspot mutations preventing proteasomal degradation are recurrently detected in myeloid malignancies with poor prognosis. It is believed that the mutant SETBP1 exerts amplified effects of wild-type SETBP1 rather than neomorphic functions. This indicates that dysregulated quantitative control of SETBP1 would result in the transformation of hematopoietic cells. However, little is known about the roles of endogenous SETBP1 in malignant and normal hematopoiesis. Thus, we integrated the analyses of primary AML and healthy samples, cancer cell lines, and a newly generated murine model, Vav1-iCre;Setbp1fl/fl. Despite the expression in long-term hematopoietic stem cells, SETBP1 depletion in normal hematopoiesis minimally alters self-renewal, differentiation, or reconstitution in vivo. Indeed, its loss does not profoundly alter transcription or chromatin accessibilities. Furthermore, although AML with high SETBP1 mRNA is associated with genetic and clinical characteristics for dismal outcomes, SETBP1 is dispensable for the development or maintenance of AML. Contrary to the evidence that SETBP1 mutations are restricted to myeloid malignancies, dependency on SETBP1 mRNA expression is not observed in AML. These unexpected results shed light on the unrecognized idea that a physiologically nonessential gene can act as an oncogene when the machinery of protein degradation is damaged.

BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state.

ATP-dependent chromatin remodeling SWI/SNF complexes exist in three subcomplexes: canonical BAF (cBAF), polybromo BAF (PBAF), and a newly described non-canonical BAF (ncBAF). While cBAF and PBAF regulate fates of multiple cell types, roles for ncBAF in hematopoietic stem cells (HSCs) have not been investigated. Motivated by recent discovery of disrupted expression of BRD9, an essential component of ncBAF, in multiple cancers, including clonal hematopoietic disorders, we evaluate here the role of BRD9 in normal and malignant HSCs. BRD9 loss enhances chromatin accessibility, promoting myeloid lineage skewing while impairing B cell development. BRD9 significantly colocalizes with CTCF, whose chromatin recruitment is augmented by BRD9 loss, leading to altered chromatin state and expression of myeloid-related genes within intact topologically associating domains. These data uncover ncBAF as critical for cell fate specification in HSCs via three-dimensional regulation of gene expression and illuminate roles for ncBAF in normal and malignant hematopoiesis.

Impact of Number of Drugs on Rehabilitation Outcomes in Patients after Traumatic Brain Injury: A Retrospective Cohort Study.

OBJECTIVE: To investigate the impact of the number of drugs on rehabilitation outcomes for patients with acute traumatic brain injury. DESIGN: Retrospective cohort study. SETTING: Hospital-based database created by the Japan Medical Data Center. PARTICIPANTS: Patients with acute traumatic brain injury admitted between April 2014 and November 2017. METHODS: Analysis of relationships among 1-5 and ≥ 6 drugs as well as clinical outcomes in 2603 patients. MAIN OUTCOME MEASUREMENTS: The primary outcome was defined as the Barthel index efficiency, and the secondary outcome was Barthel index gain and length of hospital stay. RESULTS: Median Barthel index score on admission was 40. Barthel index efficiency and Barthel index gain were significantly higher in the group that had taken 1-5 drugs than in the group that had taken ≥6 drugs on admission (median: 1.19 vs 0.50, 20.0 vs 10.0). Also, the group that had taken 1-5 drugs had a significantly shorter length of hospital stay than in the group that had taken ≥6 drugs on admission (median 11.0 vs 14.0). Moreover, multiple linear regression analysis showed that having taken ≥6 drugs on admission was independently associated with Barthel index efficiency, Barthel index gain, and length of stay. CONCLUSIONS: Taking≥6 drugs for acute traumatic brain injury was associated with lower Barthel index efficiency, lower Barthel index gain, and longer length of stay than taking 1-5 drugs.

Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition.

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias.

Enrichment of putative pancreatic progenitor cells from mice by sorting for prominin1 (CD133) and platelet-derived growth factor receptor beta.

