Efficacy and safety of implantable cardioverter-defibrillator implantation in the elderly-The I-70 Study: A randomized clinical trial.

Background: There is conflicting evidence on the efficacy of primary prevention implantable cardioverter-defibrillator (ICD) implantation in the elderly. Objective: The purpose of this study was to determine the efficacy and safety of ICD implantation in patients 70 years and older. Methods: Patients (n = 167) aged 70 years or older and eligible for ICD implantation were randomly assigned (1:1) to receive either optimal medical therapy (OMT) (n = 85) or OMT plus ICD (n = 82). Results: Of the 167 participants (mean age 76.4 years; 165 men), 144 completed the study protocol according to their assigned treatment. Average participant follow-up was 31.5 months. Mortality was similar between the 2 groups: 27 deaths in OMT vs 26 death in ICD (unadjusted hazard ratio 0.92; 95% confidence interval 0.53-1.57), but there was a trend favoring the ICD over the first 36 months of follow-up. Rates of sudden death (7 vs 5; P = .81) and all-cause hospitalization (2.65 events per participant in OMT vs 3.09 in ICD; P = .31) were not statistically significantly different. Eleven participants randomized to ICD received appropriate therapy. Five participants received an inappropriate therapy that included at least 1 ICD shock. Conclusion: The study did not recruit to target sample size, and accumulated data did not show benefit of ICD therapy in patients 70 years or older. Future studies similar in design might be feasible but will need to contend with patient treatment preference given the large number of patients who do not want an ICD implanted. Further research is needed to determine whether the ICD is effective in prolonging life among elderly device candidates.

Neratinib, a pan ERBB/HER inhibitor, restores sensitivity of PTEN-null, BRAFV600E melanoma to BRAF/MEK inhibition.

Introduction: Approximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy. Methods: To compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition. Results: RNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells. Conclusions: The findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.

Combined ion beam irradiation platform and 3D fluorescence microscope for cellular cancer research.

To improve particle radiotherapy, we need a better understanding of the biology of radiation effects, particularly in heavy ion radiation therapy, where global responses are observed despite energy deposition in only a subset of cells. Here, we integrated a high-speed swept confocally-aligned planar excitation (SCAPE) microscope into a focused ion beam irradiation platform to allow real-time 3D structural and functional imaging of living biological samples during and after irradiation. We demonstrate dynamic imaging of the acute effects of irradiation on 3D cultures of U87 human glioblastoma cells, revealing characteristic changes in cellular movement and intracellular calcium signaling following ionizing irradiation.

Discovery and optimization of narrow spectrum inhibitors of Tousled like kinase 2 (TLK2) using quantitative structure activity relationships.

The oxindole scaffold has been the center of several kinase drug discovery programs, some of which have led to approved medicines. A series of two oxindole matched pairs from the literature were identified where TLK2 was potently inhibited as an off-target kinase. The oxindole has long been considered a promiscuous kinase inhibitor template, but across these four specific literature oxindoles TLK2 activity was consistent, while the kinome profile was radically different ranging from narrow to broad spectrum kinome coverage. We synthesized a large series of analogues, utilizing quantitative structure-activity relationship (QSAR) analysis, water mapping of the kinase ATP binding sites, kinome profiling, and small-molecule x-ray structural analysis to optimize TLK2 inhibition and kinome selectivity. This resulted in the identification of several narrow spectrum, sub-family selective, chemical tool compounds including 128 (UNC-CA2-103) that could enable elucidation of TLK2 biology.

PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2.

In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C's roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

Homozygous CNP Mutation and Neurodegeneration in Weimaraners: Myelin Abnormalities and Accumulation of Lipofuscin-like Inclusions.

