Citations

Background: Previous studies in the SCN5A- encoded Na channel showed that amino acid substitutions at one position affected function of a Na channel containing a pathogenic mutation elsewhere. The first and most commonly used SCN5A cDNA clone (hH1) is the minor alternatively spliced transcript with an extra glutamine at position 1077 (Q1077), and also differs from the Na channel found in nearly all humans by virtue of a rare amino acid substitution (R1027Q). We hypothesized that R1027Q in previous studies using hH1 may have increased expression levels of the Brugada syndrome (BrS)-associated mutation (R1512W) and the common polymorphism (H558R). Methods: We generated mutations in the alternatively-spliced SCN5A transcript containing Q1077 (as in the hH1 clone) as the wild type (WT) channel to generate channels with the following amino acid substitutions in addition to Q1077ins: H558R, H558R/R1027Q, R1512W, and R1027Q/R1512W. These channels were expressed in HEK-293 cells and current densities measured by whole cell voltage clamp. Results: The current density for H558R was reduced dramatically (−7±2 pA/pF) whereas H558R/R1027Q exhibited normal current density (−297±56 pA/pF) comparable to WT (−310±52 pA/pF). The BrS mutation, R1512W-hH1, was characterized originally as having only subtle inactivation defects but normal current density. However, when we expressed R1512W in the patient’s true WT background (i.e. without R1027Q), the current density was attenuated significantly (−79±19 pA/pF) whereas the double mutant (R1027Q/R1512W) which replicated R1512W-hH1 showed nearly normal density (−180±21 pA/pF, −253±35 pA/pF for hH1, n=10–25 cells). Conclusions: R1027Q, the rare genetic variant present in the hH1 cDNA clone, rescues expression of Na current for channels with H558R and the BrS1-associated mutation, R1512W. Using the hH1 clone which contains R1027Q as background (as is the case for nearly all previous studies of SCN5A mutations) may yield erroneous conclusions regarding the functional impact assigned to putative pathogenic mutations. It may be critical to study mutations in either true WT clones or in the patient’s own genetic background. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

With rapid advances in our understanding of cancer, there is an expanding number of potential novel combination therapies, including novel–novel combinations. Identifying which combinations are appropriate and in which subpopulations are among the most difficult questions in medical research. We conducted a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)-guided systematic review of trials of novel–novel combination therapies involving immunotherapies or molecular targeted therapies in advanced solid tumors. A MEDLINE search was conducted using a modified Cochrane Highly Sensitive Search Strategy for published clinical trials between July 1, 2017, and June 30, 2020, in the top-ranked medical and oncology journals. Trials were evaluated according to a criterion adapted from previously published Food and Drug Administration guidance and other key considerations in designing trials of combinations. This included the presence of a strong biological rationale, the use of a new established or emerging predictive biomarker prospectively incorporated into the clinical trial design, appropriate comparator arms of monotherapy or supportive external data sources and a primary endpoint demonstrating a clinically meaningful benefit. Of 32 identified trials, there were 11 (34%) trials of the novel–novel combination of anti-programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) and anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) therapy, and 10 (31%) trials of anti-PD-1/PD-L1 and anti-vascular endothelial growth factor (VEGF) combination therapy. 20 (62.5%) trials were phase II trials, while 12 (37.5%) were phase III trials. Most (72%) trials lacked significant preclinical evidence supporting the development of the combination in the given indication. A majority of trials (69%) were conducted in biomarker unselected populations or used pre-existing biomarkers within the given indication for patient selection. Most studies (66%) were considered to have appropriate comparator arms or had supportive external data sources such as prior studies of monotherapy. All studies were evaluated as selecting a clinically meaningful primary endpoint. In conclusion, designing trials to evaluate novel–novel combination therapies presents numerous challenges to demonstrate efficacy in a comprehensive manner. A greater understanding of biological rationale for combinations and incorporating predictive biomarkers may improve effective evaluation of combination therapies. Innovative statistical methods and increasing use of external data to support combination approaches are potential strategies that may improve the efficiency of trial design. Designing trials to evaluate novel–novel combination therapies presents numerous challenges to demonstrate efficacy in a comprehensive manner. A greater understanding of biological rationale for combinations and incorporating predictive biomarkers may improve effective evaluation of combination therapies. Innovative statistical methods and increasing use of external data to support combination approaches are potential strategies that may improve the efficiency of trial design.

