Cystatin M/E ameliorates bone resorption through increasing osteoclastic cell estrogen influx.

In multiple myeloma (MM), increased osteoclast differentiation leads to the formation of osteolytic lesions in most MM patients. Bisphosphonates, such as zoledronic acid (ZA), are used to ameliorate bone resorption, but due to risk of serious side effects as well as the lack of repair of existing lesions, novel anti-bone resorption agents are required. Previously, the absence of osteolytic lesions in MM was strongly associated with elevated levels of cystatin M/E (CST6), a cysteine protease inhibitor, secreted by MM cells. In this study, both MM- and ovariectomy (OVX)-induced osteoporotic mouse models were used to compare the effects of recombinant mouse CST6 (rmCst6) and ZA on preventing bone loss. µCT showed that rmCst6 and ZA had similar effects on improving percent bone volume, and inhibited differentiation of non-adherent bone marrow cells into mature osteoclasts. Single-cell RNA sequencing showed that rmCst6 and not ZA treatment reduced bone marrow macrophage percentage in the MM mouse model compared to controls. Protein and mRNA arrays showed that both rmCst6 and ZA significantly inhibit OVX-induced expression of inflammatory cytokines. For OVX mice, ERα protein expression in bone was brought to sham surgery level by only rmCst6 treatments. rmCst6 significantly increased mRNA and protein levels of ERα and significantly increased total intracellular estrogen concentrations for ex vivo osteoclast precursor cell cultures. Based on these results, we conclude that CST6 improves MM or OVX bone loss models by increasing the expression of estrogen receptors as well as the intracellular estrogen concentration in osteoclast precursors, inhibiting their maturation.

Colonic epithelial hypoxia remains constant during the progression of diabetes in male UC Davis type 2 diabetes mellitus rats.

INTRODUCTION: Colonocyte oxidation of bacterial-derived butyrate has been reported to maintain synergistic obligate anaerobe populations by reducing colonocyte oxygen levels; however, it is not known whether this process is disrupted during the progression of type 2 diabetes. Our aim was to determine whether diabetes influences colonocyte oxygen levels in the University of California Davis type 2 diabetes mellitus (UCD-T2DM) rat model. RESEARCH DESIGN AND METHODS: Age-matched male UCD-T2DM rats (174±4 days) prior to the onset of diabetes (PD, n=15), within 1 month post-onset (RD, n=12), and 3 months post-onset (D3M, n=12) were included in this study. Rats were administered an intraperitoneal injection of pimonidazole (60 mg/kg body weight) 1 hour prior to euthanasia and tissue collection to estimate colonic oxygen levels. Colon tissue was fixed in 10% formalin, embedded in paraffin, and processed for immunohistochemical detection of pimonidazole. The colonic microbiome was assessed by 16S gene rRNA amplicon sequencing and content of short-chain fatty acids was measured by liquid chromatography-mass spectrometry. RESULTS: HbA1c % increased linearly across the PD (5.9±0.1), RD (7.6±0.4), and D3M (11.5±0.6) groups, confirming the progression of diabetes in this cohort. D3M rats had a 2.5% increase in known facultative anaerobes, Escherichia-Shigella, and Streptococcus (false discovery rate <0.05) genera in colon contents. The intensity of pimonidazole staining of colonic epithelia did not differ across groups (p=0.37). Colon content concentrations of acetate and propionate also did not differ across UCD-T2DM groups; however, colonic butyric acid levels were higher in D3M rats relative to PD rats (p<0.01). CONCLUSIONS: The advancement of diabetes in UCD-T2DM rats was associated with an increase in facultative anaerobes; however, this was not explained by changes in colonocyte oxygen levels. The mechanisms underlying shifts in gut microbe populations associated with the progression of diabetes in the UCD-T2DM rat model remain to be identified.

Phenolic acids prevent sex-steroid deficiency-induced bone loss and bone marrow adipogenesis in mice.