A progressive neurological disorder was observed in a male neutered Weimaraner. Clinical signs included fecal incontinence, lethargy, moderate paraparesis, proprioceptive pelvic limb ataxia, falling, cognitive decline, incoordination, decreased interest in food, changes in posture, and episodes of trance-like behavior. Neurologic signs were first observed at approximately 4 years, 10 months of age and progressed slowly. Magnetic resonance imaging showed generalized brain atrophy with areas of white matter pathology. Humane euthanasia was elected at 6 years, 7 months of age due to increasing severity of the neurological signs. Autofluorescent intracellular granules were observed in the cerebral and cerebellar cortexes, optic nerve, and cardiac muscle of the affected dog. These abnormal inclusions in the cerebral cortex and cardiac muscle immunolabeled with antibodies to mitochondrial ATP synthase subunit c protein, like that observed in the neuronal ceroid lipofuscinosis group of lysosomal storage diseases. Immunolabeling also demonstrated pronounced neuroinflammation in brain tissues. The ultrastructural appearances of the disease-related inclusion bodies in the brain and optic nerve were quite variable. The ultrastructure and locations of many of the inclusions in the nervous tissues suggested that they were derived, at least in part, from the myelin surrounding axons. The storage bodies in the cardiac muscle were located in mitochondria-rich regions and consisted of parallel arrays of membrane-like components interspersed with electron-dense flocculent material. The disease was characterized by pronounced abnormalities in the myelin of the brain and optic nerve consisting of distinctive areas of ballooning between the layers of myelin. The whole genome sequence generated from the affected dog contained a homozygous G-to-A missense mutation in CNP, which encodes proteins with CNPase enzyme activity and a structural role in myelin. The mutation predicts a Thr42Met amino acid sequence substitution. Genotyping of archived Weimaraner DNA samples identified an additional G > A variant homozygote with a clinical history and brain lesions similar to those of the proband. Of 304 Weimaraners and over 4000 other dogs of various breeds, the proband and the other Weimaraner that exhibited similar signs were the only two that were homozygous for the CNP missense variant. CNPase immunolabeling was widespread in brain tissues from normal dogs but was undetectable in the same tissues from the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the late-onset Weimaraner disorder likely results from the missense mutation that results in CNPase deficiency, leading to myelin abnormalities, accumulation of lysosomal storage bodies, and brain atrophy. Similar disorders have been associated with different CNP variants in Dalmatians and in human subjects.

Illuminating function of the understudied druggable kinome.

The human kinome, with more than 500 proteins, is crucial for cell signaling and disease. Yet, about one-third of kinases lack in-depth study. The Data and Resource Generating Center for Understudied Kinases has developed multiple resources to address this challenge including creation of a heavy amino acid peptide library for parallel reaction monitoring and quantitation of protein kinase expression, use of understudied kinases tagged with a miniTurbo-biotin ligase to determine interaction networks by proximity-dependent protein biotinylation, NanoBRET probe development for screening chemical tool target specificity in live cells, characterization of small molecule chemical tools inhibiting understudied kinases, and computational tools for defining kinome architecture. These resources are available through the Dark Kinase Knowledgebase, supporting further research into these understudied protein kinases.

Effect of next-step antidepressant treatment on suicidal ideation: findings from the VAST-D trial.

BACKGROUND: Major depressive disorder (MDD) contributes to suicide risk. Treating MDD effectively is considered a key suicide prevention intervention. Yet many patients with MDD do not respond to their initial medication and require a 'next-step'. The relationship between next-step treatments and suicidal thoughts and behaviors is uncharted. METHOD: The VA Augmentation and Switching Treatments for Depression trial randomized 1522 participants to one of three next-step treatments: Switching to Bupropion, combining with Bupropion, and augmenting with Aripiprazole. In this secondary analysis, features associated with lifetime suicidal ideation (SI) and attempts (SA) at baseline and current SI during treatment were explored. RESULTS: Compared to those with SI only, those with lifetime SI + SA were more likely to be female, divorced, or separated, unemployed; and to have experienced more childhood adversity. They had a more severe depressive episode and were more likely to respond to 'next-step' treatment. The prevalence of SI decreased from 46.5% (694/1492) at baseline to 21.1% (315/1492) at end-of-treatment. SI during treatment was associated with baseline SI; low positive mental health, more anxiety, greater severity and longer duration of current MDD episode; being male and White; and treatment with S-BUP or C-BUP as compared to A-ARI. CONCLUSION: SI declines for most patients during next-step medication treatments. But about 1 in 5 experienced emergent or worsening SI during treatment, so vigilance for suicide risk through the entire 12-week acute treatment period is necessary. Treatment selection may affect the risk of SI.

Combined kinome inhibition states are predictive of cancer cell line sensitivity to kinase inhibitor combination therapies.