Abstract Background Despite safety communications from the Food and Drug Administration (FDA) regarding the outbreak of Mycobacterium chimaera infections (MCIs) from contaminated heater-cooler devices, new cases continue to be identified. Methods We retrospectively reviewed confirmed cases of MCI that were managed at Mayo Clinic sites (Arizona, Florida, and Minnesota) from 09/2015 to 01/2021. Clinical histories including prior cardiovascular surgery were recorded. Diagnostic workup including ophthalmologic examination, imaging, and laboratory testing was reviewed. Treatment and survival outcomes on follow-up were obtained. Results Twelve patients with MCI were included. All patients had aortic valve or graft replacement. Five patients had their surgical procedures following the 10/15/2015 FDA safety communication. The mean time from surgery to symptom onset (range) was 32 (13–73) months. Ten of 11 patients who underwent ophthalmologic examination had chorioretinal abnormalities. Three patients who underwent microbial cell-free deoxyribonucleic acid sequencing tested positive for M. chimaera, which was subsequently confirmed with blood culture growth. Echocardiography and positron emission tomography/computed tomography (PET/CT) revealed evidence of prosthetic valve/graft infection in 7/12 (58.3%) and 6/10 (60.0%) of cases, respectively. Seven patients (58.3%) underwent redo cardiovascular surgery. Of these, 1 patient died 2 days postdischarge, 1 experienced spinal osteomyelitis relapse, and another had interval prosthetic valve fluorodeoxyglucose (FDG) uptake on PET/CT suspicious for recurrent infection. Among 4 patients on medical therapy only, 3 expired or transitioned to hospice during follow-up. Conclusions MCI continues to occur despite the FDA communications. Incorporation of ophthalmologic examination and use of advanced tools may improve MCI diagnosis. The mortality in these patients is high even with aggressive surgical/medical management.

Abstract Targeted therapy in prostate cancer is in its early stages and has a promising future since many genomic alterations in metastatic castrate resistant prostate cancer (mCRPC) do not have FDA approved agents for this indication. Three-dimensional culture has been shown to recapitulate patient response to therapy. We characterized a unique patient tumor-derived, GUR017M, 3D culture spheroid model and tested novel therapies based on its genomic profile. This tumor was obtained from a patient with mCRPC who progressed on enzalutamide, abiraterone, cabazitaxel, and had exposure to docetaxel, however discontinued due to toxicity. In order to characterize and test novel therapies for mCRPC, a 3-D spheroid drug assay was utilized and was able to recapitulate the GUR017M response to previous therapies (resistance to docetaxel and enzalutamide). Using a 3-D spheroid drug assay, single agents were investigated based on the GUR017M genomic profile which included cyclin D1, LYN, and FGF3/4/19 amplifications along with HRAS Q61R mutation, and TP53 splice site 672+2T>G. Single agents (abemaciclib, bafetinib, erdafitinib, tipifarnib, MK2206, APR-246) all caused dose dependent inhibition up to 100% inhibition. This rare prostate cancer model, GUR017M allows us to test novel single agents and combination strategies in 3-D culture. This patient tumor has also proven to be a successful PDTX model which allows for confirmatory testing in animal models in order to move forward with clinical trial proposals. Citation Format: Adam M. Kase, Winston Tan, John A. Copland. Characterization and targeted therapy using patient tumor-derived 3-D spheroids in a metastatic castrate resistant prostate cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2640.

