Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Practical considerations and consensus opinion for children's hospital-based inpatient hemostasis and thrombosis (HAT) consultative services: Communication from the ISTH SSC Subcommittee on Pediatric/Neonatal Thrombosis and Hemostasis.

Caring for children and adolescents with disorders of hemostasis and thrombosis (HAT) has become more specialized and requires a unique skill set that many providers are not able to obtain in standard pediatric hematology/oncology/bone marrow transplant fellowship training programs. The influx of numerous therapeutic advances and increasing medical complexity has expanded the need for experienced HAT providers and subspecialty collaboration in the inpatient setting due to the nuances in the management of patients with HAT complications and concerns. While there are data highlighting the benefits of an inpatient hemostasis, thrombosis, and anticoagulation management service in adult hospitals, there are limited pediatric data supporting such programs. In this article, we summarize the current practices of various pediatric institutions in the inpatient management of HAT patients and provide a consensus opinion for the development of a pediatric inpatient HAT service at tertiary care referral centers.

FATTY ACID SYNTHESIS IS REQUIRED FOR BREAST CANCER BRAIN METASTASIS.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

The Seismic Shift in End-of-Life Care: Palliative Care Challenges in the Era of Medical Assistance in Dying.

Background: Concerns regarding personal, professional, administrative, and institutional implications of medical assistance in dying (MAiD) are of particular interest to palliative and hospice care providers (PHCPs), who may encounter additional moral distress and professional challenges in providing end-of-life (EOL) care in the new legislative and cultural era. Objective: To explore PHCPs' encountered challenges and resource recommendations for caring for patients considering MAiD. Design: Qualitative thematic analysis of audio-recorded semistructured interviews with PHCPs. Setting/Subjects: Multidisciplinary PHCPs in acute, community, residential, and hospice care in Vancouver, Canada, with experience supporting patients who have made MAiD inquiries or requests. Measurements: Interviews were deidentified, transcribed verbatim, and coded by four researchers using a common coding scheme. Key themes were analyzed. Results: Twenty-six PHCP participants included physicians (n = 7), nurses (n = 12), social workers (n = 5), and spiritual health practitioners (n = 2). Average interview length was 52 minutes (range 35-90). Analysis revealed four broad challenges associated with providing EOL care after MAiD legalization: (1) moral ambiguity and provider distress, (2) family distress, (3) interprofessional team conflict, and (4) impact on palliative care. Participants also recommended three types of resources to support clinicians in delivering quality EOL care to patients contemplating MAiD: (1) education and training, (2) pre- and debriefing for team members, and (3) tailored bereavement support. Conclusions: PHCPs encountered multilevel MAiD-related challenges, but noted improvement in organizational policies and coordination. Resources to enhance training, pre- and debriefing, and tailored bereavement may further support PHCPs in providing high-quality EOL care as they navigate the legislative and cultural shifts.

Targeting the Hedgehog Pathway Using Itraconazole to Prevent Progression of Barrett's Esophagus to Invasive Esophageal Adenocarcinoma.

OBJECTIVE: The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma. BACKGROUND: The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC. METHODS: The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200 mg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry. RESULTS: BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively. CONCLUSION: Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium.

Evidence on physical activity and falls prevention for people aged 65+ years: systematic review to inform the WHO guidelines on physical activity and sedentary behaviour.