Success in islet transplantation-based therapies for type 1 diabetes mellitus and an extreme shortage of pancreatic islets have motivated recent efforts to develop renewable sources of islet-replacement tissue. Although pancreatic progenitor cells hold a promising potential, only a few attempts have been made at the prospective isolation of pancreatic stem/progenitor cells, because of the lack of specific markers and the development of effective cell culture methods. We found that prominin1 (also known as CD133) recognized the undifferentiated epithelial cells, whereas platelet-derived growth factor receptor beta (PDGFRbeta) was expressed on the mesenchymal cells in the mouse embryonic pancreas. We then developed an isolation method for putative stem/progenitor cells by flow cytometric cell sorting and characterized their potential for differentiation to pancreatic tissue using both in vitro and in vivo protocols. Flow cytometry and the subsequent reverse transcription-polymerase chain reaction and microarray analysis revealed pancreatic epithelial progenitor cells to be highly enriched in the prominin1(high)PDGFRbeta(-) cell population. During in vivo differentiation, these cell populations were able to differentiate into endocrine, exocrine, and ductal tissues, including the formation of an insulin-producing cell cluster. We established the prospective isolation of putative pancreatic epithelial progenitor cells by sorting for prominin1 and PDGFRbeta. Since this strategy is based on the cell surface markers common to human and rodents, these findings may lead to the development of new strategies to derive transplantable islet-replacement tissues from human pancreatic stem/progenitor cells. Disclosure of potential conflicts of interest is found at the end of this article.

Home visits by occupational therapists in acute hospital care: a systematic review.

The aim of this review was to determine the utility of home visits by occupational therapists before and after a patient is discharged from an acute care hospital. All relevant published studies were identified by searching the CENTRAL, MEDLINE, EMBASE, Occupational Therapy Systematic Evaluation of Evidence, and WHO International Clinical Trials Registry Platform databases. Randomized controlled trials were included regardless of sex, age, disease, and duration of acute hospitalization. The intervention was predischarge and postdischarge home visits made by an occupational therapist. The primary outcome was the ability to perform activities of daily living at 1 month after the intervention. We identified eight trials (including 1029 patients) that were eligible for inclusion. More than half of the trials had a low risk of bias in random sequence generation, and allocation concealment and the other half had a high risk of bias with regard to blinding of participants. However, the risk of bias in terms of blinding for outcomes assessment was low in more than half the studies. We found that home visits by an occupational therapist in a single study significantly reduced the prevalence of falls but had no significant effects on ability to perform activities of daily living, quality of life, or mood. We could not find adequate evidence to support routine home visits by an occupational therapist in the acute care. In the future, studies with larger sample sizes are needed to validate home visits by occupational therapists in patients after acute care hospitalization.

Effectiveness of acute in-hospital occupational therapy for older patients with hip fracture.

AIM: To test the hypothesis that hip fracture patients who receive occupational therapy (OT) have better functional ability than those who do not. METHODS: This retrospective observational study utilized data from the Japan Rehabilitation Database spanning 2005-2015. In-hospital hip fracture patients admitted to acute hospitals were identified. In total, 1266 patients were eligible based on the exclusion criteria. The primary outcome was motor Functional Independence Measure (FIM) efficiency. RESULTS: Patients who received OT were 50.9% of hip fracture patients. Patients who received OT had significantly higher scores in motor FIM efficiency (mean 0.79 vs 0.70; P = 0.02) and FIM effectiveness (mean 0.49 vs 0.41; P < 0.01) than the patients who did not receive OT. From multivariate linear regression analysis, OT was identified as a significant factor in motor FIM efficiency (coefficient 1.29, 95% confidence interval 1.14-1.47; P < 0.01) and FIM effectiveness (coefficient 1.07, 95% confidence interval 1.02-1.12; P < 0.01). CONCLUSIONS: The present cohort analysis showed that OT for hip fracture patients in the acute phase likely contributes to improved functional ability. Geriatr Gerontol Int 2019; 19: 611-615.

Influence of age and comorbidity on prognosis and application of adjuvant chemotherapy in elderly Japanese patients with colorectal cancer: A retrospective multicentre study.

BACKGROUND: Adjuvant therapy for colorectal cancer (CRC) in patients aged ≥75 years is supported by inadequate evidence, although such patients are increasing in number worldwide. PATIENTS AND METHODS: We assessed the influence of age and comorbidities on the prognosis of CRC in elderly patients using pooled data by the Japanese Study Group for Postoperative Follow-up of Colorectal Cancer. In total, 4598 patients (3304 with colon cancer and 1294 with rectal cancer) who underwent curative surgery from 2004 to 2006 were analysed with respect to age, Charlson comorbidity score (CS), tumour marker positivity, adjuvant therapy and prognosis. RESULTS: The number of patients aged <64, 65-74 and >75 years was 2007 (44%), 1614 (35%) and 977 (21%), respectively. Tumour location, tumour marker positivity, clinical stage, performance of adjuvant therapy, CS and overall survival (OS) were significantly different among these age groups (P < 0.0001). Among patients aged ≥75 years with stage III CRC, 35% with colon cancer and 21% with rectal cancer received adjuvant therapy; these proportions were much lower than those in younger patients. Application of adjuvant therapy was dependent on the CS in patients aged ≤74 years, but not in older patients. Sex, the carcinoembryonic antigen concentration and adjuvant therapy were significantly associated with OS in elderly patients with stage III CRC. CONCLUSION: Age and comorbidities worsened the OS of patients with CRC who underwent curative surgery. However, patients aged ≥75 years were undertreated regardless of their CS despite the possibility of OS improvement by adjuvant therapy.

