Analyzing the functional effects of DNA variants with gene editing.

Continual advancements in genomics have led to an ever-widening disparity between the rate of discovery of genetic variants and our current understanding of their functions and potential roles in disease. Systematic methods for phenotyping DNA variants are required to effectively translate genomics data into improved outcomes for patients with genetic diseases. To make the biggest impact, these approaches must be scalable and accurate, faithfully reflect disease biology, and define complex disease mechanisms. We compare current methods to analyze the function of variants in their endogenous DNA context using genome editing strategies, such as saturation genome editing, base editing and prime editing. We discuss how these technologies can be linked to high-content readouts to gain deep mechanistic insights into variant effects. Finally, we highlight key challenges that need to be addressed to bridge the genotype to phenotype gap, and ultimately improve the diagnosis and treatment of genetic diseases.

Evaluating the Acceptability and Feasibility of Collecting Passive Smartphone Data to Estimate Psychological Functioning in U.S. Service Members and Veterans: A Pilot Study.

INTRODUCTION: This study investigated the acceptability and feasibility of digital phenotyping in a military sample with a history of traumatic brain injury and co-occurring psychological and cognitive symptoms. The first aim was to evaluate the acceptability of digital phenotyping by (1a) quantifying the proportion of participants willing to download the app and rates of dropout and app discontinuation and (1b) reviewing the stated reasons for both refusing and discontinuing use of the app. The second aim was to investigate technical feasibility by (2a) characterizing the amount and frequency of transferred data and (2b) documenting technical challenges. Exploratory aim 3 sought to leverage data on phone and keyboard interactions to predict if a participant (a) is depressed and (b) has depression that improves over the course of the study. MATERIALS AND METHODS: A passive digital phenotyping app (Mindstrong Discovery) functioned in the background of the participants' smartphones and passively collected phone usage and typing kinematics data. RESULTS: Fifteen out of 16 participants (93.8%) consented to install the app on their personal smartphone devices. Four participants (26.7%) discontinued the use of the app partway through the study, primarily because of keyboard usability and technical issues. Fourteen out of 15 participants (93.3%) had at least one data transfer, and the median number of days with data was 40 out of a possible 57 days. The exploratory machine learning models predicting depression status and improvement in depression performed better than chance. CONCLUSIONS: The findings of this pilot study suggest that digital phenotyping is acceptable and feasible in a military sample and provides support for future larger investigations of this technology.

Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation.

Loss-of-function of DDX3X is a leading cause of neurodevelopmental disorders (NDD) in females. DDX3X is also a somatically mutated cancer driver gene proposed to have tumour promoting and suppressing effects. We perform saturation genome editing of DDX3X, testing in vitro the functional impact of 12,776 nucleotide variants. We identify 3432 functionally abnormal variants, in three distinct classes. We train a machine learning classifier to identify functionally abnormal variants of NDD-relevance. This classifier has at least 97% sensitivity and 99% specificity to detect variants pathogenic for NDD, substantially out-performing in silico predictors, and resolving up to 93% of variants of uncertain significance. Moreover, functionally-abnormal variants can account for almost all of the excess nonsynonymous DDX3X somatic mutations seen in DDX3X-driven cancers. Systematic maps of variant effects generated in experimentally tractable cell types have the potential to transform clinical interpretation of both germline and somatic disease-associated variation.

TEAD Inhibition Overcomes YAP1/TAZ-Driven Primary and Acquired Resistance to KRASG12C Inhibitors.

Primary/intrinsic and treatment-induced acquired resistance limit the initial response rate to and long-term efficacy of direct inhibitors of the KRASG12C mutant in cancer. To identify potential mechanisms of resistance, we applied a CRISPR/Cas9 loss-of-function screen and observed loss of multiple components of the Hippo tumor suppressor pathway, which acts to suppress YAP1/TAZ-regulated gene transcription. YAP1/TAZ activation impaired the antiproliferative and proapoptotic effects of KRASG12C inhibitor (G12Ci) treatment in KRASG12C-mutant cancer cell lines. Conversely, genetic suppression of YAP1/WWTR1 (TAZ) enhanced G12Ci sensitivity. YAP1/TAZ activity overcame KRAS dependency through two distinct TEAD transcription factor-dependent mechanisms, which phenocopy KRAS effector signaling. First, TEAD stimulated ERK-independent transcription of genes normally regulated by ERK (BIRC5, CDC20, ECT2, FOSL1, and MYC) to promote progression through the cell cycle. Second, TEAD caused activation of PI3K-AKT-mTOR signaling to overcome apoptosis. G12Ci treatment-induced acquired resistance was also caused by YAP1/TAZ-TEAD activation. Accordingly, concurrent treatment with pharmacologic inhibitors of TEAD synergistically enhanced KRASG12C inhibitor antitumor activity in vitro and prolonged tumor suppression in vivo. In summary, these observations reveal YAP1/TAZ-TEAD signaling as a crucial driver of primary and acquired resistance to KRAS inhibition and support the use of TEAD inhibitors to enhance the antitumor efficacy of KRAS-targeted therapies. SIGNIFICANCE: YAP1/TAZ-TEAD activation compensates for loss of KRAS effector signaling, establishing a mechanistic basis for concurrent inhibition of TEAD to enhance the efficacy of KRASG12C-selective inhibitor treatment of KRASG12C-mutant cancers. See related commentary by Johnson and Haigis, p. 4005.