Protein kinases are a primary focus in targeted therapy development for cancer, owing to their role as regulators in nearly all areas of cell life. Recent strategies targeting the kinome with combination therapies have shown promise, such as trametinib and dabrafenib in advanced melanoma, but empirical design for less characterized pathways remains a challenge. Computational combination screening is an attractive alternative, allowing in-silico filtering prior to experimental testing of drastically fewer leads, increasing efficiency and effectiveness of drug development pipelines. In this work, we generated combined kinome inhibition states of 40,000 kinase inhibitor combinations from kinobeads-based kinome profiling across 64 doses. We then integrated these with transcriptomics from CCLE to build machine learning models with elastic-net feature selection to predict cell line sensitivity across nine cancer types, with accuracy R2 ∼ 0.75-0.9. We then validated the model by using a PDX-derived TNBC cell line and saw good global accuracy (R2 ∼ 0.7) as well as high accuracy in predicting synergy using four popular metrics (R2 ∼ 0.9). Additionally, the model was able to predict a highly synergistic combination of trametinib and omipalisib for TNBC treatment, which incidentally was recently in phase I clinical trials. Our choice of tree-based models for greater interpretability allowed interrogation of highly predictive kinases in each cancer type, such as the MAPK, CDK, and STK kinases. Overall, these results suggest that kinome inhibition states of kinase inhibitor combinations are strongly predictive of cell line responses and have great potential for integration into computational drug screening pipelines. This approach may facilitate the identification of effective kinase inhibitor combinations and accelerate the development of novel cancer therapies, ultimately improving patient outcomes.

Factors Affecting Antidepressant Response Trajectories: A Veterans Affairs Augmentation and Switching Treatments for Improving Depression Outcomes Trial Report.

Background: In this secondary analysis of the VA Augmentation and Switching Treatments for Improving Depression Outcomes (VAST-D) study we used antidepressant response trajectories to assess the association of treatment and multiple clinical/demographic factors with the probability of response. Methods: Using data from VAST-D, a multi-site, randomized, single-blind trial with parallel-assignment to one of three treatment interventions in 1522 Veterans whose major depressive disorder was unresponsive to at least one antidepressant trial, we evaluated response patterns using group-based trajectory modeling (GBTM). A weighted multinomial logistic regression analysis with backward elimination and additional exploratory analyses were performed to evaluate the association of multiple clinical/demographic factors with the probability of inclusion into specific trajectories. Additional exploratory analyses were used to identify factors associated with trajectory group membership that could have been missed in the primary analysis. Results: GBTM showed the best fit for depression symptom change was comprised of six trajectories, with some trajectories demonstrating minimal improvement and others showing a high probability of remission. High baseline depression and anxiety severity scores decreased, and early improvement increased, the likelihood of inclusion into the most responsive trajectory in both the GBTM and exploratory analyses. Conclusion: While multiple factors influence responsiveness, the probability of inclusion into a specific depression symptom trajectory is most strongly influenced by three factors: baseline depression, baseline anxiety, and the presence of early improvement.

A Homozygous MAN2B1 Missense Mutation in a Doberman Pinscher Dog with Neurodegeneration, Cytoplasmic Vacuoles, Autofluorescent Storage Granules, and an α-Mannosidase Deficiency.

A 7-month-old Doberman Pinscher dog presented with progressive neurological signs and brain atrophy suggestive of a hereditary neurodegenerative disorder. The dog was euthanized due to the progression of disease signs. Microscopic examination of tissues collected at the time of euthanasia revealed massive accumulations of vacuolar inclusions in cells throughout the central nervous system, suggestive of a lysosomal storage disorder. A whole genome sequence generated with DNA from the affected dog contained a likely causal, homozygous missense variant in MAN2B1 that predicted an Asp104Gly amino acid substitution that was unique among whole genome sequences from over 4000 dogs. A lack of detectable α-mannosidase enzyme activity confirmed a diagnosis of a-mannosidosis. In addition to the vacuolar inclusions characteristic of α-mannosidosis, the dog exhibited accumulations of autofluorescent intracellular inclusions in some of the same tissues. The autofluorescence was similar to that which occurs in a group of lysosomal storage disorders called neuronal ceroid lipofuscinoses (NCLs). As in many of the NCLs, some of the storage bodies immunostained strongly for mitochondrial ATP synthase subunit c protein. This protein is not a substrate for α-mannosidase, so its accumulation and the development of storage body autofluorescence were likely due to a generalized impairment of lysosomal function secondary to the accumulation of α-mannosidase substrates. Thus, it appears that storage body autofluorescence and subunit c accumulation are not unique to the NCLs. Consistent with generalized lysosomal impairment, the affected dog exhibited accumulations of intracellular inclusions with varied and complex ultrastructural features characteristic of autophagolysosomes. Impaired autophagic flux may be a general feature of this class of disorders that contributes to disease pathology and could be a target for therapeutic intervention. In addition to storage body accumulation, glial activation indicative of neuroinflammation was observed in the brain and spinal cord of the proband.

