Small RMA Signatures of Acute Ischemic Stroke in L1CAM Positive Extracellular Vesicles

L1CAM-positive extracellular vesicles (L1EV) are an emerging biomarker that may better reflect ongoing neuronal damage than other blood-based biomarkers. The physiological roles and regulation of L1EVs and their small RNA cargoes following stroke is unknown. We sought to characterize L1EV small RNAs following stroke and assess L1EV RNA signatures for diagnosing stroke using weighted gene co-expression network analysis and random forest (RF) machine learning algorithms. Interestingly, small RNA sequencing of plasma L1EVs from patients with stroke and control patients (n = 28) identified micro(mi)RNAs known to be enriched in the brain. Weighted gene co-expression network analysis (WGCNA) revealed small RNA transcript modules correlated to diagnosis, initial NIH stroke scale, and age. L1EV RNA signatures associated with the diagnosis of AIS were derived from WGCNA and RF classification. These small RNA signatures demonstrated a high degree of accuracy in the diagnosis of AIS with an area under the curve (AUC) of the signatures ranging from 0.833 to 0.932. Further work is necessary to understand the role of small RNA L1EV cargoes in the response to brain injury, however, this study supports the utility of L1EV small RNA signatures as a biomarker of stroke

Small RNA Signatures of Acute Ischemic Stroke in L1CAM Positive Extracellular Vesicles

L1CAM-positive extracellular vesicles (L1EV) are an emerging biomarker that may better reflect ongoing neuronal damage than other blood-based biomarkers. The physiological roles and regulation of L1EVs and their small RNA cargoes following stroke is unknown. We sought to characterize L1EV small RNAs following stroke and assess L1EV RNA signatures for diagnosing stroke using weighted gene co-expression network analysis and random forest (RF) machine learning algorithms. Interestingly, small RNA sequencing of plasma L1EVs from patients with stroke and control patients (n = 28) identified micro(mi)RNAs known to be enriched in the brain. Weighted gene co-expression network analysis (WGCNA) revealed small RNA transcript modules correlated to diagnosis, initial NIH stroke scale, and age. L1EV RNA signatures associated with the diagnosis of AIS were derived from WGCNA and RF classification. These small RNA signatures demonstrated a high degree of accuracy in the diagnosis of AIS with an area under the curve (AUC) of the signatures ranging from 0.833 to 0.932. Further work is necessary to understand the role of small RNA L1EV cargoes in the response to brain injury, however, this study supports the utility of L1EV small RNA signatures as a biomarker of stroke

Kinase-Impaired BTK Mutations Are Susceptible to Clinical-Stage BTK and IKZF1/3 Degrader NX-2127

INTRODUCTION: Bruton’s tyrosine kinase (BTK) is a nonreceptor kinase in the B cell receptor (BCR) signaling cascade critical for B cell survival. As such, chronic lymphocytic leukemia (CLL) and other B cell cancers are sensitive to inhibition of BTK. Covalent and noncovalent inhibitors of BTK have revolutionized the treatment of these cancers. Therefore, understanding mechanisms by which acquired mutation in BTK confer drug resistance and developing new therapies to overcome resistance are critically important. RATIONALE: We recently discovered BTK mutations that confer resistance across covalent and noncovalent BTK inhibitors. In this study, we found that a group of these mutants impair BTK kinase activity despite still enabling downstream BCR signaling. We therefore set out to understand the nonenzymatic functions of BTK and explored targeted protein degradation to overcome the oncogenic scaffold function of mutant BTK. This effort included evaluation of BTK degradation in patients with CLL treated in a phase 1 clinical trial of NX-2127, a first-in-class BTK degrader (NCT04830137). RESULTS: BTK enzymatic activity assays revealed that drug resistance mutations in BTK fall into two distinct groups: kinase proficient and kinase impaired. Immunoprecipitation mass spectrometry of kinase-impaired BTK L528W (Leu528→Trp) revealed a scaffold function of BTK with downstream signaling and survival dependent on surrogate kinases that bind to kinase-impaired BTK proteoforms. To target the nonenzymatic functions of BTK, we developed NX-2127, a heterobifunctional molecule that engages the ubiquitin-proteasome system to simultaneously bind both BTK and the cereblon E3 ubiquitin ligase complex, inducing polyubiquitination and proteasome-dependent degradation of IKZF1/3 and all recurrent drug-resistant forms of mutant BTK. The activity of NX-2127 on BTK degradation was further demonstrated in patients with CLL treated in a phase 1 clinical trial of NX-2127, where \u3e80% BTK degradation was achieved and clinical responses were also seen in 79% of evaluable patients, independent of mutant BTK genotypes. CONCLUSION: We identified that BTK inhibitor resistance mutations fall into two distinct functional categories. Kinase-impaired BTK mutants disable BTK kinase activity while promoting physical interactions with other kinases to sustain downstream BCR signaling. This scaffold function of BTK was disrupted by NX-2127, a potent BTK degrader, which showed promising responses for patients with relapsed and refractory CLL, independently of mutant BTK functional category

Small RNA signatures of acute ischemic stroke in L1CAM positive extracellular vesicles

Abstract L1CAM-positive extracellular vesicles (L1EV) are an emerging biomarker that may better reflect ongoing neuronal damage than other blood-based biomarkers. The physiological roles and regulation of L1EVs and their small RNA cargoes following stroke is unknown. We sought to characterize L1EV small RNAs following stroke and assess L1EV RNA signatures for diagnosing stroke using weighted gene co-expression network analysis and random forest (RF) machine learning algorithms. Interestingly, small RNA sequencing of plasma L1EVs from patients with stroke and control patients (n = 28) identified micro(mi)RNAs known to be enriched in the brain. Weighted gene co-expression network analysis (WGCNA) revealed small RNA transcript modules correlated to diagnosis, initial NIH stroke scale, and age. L1EV RNA signatures associated with the diagnosis of AIS were derived from WGCNA and RF classification. These small RNA signatures demonstrated a high degree of accuracy in the diagnosis of AIS with an area under the curve (AUC) of the signatures ranging from 0.833 to 0.932. Further work is necessary to understand the role of small RNA L1EV cargoes in the response to brain injury, however, this study supports the utility of L1EV small RNA signatures as a biomarker of stroke

Elevated complement mediator levels in endothelial-derived plasma exosomes implicate endothelial innate inflammation in diminished brain function of aging humans.