Mediator Kinase Inhibition Impedes Transcriptional Plasticity and Prevents Resistance to ERK/MAPK-Targeted Therapy in KRAS-Mutant Cancers.

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and cell line-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, cell line-specific resistance programs.

Suicidal Behaviors Among Active-Duty US Service Members: Data from the 2018 Health-Related Behaviors Survey.

Background: This study aimed to 1) determine the prevalence of past-year suicidal ideation (SI) and attempts (SA) among active-duty SMs; 2) determine whether differences exist by age, sex, and race; and 3) assess whether prevalence estimates vary by risk profiles of mental health conditions and substance use. Methods: Data were from the 2018 Health-Related Behavior Survey (HRBS), a cross-sectional survey of active-duty SMs (n = 17,166). We used the logistic model to identify the factors of SI and SA and latent class analysis (LCA) to identify the risk profiles. Results: Among active duty SMs, 8.26% had SI and 1.25% had SA in the past year. Gender and age have been shown to influence how race might contribute to suicidal behaviors. Mental health conditions were associated with higher odds of SI and SA, as were younger ages; LGB identity; being separated, divorced, or widowed; use of e-cigarettes, dual use of e-cigarettes and cigarettes, or drugs; and history of deployment of less than 12 months. Frequencies of cigarette and e-cigarette use were also associated with SI and SA, indicating the odds were increasing by 0.3% for every additional cigarette or e-cigarette used. Five risk profiles were identified: class 1 (illegal drug use), class 2 (mental health needs with tobacco and alcohol use), class 3 (mental health conditions only), class 4 ("low risk" SMs with low levels of illegal drug use, mental health visits, tobacco use, and alcohol use), and class 5 (alcohol use). Compared to class 4 ("low risk"), all other risk profiles were associated with increased odds of suicidal behaviors. Conclusion: Despite the resources and increased access provided for mental health support, the prevalence of SI among active-duty SMs is greater than in the general population of the same age, likely due to additional military exposures and stressors.

Malaria: moving beyond the search for magic bullets.

Round table discussion on challenges and opportunities in malaria research with Elena Levashina, Dominique Soldati-Favre, Andrew Waters, Friedrich Frischknecht, and Julian Rayner.

Pilot study of attentional retraining for postpartum smoking relapse.

Introduction: Tobacco smoking is a leading cause of preventable death worldwide. The perinatal period provides a unique opportunity for intervention, as many smokers quit smoking during pregnancy but relapse postpartum. Novel relapse prevention interventions that reduce the burden of treatment attendance in this population are needed. Attentional retraining (AR) has been shown to reduce attentional biases toward smoking-related stimuli, a cognitive process implicated in smoking, AR has not been applied to perinatal smokers, and the effect of AR on craving and smoking is not clear. The goal of this study was to evaluate the delivery of AR for smoking cues in perinatal smokers utilizing a mobile intervention. Methods: This pilot study utilized Ecological Momentary Assessment (EMA) methodology delivered on a mobile device to examine the relapse process and evaluate the utility of AR in former smokers attempting to remain abstinent postpartum. AR (or Control Training) was administered to abstinent smokers (N = 17) for up to 2 weeks both before and after delivery. Results: All 17 participants completed the study. There was evidence that AR reduced attentional bias in the AR group (vs. Controls). There was no evidence that AR reduced craving. An exploratory analysis revealed that there was no evidence that AR reduced smoking during the study period. Discussion: AR using EMA methodology via a mobile device is feasible in perinatal smokers. Further research using larger samples is required to evaluate the utility of mobile AR in reducing craving and smoking.

A conserved metabolic signature associated with response to fast-acting anti-malarial agents.

IMPORTANCE: In malaria drug discovery, understanding the mode of action of lead compounds is important as it helps in predicting the potential emergence of drug resistance in the field when these drugs are eventually deployed. In this study, we have employed metabolomics technologies to characterize the potential targets of anti-malarial drug candidates in the developmental pipeline at NITD. We show that NITD fast-acting leads belonging to spiroindolone and imidazothiadiazole class induce a common biochemical theme in drug-exposed malaria parasites which is similar to another fast-acting, clinically available drug, DHA. These biochemical features which are absent in a slower acting NITD lead (GNF17) point to hemoglobin digestion and inhibition of the pyrimidine pathway as potential action points for these drugs. These biochemical themes can be used to identify and inform on the mode of action of fast drug candidates of similar profiles in future drug discovery programs.

