Adeno-to-squamous transition drives resistance to KRAS inhibition in LKB1 mutant lung cancer.

KRASG12C inhibitors (adagrasib and sotorasib) have shown clinical promise in targeting KRASG12C-mutated lung cancers; however, most patients eventually develop resistance. In lung patients with adenocarcinoma with KRASG12C and STK11/LKB1 co-mutations, we find an enrichment of the squamous cell carcinoma gene signature in pre-treatment biopsies correlates with a poor response to adagrasib. Studies of Lkb1-deficient KRASG12C and KrasG12D lung cancer mouse models and organoids treated with KRAS inhibitors reveal tumors invoke a lineage plasticity program, adeno-to-squamous transition (AST), that enables resistance to KRAS inhibition. Transcriptomic and epigenomic analyses reveal ΔNp63 drives AST and modulates response to KRAS inhibition. We identify an intermediate high-plastic cell state marked by expression of an AST plasticity signature and Krt6a. Notably, expression of the AST plasticity signature and KRT6A at baseline correlates with poor adagrasib responses. These data indicate the role of AST in KRAS inhibitor resistance and provide predictive biomarkers for KRAS-targeted therapies in lung cancer.

Modeling of clinical phenotypes in systemic lupus erythematosus based on the platelet transcriptome and FCGR2a genotype.

BACKGROUND: The clinical heterogeneity of SLE with its complex pathogenesis remains challenging as we strive to provide optimal management. The contribution of platelets to endovascular homeostasis, inflammation and immune regulation highlights their potential importance in SLE. Prior work from our group showed that the Fcγ receptor type IIa (FcγRIIa)-R/H131 biallelic polymorphism is associated with increased platelet activity and cardiovascular risk in SLE. The study was initiated to investigate the platelet transcriptome in patients with SLE and evaluate its association across FcγRIIa genotypes and distinct clinical features. METHODS: Fifty-one patients fulfilling established criteria for SLE (mean age = 41.1 ± 12.3, 100% female, 45% Hispanic, 24% black, 22% Asian, 51% white, mean SLEDAI = 4.4 ± 4.2 at baseline) were enrolled and compared with 18 demographically matched control samples. The FCGR2a receptor was genotyped for each sample, and RNA-seq was performed on isolated, leukocyte-depleted platelets. Transcriptomic data were used to create a modular landscape to explore the differences between SLE patients and controls and various clinical parameters in the context of FCGR2a genotypes. RESULTS: There were 2290 differentially expressed genes enriched for pathways involved in interferon signaling, immune activation, and coagulation when comparing SLE samples vs controls. When analyzing patients with proteinuria, modules associated with oxidative phosphorylation and platelet activity were unexpectedly decreased. Furthermore, genes that were increased in SLE and in patients with proteinuria were enriched for immune effector processes, while genes increased in SLE but decreased in proteinuria were enriched for coagulation and cell adhesion. A low-binding FCG2Ra allele (R131) was associated with decreases in FCR activation, which further correlated with increases in platelet and immune activation pathways. Finally, we were able to create a transcriptomic signature of clinically active disease that performed significantly well in discerning SLE patients with active clinical disease form those with inactive clinical disease. CONCLUSIONS: In aggregate, these data demonstrate the platelet transcriptome provides insight into lupus pathogenesis and disease activity, and shows potential use as means of assessing this complex disease using a liquid biopsy.

P2Y12 Inhibition Suppresses Proinflammatory Platelet-Monocyte Interactions.

BACKGROUND: Monocyte-platelet aggregates (MPAs) represent the crossroads between thrombosis and inflammation, and targeting this axis may suppress thromboinflammation. While antiplatelet therapy (APT) reduces platelet-platelet aggregation and thrombosis, its effects on MPA and platelet effector properties on monocytes are uncertain. OBJECTIVES: To analyze the effect of platelets on monocyte activation and APT on MPA and platelet-induced monocyte activation. METHODS: Agonist-stimulated whole blood was incubated in the presence of P-selectin, PSGL1, PAR1, P2Y12, GP IIb/IIIa, and COX-1 inhibitors and assessed for platelet and monocyte activity via flow cytometry. RNA-Seq of monocytes incubated with platelets was used to identify platelet-induced monocyte transcripts and was validated by RT-qPCR in monocyte-PR co-incubation ± APT. RESULTS: Consistent with a proinflammatory platelet effector role, MPAs were increased in patients with COVID-19. RNA-Seq revealed a thromboinflammatory monocyte transcriptome upon incubation with platelets. Monocytes aggregated to platelets expressed higher CD40 and tissue factor than monocytes without platelets (p < 0.05 for each). Inhibition with P-selectin (85% reduction) and PSGL1 (87% reduction) led to a robust decrease in MPA. P2Y12 and PAR1 inhibition lowered MPA formation (30 and 21% reduction, p < 0.05, respectively) and decreased monocyte CD40 and TF expression, while GP IIb/IIIa and COX1 inhibition had no effect. Pretreatment of platelets with P2Y12 inhibitors reduced the expression of platelet-mediated monocyte transcription of proinflammatory SOCS3 and OSM. CONCLUSIONS: Platelets skew monocytes toward a proinflammatory phenotype. Among traditional APTs, P2Y12 inhibition attenuates platelet-induced monocyte activation.

Platelet Activity and Cardiovascular Risk in CKD and Peripheral Artery Disease.

