Enhancing the health knowledge and health literacy of recently resettled refugees through classroom-based instructional methods.

Health education can elevate health literacy, which is associated with health knowledge, health-seeking behaviors and overall improved health outcomes. Refugees are particularly vulnerable to the effects of low health knowledge and literacy, which can exacerbate already poor health stemming from their displacement experience. Traditional learning methods including classroom-based instruction are typically how health-related information is presented to refugees. Through a series of interactive classes focused on specific health topics relevant to the resettled refugee population, this study evaluated the effectiveness of a classroom-based health education model in enhancing the health knowledge of recently resettled refugees. We used the Wilcoxon signed-rank test to evaluate differences in pre- and post-class knowledge through test performance. We found a significant improvement in health knowledge in two refugee groups: females and those who were employed. Culturally and socially sensitive considerations including language inclusiveness, class timing, transportation and childcare provisions are important when creating an educational program for individuals with refugee backgrounds. Developing focused approaches to instruction that enhance health knowledge could lead to better health literacy and ultimately improve health-related behaviors and outcomes in the refugee population.

Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

BACKGROUND: Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma, it remains unclear whether their mechanisms of action overlap. METHODS: We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1, D4M-UV2 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion, tetramer binding assays and ELISPOT assays were used to demonstrate the unique role of CD4+T cell help in the antitumor effects of the anti-PD-1+LAG-3 combination. RESULTS: The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+regulatory CD4+ cells (Tregs), fewer activated CD4+T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+T cell depletion was associated with fewer activated (CD69+) CD8+T cells and impaired IFNγ release but, conversely, increased numbers of activated CD8+T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors. CONCLUSIONS: Together these studies suggest that these two clinically relevant immune checkpoint inhibitor (ICI) combinations have differential effects on CD4+T cell polarization, which in turn, impacted cytotoxic CD8+T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.

URINE TOXICOLOGY PROFILES OF EMERGENCY DEPARTMENT PATIENTS WITH UNTREATED OPIOID USE DISORDER: A MULTI-SITE VIEW.

BACKGROUND: Opioid overdose deaths in 2021 were the highest ever, driven by fentanyl and polysubstance use. OBJECTIVE: The aim of the study was to characterize drug use, assessed by urine drug screens (UDSs), in patients with untreated opioid use disorder (OUD) presenting to 28 emergency departments (EDs) nationally and by region. METHODS: We analyzed UDSs from patients enrolled in the CTN-0099 ED-INNOVATION (Emergency Department-Initiated Buprenorphine Validation) trial between July 12, 2020 and March 9, 2022. Participants were adult ED patients with OUD not engaged in addiction treatment with a UDS positive for an opioid, but negative for methadone. Sites were divided into "East" and "West" regions. RESULTS: A UDS was available for all 925 enrolled participants, 543 from East and 382 from West. Fentanyl was in 702 specimens (76%) (n = 485 [89%] East vs. n = 217 [57%] West; p < 0.01) and was the only opioid in 269 (29%). After fentanyl, the most common opioids were morphine (presumably heroin; n = 411 [44%]; n = 192 [35%] East vs. n = 219 [57%] West; p < 0.01) and buprenorphine (n = 329 [36%]; n = 186 [35%] East vs. n = 143 [37%] West; p = 0.32). The most common drugs found with opioids were stimulants (n = 545 [59%]), tetrahydrocannabinol (n = 417 [45%]), and benzodiazepines (n = 151 [16%]). Amphetamine-type stimulants were more common in West (n = 209 [55%] vs. East (n = 125 [23%]). Cocaine was more common in East (n = 223 [41%]) vs. West (n = 82 [21%]). The presence of multiple drugs was common (n = 759 [82%]). CONCLUSIONS: Most participants had UDS specimens containing multiple substances; a high proportion had fentanyl, stimulants, and buprenorphine. Regional differences were noted. Given the increased risk of death with fentanyl and polysubstance use, ED providers should be providing risk reduction counseling, treatment, and referral.