Phenolic acids, such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA), can be produced from microbiome digestion of polyphenols. Previously it was found that HA and 3-3-PPA facilitate bone formation and suppress bone resorption. However, the mechanism of action by which HA and 3-3-PPA protect bone from degeneration is currently unknown. In this report, we present that HA and 3-3-PPA suppression of bone resorption is able to ameliorate bone loss in an ovariectomy (OVX) osteopenic mouse model though not to the extent of Zoledronic acid (ZA). HA and 3-3-PPA treatments were shown to significantly decrease bone marrow adipocyte-like cell formation and inhibited gene expression of key adipogenesis regulator peroxisome proliferator activated receptor gamma (PPARγ) and lipoprotein lipase (Lpl) in bone from OVX mice. In addition, ChIP experiments showed that the association between PPARγ and Lpl promoter region in preadipocyte-like cells was significantly suppressed following HA or 3-3-PPA treatment. Contrasting HA and 3-3-PPA, ZA significantly increased TRAP activity in the area close to growth plate and significantly suppressed bone cell proliferation. These data suggest that phenolics acids such as HA or 3-3-PPA may prevent bone degeneration after OVX through suppression of inflammatory milieu in the bone.

Operationalizing selection criteria for clinical trials in Alzheimer's disease: Biomarker and clinical considerations.

Alzheimer's disease (AD) staging criteria lack standardized, empirical description. Well-defined AD staging criteria are an important consideration in protocol design, influencing a more standardized inclusion/exclusion criteria and defining what constitutes meaningful differentiation among the stages. However, many trials are being designed on the basis of biomarker features and the two need to be coordinated. The Alzheimer's Association Research Roundtable (AARR) Spring 2021 meeting discussed the implementation of preclinical AD staging criteria, and provided recommendations for how they may best be incorporated into clinical trials research. Discussion also included what currently available tools for global clinical trials may best define populations in preclinical AD trials, and if are we able to differentiate preclinical from clinical stages of the disease. Well-defined AD staging criteria are key to improving early detection, diagnostics, clinical trial enrollment, and identifying statistically significant clinical changes, and researchers discussed how emerging blood biomarkers may help with more efficient screening in preclinical stages.

Cross-sectional and longitudinal assessments of function in prodromal-to-mild Alzheimer's disease: A comparison of the ADCS-ADL and A-IADL-Q scales.

Introduction: Prior observational work in a heterogeneous cohort of participants with mild cognitive impairment suggests the Amsterdam Instrumental Activities of Daily Living Questionnaire (A-IADL-Q) may have greater sensitivity for functional decline than the more established Alzheimer's Disease Cooperative Study-Activities of Daily Living (ADCS-ADL) scale. However, the relative utility of the A-IADL-Q versus the ADCS-ADL for clinical trials in early Alzheimer's disease (AD) remains uncertain. Methods: We compared baseline and longitudinal performance of the A-IADL-Q and ADCS-ADL in participants with biomarker-confirmed prodromal (pAD; n = 158) or mild (mAD; n = 283) AD enrolled in the 18-month Tauriel study of semorinemab (NCT03289143). Results: The A-IADL-Q exhibited numerically stronger discrimination between pAD and mAD participants at baseline per Cohen's d analyses and similar sensitivity to longitudinal decline across cohorts over 18 months relative to the ADCS-ADL. Discussion: The comparable performance of the ADCS-ADL and A-IADL-Q supports the utility of the A-IADL-Q in early AD clinical trials. Highlights: The Amsterdam Instrumental Activities of Daily Living Questionnaire (A-IADL-Q) may be more sensitive than the Alzheimer's Disease Cooperative Study-Activities of Daily Living Scale (ADCS-ADL) for distinguishing prodromal and mild Alzheimer's disease (AD).A-IADL-Q and ADCS-ADL are similarly sensitive to decline in early AD over 18 months.Comparable performance of these indices supports A-IADL-Q use in future AD trials.Additional AD clinical trial data could extend findings across more diverse cohorts.

Progression from Prodromal Alzheimer's Disease to Mild Alzheimer's Disease Dementia in the Verubecestat APECS Study: Adjudicating Diagnostic Transitions.

BACKGROUND: Delay of progression from prodromal Alzheimer's disease (AD) to dementia is an important outcome in AD trials. Centralized adjudication is intended to improve the consistency of dementia diagnosis but has not been scrutinized. OBJECTIVE: To evaluate centralized adjudication for determining progression to dementia compared with Site Investigator opinion or change in Clinical Dementia Rating (CDR). METHODS: We used data from the 2-year APECS trial of verubecestat versus placebo in 1,451 prodromal AD participants. Cases were triggered for central adjudication if: 1) the Site Investigator judged the participant had progressed to dementia, or 2) the participant's CDR sum-of-boxes score increased ≥2 points from baseline. Post-hoc analyses were performed on pooled treatment-group data to compare methods of assessing progression. RESULTS: 581/1,451 (40%) participants had changes triggering adjudication and most (83%) were confirmed as progression to dementia. Only 66% of those who met CDR criteria (regardless of whether they also met Site Investigator criteria) were adjudicated to have progressed to dementia and just 15% of those who met only CDR criteria were adjudicated to have progressed, representing 5% of progressors. In contrast, 99% of those who met Site Investigator criteria (regardless of whether they also met CDR criteria) were adjudicated to have progressed, and the same was true for those who met only Site Investigator criteria. CONCLUSION: A positive Site Investigator opinion is an excellent predictor for a positive adjudication decision regarding onset of dementia. Conversely, sole use of CDR sum-of-boxes change ≥2 is inadequate. The benefit of centralized adjudication appears doubtful.