Abstract The increase in fatty acid metabolism is a hallmark of cancer. Stearoyl CoA desaturase (SCD) is an endoplasmic reticulum (ER) enzyme that catalyzes D9 cis désaturation of fatty acyl-CoA substrates. One of its isoforms, SCD1, is overexpressed in several cancers including breast cancer. The higher expression of SCD1 in breast cancer patients correlates with significantly shorter relapse-free and overall survival rates, indicating that SCD1 may be a therapeutic target. We have previously developed and published a novel SCD1 inhibitor, SSI4. Metabolic reprogramming facilitates tumor growth by promoting immune suppression in tumors. Fundamental metabolic processes like fatty acid (FA) metabolism are involved in T cell activation and differentiation and their modulation can differentially affect the development of T helper cell lineages. FA synthesis and oxidation are essential for the development and functioning of CD8+ T memory cells and are contributing factors that influence CD4+ T effector and Treg cell development. These associations between metabolism and immune cells lead us to our hypothesis that aberrant de novo lipogenesis is linked to immune suppression. Thus, SCD1 inhibitors should increase anti-tumor immunity. We evaluated the effect of SSI4-mediated SCD1 inhibition in two mouse triple negative breast cancer cell lines. The data showed that SSI4 stimulates calreticulin (CRT) translocation to cell membrane. CRT is an ER resident protein that translocates to the cell surface under ER stress conditions. Cell surface expression of CRT is known to induce immune responses including phagocytosis, translocation of cancer antigen to the cell surface, and apoptosis. We are currently studying the effect of CRT translocation in our model. A major focus of our project is to assess the efficacy of combination treatment of SSI4 and an immune checkpoint blockade. To this end, this combination therapy synergistically blocked E0771 tumor growth in vivo. Further, to understand the immune responses generated, we performed immunohistochemical analysis of the tumors (day 20 post-treatment) indicating that the combination therapy resulted in higher membrane localized CRT, supporting our rationale that CRT translocation is involved in mediating the anti-tumor responses. Flow cytometry analysis indicated that the combination treatment increased immune cell infiltration. Specifically, SSI4 treatment induced infiltration of double negative T cells, natural killer cells and macrophages, suggesting that it promoted a pro-inflammatory immune response. SSI4 treatment down regulated immune suppressive cells including Tregs, dendritic cells and mesenchymal derived stem cells. The response of cancers with low and intermediate tumor mutational burden such as breast cancer to immune checkpoint inhibition is limited. Our data indicates that combination treatment of SSI4 and anti PD1 is a promising therapeutic strategy. Citation Format: Sneha Vivekanandhan, Justyna Trynda, Laura A. Marlow, Barath shreeder, James L. Miller, Adam M. Kase, Winston Tan, Keith L. Knutson, John A. Copland. SCD-1 blockade sensitizes triple negative breast cancer to immune checkpoint inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1484.

Background: While NKX2-5 plays pivotal roles in human cardiac development and disease, no ChIP-Seq studies of NKX2-5 in human cardiac tissues currently exist, resulting in an incomplete understanding of its direct gene targets. Modern computational methods which identify binding sites using transcription factor motif and tissue-specific experimental data can help to fill this knowledge gap. Objective: To use computational methods to identify likely NKX2-5 binding sites and downstream gene targets using human heart-specific experimental data. Methods: Human cardiomyocyte DNAse hypersensitivity data (2 replicates) were downloaded from the Encyclopedia of DNA Elements (ENCODE) database. The position weight matrix (PWM) representing the transcription factor motif of NKX2-5 was obtained from JASPAR. We applied the Protein Interaction Quantification (PIQ) algorithm to detect NKX2-5 binding sites using the PWM and DNAse hypersensitivity data as inputs. RNA-Seq data from 108 human heart-specific samples (atrial appendages and left ventricles) were downloaded from the Genotype-Tissue Expression (GTEx) database. Protein-coding genes significantly expressed in the heart (RPKM <= 1 based on the GTEx RNA-Seq data) that were within 100kb of the predicted binding sites were then identified. Pathway analysis of these genes was performed using Ingenuity Pathway Analysis (IPA). Results: 1283 binding sites for NKX2-5 were discovered by PIQ. Of 12698 protein-coding, heart-expressed genes, 625 were within 100kb of these binding sites. The identified genes were highly enriched in physiologic categories like “Vasculogenesis” and “Development of Cardiovascular Tissue” (p = 2.36x10-9 and 2.31x10-8 respectively). Notable genes included: cardiac transcription factors (MEF2A, TBX20); growth factors (TGFB2, BMP2); muscle and ion channel function (ACTA2, BIN1), and; calcium signaling (CALM2, CAMK2D). Conclusion: By using computational analyses of transcription factor motif and human heart-specific experimental data, we have identified candidate downstream targets of NKX2-5. Future work will include validation studies in an external cohort and analysis of associations between these candidate genes and cardiac disease.