Risk Factors for Common Kidney Stones Are Correlated with Kidney Function Independent of Stone Composition.

INTRODUCTION: Kidney stone type varies with age, sex, season, and medical conditions. Lower estimate glomerular filtration rate (eGFR) leads to changes in urine chemistry, and risk factors for kidney stones are thought to vary by stone type. We explore the association between eGFR, urine risk factors, and common stone compositions. METHODS: This was a retrospective cohort study of 811 kidney stone patients seen at Yale Medicine between 1994 and 2021 with serum chemistries and 24-h urine chemistries matched within 1 year of baseline stone analysis. Patients' eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) 2021 equation. Demographics and medical history were compared by χ2 tests. 24-h urine chemistries and stone analyses were analyzed by one-way ANOVA. Linear regressions were performed to control for demographics, comorbidities, and stone composition. RESULTS: With lower eGFR, the proportion of calcium stones declined while uric acid (UA) stones increased. On univariable analysis, lower eGFR was associated with lower urine pH, calcium, citrate, UA, magnesium, phosphorus, and ammonium. On multivariable analysis, controlling for age, sex, ethnicity, body mass index, comorbidities, and stone type, these factors remained significant. Stone formers with lower eGFR had elevated supersaturation for UA, but reduced supersaturations for calcium-containing stones. Though urine oxalate was significant on univariable analysis, it was not on multivariable analysis. CONCLUSION: Changes in urine parameters are strongly correlated with eGFR regardless of stone type. Renal function may play a key role in modulating kidney stone risk factors. Strategies to mitigate stone risk may need to vary with kidney function, especially when patient urine or stone composition data are unavailable.

Efficacy of Osimertinib in Patients with Lung Cancer Positive for Uncommon EGFR Exon 19 Deletion Mutations.

PURPOSE: The uncommon EGFR exon 19 deletion (ex19del), L747_A750>P, demonstrates reduced sensitivity to osimertinib compared with the common ex19del, E746_A750del in preclinical models. The clinical efficacy of osimertinib in patients with non-small cell lung cancer harboring L747_A750>P and other uncommon ex19dels is not known. EXPERIMENTAL DESIGN: The AACR GENIE database was interrogated to characterize the frequency of individual ex19dels relative to other variants, and a multicenter retrospective cohort was used to compare clinical outcomes for patients with tumors harboring E746_A750del, L747_A750>P, and other uncommon ex19dels who received osimertinib in the first line (1L) or in second or later lines of therapy and were T790M+ (≥2L). RESULTS: ex19dels comprised 45% of EGFR mutations, with 72 distinct variants ranging in frequency from 28.1% (E746_A750del) to 0.03%, with L747_A750>P representing 1.8% of the EGFR mutant cohort. In our multi-institutional cohort (N = 200), E746_A750del was associated with significantly prolonged progression-free survival (PFS) with 1L osimertinib versus L747_A750>P [median 21.3 months (95% confidence interval, 17.0-31.7) vs. 11.7 months (10.8-29.4); adjusted HR 0.52 (0.28-0.98); P = 0.043]. Osimertinib efficacy in patients with other uncommon ex19dels varied on the basis of the specific mutation present. CONCLUSIONS: The ex19del L747_A750>P is associated with inferior PFS compared with the common E746_A750del mutation in patients treated with 1L osimertinib. Understanding differences in osimertinib efficacy among EGFR ex19del subtypes could alter management of these patients in the future.

Nocturnal Dipping and Kidney Function Decline: Findings From the CKD in Children Study.