We test the hypothesis that endothelial cells adopt an inflammatory phenotype in functionally intact aged human subjects with radiographic evidence of white matter hyperintensity (WMH) suggestive of small cerebrovascular disease. Components of all three complement effector pathways and regulatory proteins were quantified in extracts of plasma endothelial-derived exosomes (EDE) of 11 subjects (age 70-82) with and 15 without evidence of WMH on MRI. Group differences and associations with plasma markers of immune activation (IL6, ICAM1), cognition and neuroimaging were calculated via regression modelling. EDE complement factors within the alternative and classical pathways were found to be higher and regulatory proteins lower in subjects with WMH. EDE levels of some complement components demonstrated significant associations with cognitive slowing and elevated systolic blood pressure. The inhibitor of the membrane attack complex, CD46, showed a significant positive association with cerebral grey matter volume. Plasma inflammatory markers, IL6 and ICAM1, were positively associated with EDE levels of several complement components. These findings provide the first in vivo evidence of the association of endothelial cell inflammation with white matter disease, age-associated cognitive changes, and brain degeneration in functionally normal older individuals. Future endothelial biomarker development may permit recognition of early or preclinical stages of vascular contributions to cognitive impairment and dementia

Lower White Matter Volume and Worse Executive Functioning Reflected in Higher Levels of Plasma GFAP among Older Adults with and Without Cognitive Impairment.

ObjectiveThere are minimal data directly comparing plasma neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) in aging and neurodegenerative disease research. We evaluated associations of plasma NfL and plasma GFAP with brain volume and cognition in two independent cohorts of older adults diagnosed as clinically normal (CN), mild cognitive impairment (MCI), or Alzheimer's dementia.MethodsWe studied 121 total participants (Cohort 1: n = 50, age 71.6 ± 6.9 years, 78% CN, 22% MCI; Cohort 2: n = 71, age 72.2 ± 9.2 years, 45% CN, 25% MCI, 30% dementia). Gray and white matter volumes were obtained for total brain and broad subregions of interest (ROIs). Neuropsychological testing evaluated memory, executive functioning, language, and visuospatial abilities. Plasma samples were analyzed in duplicate for NfL and GFAP using single molecule array assays (Quanterix Simoa). Linear regression models with structural MRI and cognitive outcomes included plasma NfL and GFAP simultaneously along with relevant covariates.ResultsHigher plasma GFAP was associated with lower white matter volume in both cohorts for temporal (Cohort 1: β = -0.33, p = .002; Cohort 2: β = -0.36, p = .03) and parietal ROIs (Cohort 1: β = -0.31, p = .01; Cohort 2: β = -0.35, p = .04). No consistent findings emerged for gray matter volumes. Higher plasma GFAP was associated with lower executive function scores (Cohort 1: β = -0.38, p = .01; Cohort 2: β = -0.36, p = .007). Plasma NfL was not associated with gray or white matter volumes, or cognition after adjusting for plasma GFAP.ConclusionsPlasma GFAP may be more sensitive to white matter and cognitive changes than plasma NfL. Biomarkers reflecting astroglial pathophysiology may capture complex dynamics of aging and neurodegenerative disease

Plasma Glial Fibrillary Acidic Protein Levels Differ Along the Spectra of Amyloid Burden and Clinical Disease Stage.

BackgroundMeasuring plasma glial fibrillary acidic protein (GFAP) alongside cortical amyloid-β (Aβ) may shed light on astrocytic changes in aging and Alzheimer's disease (AD).ObjectiveTo examine associations between plasma GFAP and cortical Aβ deposition in older adults across the typical aging-to-AD dementia spectrum.MethodsWe studied two independent samples from UCSF (Cohort 1, N = 50; Cohort 2, N = 37) covering the spectra of clinical severity (CDR Sum of Boxes; CDR-SB) and Aβ-PET burden. Aβ-PET was completed with either florbetapir or Pittsburgh Compound B and standardized uptake value ratios were converted to the Centiloid (CL) scale for analyses. All participants with CDR-SB > 0 were Aβ-PET positive, while clinically normal participants (CDR-SB = 0) were a mix of Aβ-PET positive and negative. Regression analyses evaluated main effect and interaction associations between plasma GFAP, Aβ-PET, and clinical severity.ResultsIn both cohorts, plasma GFAP increased linearly with Aβ-PET CLs in clinically normal older adults. In Cohort 2, which included participants with more severe clinical dysfunction and Aβ-PET burden, the association between Aβ and GFAP became curvilinear (inverted U-shape; quadratic model R2 change = 0.165, p = 0.009), and Aβ-PET interacted with CDR-SB (R2 change = 0.164, p = 0.007): older adults with intermediate functional impairment (CDR-SB = 0.5-4.0) showed a weak (negative) association between Aβ-PET CLs and plasma GFAP, while older adults with dementia (CDR-SB > 4.0) showed a strong, negative association of higher Aβ-PET CLs with lower plasma GFAP.ConclusionThe relationship between astrocytic integrity and cortical Aβ may be highly dynamic, with linear, positive associations early in disease that diverge in more severe disease stages

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients