Lin, Steven H.

Atherosclerotic and high-risk plaques are predominantly localized to vessel wall regions with non-laminar disturbed blood flow (d-flow), leading to the principle that d-flow-, but not laminar flow (l-flow)-driven endothelial cells (EC) dysfunction plays a role in this process. LATS1/2 and YAP are components of the Hippo signaling pathway that are involved in cellular responses to l-flow. The l-flow-mediated inhibition of LATS1/2 activity triggers YAP activation and maintains vascular structure. However, the role of Hippo signaling under d-flow remains unclear. Although both l- and d-flow increased YAP activity, only d-flow decreased LATS1/2 expression. Since the effects of l- and d-flow on EC differ, we studied the role of EC LATS1/2 using EC-specific Lats1 and 2 knock-out (EKO) mice. Within 14 days after inducing Lats1/2 deletion in tamoxifen-inducible Lats1 homo / Lats2 homo EKO mice, all the mice (27/27) died of severe systemic edema, accompanied by increased EC apoptosis and vascular permeability. We generated Lats1 he t / Lats2 homo -EKO mice ( Lats1 het /2 homo -EKO) and induced hypercholesteremia with AAV8-PCSK9 injections and a high-fat diet (HFD). After HFD, we detected plaque erosion-like plaque lesions at the d-flow areas, which revealed strongly fibrin/fibrinogen-positive organized thrombus (OTh) formation without sizeable necrotic cores. In Lats1 het /2 homo -EKO, we found significant increases in 1) EC proliferation, 2) EC apoptosis with senescent phenotype, 3) tissue factor (TF) expression, and 4) inflammation. Therefore, the phenotype observed in Lats1 het /2 homo -EKO represents a unique senescence-associated stemness (SAS) phenotype, which allows EC to escape senescence-induced cell cycle arrest. The EC proliferation induced by depletion of LATS1/2 was YAP-dependent. In contrast, EC senescence induced by CD38-mediated NAD + depletion was YAP-independent. These findings indicate that downregulating LATS1/2 expression-induced SAS leads to OTh via both YAP-dependent and independent mechanisms. OTh plays a key role in human atherosclerosis but is rarely observed in the mouse atherosclerosis model. Lats1 het /2 homo -EKO can provide a valuable platform for studying the mechanism and unique role of EC SAS-induced OTh formation.

Abstract Cancer Associated Macrophage Like cells (CAMLs) are antigen presenting circulating stromal cells found in the blood of cancer patients (pts). Recently, PD-L1 expression on CAMLs was described in breast (BC), lung (LC) and renal cell carcinomas (RCC), appearing to parallel the inflammatory PD-L1 state of the tumor microenvironment, and acting as a possible predictive biomarker for PD-L1/PD-1 immunotherapeutics (IMTs). We monitored CAML PD-L1 from 48 pts in an array of solid tumors, prior PD-L1/PD-1 IMTs induction, to evaluate CAML’s ability to predict response by progression free survival (PFS) and overall survival (OS). Three single blind multi-year prospective studies were run to test PD-L1 CAML expression against PFS & OS, prior to induction of therapy in metastatic (m) LC (n=24), mBC (n=16) or mRCC (n=8). We recruited pts with pathologically confirmed progressing disease after at least one failed prior line of therapy. Blood samples (7.5 mL) were taken prior to start of new therapeutic regimes, i.e. pembrolizumab (n=36) or nivolumab (n=12). Whole blood was filtered by CellSieve microfilters to collect CAMLs, stain for PD-L1, and then scored as a binary high or low, to evaluate PFS & OS hazard ratios (HRs) by censored univariate & multivariate analysis at 24 months. CAMLs were found in 98% of all tested samples. Overall, pts with high CAML PD-L1 expression treated with IMT had significantly better PFS (HR=2.9, 95%CI=1.4–6.1, p=0.0067) and better OS (HR=2.7, 95%CI=1.2–6.1, p=0.0317) vs patients with low CAML PD-L1. These data suggests that PD-L1 expression in circulating CAMLs appears to predict pts with increased benefit to PD-L1/PD-1 IMTs in an array of tumor subtypes, though larger studies are needed to validate these findings. Private Funding by Creatv MicroTech, Inc. and supported grants by NCI-NIH (R43CA206840), and the Defense Advanced Research Projects Agency (DARPA)(W911NF-14-C-0098).