Severe Acute Respiratory Syndrome Coronavirus-2 Lambda Variant Collected from a Child from Arkansas and Sequenced.

An eleven-year-old tested positive for SARS-CoV-2 Lambda variant. Sequencing was performed on the Oxford Nanopore and the Illumina NextSeq 500. Both platforms identified all 7 of the synonymous mutations in the sample, while all 28 nonsynonymous mutations were identified from Oxford Nanopore and 20 nonsynonymous mutations were identified from Illumina.

Human Milk Oligosaccharides Impact Cellular and Inflammatory Gene Expression and Immune Response.

Human milk harbors complex carbohydrates, including human milk oligosaccharides (HMOs), the third most abundant component after lactose and lipids. HMOs have been shown to impact intestinal microbiota, modulate the intestinal immune response, and prevent pathogenic bacterial binding by serving as decoy receptors. However, the direct effect of HMOs on intestinal function and immunity remains to be elucidated. To address this knowledge gap, 21-day-old germ-free mice (C57BI/6) were orally gavaged with 15 mg/day of pooled HMOs for 7 or 14 days and euthanized at day 28 or 35. A set of mice was maintained until day 50 to determine the persistent effects of HMOs. Control groups were maintained in the isolators for 28, 35, or 50 days of age. At the respective endpoints, intestinal tissues were subjected to histomorphometric and transcriptomic analyses, while the spleen and mesenteric lymph nodes (MLNs) were subjected to flow cytometric analysis. The small intestine (SI) crypt was reduced after HMO treatment relative to control at days 28 and 35, while the SI villus height and large intestine (LI) gland depth were decreased in the HMO-treated mice relative to the control at day 35. We report significant HMO-induced and location-specific gene expression changes in host intestinal tissues. HMO treatment significantly upregulated genes involved in extracellular matrix, protein ubiquitination, nuclear transport, and mononuclear cell differentiation. CD4+ T cells were increased in both MLNs and the spleen, while CD8+ T cells were increased in the spleen at day 50 in the HMO group in comparison to controls. In MLNs, plasma cells were increased in HMO group at days 28 and 35, while in the spleen, only at day 28 relative to controls. Macrophages/monocytes and neutrophils were lower in the spleen of the HMO group at days 28, 35, and 50, while in MLNs, only neutrophils were lower at day 50 in the 14-day HMO group. In addition, diphtheria toxoid and tetanus toxoid antibody-secreting cells were higher in HMO-supplemented group compared to controls. Our data suggest that HMOs have a direct effect on gastrointestinal tract metabolism and the immune system even in the absence of host microbiota.

Nox4 expression in osteo-progenitors controls bone development in mice during early life.

Tightly regulated and cell-specific NADPH-oxidases (Nox) represent one of the major sources of reactive oxygen species (ROS) signaling molecules that are involved in tissue development and stem cell self-renewal. We have characterized the role of Nox4 in osteo-progenitors during postnatal bone development. Nox4 expression in bone and ROS generation were increased during early osteoblast differentiation and bone development. Stromal osteoblastic cell self-renewal, proliferation and ROS production were significantly lower in samples from whole-body Nox4 knockout mice (Nox4-/-) and conditional knockout (CKO) mice with depletion of Nox4 in the limb bud mesenchyme compared with those from control mice (Nox4fl/fl), but they were reversed after 9 passages. In both sexes, bone volume, trabecular number and bone mineral density were significantly lower in 3-week old CKO and Nox4-/- mice compared with Nox4fl/fl controls. This was reflected in serum levels of bone formation markers alkaline phosphatase (ALP) and procollagen 1 intact N-terminal propeptide (P1NP). However, under-developed bone formation in 3-week old CKO and Nox4-/- mice quickly caught up to levels of control mice by 6-week of age, remained no different at 13-week of age, and was reversed in 32-week old male mice. Osteoclastogenesis showed no differences among groups, however, CTX1 reflecting osteoclast activity was significantly higher in 3-week old male CKO and Nox4-/- mice compared with control mice, and significantly lower in 32-week old Nox4-/- mice compared with control mice. These data suggest that Nox4 expression and ROS signaling in bone and osteoblastic cells coordinately play an important role in osteoblast differentiation, proliferation and maturation.

