Citations

Mitochondrial antioxidant genes are major regulators of oxidative stress in the vasculature. Previous data has shown that reducing antioxidant capacity accelerates atherosclerotic plaque size, however, it is unclear whether reducing mitochondrial antioxidant capacity alters plaque composition. Therefore, we tested the hypothesis that reductions of mitochondrial antioxidant capacity will increase osteogenic markers and intimal plaque calcification in aorta from hypercholesterolemic mice. We used Ldlr -/- ApoB 100/100 (LA) mice that were either wild-type (LA-MnSOD +/+ ) or heterozygous (LA-MnSOD +/- ) for manganese superoxide dismutase that were fed a Western Diet (TD88137) for 3 or 6 months. Changes in changes gene expression were assessed in aortic arch using qRT-PCR, plaque calcium levels were examined using histological evaluation from aortic cryosections, and endothelial function was measured using isolated organ bath chambers ( ex vivo ). While expression of SP7 (an osteogenic transcription factor) was unchanged between LA-MnSOD +/+ (1.0±0.6) and LA-MnSOD +/- (0.95±0.3) at 3 months, SP7 levels were dramatically increased in LA-MnSOD +/+ (8.0±2.8) at 6 months and increased further in LA-MnSOD +/+ mice (17.5±5.9; p<0.05). Alizarin Red staining for calcium showed increased calcium burden in plaques of 6 month LA-MnSOD +/- compared to LA-MnSOD +/+ (3636±669 pixels versus 649±206 pixels; p<0.05). Vasomotor function in aorta was unchanged between LA-MnSOD +/+ and LA-MnSOD +/- mice, suggesting that the transcriptional and functional changes related to calcification in LA-MnSOD +/- mice occur independently of nitric oxide signaling. In conclusion, our data strongly support our working hypothesis that reductions in mitochondrial antioxidant capacity significantly accelerate osteogenic calcification in aorta from hypercholesterolemic mice, and suggest that targeting mitochondrial oxidative stress may be a viable therapeutic strategy to slow vascular calcification.

Cardiovascular calcification is often an active process involving re-differentiation of cells to an osteoblast-like phenotype, and is strongly associated with aging. Sirtuins are histone and protein deacetylases that play a critical role in a number of age-related diseases, and SIRT6-deficient mice have the most profound progeroid phenotype. The role of SIRT6 in the regulation of cardiovascular calcification, however, is poorly understood. We hypothesized that age-related reductions in sirtuins promote osteogenic differentiation in cells from aortic valve and aorta. We used aortic valve interstitial cells isolated from SIRT6-deficient mice, pigs, or humans, and vascular smooth muscle cells from mouse aorta to determine whether osteogenic differentiation of cardiovascular cells is regulated by SIRT6 levels. In line with our hypothesis, we found increased p-SMAD1/5/8 levels in cells from SIRT6-deficient aortic valve and aorta. Second, global SIRT inhibition with NAD+-depletion increased histone acetylation and protein levels of the osteogenic proteins Sp7 and Runx2. Third, inhibition of SIRTs enhanced the transcriptional responses of Sp7 and Runx2 when cells were treated with BMP2. Fourth, knockdown of SIRT6 in mouse and human aorta and aortic valve cells by RNA interference increased Sp7 expression. Finally, chronic pharmacological inhibition of SIRTs promoted calcified nodule formation and calcium deposition in cultured mouse/swine aortic valve cells. Collectively, our data suggest that reductions in SIRT6 levels favor amplification of pro-osteogenic signaling and osteogenic differentiation of cardiovascular cells and promote the formation of calcified nodules in vitro . These data also suggest that sirtuin re-activation may be a useful therapy to slow initiation and/or progression of cardiovascular calcification in vivo.

Thrombin plays a major role in coagulation cascade and participates in regulation of vascular function through its effects on protease-activated receptors (PARs). The effect of thrombin inhibition on vascular and aortic valve function during prolonged hypercholesterolemia, however, remains unknown. In the present study, we tested the hypothesis that direct thrombin inhibition (DTI, dabigatran etexilate) slows progression of endothelial and aortic valve dysfunction in ldlr-deficient, apolipoprotein B100-only (LA) mice. Carotid arteries from LA mice were used for studies of endothelial function in isolated organ baths, and aortic valve function was evaluated using echocardiography. In placebo-treated mice, maximum relaxation to acetylcholine was reduced following 6 and 9 months of hypercholesterolemia (maximum relaxation to acetylcholine (MR ACH ) =75%± 3.7, and 67%± 3.6, respectively), but not affected by DTI (75%± 2.5, and 62%± 3.2, respectively). Relaxation to exogenous NO donors was also unaffected by DTI. Aortic valve function progressively deteriorated at 3, 6, and 9 months in placebo-treated mice (2.1 m/s± 0.04, 2.5 m/s± 0.1, and 2.9m/s ± 0.1, respectively), and was not affected by DTI (2.0m/s± 0.1, 2.5m/s± 0.1 and 3.0m/s ± 0.4, respectively). While PAR receptor expression was detected in aorta and aortic valve tissue, expression of SPHK1 (an indicator of PAR1 activity) was not altered by DTI. Expression of CTGF in aortic valves, however, tended to be reduced by DTI. In conclusion, our data suggest that DTI does not prevent vasomotor or aortic valve dysfunction in mice with prolonged hypercholesterolemia, but may attenuate pro-fibrotic signaling in aortic valves.