Senescence induced by various stresses reprograms cancer cells to acquire senescence-associated (SA) cell growth (SACG), which allows them to escape senescence-induced cell cycle arrest with enhanced growth potential. ERK5 is a dual kinase-transcription factor, containing an N-terminal kinase domain and transactivation domains in the C-terminal half. Recent data have raised questions regarding the functional role of ERK5 kinase inhibitors in cancer and inflammatory diseases. We aimed to investigate how ERK5 reprograms myeloid cells (MC) to the SA secretory phenotype (SASP) and SACG, consequently leading to atherosclerosis (AthS). AthS was inhibited in ERK5 S496A knock-in (KI) mice. Single-cell analysis of p53 and Ki67 with imaging mass cytometry revealed three groups of MCs with different p53 and Ki67 expression patterns in the plaque. One group showed that p53 and Ki67 were co-expressed lineally, suggesting that this group of cells escaped from the growth-suppression effects of p53, which is also known as a senescence marker. The % of ERK5 S496A KI cells in this group was significantly lower than in wild-type (WT). Also, we found an increase of SA β-gal (SAβ-gal) + Ki67 + and 53BP1 + Ki67 + cells in bone narrow-derived macrophage (BMDMs) isolated from hypercholesterolemia (H-chol) WT mice, suggesting H-chol-induced MC SACG. MC SACG were significantly inhibited in ERK5 S496A KI mice, supporting the role of ERK5 S496 phosphorylation on SACG. RNA sequencing analysis identified the ERK5 S496 phosphorylation-dependent AHR induction by H-chol, and AHR depletion significantly inhibited SACG. We also discovered a critical effect of ERK5 S496 phosphorylation on SUMOylation at a novel site of NRF2 (i.e., K518), which inhibited NRF2 transcriptional activity without affecting ERK5 kinase activity and antagonized oxidized LDL (oxLDL)-induced SASP. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) inhibited oxLDL-induced ERK5 S496 phosphorylation, suggesting that ERK5 S496 phosphorylation was involved at least in part of the effects of these inhibitors. We have discovered a novel mechanism in which ERK5 S496 phosphorylation directly inhibits NRF2 activity by upregulating NRF2 K518 SUMOylation and AHR induction, subsequently inducing SACG and AthS.

Background: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. Methods: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow–derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. Results: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow–derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)–induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. Conclusions: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)–expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti–PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti–PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti–PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

Background: There is a paucity of data regarding the association between radiation exposure of heart substructures and the incidence of major coronary events (MCEs) in patients with esophageal cancer (ESOC) undergoing chemoradiation therapy. We studied radiation dosimetric determinants of MCE risk and measured their impact on patient prognosis using a cohort of ESOC patients treated at a single institution. Methods: Between March 2005 and October 2015, 355 ESOC patients treated with concurrent chemoradiotherapy were identified from a prospectively maintained and institutional-regulatory-board-approved clinical database. Dose-distribution parameters of the whole heart, the atria, the ventricles, the left main coronary artery, and three main coronary arteries were extracted for analysis. Results: Within a median follow-up time of 67 months, 14 patients experienced MCEs at a median of 16 months. The incidence of MCEs was significantly associated with the left anterior descending coronary artery (LAD) receiving ≥30 Gy (V30Gy) (p = 0.048). Patients receiving LAD V30Gy ≥ 10% of volume experienced a higher incidence of MCEs versus the LAD V30Gy < 10% group (p = 0.044). The relative rate of death increased with the left main coronary artery (LMA) mean dose (Gy) (p = 0.002). Furthermore, a mutual promotion effect of hyperlipidemia and RT on MCEs was observed. Conclusion: Radiation dose to coronary substructures is associated with MCEs and overall survival in patients with ESOC. In this study, the doses to these substructures appeared to be better predictors of toxicity outcomes than mean heart dose (MHD) or whole-heart V30Gy. These findings have implications for reducing coronary events through radiation therapy planning.

Background To determine whether antibiotic treatment is a risk factor for immune-related adverse events (irAEs) across different patients with cancer receiving anti-PD-1/PD-L1 therapies. Methods The retrospective analysis includes clinical information from 767 patients with cancer treated at Hunan Cancer Hospital from 2017 to 2020. The pharmacovigilance data analysis includes individual cases of 38,705 safety reports from the US Food and Drug Administration Adverse Event Reporting System (FAERS) from 2014 to 2020, and 25,122 cases of safety reports from the World Health Organization database VigiBase from 2014 to 2019. All cases that received anti-PD-1/PD-L1 treatment were included. Multiomics data from patients across 25 cancer types were download from The Cancer Genome Atlas. Logistic regression and propensity score algorithm was employed to calculate OR of irAEs. Results Retrospective analysis of in-house patients showed that irAE potential risks are higher in all cancer (OR 2.12, 95% CI 1.38 to 3.22, false discovery rate (FDR) adjusted-p=1.93×10−3) and patients with lung cancer (OR 3.16, 95% CI 1.67 to 5.95, FDR adjusted-p=1.93×10−3) when using antibiotics. Potential risk of irAEs in patients with lung cancer with antibiotic treatment is significantly higher in FAERS (OR 1.39, 95% CI 1.21 to 1.59; FDR adjusted-p=1.62×10−5) and VigiBase (OR 1.32, 95% CI 1.09 to 1.59, FDR adjusted-p=0.05). Mechanistically, decreased microbial diversity caused by antibiotics use may increase the irAE risk through mediating the irAE-related factors. Conclusions Our study is the first to comprehensively demonstrate the associations of irAEs and antibiotic during anti-PD-1/PD-L1 therapy across a wide spectrum of cancers by analyzing multisource data. Administration of antibiotics should be carefully evaluated in patients with cancer treated by anti-PD-1/PD-L1 to avoid potentially increasing irAE risk.