BACKGROUND: Exercise prevents falls in older adults. Regular updates of estimated effects of exercise on falls are warranted given the number of new trials, the increasing number of older people globally and the major consequences of falls and fall-related injuries. METHODS: This update of a 2019 Cochrane Review was undertaken to inform the World Health Organization guidelines on physical activity and sedentary behaviour. Searches were conducted in six databases. We included randomised controlled trials evaluating effects of any form of physical activity as a single intervention on falls in people aged 60+ years living in the community. Analyses explored dose-response relationships. The certainty of the evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE). RESULTS: This review included 116 studies, involving 25,160 participants; nine new studies since the 2019 Cochrane Review. Exercise reduces the rate of falls by 23% (pooled rate ratio (RaR) 0.77, 95% confidence interval (CI) 0.71 to 0.83, 64 studies, high certainty evidence). Subgroup analysis showed variation in effects of different types of exercise (p < 0.01). Rate of falls compared with control is reduced by 24% from balance and functional exercises (RaR 0.76, 95% CI 0.70 to 0.82, 39 studies, high certainty evidence), 28% from programs involving multiple types of exercise (commonly balance and functional exercises plus resistance exercises, RaR 0.72, 95% CI 0.56 to 0.93, 15 studies, moderate certainty evidence) and 23% from Tai Chi (RaR 0.77, 95% CI 0.61 to 0.97, 9 studies, moderate certainty evidence). The effects of programs that primarily involve resistance training, dance or walking remain uncertain. Interventions with a total weekly dose of 3+ h that included balance and functional exercises were particularly effective with a 42% reduction in rate of falls compared to control (Incidence Rate Ratio (IRR) 0.58, 95% CI 0.45 to 0.76). Subgroup analyses showed no evidence of a difference in the effect on falls on the basis of participant age over 75 years, risk of falls as a trial inclusion criterion, individual versus group exercise, or whether a health professional delivered the intervention. CONCLUSIONS: Given the strength of this evidence, effective exercise programs should now be implemented at scale.

Childhood asthma increases respiratory morbidity, but not all-cause mortality in adulthood: The Busselton Health Study.

BACKGROUND: Long-term childhood asthma studies that investigate adult outcomes other than lung function are lacking. This study examines the associations of childhood asthma and the occurrence of respiratory events and all-cause mortality in adulthood. METHODS: A cohort of 4430 school children (aged to 17 years) who attended the Busselton Health Study between 1967 and 1983 were analysed. Self-reported history of asthma was determined using questionnaires. Participants were followed until 2014 for respiratory disease-related events (hospital admissions or death) and all-cause mortality using the Western Australia Data Linkage System. Cox regression models were used to investigate the impact of childhood asthma on respiratory events and all-cause mortality in adulthood. A subgroup of 2153 participants who re-attended a survey in young adulthood was also analysed. RESULTS: A total of 462 (10%) of the cohort had childhood asthma. During follow-up 791 participants experienced a respiratory event and 140 participants died. Childhood asthma was associated with an increased risk of respiratory events in adulthood (unadjusted HR 1.84, 95% CI 1.52 to 2.23; P < 0.0001). The result remained significant after adjusting for adult-onset asthma, FEV1, body mass index, smoking, dusty job, hay fever, and respiratory symptoms (adjusted HR 1.68, 95% CI 1.07 to 2.64; P = 0.0247). Childhood asthma was not associated with all-cause mortality in adulthood (unadjusted HR 1.08, 95% CI 0.63 to 1.84; P = 0.7821). CONCLUSION: Childhood asthma is associated with increased risk of respiratory disease-related hospital admissions and death but not all-cause mortality in adulthood.

Early epigenomic and transcriptional changes reveal Elk-1 transcription factor as a therapeutic target in Huntington's disease.

Huntington's disease (HD) is a chronic neurodegenerative disorder characterized by a late clinical onset despite ubiquitous expression of the mutant Huntingtin gene (HTT) from birth. Transcriptional dysregulation is a pivotal feature of HD. Yet, the genes that are altered in the prodromal period and their regulators, which present opportunities for therapeutic intervention, remain to be elucidated. Using transcriptional and chromatin profiling, we found aberrant transcription and changes in histone H3K27acetylation in the striatum of R6/1 mice during the presymptomatic disease stages. Integrating these data, we identified the Elk-1 transcription factor as a candidate regulator of prodromal changes in HD. Exogenous expression of Elk-1 exerted beneficial effects in a primary striatal cell culture model of HD, and adeno-associated virus-mediated Elk-1 overexpression alleviated transcriptional dysregulation in R6/1 mice. Collectively, our work demonstrates that aberrant gene expression precedes overt disease onset in HD, identifies the Elk-1 transcription factor as a key regulator linked to early epigenetic and transcriptional changes in HD, and presents evidence for Elk-1 as a target for alleviating molecular pathology in HD.