CD36 is a microvascular endothelial cell (MVEC) receptor mediating angiostatic activity of TSP-1 and related proteins. We previously reported that lysophosphatidic acid (LPA), a biologically active lipid signaling mediator, inhibited MVEC CD36 transcription via PKD-1 signaling. Moreover, CD36 transcriptional repression abolished endothelial cell responses to TSP-1 in vitro and in vivo . We now show with luciferase transfection assays that LPA exposure significantly suppressed CD36 promoter activity in MVEC. Co-exposure to class II HDAC inhibitors SAHA or TSA reversed the inhibition of promoter activity and restored transcription. HDAC7 knockdown by shRNA also attenuated LPA-suppressed CD36 promoter activity and mRNA levels. Mechanistically, FoxO1 was found to directly interact with putative FoxO1 binding sites (FHRE) in proximal promoter in vitro by avidin-biotin-conjugated DNA-binding assay, and in vivo by chromatin IP assay. Furthermore, FHRE mutations significantly attenuated the promoter activity. Western blots and immunofluorescence microscopy showed increased nuclear accumulation of PKD-1, FoxO1 and HADC7 after MVEC were exposed to LPA. Co-IP of nuclear extracts showed a physical interaction of HDAC7 with FoxO1 that was attenuated with PKD-1 knockdown. These data demonstrate that HDAC7 modulation by LPA receptor/PKD-1 signaling pathway represses FoxO1-mediated CD36 transcriptional activity. Additionally, angiogenic transcription profiling of endothelial cells revealed significantly increased mRNA expression of 17 angiogenic genes in response to LPA, including ephrin B2 expression that was confirmed by Western Blots. Transduction of constitutively active PKD-1 (PKD-CA) into EC also increased ephrin B2 expression. Functionally, PKD-CA overexpression promoted branching morphogenesis of arterial endothelial cells in a three-dimensional spheroid assay. The data indicate that PKD-1 is a potent angiogenic and arteriogenic kinase in EC by down-regulating CD36 transcription and promoting arteriogenesis. PKD-1-FoxO1-CD36 signaling axis may have potential as an important target in cardiovascular ischemia and malignant tumors.

Introduction While progression of calcific aortic valve stenosis (CAVS) is known to be strongly associated with activation of pro-inflammatory and pro-osteogenic pathways, effective treatments for CAVS have remained elusive. TNF-α is a pleiotropic inflammatory cytokine and is upregulated in patients with CAVS. Hence, we hypothesized that TNF-α inhibition, using the neutralizing antibody infliximab, can slow the progression of CAVS in vivo . Methods We used Ldlr -/- Apob 100/100 mice, which develop robust CAVS in an age-dependent manner, as a model. Mice were fed a Western-type diet for 6 months, then randomized into one of two groups: 1) twice-weekly infliximab injections (10 mg/kg intraperitoneally; n=10), and 2) saline injections (n=9). Aortic valve function was evaluated using echocardiography, and gene expression in aortic valves was measured using quantitative real-time PCR. Data are presented as mean ± SEM. Results During the 3 months preceding treatment initiation, aortic valve cusp separation distance decreased progressively in both groups (reduction by 0.082±0.011 mm in control group, 0.095±0.016 mm in infliximab group; p =0.27). Three months after initiation of treatment, cusp separation continued to decrease in control mice (by 0.108±0.021 mm), while progression of valve dysfunction was slower by 42% in the infliximab group (reduction of 0.063±0.009 mm; p =0.03 vs control). Expression of the pro-inflammatory genes Vcam1 and Nos2 was significantly reduced in valves of the infliximab group (by 37% and 60%, respectively). Moreover, endothelium-dependent vasodilation was improved by infliximab (improvement in maximum relaxation to acetylcholine in infliximab group 18±6% compared to control). Conclusions This study is the first to report that in vivo inhibition of TNF-α slows progression of CAVS in mice with established valve disease. This beneficial effect was accompanied by reduction in pro-inflammatory gene expression and improvement of systemic endothelial function, suggesting infliximab may exert its effects in part by increasing nitric oxide bioavailability. The findings warrant further investigation into the role of TNF-α in initiation and progression of CAVS, as well as its potential as a therapeutic target in human CAVS.

