Citations

Disrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which disrupted NCT causes neurodegeneration remain unclear. In a Drosophila screen, we identified ref(2)P/p62 , a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. Our data suggest that the C9orf72 -HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD.

Abstract Background: Clinical use of breast cancer risk prediction requires simplified models. We evaluate a simplified version of the validated Rosner–Colditz model and add percent mammographic density (MD) and polygenic risk score (PRS), to assess performance from ages 45–74. We validate using the Mayo Mammography Health Study (MMHS). Methods: We derived the model in the Nurses' Health Study (NHS) based on: MD, 77 SNP PRS and a questionnaire score (QS; lifestyle and reproductive factors). A total of 2,799 invasive breast cancer cases were diagnosed from 1990–2000. MD (using Cumulus software) and PRS were assessed in a nested case–control study. We assess model performance using this case–control dataset and evaluate 10-year absolute breast cancer risk. The prospective MMHS validation dataset includes 21.8% of women age <50, and 434 incident cases identified over 10 years of follow-up. Results: In the NHS, MD has the highest odds ratio (OR) for 10-year risk prediction: ORper SD = 1.48 [95% confidence interval (CI): 1.31–1.68], followed by PRS, ORper SD = 1.37 (95% CI: 1.21–1.55) and QS, ORper SD = 1.25 (95% CI: 1.11–1.41). In MMHS, the AUC adjusted for age + MD + QS 0.650; for age + MD + QS + PRS 0.687, and the NRI was 6% in cases and 16% in controls. Conclusion: A simplified assessment of QS, MD, and PRS performs consistently to discriminate those at high 10-year breast cancer risk. Impact: This simplified model provides accurate estimation of 10-year risk of invasive breast cancer that can be used in a clinical setting to identify women who may benefit from chemopreventive intervention. See related commentary by Tehranifar et al., p. 587

Abstract Glioblastoma (GBM) is a highly malignant type of adult brain tumor with a poor prognosis. Temozolomide (TMZ), a DNA alkylating agent, has been widely used as an effective first-line chemotherapeutic agent for the treatment of GBM patients. The efficacy of TMZ in GBM depends on the absence of the DNA repair protein MGMT which reverses the DNA damage induced by TMZ. The MGMT promoter is hypermethylated in about 45% of GBMs, resulting in lack of MGMT expression and increased responsiveness to TMZ. TMZ is less effective in MGMT unmethylated GBMs. We propose that EGFR inhibition downregulates MGMT and sensitizes glioma cells to TMZ and a combination of pretreatment with erlotinib followed by TMZ could be a useful therapeutic approach in MGMT expressing GBMs. As our experimental model we used multiple MGMT unmethylated lines from the Mayo Clinic patient derived xenografts (PDXs) panel. Our data demonstrate that exposure of cells to EGFR tyrosine kinase inhibitor erlotinib for 48h results in downregulation of MGMT at the mRNA and protein level. Additionally, EGFR inhibition activates the AP-1 transcription factor and overexpression of AP-1 components Fos and Jun results in decreased MGMT expression in TMZ resistant PDXs, suggesting that AP-1 acts as a transcriptional repressor of MGMT. We further identified that the mice implanted with TMZ resistant PDXs pretreated with afatinib followed by TMZ treatment survived longer compared to those treated with TMZ alone. Thus, the use of EGFR inhibition may enhance the sensitivity of MGMT unmethylated GBMs to TMZ.