Background Uterine leiomyomas, also known as fibroids (UF), are benign neoplasms of the uterus. UF consist of smooth muscle cells and fibroblasts, and display enriched extracellular matrix (ECM). Reports estimate a cumulative incidence of 70% in premenopausal women, with annual total health care costs attributed to UF exceeding 34 billion dollars in the United States alone. In spite of their prevalence, the underlying processes that drive UF development and growth are still poorly understood. Accumulating evidence implicates the canonical Hippo signaling pathway effectors Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding domain (TAZ) as key drivers of fibroblast biology. Thus, we seek to assess the differences in YAP/TAZ localization, ECM deposition, and gene expression between UF and normal myometrium (Myo). Methods Matched samples of UF and Myo were obtained from premenopausal women with IRB approval. Immunohistochemistry (N=5) was performed on OCT embedded tissue with YAP/TAZ antibody. For cell culture, UF and Myo were minced and digested with collagenase type 4. Cells (passages 1–4) were plated on plastic at confluence (N=4 for both). Immunostaining for YAP/TAZ, Fibronectin (FN), Collagen Type I (COL1), and Collagen Type III (COL3) were performed (N=4–5). Transcript levels were analyzed by treating cells with control or YAP/TAZ siRNA, and performing subsequent cDNA synthesis and qPCR reactions (N=4). Kruskall‐Wallis and Mann Whitney tests were used as appropriate. Results YAP/TAZ nuclear localization was upregulated in UF when compared to Myo tissue by immunofluorescence imaging. In freshly isolated cells cultured at confluence on tissue culture plastic, baseline YAP/TAZ nuclear localization was increased in UF when compared to Myo (p=0.03). UFs also exhibited a trend toward increased FN, COL1, and COL3 deposition when compared to Myo. UF displayed increased transcript levels for several matrix and pro‐fibrotic signaling genes (COL1A1, COL3A1, CTGF, EDN1, FN1) compared to the matched Myo. Following siRNA treatment for YAP/TAZ gene expression was reduced 40% and 50% for EDN1 (encoding endothelin‐1), as well as 70% and 65% for CTGF (encoding connective tissue growth factor), in UF and Myo respectively. Conclusions UF nuclear YAP/TAZ is increased at baseline in vivo as well as in vitro . UF also display upregulated deposition of the ECM proteins. Additionally, there are increased transcript levels of profibrotic genes in UF compared to Myo. Moreover, the observed decreases in gene expression following siRNA knockdown of YAP/TAZ suggests a role for the Hippo signaling pathway in regulation of certain profibrotic genes in UF. Together these results suggest that enhanced baseline YAP/TAZ localization in UF may drive upregulated expression of pro‐fibrotic genes promoting UF progression. Support or Funding Information Center for Biomedical Discovery‐Department of Obstetrics and Gynecology Team Science Pilot Award Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Objective To compare the prevalence and patterns of anomalous coronary artery origin from the opposite sinus (ACAOS) in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Methods Retrospective review of consecutive patients with surgically excised BAV and TAV was performed from 1994 to 2015. Clinical notes, echocardiograms, coronary angiograms, CT angiographies, and pathology reports were reviewed. ACAOS included right coronary artery from the left cusp, left circumflex artery from the right cusp and left main or left anterior descending artery from the right cusp. Results 2371 (years 1994–2015) and 1679 (years 2009–2015) consecutive patients with pathology-confirmed BAV and TAV, respectively, and defined preoperative coronary anatomy were identified. A left dominant coronary circulation was present in 386 (18%) patients with BAV and 179 (11%) patients with TAV (p<0.001). ACAOS was identified in 43 (1.8%) patients with BAV and 15 (0.9%) patients with TAV, p=0.02. Among patients with BAV and ACAOS, the most common phenotype was right-left fusion (n=34, 79%) with present raphe (n=36, 84%), with no association between BAV phenotype and ACAOS type. On multivariate analysis, BAV status and size of the mid-ascending aorta were independently associated with ACAOS (OR 3.29; CI 1.26 to 8.6; p=0.02; OR 0.93; CI 0.87 to 0.98; p=0.01; respectively). Only two patients with ACAOS, one with BAV and one with TAV, had a perioperative coronary ischaemic event. Conclusions The prevalence of the potentially malignant ACAOS is significantly higher (threefold higher odds) in patients with BAV as compared with TAV, yet remains uncommon in absolute terms. Most patients with BAV and ACAOS had right-left cusp fusion and present raphe. Perioperative coronary events are rare in patients with ACAOS.