Short-Term Metformin Treatment Enriches Bacteroides dorei in an Obese Liver Steatosis Zucker Rat Model.

Obesity is the leading cause of health-related diseases in the United States and World. Previously, we reported that obesity can change gut microbiota using the Zucker rat model. Metformin is an oral anti-hyperglycemic agent approved by the FDA to treat type 2 diabetes (T2D) in adults and children older than 10 years of age. The correlation of short-term metformin treatment and specific alterations to the gut microbiota in obese models is less known. Short-term metformin has been shown to reduce liver steatosis. Here we investigate the effects of short-term metformin treatment on population of gut microbiota profile in an obese rat model. Five week old obese (n = 12) female Zucker rats after 1 week of acclimation, received AIN-93 G diet for 8 weeks and then rats were randomly assigned into two groups (6 rats/group): (1) obese without metformin (ObC), or (2) obese with metformin (ObMet). Metformin was mixed with AIN-93G diet at 1,000 mg/kg of diet. Rats were weighed twice per week. All rats were sacrificed at the end of metformin treatment at 10 weeks and fecal samples were collected and kept at -80°C. Total microbial DNA was collected directly from the fecal samples used for shotgun-metagenomics sequencing and subsequently analyzed using MetaPlAn and HUMAnN. After stringent data filtering and quality control we found significant differences (p = 0.0007) in beta diversity (Aitchison distances) between the ObC vs. ObMet groups. Supervised and unsupervised analysis of the log-ratios Bacteroides dorei and B. massiliensis vs. all other Bacteroides spp., revealed that B. dorei and B. massiliensis were enriched in the ObMet group, while the remaining Bacteroides spp. where enriched in the ObC group (p = 0.002). The contributional diversity of pathways is also significantly associated by treatment group (p = 0.008). In summary, in the obese Zucker rat model, short-term metformin treatment changes the gut microbiota profile, particularly altering the composition Bacteroides spp. between ObC and ObMet.

Bacterial and Fungal Adaptations in Cecum and Distal Colon of Piglets Fed With Dairy-Based Milk Formula in Comparison With Human Milk.

Exclusive breastfeeding is recommended to newborns during the first 6 months of life, whereas dairy-based infant formula is an alternative nutrition source offered to infants. Several studies demonstrated that breastfed infants have a different gut bacterial composition relative to formula-fed infants. In addition, animal models have shown that human milk (HM)-fed piglets had a distinct intestinal bacterial composition compared with milk formula (MF)-fed piglets. However, the gut fungal composition and the interactions with the bacterial community in breastfed compared with formula-fed infants remain to be investigated. In an attempt to evaluate such differences, we used an animal model to perform a shotgun metagenomics analysis on the cecal and distal colon contents of neonatal piglets fed with pasteurized HM or a dairy-based infant formula (MF) during the first 21 days of life. At postnatal day 21 (PND 21), a subset of piglets from each diet group (n = 11 per group) was euthanized. The remaining piglets in each group were weaned to a solid diet and euthanized at PND 51 (n = 13 per group). Large intestine contents (i.e., cecum and distal colon) were subjected to shotgun metagenomics analysis. The differential taxonomic composition of bacteria and fungi and the predicted functional gene profiling were evaluated. Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria are the most abundant bacterial phyla observed in piglets at PND 21 and PND 51. In the large intestine at PND 21 and PND 51, Proteobacteria phylum was significantly higher in MF-fed group, and species Burkholderiales bacterium of phyla was significantly higher in MF group relative to HM group. In addition, in HM group, several Lactobacillus spp. and Bacteroides spp. were higher relative to MF group in the large intestine at PND 21 and PND 51. Fungal genus Aspergillus was higher in MF, whereas Malassezia was lower relative to HM group. Persistent effects of the neonatal diets were observed at PND 51, where alpha- and beta-diversity differences were detected for bacterial and fungal species in the large intestine. Overall, our findings indicate that neonatal diet affects the large intestinal microbial community during the exclusive milk-feeding period, as well as after the introduction of the complementary food.