Searching for answers: expert pattern recognition and planning.

Does expertise mostly stem from pattern recognition or look-ahead search? van Opheusden et al. contribute to this important debate in cognitive psychology and artificial intelligence (AI) with a multi-method, multi-experiment study and a new model. Using a novel, relatively simple board game, they show that players increase depth of search when improving their skill.

Association between burnout and insomnia in U.S. Air Force Pararescue personnel: A cross-sectional study.

Studies have examined burnout and its impact on health, to include its influence on sleep. While many studies report a significant relationship between burnout and insomnia in civilian populations, no studies have examined this relationship in a military population. The United States Air Force (USAF) Pararescue personnel are an elite combat force who are specially trained to conduct both first-line combat and full spectrum personnel recovery and may be at high risk of burnout and insomnia. The current study investigated the association between dimensions of burnout and insomnia, and also examined potential moderators of the associations. A cross-sectional survey was administered to 203 Pararescue personnel (Mean Age = 32.1 years; 100% Male; 90.1% Caucasian) recruited from six US bases. The survey included measures of three dimensions of burnout (emotional exhaustion, depersonalization, personal achievement), insomnia, psychological flexibility, and social support. Emotional exhaustion was significantly associated with insomnia with a moderate to large effect size, when controlling for covariates. Depersonalization, but not personal achievement, was also significantly associated with insomnia. There was no evidence that associations between burnout and insomnia were moderated by psychological flexibility or social support. These findings help to identify individuals at risk of insomnia and may ultimately be useful in developing interventions for insomnia in this population.

Dual-pharmacophore artezomibs hijack the Plasmodium ubiquitin-proteasome system to kill malaria parasites while overcoming drug resistance.

Artemisinins (ART) are critical anti-malarials and despite their use in combination therapy, ART-resistant Plasmodium falciparum is spreading globally. To counter ART resistance, we designed artezomibs (ATZs), molecules that link an ART with a proteasome inhibitor (PI) via a non-labile amide bond and hijack parasite's own ubiquitin-proteasome system to create novel anti-malarials in situ. Upon activation of the ART moiety, ATZs covalently attach to and damage multiple parasite proteins, marking them for proteasomal degradation. When damaged proteins enter the proteasome, their attached PIs inhibit protease function, potentiating the parasiticidal action of ART and overcoming ART resistance. Binding of the PI moiety to the proteasome active site is enhanced by distal interactions of the extended attached peptides, providing a mechanism to overcome PI resistance. ATZs have an extra mode of action beyond that of each component, thereby overcoming resistance to both components, while avoiding transient monotherapy seen when individual agents have disparate pharmacokinetic profiles.

Integrating cognitive bias modification for pain and opioid cues into medication for opioid use disorder clinical care: Feasibility, acceptability, and preliminary results.

BACKGROUND: Despite high co-occurrence, chronic pain is often unaddressed in treatment for opioid use disorder (OUD) and little is known about mechanisms that may underlie associations between pain and opioid use. Using an attentional bias (AB) task with both pain and opioid cues, we evaluated a cognitive bias modification (CBM) task administered during regularly scheduled medications for OUD (mOUD) dosing visits. The current study evaluated the feasibility, acceptability, and preliminary efficacy of the CBM task. Outcomes for AB tasks used traditional mean-based score and trial-level bias scores (TLBS). METHODS: In a double-blind, randomized controlled trial, 28 individuals with OUD and chronic pain engaged in mOUD were randomized to either CBM or an AB control condition and completed up to three tasks per week for four weeks. Standard AB task was completed at baseline and post-treatment. Participants completed feasibility and acceptability measures, and preliminary efficacy (i.e., change in AB) was assessed using ANOVA models. RESULTS: Participants attended 83.3% of scheduled sessions and generally reported the task was enjoyable, credible, and easy to complete. Preliminary results demonstrated a condition by time interaction highlighting a reduction in AB in the CBM group but not the control group in opioid TLBS variability (F[1,26]=5.01, p = .034) and pain TLBS towards (F[1,26]=6.42, p = .018) and pain TLBS variability (F[1,26]=5.24, p = .03). CONCLUSIONS: The current study supports integrating brief, computer-based tasks designed to reduce AB into mOUD clinical care. The preliminary results suggest that TLBS outcomes may be more sensitive to capture changes in AB; however, larger studies are required.

Regulators of male and female sexual development are critical for the transmission of a malaria parasite.

Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite.