Krüppel-like Factor 5 Promotes Sonic Hedgehog Signaling and Neoplasia in Barrett's Esophagus and Esophageal Adenocarcinoma.

Krüppel-like Factor 5 (KLF5) is a zinc-finger transcription factor associated with cell cycle progression and cell survival. KLF5 plays a key role in mammalian intestinal epithelium development and maintenance, expressed at high levels in basal proliferating cells and low levels in terminally differentiated cells. Considering Barrett's esophagus (BE) and esophageal adenocarcinoma's (EAC) histopathological similarities to intestinal epithelium, we sought to determine KLF5's role in BE and EAC, as well as KLF5's possible connection to the sonic hedgehog (SHH) pathway which is highly active in BE and EAC development. Low levels of KLF5 mRNA were found in BE cell lines and tissue- similar to what has been reported in differentiated intestinal epithelium. In contrast, higher KLF5 levels were observed in EAC cells and tissues. KLF5 knockdown in EAC cells caused significant decreases in cell migration, proliferation, and EAC-associated gene expression. Moreover, KLF5 knockdown led to decreased SHH signaling. These results suggest that KLF5 is connected to the SHH pathway in BE and EAC and may represent a potential drug target in EAC; further studies are now indicated to verify these findings and elucidate underlying mechanisms involved.

Compound haploinsufficiency of Dok2 and Dusp4 promotes lung tumorigenesis.

Recurrent broad-scale heterozygous deletions are frequently observed in human cancer. Here we tested the hypothesis that compound haploinsufficiency of neighboring genes at chromosome 8p promotes tumorigenesis. By targeting the mouse orthologs of human DOK2 and DUSP4 genes, which were co-deleted in approximately half of human lung adenocarcinomas, we found that compound-heterozygous deletion of Dok2 and Dusp4 in mice resulted in lung tumorigenesis with short latency and high incidence, and that their co-deletion synergistically activated MAPK signaling and promoted cell proliferation. Conversely, restoration of DOK2 and DUSP4 in lung cancer cells suppressed MAPK activation and cell proliferation. Importantly, in contrast to downregulation of DOK2 or DUSP4 alone, concomitant downregulation of DOK2 and DUSP4 was associated with poor survival in human lung adenocarcinoma. Therefore, our findings lend in vivo experimental support to the notion that compound haploinsufficiency, due to broad-scale chromosome deletions, constitutes a driving force in tumorigenesis.

Preleukemic and second-hit mutational events in an acute myeloid leukemia patient with a novel germline RUNX1 mutation.

BACKGROUND: Germline mutations in the RUNX1 transcription factor give rise to a rare autosomal dominant genetic condition classified under the entity: Familial Platelet Disorders with predisposition to Acute Myeloid Leukaemia (FPD/AML). While several studies have identified a myriad of germline RUNX1 mutations implicated in this disorder, second-hit mutational events are necessary for patients with hereditary thrombocytopenia to develop full-blown AML. The molecular picture behind this process remains unclear. We describe a patient of Malay descent with an unreported 7-bp germline RUNX1 frameshift deletion, who developed second-hit mutations that could have brought about the leukaemic transformation from a pre-leukaemic state. These mutations were charted through the course of the treatment and stem cell transplant, showing a clear correlation between her clinical presentation and the mutations present. CASE PRESENTATION: The patient was a 27-year-old Malay woman who presented with AML on the background of hereditary thrombocytopenia affecting her father and 3 brothers. Initial molecular testing revealed the same novel RUNX1 mutation in all 5 individuals. The patient received standard induction, consolidation chemotherapy, and a haploidentical stem cell transplant from her mother with normal RUNX1 profile. Comprehensive genomic analyses were performed at diagnosis, post-chemotherapy and post-transplant. A total of 8 mutations (RUNX1, GATA2, DNMT3A, BCORL1, BCOR, 2 PHF6 and CDKN2A) were identified in the pre-induction sample, of which 5 remained (RUNX1, DNMT3A, BCORL1, BCOR and 1 out of 2 PHF6) in the post-treatment sample and none were present post-transplant. In brief, the 3 mutations which were lost along with the leukemic cells at complete morphological remission were most likely acquired leukemic driver mutations that were responsible for the AML transformation from a pre-leukemic germline RUNX1-mutated state. On the contrary, the 5 mutations that persisted post-treatment, including the germline RUNX1 mutation, were likely to be part of the preleukemic clone. CONCLUSION: Further studies are necessary to assess the prevalence of these preleukemic and secondary mutations in the larger FPD/AML patient cohort and establish their prognostic significance. Given the molecular heterogeneity of FPD/AML and other AML subtypes, a better understanding of mutational classes and their involvement in AML pathogenesis can improve risk stratification of patients for more effective and targeted therapy.