Progression of diabetes is associated with changes in the ileal transcriptome and ileal-colon morphology in the UC Davis Type 2 Diabetes Mellitus rat.

Deterioration in glucose homeostasis has been associated with intestinal dysbiosis, but it is not known how metabolic dysregulation alters the gastrointestinal environment. We investigated how the progression of diabetes alters ileal and colonic epithelial mucosal structure, microbial abundance, and transcript expression in the University of California Davis Type 2 Diabetes Mellitus (UCD-T2DM) rat model. Male UCD-T2DM rats (age ~170 days) were included if <1-month (n = 6, D1M) or 3-month (n = 6, D3M) post-onset of diabetes. Younger nondiabetic UCD-T2DM rats were included as a nondiabetic comparison (n = 6, ND, age ~70 days). Ileum villi height/crypt depths and colon crypt depths were assessed by histology. Microbial abundance of colon content was measured with 16S rRNA sequencing. Ileum and colon transcriptional abundances were analyzed using RNA sequencing. Ileum villi height and crypt depth were greater in D3M rats compared to ND. Colon crypt depth was greatest in D3M rats compared to both ND and D1M rats. Colon abundances of Akkermansia and Muribaculaceae were lower in D3M rats relative to D1M, while Oscillospirales, Phascolarctobacterium, and an unidentified genus of Lachnospiraceae were higher. Only two transcripts were altered by diabetes advancement within the colon; however, 2039 ileal transcripts were altered. Only colonic abundances of Sptlc3, Enpp7, Slc7a15, and Kctd14 had more than twofold changes between D1M and D3M rats. The advancement of diabetes in the UCD-T2DM rat results in a trophic effect on the mucosal epithelia and was associated with regulation of gastrointestinal tract RNA expression, which appears more pronounced in the ileum relative to the colon.

Building clinically relevant outcomes across the Alzheimer's disease spectrum.

Demonstrating that treatments are clinically meaningful across the Alzheimer's disease (AD) continuum is critical for meeting our goals of accelerating a cure by 2025. While this topic has been a focus of several Alzheimer's Association Research Roundtable (AARR) meetings, there remains no consensus as to what constitutes a "clinically meaningful outcome" in the eyes of patients, clinicians, care partners, policymakers, payers, and regulatory bodies. Furthermore, the field has not come to agreement as to what constitutes a clinically meaningful treatment effect at each stage of disease severity. The AARR meeting on November 19-20, 2019, reviewed current approaches to defining clinical meaningfulness from various perspectives including those of patients and care partners, clinicians, regulators, health economists, and public policymakers. Participants discussed approaches that may confer clinical relevance at each stage of the disease continuum and fostered discussion about what should guide us in the future.

Multi-omics data integration reveals correlated regulatory features of triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer with very little treatment options. TNBC is very heterogeneous with large alterations in the genomic, transcriptomic, and proteomic landscapes leading to various subtypes with differing responses to therapeutic treatments. We applied a multi-omics data integration method to evaluate the correlation of important regulatory features in TNBC BRCA1 wild-type MDA-MB-231 and TNBC BRCA1 5382insC mutated HCC1937 cells compared with non-tumorigenic epithelial breast MCF10A cells. The data includes DNA methylation, RNAseq, protein, phosphoproteomics, and histone post-translational modification. Data integration methods identified regulatory features from each omics method that had greater than 80% positive correlation within each TNBC subtype. Key regulatory features at each omics level were identified distinguishing the three cell lines and were involved in important cancer related pathways such as TGFß signaling, PI3K/AKT/mTOR, and Wnt/beta-catenin signaling. We observed overexpression of PTEN, which antagonizes the PI3K/AKT/mTOR pathway, and MYC, which downregulates the same pathway in the HCC1937 cells relative to the MDA-MB-231 cells. The PI3K/AKT/mTOR and Wnt/beta-catenin pathways are both downregulated in HCC1937 cells relative to MDA-MB-231 cells, which likely explains the divergent sensitivities of these cell lines to inhibitors of downstream signaling pathways. The DNA methylation and RNAseq data is freely available via GEO GSE171958 and the proteomics data is available via the ProteomeXchange PXD025238.

Mapping methylation quantitative trait loci in cardiac tissues nominates risk loci and biological pathways in congenital heart disease.