Introduction: Normally, blood pressure (BP) declines by at least 10% from daytime to nighttime. In adults, blunted nocturnal dipping has been associated with more rapid decline in kidney function. Nondipping is prevalent in children with chronic kidney disease (CKD). We sought to determine whether nondipping is associated with proteinuria and progression to kidney failure in children with CKD. Methods: In the prospective CKD in children (CKiD) cohort, Cox proportional hazards models were used to evaluate the relationship between baseline nondipping and progression to kidney failure. Linear mixed effects models were used to evaluate the relationship between nondipping and changes in iohexol glomerular filtration rate (GFR) and urine protein-to-creatinine ratio (log-UPCR, mg/mg) over time. Results: Among 620 participants, mean age was 11 (± 4) years, mean iohexol GFR was 52 (± 22) ml/min per 1.73 m2, and 40% were nondippers at baseline. There were 169 kidney failure events during 2.9 years (median) of follow-up. Dipping status was not significantly associated with kidney failure overall (hazard ratio [HR] 1.08; 95% confidence interval [CI] 0.77, 1.51) or in those with (HR 1.21; 95% CI 0.53, 2.77) or without (HR 1.05; 95% CI 0.71, 1.55) glomerular disease. Dipping status did not modify the relationship between time and change in iohexol GFR or log (UPCR) from baseline (interaction P values = 0.20 and 0.054, respectively). Conclusion: Nondipping is not associated with end-stage kidney disease, GFR decline, or change in proteinuria within the CKiD cohort.

Biochemical and structural basis for differential inhibitor sensitivity of EGFR with distinct exon 19 mutations.

Tyrosine kinase inhibitors (TKIs) are used to treat non-small cell lung cancers (NSCLC) driven by epidermal growth factor receptor (EGFR) mutations in the tyrosine kinase domain (TKD). TKI responses vary across tumors driven by the heterogeneous group of exon 19 deletions and mutations, but the molecular basis for these differences is not understood. Using purified TKDs, we compared kinetic properties of several exon 19 variants. Although unaltered for the second generation TKI afatinib, sensitivity varied significantly for both the first and third generation TKIs erlotinib and osimertinib. The most sensitive variants showed reduced ATP-binding affinity, whereas those associated with primary resistance retained wild type ATP-binding characteristics (and low KM, ATP). Through crystallographic and hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies, we identify possible origins for the altered ATP-binding affinity underlying TKI sensitivity and resistance, and propose a basis for classifying uncommon exon 19 variants that may have predictive clinical value.

Single-cell Characterization of the Cellular Landscape of Acral Melanoma Identifies Novel Targets for Immunotherapy.

PURPOSE: Acral melanoma is a rare subtype of melanoma that arises on the non-hair-bearing skin of the palms, soles, and nail beds. In this study, we used single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape of acral melanoma and identify novel immunotherapeutic targets. EXPERIMENTAL DESIGN: We performed scRNA-seq on nine clinical specimens (five primary, four metastases) of acral melanoma. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by analysis of The Cancer Genome Atlas (TCGA) and single-cell datasets. Cell-cell interactions were inferred and compared with those in nonacral cutaneous melanoma. RESULTS: Multiple phenotypic subsets of T cells, natural killer (NK) cells, B cells, macrophages, and dendritic cells with varying levels of activation/exhaustion were identified. A comparison between primary and metastatic acral melanoma identified gene signatures associated with changes in immune responses and metabolism. Acral melanoma was characterized by a lower overall immune infiltrate, fewer effector CD8 T cells and NK cells, and a near-complete absence of γδ T cells compared with nonacral cutaneous melanomas. Immune cells associated with acral melanoma exhibited expression of multiple checkpoints including PD-1, LAG-3, CTLA-4, V-domain immunoglobin suppressor of T cell activation (VISTA), TIGIT, and the Adenosine A2A receptor (ADORA2). VISTA was expressed in 58.3% of myeloid cells and TIGIT was expressed in 22.3% of T/NK cells. CONCLUSIONS: Acral melanoma has a suppressed immune environment compared with that of cutaneous melanoma from nonacral skin. Expression of multiple, therapeutically tractable immune checkpoints were observed, offering new options for clinical translation.