Whole cell vaccination using immunogenic cell death by an oncolytic adenovirus is effective against a colorectal cancer model.

Cancer vaccine application is limited to specific cancer types because few cancer-associated antigens are known to induce tumor rejection. Accordingly, we assessed the utility of Ad881, an oncolytic adenovirus in which viral replication was strictly regulated by the cancer-specific midkine promoter, as a cancer vaccine in a murine colorectal cancer model lacking specific cancer-associated antigens. In CT26 and CMT93 cells, Ad881 (multiplicity of infection: 100 or 1,000) showed stronger cytotoxicity and oncolysis in vitro than its equivalent replication-defective adenovirus, Ad884. CT26 cells (1 × 104) infected with Ad881 (multiplicity of infection: 1,000) for 24 hours were suitable as vaccine antigens without tumor formation in our model. Repeated vaccinations, but not single vaccination, induced a greater prophylactic immune response. The percentage of mice that rejected the tumor challenge was 0, 4, and 38% after no vaccination, single vaccination, and repeated vaccinations, respectively. Immunogenic cell death marker high-mobility group box 1 protein (HMGB1) and adenosine triphosphate in culture medium were higher after Ad881 infection (24.3 ng/ml and 48.2 nmol/l, respectively) than after Ad884 infection (8.6 ng/ml and 15.4 nmol/l, respectively) or oxaliplatin treatment (3.7 ng/ml and 1.8 nmol/l, respectively). These results indicate that repeated whole cell vaccination using an oncolytic adenovirus may be a potent approach to evoke immunogenic cell death.

α-Actinin-4 enhances colorectal cancer cell invasion by suppressing focal adhesion maturation.

α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion.

Phosphatidylinositol 4-phosphate in the Golgi apparatus regulates cell-cell adhesion and invasive cell migration in human breast cancer.

Downregulation of cell-cell adhesion and upregulation of cell migration play critical roles in the conversion of benign tumors to aggressive invasive cancers. In this study, we show that changes in cell-cell adhesion and cancer cell migration/invasion capacity depend on the level of phosphatidylinositol 4-phosphate [PI(4)P] in the Golgi apparatus in breast cancer cells. Attenuating SAC1, a PI(4)P phosphatase localized in the Golgi apparatus, resulted in decreased cell-cell adhesion and increased cell migration in weakly invasive cells. In contrast, silencing phosphatidylinositol 4-kinase IIIß, which generates PI(4)P in the Golgi apparatus, increased cell-cell adhesion and decreased invasion in highly invasive cells. Furthermore, a PI(4)P effector, Golgi phosphoprotein 3, was found to be involved in the generation of these phenotypes in a manner that depends on its PI(4)P-binding ability. Our results provide a new model for breast cancer cell progression in which progression is controlled by PI(4)P levels in the Golgi apparatus.

Regulated Nodal signaling promotes differentiation of the definitive endoderm and mesoderm from ES cells.

Nodal signaling induces the formation of the endoderm and mesoderm during gastrulation. Nodal expression persists until the definitive endoderm progenitor has completely formed, and disappears thereafter. A tightly regulated Nodal expression system is essential for the differentiation of embryonic stem (ES) cells into distinct tissue lineages. On this basis, we established an ES cell differentiation system with the tetracycline-regulated expression of Nodal. The upregulated Nodal signaling pathway and its downstream transcriptional targets induced the specification of ES cells into definitive endoderm and mesoderm derivatives, and the subsequent downregulation of Nodal signaling promoted further maturation of the gut tube both in vitro and in vivo. Sustained expression of the Nodal gene inhibited the maturation of the definitive endoderm owing to persistent Oct3 and/or Oct4 expression and teratoma formation. Furthermore, quantitative single cell analysis by flow cytometry using CXCR4, VEGFR2 and PDGFR-alpha indicated that this protocol for definitive endoderm and mesoderm differentiation is superior to any other available protocol. Our findings also indicated that the Nodal or Nodal-related molecules secreted from Nodal-expressing ES cells could cause genetically unmanipulated ES cells to induce the expression of the Nodal signaling pathway and its downstream targets, which consequently leads to the differentiation of the ES cells into definitive endoderm and mesoderm. Our differentiation system, using tightly regulated Nodal expression, enabled us to investigate the mechanism of ES cell differentiation into definitive endoderm or mesoderm derivatives. Our findings also demonstrate that Nodal-expressing ES cells might be a source of highly active proteins that could be used for developing endoderm or mesoderm tissues in regenerative medicine.