BACKGROUND: Most congenital heart defects (CHDs) result from complex interactions among genetic susceptibilities, epigenetic modifications, and maternal environmental exposures. Characterizing the complex relationship between genetic, epigenetic, and transcriptomic variation will enhance our understanding of pathogenesis in this important type of congenital disorder. We investigated cis-acting effects of genetic single nucleotide polymorphisms (SNPs) on local DNA methylation patterns within 83 cardiac tissue samples and prioritized their contributions to CHD risk by leveraging results of CHD genome-wide association studies (GWAS) and their effects on cardiac gene expression. RESULTS: We identified 13,901 potential methylation quantitative trait loci (mQTLs) with a false discovery threshold of 5%. Further co-localization analyses and Mendelian randomization indicated that genetic variants near the HLA-DRB6 gene on chromosome 6 may contribute to CHD risk by regulating the methylation status of nearby CpG sites. Additional SNPs in genomic regions on chromosome 10 (TNKS2-AS1 gene) and chromosome 14 (LINC01629 gene) may simultaneously influence epigenetic and transcriptomic variations within cardiac tissues. CONCLUSIONS: Our results support the hypothesis that genetic variants may influence the risk of CHDs through regulating the changes of DNA methylation and gene expression. Our results can serve as an important source of information that can be integrated with other genetic studies of heart diseases, especially CHDs.

Engineering of Saposin C Protein Chimeras for Enhanced Cytotoxicity and Optimized Liposome Binding Capability.

Saposin C (sapC) is a lysosomal, peripheral-membrane protein displaying liposome fusogenic capabilities. Proteoliposomes of sapC and phosphatidylserine have been shown to be toxic for cancer cells and are currently on clinical trial to treat glioblastoma. As proof-of-concept, we show two strategies to enhance the applications of sapC proteoliposomes: (1) Engineering chimeras composed of sapC to modulate proteoliposome function; (2) Engineering sapC to modify its lipid binding capabilities. In the chimera design, sapC is linked to a cell death-inducing peptide: the BH3 domain of the Bcl-2 protein PUMA. We show by solution NMR and dynamic light scattering that the chimera is functional at the molecular level by fusing liposomes and by interacting with prosurvival Bcl-xL, which is PUMA's known mechanism to induce cell death. Furthermore, sapC-PUMA proteoliposomes enhance cytotoxicity in glioblastoma cells compared to sapC. Finally, the sapC domain of the chimera has been engineered to optimize liposome binding at pH close to physiological values as protein-lipid interactions are favored at acidic pH in the native protein. Altogether, our results indicate that the properties of sapC proteoliposomes can be modified by engineering the protein surface and by the addition of small peptides as fusion constructs.

Durable Suppression of Acquired MEK Inhibitor Resistance in Cancer by Sequestering MEK from ERK and Promoting Antitumor T-cell Immunity.

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAF non-V600, and BRAF V600 mutations. Tumor cell-intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8+ T cells, and durable tumor regression elicited by this combination requires CD8+ T cells, which can be reinvigorated by anti-PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy. SIGNIFICANCE: Type I RAFi + MEKi are indicated only in certain BRAF V600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8+ T cells are mechanisms that may be further exploited.This article is highlighted in the In This Issue feature, p. 521.

Cognitive outcomes in trials of two BACE inhibitors in Alzheimer's disease.

INTRODUCTION: The APECS and AMARANTH trials showed that beta-secretase (BACE) inhibitors verubecestat and lanabecestat failed to slow cognitive and functional decline in individuals with prodromal or early Alzheimer's disease. Here, the performance on secondary and exploratory cognitive measures in both studies is reported. METHODS: APECS (verubecestat) and AMARANTH (lanabecestat) were randomized, double-blind, placebo-controlled, parallel-group, 104-week clinical trials conducted by different sponsors. Measures included the 3-Domain Composite Cognition Score (CCS-3D), Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), Letter/Category Fluency, and Digit Symbol Coding. RESULTS: Verubecestat showed worsening on the CCS-3D Total Score, Episodic Memory, and Attention/Processing Speed domains. Lanabecestat showed worsening on the RBANS Total Score, Immediate Memory, and Visuospatial/Constructional Indexes. Both BACE inhibitors showed worsening on Digit Symbol Coding and improvements on Letter/Category Fluency. DISCUSSION: In both studies, many measures showed treatment-associated cognitive worsening, whereas verbal fluency tasks showed improvement.