3039 Background: Patients with multiple organ metastases have poorer prognoses than those with a single organ metastasis, are frequently associated with drug resistance, and have higher tumor burden. Engorged (≥50um) Cancer Associated Macrophage-Like Cells (CAMLs) are a circulating stromal cell subtype detected in the blood of patients with solid tumors at high risk for recurrence or progression. While numerous studies have shown that ≥50um CAMLs predict poor clinical outcomes, meta-analysis of these studies have also suggested that hyper engorged CAMLs ≥100um (heCAMLs) may be associated with multifocal metastatic disease and even worse outcomes. In this prospective study, we evaluated the presence of heCAMLs in patients with metastatic disease and demonstrated a strong relationship with multi organ spread, which also correlated with shorter Progression Free Survival (PFS) and Overall Survival (OS). Methods: We prospectively recruited 151 patients with metastatic (m) mbreast (n = 58), mlung (n = 34), mprostate (n = 39), and mrenal (n = 20) cancers. Peripheral blood was collected prior to the induction of new treatment for metastatic cancer. Cells were isolated following standard CellSieve techniques, then imaged and measured in ZenBlue. Multi organ metastasis was defined as spread to ≥2 distant organ sites, or any spread to the brain. Single factor ANOVA was conducted to compare heCAML presence in multi organ metastatic patients versus patients with single organ site metastasis. Univariate and multivariate analysis was run to evaluate for PFS and OS against heCAMLs, and all known clinical parameters. Results: 150 viable samples (excluding 1 failed sample) were obtained. Multi organ metastases were present in 55% (n = 83/150) of patients. heCAMLs were found in 59% (n = 49/83) of the multi organ metastatic population, but only in 16% (n = 11/67) of the single site metastatic cohort (p < 0.001). heCAML presence appeared to differentiate multi organ vs single organ metastases in mbreast (85% vs. 52%, p = 0.006), mlung (71% vs. 26%, p = 0.025), mprostate (75% vs. 37%, p = 0.029), and mRCC (88% vs. 36%, p = 0.025). Further, in all n = 150 patients, heCAML presence predicted a significantly shorter median PFS of 4.5 versus 7.2 months, 24 month PFS (HR = 1.67, 95%CI = 1.13-2.45, p = 0.013), and significantly shorter median OS of 13.1 versus 20.4 months, 24 month OS (HR = 2.05, 95%CI = 1.24-3.39, p = 0.008). Conclusions: We examined a non-invasive prognostic blood based assay to determine its relationship to multi organ metastatic spread as well as its prognostic value in several solid cancers. These results showed patients with heCAMLs had higher rates of multi organ metastases, and appear to predict for shorter PFS and OS. Studies of larger cohorts are needed for prospective validation of these initial findings.

Abstract Background Accumulated evidence supports the existence of sex-associated differences in immune systems. Understanding the role of sex in immune-related adverse events (irAEs) is important for management of irAE in patients receiving immunotherapy. Methods We performed meta-analysis on published clinical study data and multivariable logistic regression on pharmacovigilance data and applied a propensity algorithm to The Cancer Genome Atlas omics data. We further validated our observations in 2 independent in-house cohorts of 179 and 767 cancer patients treated with immune checkpoint inhibitors. Results A meta-analysis using 13 clinical studies that reported on 1096 female patients (36.8%, 95% confidence interval [CI] = 35.0% to 38.5%) and 1886 male patients (63.2%, 95% CI = 61.5% to 65.0%) demonstrated no statistically significant irAE risk difference between the sexes (odds ratio [OR] = 1.19, 95% CI = 0.91 to 1.54, 2-sided P = .21). Multivariable logistic regression analysis of 12 225 patients from the Food and drug administration Adverse Event Reporting System (FAERS) and 10 979 patients from VigiBase showed no statistically significant difference in irAEs by sex. A propensity score algorithm used on multi-omics data for 6019 patients from The Cancer Genome Atlas found no statistically significant difference by sex for irAE-related factors or pathways. The retrospective analysis of 2 in-house patient cohorts validated these results (OR = 1.55, 95% CI = 0.98 to 2.47, false discovery rate = 0.13, for cohort 1; OR = 1.16, 95% CI = 0.86 to 1.57, false discovery rate = 0.39, for cohort 2). Conclusions We observed minimal sex-associated differences in irAEs among cancer patients who received immune checkpoint inhibitor therapy. It may be unnecessary to consider sex effects for irAE management in clinical practice.