Faculty Bibliography

IMPORTANCE:Biomarkers may improve prediction of cardiovascular events for patients with stable coronary artery disease (CAD), but their importance in addition to clinical tests of inducible ischemia and CAD severity is unknown. OBJECTIVES:To evaluate the prognostic value of multiple biomarkers in stable outpatients with obstructive CAD and moderate or severe inducible ischemia. DESIGN AND SETTING:The ISCHEMIA and ISCHEMIA CKD trials randomized 5,956 participants with CAD to invasive or conservative management from July 2012 to January 2018; 1,064 participated in the biorepository. MAIN OUTCOME MEASURES:Primary outcome was cardiovascular death, myocardial infarction (MI), or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. Secondary outcome was cardiovascular death or MI. Improvements in prediction were assessed by cause-specific hazard ratios (HR) and area under the receiver operating characteristics curve (AUC) for an interquartile increase in each biomarker, controlling for other biomarkers, in a base clinical model of risk factors, left ventricular ejection fraction (LVEF) and ischemia severity. Secondary analyses were performed among patients in whom core-lab confirmed severity of CAD was ascertained by computed cardiac tomographic angiography (CCTA). EXPOSURES:Baseline levels of interleukin-6 (IL-6), high sensitivity troponin T (hsTnT), growth differentiation factor 15 (GDF-15), N-terminal pro-B-type natriuretic peptide (NT-proBNP), lipoprotein a (Lp[a]), high sensitivity C-reactive protein (hsCRP), Cystatin C, soluble CD 40 ligand (sCD40L), myeloperoxidase (MPO), and matrix metalloproteinase 3 (MMP3). RESULTS:Among 757 biorepository participants, median (IQR) follow-up was 3 (2-5) years, age was 67 (61-72) years, and 144 (19%) were female; 508 had severity of CAD by CCTA available. In an adjusted multimarker model with hsTnT, GDF-15, NT-proBNP and sCD40L, the adjusted HR for the primary outcome per interquartile increase in each biomarker was 1.58 (95% CI 1.22, 2.205), 1.60 (95% CI 1.16, 2.20), 1.61 (95% 1.22, 2.14), and 1.46 (95% 1.12, 1.90), respectively. The adjusted multimarker model also improved prediction compared with the clinical model, increasing the AUC from 0.710 to 0.792 (P < .01) and 0.714 to 0.783 (P < .01) for the primary and secondary outcomes, respectively. Similar findings were observed after adjusting for core-lab confirmed atherosclerosis severity. CONCLUSIONS AND RELEVANCE:Among ISCHEMIA biorepository participants, biomarkers of myocyte injury/distension, inflammation, and platelet activity improved cardiovascular event prediction in addition to risk factors, LVEF, and assessments of ischemia and atherosclerosis severity. These biomarkers may improve risk stratification for patients with stable CAD.

Lung cancer treatment has benefited greatly through advancements in immunotherapies. However, immunotherapy often fails in patients with specific mutations like KEAP1, which are frequently found in lung adenocarcinoma. We established an antigenic lung cancer model and used it to explore how Keap1 mutations remodel the tumor immune microenvironment. Using single-cell technology and depletion studies, we demonstrate that Keap1-mutant tumors diminish dendritic cell and T cell responses driving immunotherapy resistance. This observation was corroborated in patient samples. CRISPR-Cas9-mediated gene targeting revealed that hyperactivation of the NRF2 antioxidant pathway is responsible for diminished immune responses in Keap1-mutant tumors. Importantly, we demonstrate that combining glutaminase inhibition with immune checkpoint blockade can reverse immunosuppression, making Keap1-mutant tumors susceptible to immunotherapy. Our study provides new insight into the role of KEAP1 mutations in immune evasion, paving the way for novel immune-based therapeutic strategies for KEAP1-mutant cancers.

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of β-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.