Abstract Background: Tumor infiltrating lymphocytes (TILs) play an important, but incompletely understood role in chemotherapy response and prognosis. In breast cancer, there appear to be distinct immune responses by subtype, but most studies have used limited numbers of protein markers or bulk sequencing of RNA to characterize immune response, in which spatial organization cannot be assessed. Methods: To identify immune phenotypes of Basal-like vs. Luminal breast cancer we used the GeoMx® (NanoString) platform to perform digital spatial profiling (DSP) of immune-related proteins in tumor whole sections and tissue microarrays (TMA). Visualization of CD45, CD68, or pan-Cytokeratin by immunofluorescence was used to select regions of interest (ROIs) in formalin-fixed paraffin embedded tissue sections. 44 antibodies representing stromal markers and multiple immune cell types were applied to quantify the tumor microenvironment. Results: In whole tumor slides, immune hot spots (CD45+) had increased expression of many immune markers, suggesting a diverse and robust immune response. In epithelium-enriched areas, immune signals were also detectable and varied by subtype, with Regulatory T cell (Treg) markers (CD4, CD25, FOXP3) being higher in Basal-like vs. Luminal breast cancer. Extending these findings to TMAs with more patients (n=75), we confirmed subtype-specific immune profiles, including enrichment of Treg markers in Basal-likes. Conclusion: This work demonstrated that immune responses can be detected in epithelium-rich tissue, and that TMAs are a viable approach for obtaining important immunoprofiling data. In addition, we found that immune marker expression is associated with BC subtype, suggesting possible prognostic or targetable differences. Citation Format: Andrea Walens, Linnea T. Olsson, Xiaohua Gao, Alina M. Hamilton, Erin L. Kirk, Stephanie M. Cohen, Bentley R. Midkiff, Yongjuan Xia, Mark E. Sherman, Nana Nikolaishvili-Feinberg, Katherine A. Hoadley, Melissa A. Troester, Benjamin C. Calhoun. Protein-based immune profiles of basal-like vs luminal breast cancers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-090.

Abstract Background Coronary artery disease is the leading cause of death among elderly men and women worldwide. The aging society worldwide will lead to increasing numbers of elderly patients with multivessel coronary artery disease. Although age is recognized as one of the most important factors in a decision-making for revascularization of multivessel coronary artery disease, the very long-term outcomes in patients undergoing revascularization by percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) is still unclear. Objectives The aim of the present study was to investigate the association between revascularization strategies and 10-year outcomes in elderly patients. Methods The SYNTAX Extended Survival (SYNTAXES) study (NCT 03417050) is an investigator-driven extension of follow-up of a multicentre, randomised controlled trial done in 85 hospitals across 18 North American and European countries, enrolling 1,800 patients with de novo three-vessel disease (3VD) and/or left main coronary artery disease (LMCAD) randomized to revascularization strategy with CABG versus PCI in the SYNTAX trial. Patients were divided into two groups according to the prespecified threshold of 70 years old; elderly patients (>70 years) and non-elderly patients (≤70 years). The primary endpoint of this study was all-cause death at 10 years. Results Out of 1,800 patients, 575 patients (31.9%) were classified as elderly (>70 years). The mean age ± standard deviation (SD) of the elderly patients and the non-elderly patients was 75.8±3.6 years and 60.1±7.4 years, respectively. Of note, elderly patients were more frequently female than non-elderly patients (33.6% vs. 17.1%, p<0.001). As expected, the elderly patients had higher prevalence of chronic kidney disease (43.4% vs. 7.9%, p<0.001), had higher anatomical SYNTAX score (30.2±11.8 vs 28.0±11.2 p<0.001) when compared to those of the non-elderly patients. Up to 10 years, all-cause death occurred in 42.7% and 18.9% in the elderly and non-elderly patients, respectively (Log-rank p<0.001). The cubic spline curve showed an exponentially increase in all-cause death at 10 years according to the increase of age both in the PCI arm and the CABG arm. At 10 years, there was no significant difference in the risk of all-cause death between CABG vs. PCI either in elderly patients (41.5% vs. 44.0%; Log-rank p=0.53) or non-elderly patients (16.6% vs. 21.1%; Log-rank p=0.051). Conclusion CABG and PCI were equipoise in terms of risk of all-cause death at 10 years in patients with de novo 3VD and/or LMCAD irrespective of their age when stratified according to the prespecified threshold of 70 years old. Kaplan-Meier curves Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): German Foundation of Heart Research