[89Zr]ZrDFO-CR011 PET Correlates with Response to Glycoprotein Nonmetastatic Melanoma B-targeted Therapy in Triple-negative Breast Cancer.

There is a need for prognostic markers to select patients most likely to benefit from antibody-drug conjugate (ADC) therapy. We quantified the relationship between pretreatment PET imaging of glycoprotein nonmetastatic melanoma B (gpNMB) with 89Zr-labeled anti-gpNMB antibody ([89Zr]ZrDFO-CR011) and response to ADC therapy (CDX-011) in triple-negative breast cancer. First, we compared different PET imaging metrics and found that standardized uptake values (SUV) and tumor-to-heart SUV ratios were sufficient to delineate differences in radiotracer uptake in the tumor of four different cell- and patient-derived tumor models and achieved high standardized effect sizes. These tumor models with varying levels of gpNMB expression were imaged with [89Zr]ZrDFO-CR011 followed by treatment with a single bolus injection of CDX-011. The percent change in tumor volume relative to baseline (% CTV) was then correlated with SUVmean of [89Zr]ZrDFO-CR011 uptake in the tumor. All gpNMB-positive tumor models responded to CDX-011 over 6 weeks of treatment, except one patient-derived tumor regrew after 4 weeks of treatment. As expected, the gpNMB-negative tumor increased in volume by 130 ± 59% at endpoint. The magnitude of pretreatment SUV had the strongest inverse correlation with the % CTV at 2-4 weeks after treatment with CDX-011 (Spearman ρ = -0.8). However, pretreatment PET imaging with [89Zr]ZrDFO-CR011 did not inform on which tumor types will regrow over time. Other methods will be needed to predict resistance to treatment.

Melanoma brain metastases: Biological basis and novel therapeutic strategies.

The development of brain metastases is the deadliest complication of advanced melanoma and has long been associated with a dismal prognosis. The recent years have seen incredible progress in the development of therapies for melanoma brain metastases (MBM), with both targeted therapies (the BRAF-MEK inhibitor combination) and immune checkpoint inhibitors (the anti-CTLA-4, anti-PD-1 combination) showing impressive levels of activity. Despite this, durations of response for these therapies remain lower at intracranial sites of metastasis compared to extracranial metastases and it has been suggested that there are unique features of the brain microenvironment that contribute to therapeutic escape. In this review, we outline the latest research into the biology and pathophysiology of melanoma brain metastasis development and progression. We then discuss the current status of clinical trial that are open to patients with MBM and end by describing the ongoing challenges for the field.

Single-Cell Characterization of the Immune Microenvironment of Melanoma Brain and Leptomeningeal Metastases.

PURPOSE: Melanoma brain metastases (MBM) and leptomeningeal melanoma metastases (LMM) are two different manifestations of melanoma CNS metastasis. Here, we used single-cell RNA sequencing (scRNA-seq) to define the immune landscape of MBM, LMM, and melanoma skin metastases. EXPERIMENTAL DESIGN: scRNA-seq was undertaken on 43 patient specimens, including 8 skin metastases, 14 MBM, and 19 serial LMM specimens. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by IHC and flow cytometry. Association analyses were undertaken to identify immune cell subsets correlated with overall survival. RESULTS: The LMM microenvironment was characterized by an immune-suppressed T-cell landscape distinct from that of brain and skin metastases. An LMM patient with long-term survival demonstrated an immune repertoire distinct from that of poor survivors and more similar to normal cerebrospinal fluid (CSF). Upon response to PD-1 therapy, this extreme responder showed increased levels of T cells and dendritic cells in their CSF, whereas poor survivors showed little improvement in their T-cell responses. In MBM patients, therapy led to increased immune infiltrate, with similar T-cell transcriptional diversity noted between skin metastases and MBM. A correlation analysis across the entire immune landscape identified the presence of a rare population of dendritic cells (DC3) that was associated with increased overall survival and positively regulated the immune environment through modulation of activated T cells and MHC expression. CONCLUSIONS: Our study provides the first atlas of two distinct sites of melanoma CNS metastases and defines the immune cell landscape that underlies the biology of this devastating disease.