BACKGROUND/UNASSIGNED:Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. METHODS/UNASSIGNED: RESULTS/UNASSIGNED: CONCLUSIONS/UNASSIGNED:Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

Approximately 30% of early-stage lung adenocarcinoma patients present with disease progression after successful surgical resection. Despite efforts of mapping the genetic landscape, there has been limited success in discovering predictive biomarkers of disease outcomes. Here we performed a systematic multi-omic assessment of 143 tumors and matched tumor-adjacent, histologically-normal lung tissue with long-term patient follow-up. Through histologic, mutational, and transcriptomic profiling of tumor and adjacent-normal tissue, we identified an inflammatory gene signature in tumor-adjacent tissue as the strongest clinical predictor of disease progression. Single-cell transcriptomic analysis demonstrated the progression-associated inflammatory signature was expressed in both immune and non-immune cells, and cell type-specific profiling in monocytes further improved outcome predictions. Additional analyses of tumor-adjacent transcriptomic data from The Cancer Genome Atlas validated the association of the inflammatory signature with worse outcomes across cancers. Collectively, our study suggests that molecular profiling of tumor-adjacent tissue can identify patients at high risk for disease progression.

Reciprocal interactions between non-myocytes and cardiomyocytes regulate cardiac growth and differentiation. Here, we report that the transcription factor Ebf1 is highly expressed in non-myocytes and potently regulates heart development. Ebf1-deficient hearts display myocardial hypercellularity and reduced cardiomyocyte size, ventricular conduction system hypoplasia, and conduction system disease. Growth abnormalities in Ebf1 knockout hearts are observed as early as embryonic day 13.5. Transcriptional profiling of Ebf1-deficient embryonic cardiac non-myocytes demonstrates dysregulation of Polycomb repressive complex 2 targets, and ATAC-Seq reveals altered chromatin accessibility near many of these same genes. Gene set enrichment analysis of differentially expressed genes in cardiomyocytes isolated from E13.5 hearts of wild-type and mutant mice reveals significant enrichment of MYC targets and, consistent with this finding, we observe increased abundance of MYC in mutant hearts. EBF1-deficient non-myocytes, but not wild-type non-myocytes, are sufficient to induce excessive accumulation of MYC in co-cultured wild-type cardiomyocytes. Finally, we demonstrate that BMP signaling induces Ebf1 expression in embryonic heart cultures and controls a gene program enriched in EBF1 targets. These data reveal a previously unreported non-cell-autonomous pathway controlling cardiac growth and differentiation.

Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDRs) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend life span in DC cells. Of the cellular clones isolated due to increased life span, 34% had a guide RNA (gRNA) targeting CEBPB, while gRNAs targeting WSB1, MED28, and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA sequencing to compare DC and control cells. Expression of inflammatory genes was found to be significantly elevated (P < 0.0001) in addition to a key subset of these inflammation-related genes [IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5]. which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere length-dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB's contribution in DC cells' senescence is ROS independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.

BACKGROUND:High rates of vaccination and natural infection drive immunity and redirect selective viral adaptation. Updated boosters are installed to cope with drifted viruses, yet data on adaptive evolution under increasing immune pressure in a real-world situation are lacking. METHODS:Cross-sectional study to characterise SARS-CoV-2 mutational dynamics and selective adaptation over >1 year in relation to vaccine status, viral phylogenetics, and associated clinical and demographic variables. FINDINGS/RESULTS:The study of >5400 SARS-CoV-2 infections between July 2021 and August 2022 in metropolitan New York portrayed the evolutionary transition from Delta to Omicron BA.1-BA.5 variants. Booster vaccinations were implemented during the Delta wave, yet booster breakthrough infections and SARS-CoV-2 re-infections were almost exclusive to Omicron. In adjusted logistic regression analyses, BA.1, BA.2, and BA.5 had a significant growth advantage over co-occurring lineages in the boosted population, unlike BA.2.12.1 or BA.4. Selection pressure by booster shots translated into diffuse adaptive evolution in Delta spike, contrasting with strong, receptor-binding motif-focused adaptive evolution in BA.2-BA.5 spike (Fisher Exact tests; non-synonymous/synonymous mutation rates per site). Convergent evolution has become common in Omicron, engaging spike positions crucial for immune escape, receptor binding, or cleavage. INTERPRETATION/CONCLUSIONS:Booster shots are required to cope with gaps in immunity. Their discriminative immune pressure contributes to their effectiveness but also requires monitoring of selective viral adaptation processes. Omicron BA.2 and BA.5 had a selective advantage under booster vaccination pressure, contributing to the evolution of BA.2 and BA.5 sublineages and recombinant forms that predominate in 2023. FUNDING/BACKGROUND:The study was supported by NYU institutional funds and partly by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center.