Abstract Background The SYNTAX Extended Survival (SYNTAXES) study is an investigator-driven extension of follow-up of the SYNTAX trial, which was a non-inferiority trial that compared percutaneous coronary intervention (PCI) using first-generation paclitaxel-eluting stents with coronary artery bypass grafting (CABG) in patients with de-novo three-vessel and left main coronary artery disease. The SYNTAXES study is the first randomized trial that reported the complete 10-year data on all-cause death in patients with complex coronary artery disease. Purpose Patients with coronary artery disease (CAD) and concomitant diabetes mellitus (DM) or chronic kidney disease (CKD) are more susceptible to major adverse cardiovascular and cerebrovascular events. However, to date, the long-term prognosis and which revascularization strategy was associated with better clinical outcomes for patients with complex coronary artery disease and concomitant with DM and CKD have not been documented. Methods In this sub-analysis of the SYNTAXES trial, a total of 1,638 patients were classified into four subgroups according to the DM and CKD status: DM−/CKD− (n=999, 60.1%), DM+/CKD− (n=323, 19.7%), DM−/CKD+ (n=231, 14.1%), and DM+/CKD+ (n=85, 5.2%). The treatment effects of PCI and CABG were analyzed in each subgroup. The primary endpoint was all-cause death at 10 years. Results Compared with the DM−/CKD− patients, patients with DM+/CKD+ were older, more often had a history of stroke, hypertension, heart failure, and were more frequently presented with total occlusion, bifurcation lesion and three-vessel disease. At 10 years, patients with DM+/CKD+ had a 3.94-fold higher incidence of all-cause mortality compared with DM−/CKD− individuals (54.1% versus 18.9%, 95% CI [2.85–5.44]). Patients with DM−/CKD+ (38.1%, HR 2.36; 95% CI [1.83–5.44]) or DM+/CKD− (28.2%, HR 1.61; 95% CI [1.26–2.07]) had intermediate risk profile. For DM+/CKD+ patients, compared with PCI, those who underwent CABG were associated with lower incidence of all-cause mortality (64.3% versus 44.2%, adjusted HR 0.52; 95% CI [0.27–0.99], p=0.047, pinteraction=0.443). The number of needed-to-treat to reduce mortality for CABG was 12. Conclusion In the SYNTAX population, patients with DM and CKD are at markedly increased risk of long-term mortality rate compared with patients one or neither of these risk factors. For patients with both comorbidities, CABG was associated with better clinical outcome compared with PCI. These findings should be interpreted as hypothesis-generating. Figure 1. Kaplan-Meier curves showing the clinical events according to treatment and DM/CKD status. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Boston Scientific Corporation