Combined kinome inhibition states are predictive of cancer cell line sensitivity to kinase inhibitor combination therapies.

Protein kinases are a primary focus in targeted therapy development for cancer, owing to their role as regulators in nearly all areas of cell life. Kinase inhibitors are one of the fastest growing drug classes in oncology, but resistance acquisition to kinase-targeting monotherapies is inevitable due to the dynamic and interconnected nature of the kinome in response to perturbation. Recent strategies targeting the kinome with combination therapies have shown promise, such as the approval of Trametinib and Dabrafenib in advanced melanoma, but similar empirical combination design for less characterized pathways remains a challenge. Computational combination screening is an attractive alternative, allowing in-silico screening prior to in-vitro or in-vivo testing of drastically fewer leads, increasing efficiency and effectiveness of drug development pipelines. In this work, we generate combined kinome inhibition states of 40,000 kinase inhibitor combinations from kinobeads-based kinome profiling across 64 doses. We then integrated these with baseline transcriptomics from CCLE to build robust machine learning models to predict cell line sensitivity from NCI-ALMANAC across nine cancer types, with model accuracy R2 ~ 0.75-0.9 after feature selection using elastic-net regression. We further validated the model's ability to extend to real-world examples by using the best-performing breast cancer model to generate predictions for kinase inhibitor combination sensitivity and synergy in a PDX-derived TNBC cell line and saw reasonable global accuracy in our experimental validation (R2 ~ 0.7) as well as high accuracy in predicting synergy using four popular metrics (R2 ~ 0.9). Additionally, the model was able to predict a highly synergistic combination of Trametinib (MEK inhibitor) and Omipalisib (PI3K inhibitor) for TNBC treatment, which incidentally was recently in phase I clinical trials for TNBC. Our choice of tree-based models over networks for greater interpretability also allowed us to further interrogate which specific kinases were highly predictive of cell sensitivity in each cancer type, and we saw confirmatory strong predictive power in the inhibition of MAPK, CDK, and STK kinases. Overall, these results suggest that kinome inhibition states of kinase inhibitor combinations are strongly predictive of cell line responses and have great potential for integration into computational drug screening pipelines. This approach may facilitate the identification of effective kinase inhibitor combinations and accelerate the development of novel cancer therapies, ultimately improving patient outcomes.

Combined CDK4/6 and ERK1/2 Inhibition Enhances Antitumor Activity in NF1-Associated Plexiform Neurofibroma.

PURPOSE: Plexiform neurofibromas (PNF) are peripheral nerve sheath tumors that cause significant morbidity in persons with neurofibromatosis type 1 (NF1), yet treatment options remain limited. To identify novel therapeutic targets for PNF, we applied an integrated multi-omic approach to quantitatively profile kinome enrichment in a mouse model that has predicted therapeutic responses in clinical trials for NF1-associated PNF with high fidelity. EXPERIMENTAL DESIGN: Utilizing RNA sequencing combined with chemical proteomic profiling of the functionally enriched kinome using multiplexed inhibitor beads coupled with mass spectrometry, we identified molecular signatures predictive of response to CDK4/6 and RAS/MAPK pathway inhibition in PNF. Informed by these results, we evaluated the efficacy of the CDK4/6 inhibitor, abemaciclib, and the ERK1/2 inhibitor, LY3214996, alone and in combination in reducing PNF tumor burden in Nf1flox/flox;PostnCre mice. RESULTS: Converging signatures of CDK4/6 and RAS/MAPK pathway activation were identified within the transcriptome and kinome that were conserved in both murine and human PNF. We observed robust additivity of the CDK4/6 inhibitor, abemaciclib, in combination with the ERK1/2 inhibitor, LY3214996, in murine and human NF1(Nf1) mutant Schwann cells. Consistent with these findings, the combination of abemaciclib (CDK4/6i) and LY3214996 (ERK1/2i) synergized to suppress molecular signatures of MAPK activation and exhibited enhanced antitumor activity in Nf1flox/flox;PostnCre mice in vivo. CONCLUSIONS: These findings provide rationale for the clinical translation of CDK4/6 inhibitors alone and in combination with therapies targeting the RAS/MAPK pathway for the treatment of PNF and other peripheral nerve sheath tumors in persons with NF1.