Deintensification or No Statin Treatment Is Associated With Higher Mortality in Patients With Ischemic Stroke or Transient Ischemic Attack.

Background and Purpose: Practice guidelines recommend that most patients receive moderate- or high-potency statins after ischemic stroke or transient ischemic attack (TIA) of atherosclerotic origin. We tested the association of different patterns of potency for prescribed statin therapy­assessed before admission and at hospital discharge for ischemic stroke or TIA­on mortality in a large, nationwide sample of US Veterans. Methods: The study population included patients with an ischemic stroke or TIA occurring during 2011 at any of the 134 Veterans Health Administration facilities. We used electronic outpatient pharmacy files to identify statin dose at hospital admission and within 7 days after hospital discharge. We categorized statin dosing as low, moderate, or high potency; moderate or high potency was considered at goal. We created 6 mutually exclusive groups to reflect patterns of statin potency from hospital admission to discharge: goal to goal, low to goal, goal to low or goal to none (deintensification), none to none, none to low, and low to low. We used logistic regression to compare 30-day and 1-year mortality across statin potency groups. Results: The population included 9380 predominately White (71.1%) men (96.3%) who were hospitalized for stroke or TIA. In this sample, 34.1% of patients (n=3194) were discharged off a statin medication. Deintensification occurred in 14.0% of patients (n=1312) and none to none in 20.5% (n=1924). Deintensification and none to none were associated with a higher odds of mortality as compared with goal to goal (adjusted odds ratio 1-year mortality: deintensification versus goal to goal, 1.26 [95% CI, 1.02­1.57]; none to none versus goal to goal, 1.59 [95% CI, 1.30­1.93]). Adjustments for differences in baseline characteristics using propensity weighted scores demonstrated similar results. Conclusions: Underutilization of statins, including no treatment or underdosing after stroke (deintensification), was observed in approximately one-third of veterans with ischemic stroke or TIA and was associated with higher mortality when compared with patients who were at goal for statin prescription dosing.

Targeted Therapy Given after Anti-PD-1 Leads to Prolonged Responses in Mouse Melanoma Models through Sustained Antitumor Immunity.

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti-PD-1)→ TT (ceritinib-trametinib or dabrafenib-trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.

Association of AKI-D with Urinary Findings and Baseline eGFR in Hospitalized COVID-19 Patients.

Background: AKI is common in patients hospitalized with coronavirus disease 2019 (COVID-19). Risk factors for AKI requiring dialysis (AKI-D) are not fully understood. We aimed to identify risk factors associated with AKI-D and AKI not requiring dialysis (AKI-ND). Methods: We reviewed electronic health records of 3186 patients aged ≥18 years old who were hospitalized with COVID-19 across six hospitals. Patient characteristics, urinalysis findings, and inflammatory markers were analyzed for association with in-hospital AKI status (AKI-D, AKI-ND, or no AKI), and we subsequently evaluated mortality. Results: After adjustment for multiple covariates, higher baseline eGFR was associated with 30% lower odds of AKI-D and 11% lower odds of AKI-ND (for AKI-D, OR, 0.70; 95% CI, 0.64 to 0.77; for AKI-ND, OR, 0.89; 95% CI, 0.85 to 0.92). Patients with obesity and those who were Latino had increased odds of AKI-D, whereas patients with congestive heart failure or diabetes with complications had increased odds of AKI-ND. Females had lower odds of in-hospital AKI (for AKI-D, OR, 0.28; 95% CI, 0.17 to 0.46; for AKI-ND, OR, 0.83; 95% CI, 0.70 to 0.99). After adjustment for covariates and baseline eGFR, 1-4+ protein on initial urinalysis was associated with a nine-fold increase in odds of AKI-D (OR, 9.00; 95% CI, 2.16 to 37.38) and more than two-fold higher odds of AKI-ND (OR, 2.28; 95% CI, 1.66 to 3.13). Findings of 1-3+ blood and trace glucose on initial urinalysis were also associated with increased odds of both AKI-D and AKI-ND. AKI-D and AKI-ND were associated with in-hospital death (for AKI-D, OR, 2.64; 95% CI, 1.13 to 6.17; for AKI-ND, OR, 2.44; 95% CI, 1.77 to 3.35). Conclusions: Active urine sediments, even after adjustment for baseline kidney function, and reduced baseline eGFR are significantly associated with increased odds of AKI-D and AKI-ND. In-hospital AKI was associated with in-hospital death. These findings may help prognosticate patients hospitalized with COVID-19.

Association of Demographic and Early-Life Socioeconomic Factors by Birth Cohort With Dementia Incidence Among US Adults Born Between 1893 and 1949.

Importance: Early-life factors may be important for later dementia risk. The association between a more advantaged early-life environment, as reflected through an individual's height and socioeconomic status indicators, and decreases in dementia incidence by birth cohort is unknown. Objectives: To examine the association of birth cohort and early-life environment with dementia incidence among participants in the Adult Changes in Thought study from 1994 to 2015. Design, Setting, and Participants: This prospective cohort study included 4277 participants from the Adult Changes in Thought study, an ongoing longitudinal population-based study of incident dementia in a random sample of adults 65 years and older who were born between 1893 and 1949 and are members of Kaiser Permanente Washington in the Seattle region. Participants in the present analysis were followed up from 1994 to 2015. At enrollment, all participants were dementia-free and completed a baseline evaluation. Subsequent study visits were held every 2 years until a diagnosis of dementia, death, or withdrawal from the study. Participants were categorized by birth period (defined by historically meaningful events) into 5 cohorts: pre-World War I (1893-1913), World War I and Spanish influenza (1914-1920), pre-Great Depression (1921-1928), Great Depression (1929-1939), and World War II and postwar (1940-1949). Participants' height, educational level, childhood financial stability, and childhood household density were examined as indicators of early-life environment, and later-life vascular risk factors for dementia were assessed. Cox proportional hazards regression models, adjusted for competing survival risk, were used to analyze data. Data were analyzed from June 1, 2018, to April 29, 2020. Main Outcomes and Measures: Participants completed the Cognitive Abilities Screening Instrument every 2 years to assess global cognition. Those with scores indicative of cognitive impairment completed an evaluation for dementia, with dementia diagnoses determined during consensus conferences using criteria from the Diagnostic and Statistical Manual of Mental Disorders, 4th edition. Results: Among 4277 participants, the mean (SD) age was 74.5 (6.4) years, and 2519 participants (58.9%) were women. The median follow-up was 8 years (interquartile range, 4-12 years), with 730 participants developing dementia over 24 378 person-years. The age-specific dementia incidence was lower for those born in 1929 and later compared with those born earlier. Compared with participants born in the pre-Great Depression years (1921-1928), the age- and sex-adjusted hazard ratio was 0.67 (95% CI, 0.53-0.85) for those born in the Great Depression period (1929-1939) and 0.62 (95% CI, 0.29-1.31) for those born in the World War II and postwar period (1940-1949). Although indicators of a more advantaged early-life environment and higher educational level (college or higher) were associated with a lower incidence of dementia, these variables did not explain the association between birth cohort and dementia incidence, which remained when vascular risk factors were included and were similar by sex. Conclusions and Relevance: Age-specific dementia incidence was lower in participants born after the mid-1920s compared with those born earlier. In this population, the decrease in dementia incidence may reflect societal-level changes or individual differences over the life course rather than early-life environment, as reflected through recalled childhood socioeconomic status and measured height, educational level, and later-life vascular risk.