OBJECTIVE:Early-stage lung adenocarcinoma is treated with local therapy alone, although patients with grade 3 stage I lung adenocarcinoma have a 50% 5-year recurrence rate. Our objective is to determine if analysis of the tumor microenvironment can create a predictive model for recurrence. METHODS:Thirty-four patients with grade 3 stage I lung adenocarcinoma underwent surgical resection. Digital spatial profiling was used to perform genomic (n = 31) and proteomic (n = 34) analyses of pancytokeratin positive and negative tumor cells. K-means clustering was performed on the top 50 differential genes and top 20 differential proteins, with Kaplan-Meier recurrence curves based on patient clustering. External validation of high-expression genes was performed with Kaplan-Meier plotter. RESULTS:There were no significant clinicopathologic differences between patients who did (n = 14) and did not (n = 20) have recurrence. Median time to recurrence was 806 days; median follow-up with no recurrence was 2897 days. K-means clustering of pancytokeratin positive genes resulted in a model with a Kaplan-Meier curve with concordance index of 0.75. K-means clustering for pancytokeratin negative genes was less successful at differentiating recurrence (concordance index 0.6). Genes upregulated or downregulated for recurrence were externally validated using available public databases. Proteomic data did not reach statistical significance but did internally validate the genomic data described. CONCLUSIONS:Genomic difference in lung adenocarcinoma may be able to predict risk of recurrence. After further validation, stratifying patients by this risk may help guide who will benefit from adjuvant therapy.

PURPOSE:We previously showed that elevated frequencies of peripheral blood CD3+CD4+CD127-GARP-CD38+CD39+ T cells were associated with checkpoint immunotherapy resistance in patients with metastatic melanoma. In the present study, we sought to further investigate this population of ectoenzyme-expressing T cells (Teee). EXPERIMENTAL DESIGN:Teee derived from the peripheral blood of patients with metastatic melanoma were evaluated by bulk RNA-sequencing (RNA-seq) and flow cytometry. The presence of Teee in the tumor microenvironment was assessed using publically available single-cell RNA-seq datasets of melanoma, lung, and bladder cancers along with multispectral immunofluorescent imaging of melanoma patient formalin-fixed, paraffin-embedded specimens. Suppressive function of Teee was determined by an in vitro autologous suppression assay. RESULTS:Teee had phenotypes associated with proliferation, apoptosis, exhaustion, and high expression of inhibitory molecules. Cells with a Teee gene signature were present in tumors of patients with melanoma, lung, and bladder cancers. CD4+ T cells co-expressing CD38 and CD39 in the tumor microenvironment were preferentially associated with Ki67- CD8+ T cells. Co-culture of patient Teee with autologous T cells resulted in decreased proliferation of target T cells. High baseline intratumoral frequencies of Teee were associated with checkpoint immunotherapy resistance and poor overall survival in patients with metastatic melanoma. CONCLUSIONS:These results demonstrate that a novel population of CD4+ T cells co-expressing CD38 and CD39 is found both in the peripheral blood and tumor of patients with melanoma and is associated with checkpoint immunotherapy resistance.