Diverse genetic-driven immune landscapes dictate tumor progression through distinct mechanisms.

Multiple immune-cell types can infiltrate tumors and promote progression and metastasis through different mechanisms, including immunosuppression. How distinct genetic alterations in tumors affect the composition of the immune landscape is currently unclear. Here, we characterized the immune-cell composition of prostate cancers driven by the loss of the critical tumor suppressor gene Pten, either alone or in combination with the loss of Trp53, Zbtb7a or Pml. We observed a striking quantitative and qualitative heterogeneity that was directly dependent on the specific genetic events in the tumor and ranged from 'cold', noninflamed tumors to massively infiltrated landscapes-results with important therapeutic implications. Further, we showed these qualitative differences in transcriptomic analysis of human prostate cancer samples. These data suggest that patient stratification on the basis of integrated genotypic-immunophenotypic analyses may be necessary for successful clinical trials and tailored precision immunological therapies.

An aberrant SREBP-dependent lipogenic program promotes metastatic prostate cancer.

Lipids, either endogenously synthesized or exogenous, have been linked to human cancer. Here we found that PML is frequently co-deleted with PTEN in metastatic human prostate cancer (CaP). We demonstrated that conditional inactivation of Pml in the mouse prostate morphs indolent Pten-null tumors into lethal metastatic disease. We identified MAPK reactivation, subsequent hyperactivation of an aberrant SREBP prometastatic lipogenic program, and a distinctive lipidomic profile as key characteristic features of metastatic Pml and Pten double-null CaP. Furthermore, targeting SREBP in vivo by fatostatin blocked both tumor growth and distant metastasis. Importantly, a high-fat diet (HFD) induced lipid accumulation in prostate tumors and was sufficient to drive metastasis in a nonmetastatic Pten-null mouse model of CaP, and an SREBP signature was highly enriched in metastatic human CaP. Thus, our findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis.

CEBPA mutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay.

AIM: The presence of biallelic CEBPA mutations is a favourable prognostic feature in acute myeloid leukaemia (AML). CEBPA mutations are currently identified through conventional capillary sequencing (CCS). With the increasing adoption of next-generation sequencing (NGS) platforms, challenges with regard to amplification efficiency of CEBPA due to the high GC content may be encountered, potentially resulting in suboptimal coverage. Here, the performance of an amplicon-based NGS method using a laboratory-developed CEBPA-specific Nextera XT (CEBNX) was evaluated. METHODS: Mutational analyses of the CEBPA gene of 137 AML bone marrow or peripheral blood retrospective specimens were performed by the amplification of the CEBPA gene using the Expand Long Range dNTPack and the amplicons processed by CCS and NGS. CEBPA-specific libraries were then constructed using the Nextera XT V.2 kit. All FASTQ files were then processed with the MiSeq Reporter V.2.6.2.3 using the PCR Amplicon workflow via the customised CEBPA-specific manifest file. The variant calling format files were analysed using the Illumina Variant Studio V.2.2. RESULTS: A coverage per base of 3631X to 28184X was achieved. 22 samples (16.1%) were found to contain CEBPA mutations, with variant allele frequencies (VAF) ranging from 3.8% to 58.2%. Taking CCS as the 'gold standard', sensitivity and specificity of 97% and 97% was achieved. For the transactivation domain 2 polymorphism (c.584_589dupACCCGC/p.His195_Pro196dup), the CEBNX achieved 100% sensitivity and 100% specificity relative to CCS. CONCLUSIONS: Our laboratory-developed CEBNX workflow shows high coverage and thus overcomes the challenges associated with amplification efficiency and low coverage of CEBPA. Therefore, our assay is suitable for deployment in the clinical laboratory.