Abstract Background Periprocedural myocardial infarction (PMI) occurs frequently after both percutaneous coronary intervention (PCI) and bypass grafting surgery (CABG) in patients with complex coronary artery disease (CAD), and PMI has been shown to have a detrimental impact on mortality. On the other hand, long-term impact of PMI on mortality has not been fully evaluated. Purpose This study aimed to assess the impact of PMI according to SCAI definition on 10-year all-cause death in patients with complex CAD. Methods The SYNTAX Extended Survival (SYNTAXES) study evaluated vital status up to 10 years in 1800 patients with de novo three-vessel disease and/or left main coronary artery disease randomized to treatment with CABG or PCI in the SYNTAX trial. Blood was sampled for creatine kinase (CK) pre- and post-revascularisation, and the cardiac specific MB iso-enzyme (CK-MB) was determined only if the CK ratio ≥2 x the upper limit of normal (ULN). If the CK ratio <2 ULN, CK-MB assessment was not mandated. In this analysis, patients with at least one blood sampling within 48 hours of the procedure were included. PMI was defined as follows; peak CK-MB measured within 48 hours of the procedure ≥10 x ULN, or ≥5 x ULN with new Q-waves in 2 contiguous leads or new persistent left bundle branch block. Results Of 1800 patients, 1679 (93.2%) patients were included. Of 877 patients treated with PCI, PMI occurred in 26 patients (3.0%), whereas 14 (1.7%) PMIs were observed in 802 patients treated with CABG. Compared with patients without PMI, patients with PMI presented with unstable angina more frequently (45.0% vs. 28.7, p=0.033), and had a higher rate of bifurcation lesion (87.5% vs. 72.5, p=0.046). PMI was associated with a higher all-cause mortality at 10 years compared with no PMI (55.3% vs. 25.4%; Log-rank p<0.001, Figure), which was mainly driven by a high mortality rate within 1 year. In patients undergoing PCI, the mortality rates were significantly higher in patients with PMI not only within 1 year (Log-rank p<0.001) but also beyond one year (Log-rank p=0.016), compare to patients without PMI (Figure). On the other hand, in patients undergoing CABG, a higher mortality rate in patients with PMI was observed until 1 year (Log-rank p<0.001), but the impact of PMI on mortality beyond one year after CABG subsided (Log-rank p=0.308) (Figure 1). Conclusion PMI was associated with a poor prognosis at 10 years. The impact of PMI on mortality was strong within one year. Of note, the impact of PMI on mortality persisted beyond 1 year only in patients undergoing PCI. Patients who were treated with PCI and suffered PMI need careful follow-up beyond one year after revascularization. Figure 1 Funding Acknowledgement Type of funding source: None

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300 LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300 LSECΔ/Δ mice compared to p300 fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300 LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300 LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

Abstract Background: Accumulating evidence suggests a relationship between endometrial cancer and ovarian cancer. Independent genome-wide association studies (GWAS) for endometrial cancer and ovarian cancer have identified 16 and 27 risk regions, respectively, four of which overlap between the two cancers. We aimed to identify joint endometrial and ovarian cancer risk loci by performing a meta-analysis of GWAS summary statistics from these two cancers. Methods: Using LDScore regression, we explored the genetic correlation between endometrial cancer and ovarian cancer. To identify loci associated with the risk of both cancers, we implemented a pipeline of statistical genetic analyses (i.e., inverse-variance meta-analysis, colocalization, and M-values) and performed analyses stratified by subtype. Candidate target genes were then prioritized using functional genomic data. Results: Genetic correlation analysis revealed significant genetic correlation between the two cancers (rG = 0.43, P = 2.66 × 10−5). We found seven loci associated with risk for both cancers (PBonferroni < 2.4 × 10−9). In addition, four novel subgenome-wide regions at 7p22.2, 7q22.1, 9p12, and 11q13.3 were identified (P < 5 × 10−7). Promoter-associated HiChIP chromatin loops from immortalized endometrium and ovarian cell lines and expression quantitative trait loci data highlighted candidate target genes for further investigation. Conclusions: Using cross-cancer GWAS meta-analysis, we have identified several joint endometrial and ovarian cancer risk loci and candidate target genes for future functional analysis. Impact: Our research highlights the shared genetic relationship between endometrial cancer and ovarian cancer. Further studies in larger sample sets are required to confirm our findings.

Abstract PrimPol has been recently identified as a DNA damage tolerant polymerase that plays an important role in replication stress response. However, the regulatory mechanisms of PrimPol are not well defined. In this study, we identify that the deubiquitinase USP36 interferes with degradation of PrimPol to regulate the replication stress response. Mechanistically, USP36 is deubiquitinated following DNA replication stress, which in turn facilitates its upregulation and interaction with PrimPol. USP36 deubiquitinates K29-linked polyubiquitination of PrimPol and increases its protein stability. Depletion of USP36 results in replication stress-related defects and elevates cell sensitivity to DNA-damage agents, such as cisplatin and olaparib. Moreover, USP36 expression positively correlates with the level of PrimPol protein and poor prognosis in patient samples. These findings indicate that the regulation of PrimPol K29-linked ubiquitination by USP36 plays a critical role in DNA replication stress and chemotherapy response.