Introduction: Platelet dysfunction and cardiovascular risk are well-recognized features of chronic kidney disease (CKD). Platelets drive the development and progression of cardiovascular disease (CVD). The relationships between kidney function, platelet activity, and cardiovascular risk are poorly defined. Methods: We compared platelet activity and incident cardiovascular events by CKD status (estimated glomerular filtration rate [eGFR] < 60 ml/min per 1.73 m2) using data from the Platelet Activity and Cardiovascular Events study, a prospective cohort study that enrolled adults with peripheral artery disease (PAD) undergoing lower extremity revascularization. Platelet activity was measured using light transmission aggregometry (LTA) in response to submaximal dose agonist stimulation, and the subjects were followed for incident adverse cardiovascular events for a median of 18 months. Results: Overall, 113 of 285 (40%) subjects had CKD. Subjects with, versus without, CKD had higher platelet aggregation in response to stimulation with adenosine diphosphate (ADP), serotonin, epinephrine, and arachidonic acid (AA) + ex vivo aspirin (P < 0.05 for each). Following multivariable adjustment, subjects with CKD had elevated risk for myocardial infarction (MI) (adjusted hazard ratio 2.2, 95% confidence interval [1.02-4.9]) and major adverse cardiovascular events (MACE) (1.9 [1.2-3.3]) compared to those without CKD. Platelet aggregation in response to submaximal dose agonist stimulation mediated 7% to 26% of the excess risk for cardiovascular events associated with CKD. Conclusion: Among subjects with PAD undergoing lower extremity revascularization, CKD is associated with increased platelet activity that mediates, in part, elevated cardiovascular risk.

More frequent olive oil intake is associated with reduced platelet activation in obesity.

BACKGROUND AND AIMS: Obesity is an independent risk factor for atherosclerotic cardiovascular disease (CVD), and platelet hyperactivation in obesity may contribute to this association. Olive oil consumption is associated with lower cardiovascular disease (CVD) risk in the general population. However, little is known for individuals with obesity. We investigated whether olive oil intake is associated with platelet activation in obesity. METHODS AND RESULTS: We assessed platelet activation (surface P-selectin expression) with and without thrombin exposure and diet composition in 63 patients with severe obesity. Among 63 subjects with obesity, the mean age was 32.2 ± 8.0 years and BMI 44.1 ± 8.5 kg/m2. Olive oil intake was stratified into <1 time/week (n = 21), 1-3 times/week (n = 18), ≥4 times/week (n = 24). Strata did not differ by age, BMI or platelet count. Unstimulated P-selectin expression did not differ by olive oil consumption. Subjects with more frequent olive oil intake exhibited lower P-selectin expression on submaximal thrombin exposure. CONCLUSIONS: More frequent olive oil intake is associated with reduced thrombin-induced platelet activation in obesity.

Platelets amplify endotheliopathy in COVID-19.

Given the evidence for a hyperactive platelet phenotype in COVID-19, we investigated effector cell properties of COVID-19 platelets on endothelial cells (ECs). Integration of EC and platelet RNA sequencing revealed that platelet-released factors in COVID-19 promote an inflammatory hypercoagulable endotheliopathy. We identified S100A8 and S100A9 as transcripts enriched in COVID-19 platelets and were induced by megakaryocyte infection with SARS-CoV-2. Consistent with increased gene expression, the heterodimer protein product of S100A8/A9, myeloid-related protein (MRP) 8/14, was released to a greater extent by platelets from COVID-19 patients relative to controls. We demonstrate that platelet-derived MRP8/14 activates ECs, promotes an inflammatory hypercoagulable phenotype, and is a significant contributor to poor clinical outcomes in COVID-19 patients. Last, we present evidence that targeting platelet P2Y12 represents a promising candidate to reduce proinflammatory platelet-endothelial interactions. Together, these findings demonstrate a previously unappreciated role for platelets and their activation-induced endotheliopathy in COVID-19.

Hydroxychloroquine is associated with lower platelet activity and improved vascular health in systemic lupus erythematosus.

OBJECTIVE: Hydroxychloroquine (HCQ) is a mainstay of therapy in the treatment of SLE. The effect of HCQ on platelets and vascular health is uncertain. We investigated the relationship between HCQ use and dose with platelet activity, platelet transcriptomics and vascular health in patients with SLE. METHODS: Platelet aggregation, platelet mRNA expression and vascular health (sublingual capillary perfused boundary region (PBR), red blood cell filling (RBCF) and brachial artery reactivity testing) were analysed by HCQ use and dose. RESULTS: Among 132 subjects with SLE (age: 39.7±12.9 years, 97% female), 108 were on HCQ. SLE disease activity was similar between subjects on and off HCQ. Platelet aggregation in response to multiple agonists was significantly lower in patients on HCQ. There were inverse relationships between HCQ dose and gene expression pathways of platelet activity. Gene expression of P-selectin (SELP) was inversely correlated with HCQ dose (r=-0.41, p=0.003), which was validated at the protein level. Subjects on HCQ had improved vascular function correlating with HCQ dose as measured by lower PBR (r=-0.52, p=0.007), higher RBCF (r=0.55, p=0.004) and greater brachial artery reactivity (r=0.43, p=0.056). CONCLUSION: HCQ use was associated with decreased platelet activation and activation-related transcripts and improved vascular health in SLE.