Esophageal Adenocarcinoma-Derived Extracellular Vesicle MicroRNAs Induce a Neoplastic Phenotype in Gastric Organoids.

There have been no reports describing the effects of cancer cell-derived extracellular vesicles (EVs) on three-dimensional organoids. In this study, we delineated the proneoplastic effects of esophageal adenocarcinoma (EAC)-derived EVs on gastric organoids (gastroids) and elucidated molecular mechanisms underlying these effects. EVs were identified using PKH-67 staining. Morphologic changes, Ki-67 immunochemistry, cell viability, growth rates, and expression levels of miR-25 and miR-210, as well as of their target mRNAs, were determined in gastroids co-cultured with EAC-derived extracellular vesicles (c-EVs). C-EVs were efficiently taken up by gastroids. Notably, c-EV-treated gastroids were more crowded, compact, and multilayered and contained smaller lumens than did those cultured in organoid medium alone or in EAC-conditioned medium that had been depleted of EVs. Moreover, c-EV-treated gastroids manifested increased proliferation and cellular viability relative to medium-only or EV-depleted controls. Expression levels of miR-25 and miR-210 were significantly higher, and those of PTEN and AIFM3 significantly lower, in c-EV-treated versus medium-only or EV-depleted control groups. Inhibitors of miR-25 and miR-210 reversed the increased cell proliferation induced by c-exosomes in co-cultured gastroids by lowering miR-25 and miR-210 levels. In conclusion, we have constructed a novel model system featuring the co-culture of c-EVs with three-dimensional gastroids. Using this model, we discovered that cancer-derived EVs induce a neoplastic phenotype in gastroids. These changes are due, at least in part, to EV transfer of miR-25 and miR-210.

Identifying large indels in targeted next generation sequencing assays for myeloid neoplasms: a cautionary tale of the ZRSR1 pseudogene.

Targeted next generation sequencing platforms have been increasingly utilised for identification of novel mutations in myeloid neoplasms, such as acute myeloid leukaemia (AML), and hold great promise for use in routine clinical diagnostics. In this study, we evaluated the utility of an open source variant caller in detecting large indels in a targeted sequencing of AML samples. While we found that this bioinformatics pipeline has the potential to accurately capture large indels (>20 bp) in patient samples, we highlighted the pitfall of a confounding ZRSR1 pseudogene that led to an erroneous ZRSR2 variant call. We further discuss possible clinical implications of the ZRSR1 pseudogene in myeloid neoplasms based on its molecular features. Knowledge of the confounding ZRSR1 pseudogene in ZRSR2 sequencing assays could be particularly important in AML diagnostics because the detection of ZRSR2 in AML patients is highly specific for an s-AML diagnosis.

Single-cell genomic profiling of acute myeloid leukemia for clinical use: A pilot study.

Although bulk high-throughput genomic profiling studies have led to a significant increase in the understanding of cancer biology, there is increasing awareness that bulk profiling approaches do not completely elucidate tumor heterogeneity. Single-cell genomic profiling enables the distinction of tumor heterogeneity, and may improve clinical diagnosis through the identification and characterization of putative subclonal populations. In the present study, the challenges associated with a single-cell genomics profiling workflow for clinical diagnostics were investigated. Single-cell RNA-sequencing (RNA-seq) was performed on 20 cells from an acute myeloid leukemia bone marrow sample. Putative blasts were identified based on their gene expression profiles and principal component analysis was performed to identify outlier cells. Variant calling was performed on the single-cell RNA-seq data. The present pilot study demonstrates a proof of concept for clinical single-cell genomic profiling. The recognized limitations include significant stochastic RNA loss and the relatively low throughput of the current proposed platform. Although the results of the present study are promising, further technological advances and protocol optimization are necessary for single-cell genomic profiling to be clinically viable.