Dabrafenib inhibits the growth of BRAF-WT cancers through CDK16 and NEK9 inhibition.

Although the BRAF inhibitors dabrafenib and vemurafenib have both proven successful against BRAF-mutant melanoma, there seem to be differences in their mechanisms of action. Here, we show that dabrafenib is more effective at inhibiting the growth of NRAS-mutant and KRAS-mutant cancer cell lines than vemurafenib. Using mass spectrometry-based chemical proteomics, we identified NEK9 and CDK16 as unique targets of dabrafenib. Both NEK9 and CDK16 were highly expressed in specimens of advanced melanoma, with high expression of both proteins correlating with a worse overall survival. A role for NEK9 in the growth of NRAS- and KRAS-mutant cell lines was suggested by siRNA studies in which silencing was associated with decreased proliferation, cell cycle arrest associated with increased p21 expression, inhibition of phospho-CHK1, decreased CDK4 expression, and the initiation of a senescence response. Inhibition of CDK4 but not CHK1 recapitulated the effects of NEK9 silencing, indicating this to be the likely mechanism of growth inhibition. We next turned our attention to CDK16 and found that its knockdown inhibited the phosphorylation of the Rb protein at S780 and increased expression of p27. Both of these effects were phenocopied in NRAS- and KRAS-mutant cancer cells by dabrafenib, but not vemurafenib. Combined silencing of NEK9 and CDK16 was associated with enhanced inhibition of melanoma cell proliferation. In summary, we have identified dabrafenib as a potent inhibitor of NEK9 and CDK16, and our studies suggest that inhibition of these kinases may have activity against cancers that do not harbor BRAF mutations.

Disruption of NAD(+) binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions.

AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD(+) cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD(+) binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD(+) binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD(+) (Km = 741 ± 257 µmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD(+) binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD(+) binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD(+) binding center in GAPDH interactions with its intranuclear partners.

The Novel ATP-Competitive MEK/Aurora Kinase Inhibitor BI-847325 Overcomes Acquired BRAF Inhibitor Resistance through Suppression of Mcl-1 and MEK Expression.

Resistance to BRAF inhibitors is a major clinical problem. Here, we evaluate BI-847325, an ATP-competitive inhibitor of MEK and Aurora kinases, in treatment-naïve and drug-resistant BRAF-mutant melanoma models. BI-847325 potently inhibited growth and survival of melanoma cell lines that were both BRAF inhibitor naïve and resistant in 2D culture, 3D cell culture conditions, and in colony formation assays. Western blot studies showed BI-847325 to reduce expression of phospho-ERK and phospho-histone 3 in multiple models of vemurafenib resistance. Mechanistically, BI-847325 decreased the expression of MEK and Mcl-1 while increasing the expression of the proapoptotic protein BIM. Strong suppression of MEK expression was observed after 48 hours of treatment, with no recovery following >72 hours of washout. siRNA-mediated knockdown of Mcl-1 enhanced the effects of BI-847325, whereas Mcl-1 overexpression reversed this in both 2D cell culture and 3D spheroid melanoma models. In vivo, once weekly BI-847325 (70 mg/kg) led to durable regression of BRAF-inhibitor naïve xenografts with no regrowth seen (>65 days of treatment). In contrast, treatment with the vemurafenib analog PLX4720 was associated with tumor relapse at >30 days. BI-847325 also suppressed the long-term growth of xenografts with acquired PLX4720 resistance. Analysis of tumor samples revealed BI-847325 to induce apoptosis associated with suppression of phospho-ERK, total MEK, phospho-Histone3, and Mcl-1 expression. Our studies indicate that BI-847325 is effective in overcoming BRAF inhibitor resistance and has long-term inhibitory effects upon BRAF-mutant melanoma in vivo, through a mechanism associated with the decreased expression of both MEK and Mcl-1.