Export of macroinvertebrate prey from tidal freshwater wetlands provides a significant energy subsidy for outmigrating juvenile salmon.

Tidal freshwater wetlands linking terrestrial, riverine, and saline habitats are critical areas for material processing and exchange. Once historically widespread, herbaceous marsh and forested tidal freshwater wetlands especially are now highly degraded worldwide. Additionally, quantitative assessments of hydrology and material exchange from these systems are lacking compared to lotic and estuarine (saltmarsh) habitats. Here we investigate macroinvertebrate and energy export from tidal marsh and forested wetlands and consider potential benefits from this ecological process to endangered Pacific salmon in a large tidal freshwater system, the Columbia River (USA). Macroinvertebrate (salmon prey) concentration, water velocity, and discharge were measured at several wetland habitat types (forested swamp, emergent marsh, and restored marsh). We used these data to compute prey flux and transport metrics. Then, applying literature values to calculate prey energy equivalents and juvenile salmon metabolic requirements, we estimated the potential energy subsidy available to juvenile salmon. Numerically, larval stages of aquatic insects were the predominant type of prey exported from the wetlands, with Diptera chironomid fly abundance exceeding other groups. Energetically, however, non-chironomid dipterans and hemipteran prey comprised most of energy transport due to their higher energetic content (energy density × mean weight). We determined the prey energy transported from the sampled tidal channels was sufficient to meet energetic needs of tens to thousands of juvenile salmon per day, depending on prey production and hydrography. The prey taxonomic composition differed among organisms exiting forested swamp, emergent marsh, and restored marsh habitats with corresponding differences in energy transport, but all habitat types supported similar numbers of juvenile salmon. We conclude that macroinvertebrate prey exported from varied tidal freshwater wetlands likely provide significant benefits to juvenile salmon over a larger ecological footprint than the wetland area would suggest.

Association of in-competition injury risk and the degree of rapid weight cutting prior to competition in division I collegiate wrestlers.

OBJECTIVES: Weight cutting is thought to offer a competitive advantage in wrestling. Dehydration has deleterious effects on physical and cognitive function, which may increase the risk of injury. The purpose of the study was to investigate whether the degree of weight cutting was associated with injury risk. METHODS: Data were collected prospectively in a cohort of collegiate wrestlers over seven seasons. Changes in weight, body fat and lean mass were measured during the preseason, at midseason and before competition. Cox proportional-hazard ratios were calculated for risk of in-competition injury. RESULTS: Among 67 unique division 1 collegiate wrestlers (163 athlete seasons), there were 53 unique injuries affecting 46 athletes. There was no difference in absolute weight change, per cent weight change, per cent body fat change or per cent lean mass change between injured and non-injured wrestlers from the preseason to midseason measurements. From midseason to competition weight, change in body weight was -7.0%±3.2% (-5.3 kg±2.6) in injured athletes compared with -5.7%±3.3% (-4.3 kg±2.5) in non-injured athletes. For every kilogram of body weight lost, wrestlers had a 14% increased hazard of injury (HR 1.14, 95% CI 1.04 to 1.25, p=0.004). For every 1% of body weight lost, wrestlers had an 11% increased hazard of injury (HR 1.11, 95% CI 1.03 to 1.19, p=0.005). CONCLUSION: Rapid weight cutting was associated with a higher risk of in-competition injuries in division 1 collegiate wrestlers. For every per cent in body weight lost, wrestlers had an 11% increased hazard of injury during competition.

Small-molecule inhibition of the archetypal UbiB protein COQ8.

Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.

Discovery and Optimization of Narrow Spectrum Inhibitors of Tousled Like Kinase 2 (TLK2) Using Quantitative Structure Activity Relationships.

The oxindole scaffold has been the center of several kinase drug discovery programs, some of which have led to approved medicines. A series of two oxindole matched pairs from the literature were identified where TLK2 was a potent off-target kinase. The oxindole has long been considered a promiscuous inhibitor template, but across these 4 specific literature oxindoles TLK2 activity was consistent, while the kinome profile was radically different from narrow to broad spectrum coverage. We synthesized a large series of analogues and through quantitative structure-activity relationship (QSAR) analysis, water mapping of the kinase ATP binding sites, small-molecule x-ray structural analysis and kinome profiling, narrow spectrum, sub-family selective, chemical tool compounds were identified to enable elucidation of TLK2 biology.