The impact of racially-targeted food marketing and attentional biases on consumption in Black adolescent females with and without obesity: Pilot data from the Black Adolescent & Entertainment (BAE) study.

Unhealthy food advertisements ("advertisements" hereafter referred to as "ads") are linked to poor diet and obesity, and food companies disproportionally target Black youth. Little is known about the mechanisms whereby food ads influence diet. One possibility may be racially-targeted ads that appeal to Black youth. Those with food-related attentional biases may be especially vulnerable. The objective of this project was to assess the feasibility and initial effects of a pilot study testing the influence of racially-targeted food ads and food-related attentional biases on eating behaviors among a sample of Black adolescent females. Feasibility of recruitment, retention, and procedures were examined. Participants (N = 41, 12-17y) were randomized to view a television episode clip of the Big Bang Theory embedded with either four 30-second racially-targeted food ads or neutral ads. A computer dot probe task assessed food-related attentional biases. The primary outcome was caloric consumption from a laboratory test meal. Interactions based on weight and ethnic identity were also examined. Analyses of variance and regressions were used to assess main and interaction effects. Exposure to racially-targeted food ads (versus neutral ads) did not affect energy consumption (p > .99). Although not statistically significant, adolescents with obesity consumed nearly 240 kcal more than non-overweight adolescents (p = 0.10). There were no significant preliminary effects related to food-related attentional biases or ethnic identity (ps = 0.22-0.79). Despite a non-significant interaction, these data provide preliminary support that adolescents with obesity may be particularly vulnerable to racially-targeted food ads. An adequately powered trial is necessary to further elucidate the associations among racially-targeted food ads among Black adolescent girls with obesity.

Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-Mutant Pancreatic Cancer.

Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK-MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K-AKT-mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor-based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC. SIGNIFICANCE: CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer.

The symmetry of the left and right tibial plateau: a comparison of 200 tibial plateaus.

PURPOSE: This study aims to investigate the symmetry of the left and right tibial plateau in young healthy individuals to determine whether left-right mirroring can be reliably used to optimize preoperative 3D virtual planning for patients with tibial plateau fractures. METHODS: One hundred healthy subjects, without previous knee surgery, severe knee trauma, or signs of osteoarthritis were included for a previous dynamic imaging study of the knee. The subjects underwent a CT scan, scanning the left and right knee with a slice thickness of 0.8 mm. 3D surface models of the femur, patella, and tibia were created using a convolutional neural network. The 3D models of the left and right tibias were exported to MATLAB © and the tibias were mirrored. The mirrored tibias were superimposed on the contralateral tibia using a coherent point drift surface matching algorithm. Correspondence points on both surfaces were established, the mean root squared distance was calculated and visualized in a boxplot and heatmaps. RESULTS: The overall mean difference between correspondence points on the left and right tibial plateau is 0.6276 ± 0.0343 mm. The greatest differences between correspondence points were seen around two specific surfaces on the outside of the tibial plateau; where the distal tibia was cut 15 mm below the tibial plateau and around the tibiofibular joint. CONCLUSIONS: The differences between the left and right tibial plateau are small and therefore, we can be confident that the mirrored contralateral, unfractured, tibial plateau can be used as a template for 3D virtual preoperative planning for young patients without previous damage to the knee.

Functional and biological heterogeneity of KRASQ61 mutations.

Missense mutations at the three hotspots in the guanosine triphosphatase (GTPase) RAS-Gly12, Gly13, and Gln61 (commonly known as G12, G13, and Q61, respectively)-occur differentially among the three RAS isoforms. Q61 mutations in KRAS are infrequent and differ markedly in occurrence. Q61H is the predominant mutant (at 57%), followed by Q61R/L/K (collectively 40%), and Q61P and Q61E are the rarest (2 and 1%, respectively). Probability analysis suggested that mutational susceptibility to different DNA base changes cannot account for this distribution. Therefore, we investigated whether these frequencies might be explained by differences in the biochemical, structural, and biological properties of KRASQ61 mutants. Expression of KRASQ61 mutants in NIH 3T3 fibroblasts and RIE-1 epithelial cells caused various alterations in morphology, growth transformation, effector signaling, and metabolism. The relatively rare KRASQ61E mutant stimulated actin stress fiber formation, a phenotype distinct from that of KRASQ61H/R/L/P, which disrupted actin cytoskeletal organization. The crystal structure of KRASQ61E was unexpectedly similar to that of wild-type KRAS, a potential basis for its weak oncogenicity. KRASQ61H/L/R-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines exhibited KRAS-dependent growth and, as observed with KRASG12-mutant PDAC, were susceptible to concurrent inhibition of ERK-MAPK signaling and of autophagy. Our results uncover phenotypic heterogeneity among KRASQ61 mutants and support the potential utility of therapeutic strategies that target KRASQ61 mutant-specific signaling and cellular output.