Increases in mitochondrial reactive oxygen species (ROS) can contribute to endothelial dysfunction in conduit arteries by reducing nitric oxide bioavailability. SIRT3 is a protein deacetylase localized to mitochondria and regulates activity of numerous electron transport chain enzymes, and can be inactivated by increases in mitochondrial ROS. Whether reductions in SIRT3 are an independent contributor to endothelial dysfunction, however, are unknown. Therefore, we hypothesized that SIRT3 null mice (SIRT3 -/- ) will have impaired relaxation to acetylcholine (MR Ach ) compared to wild-type littermates (SIRT3 +/+ ), and that this difference will be exacerbated by Western diet feeding for 3 months (TD88137, 42% of kcal from fat, 0.2% cholesterol). Endothelial function was measured using isolated organ bath chambers, and endothelium-dependent (MR Ach ) and endothelium-independent (sodium nitroprusside, MR SNP ) relaxation were measured. In contrast to our hypothesis, MR ACH was virtually identical between SIRT3 +/+ and SIRT3 -/- fed a standard chow diet (MR ACH = 68±2% and 69±3%; respectively), and MR SNP also remained unchanged between the two genotypes. In groups fed a Western diet, however, endothelium-dependent relaxation was significantly impaired in SIRT3 -/- mice compared to SIRT3 +/+ littermates (MR ACH = 49±4% and 58±3%, respectively; p<0.05). MR SNP was not altered by either Western diet feeding or SIRT3 deficiency. Interestingly, impairments in MR Ach in SIRT3 -/- mice fed a Western diet were not rescued with acute incubation of apocynin (an NAD(P)H oxidase inhibitor) or mito-tempol (a mitochondria-targeted antioxidant), suggesting that chronic antioxidant and/or acetyltransferase inhibition may be required to improve vasomotor function in this model. Collectively, our data suggest that SIRT3 plays a major role in regulating endothelial function with dietary stress, and may be a useful therapeutic target to reduce cardiovascular risk.

Abstract Background: Survival advantages with neoadjuvant chemotherapy (NACT) have been observed in patients (pts) with locally advanced breast cancer (BC) who achieve pathological complete response (pCR). Predicting risk of early recurrence in pts without pCR remains difficult. Circulating tumor DNA (ctDNA) may be a biomarker of neoadjuvant therapy response and recurrence. This study applied tumor genotyping of peripheral blood in pts with HER2− tumor BRCA-mutated (tBRCAm) BC treated with neoadjuvant niraparib. The relationship between ctDNA and tumor response was also characterized, with a focus on circulating TP53 mutant allelic frequency (MAF). Methods: Blood was collected from 21 pts enrolled in the study (NCT03329937) evaluating neoadjuvant niraparib in pts with localized HER2− tBRCAm BC. Pts received niraparib 200 mg once daily for two 28-day cycles. After 2 cycles, pts underwent surgery or received up to 6 cycles of niraparib and/or NACT prior to surgery. Blood samples were collected at screening, Cycle 1 Day 28 (C1D28), Cycle 2 Day 28 (C2D28), and pre-surgery. Ultrasound (USG) was done after each cycle and MRI after Cycle 2. Next-generation sequencing of cell-free plasma DNA was carried out using a proprietary target capture and analysis (Paweletz, Clin Cancer Res 2016). Results: ctDNA and TP53 mutations (TP53m) were detected at screening in 10/21 pts (47.6%). Across all 21 pts there was a correlation between TP53 MAF and baseline tumor stage (T) (mean [standard deviation], T1 0 [0], T2 0.05 [0.09], and T3 0.21 [0.12]; P=0.005). Linear regression modelling showed a positive correlation between tumor volume and baseline TP53 MAF (R2=0.206, P=0.0386). Baseline TP53 MAF also appeared to be associated with advanced disease stage (Table 1). Depletion of detectable TP53m from baseline was evident by C1D28 and persisted to pre-surgery (Table 2). This corresponded with decreases in tumor volume at C1D28 and further decreases at C2D28. Of 11 pts analyzed, 6 pts with &gt;90% tumor volume decreases by MRI or USG at C2D28 had sustained TP53m depletion. Three pts had pCR; of these, 2 had sustained TP53m depletion (data unavailable for third pCR pt). Two pts with less robust tumor responses had increased pre-surgery TP53 MAF. Conclusions: We report the potential utility of ctDNA as a predictive biomarker for neoadjuvant niraparib response in pts with HER2− tBRCAm BC. Although sample size limited analysis of genes other than TP53, baseline ctDNA strongly correlated with tumor volume, and TP53 MAF was associated with tumor volume and trended towards association with disease stage. Longitudinal analysis of TP53 MAF suggested a correlation with niraparib response. Further study of ctDNA dynamics during NACT and correlation with pCR and long-term clinical outcomes is warranted. Funding: GlaxoSmithKline (GSK) study 213355; NCT03329937. Medical writing support was provided by Fishawack Indicia, part of Fishawack Health, funded by GSK. Table 1.Baseline TP53 MAF association with disease stage and subtypeTP53 MAFDisease Stage (I–III)Disease SubtypeStage IStage IIStage IIIHR+TNBC(n=8)(n=10)(n=3)(n=6)(n=15)Mean (SD)0.01 (0.02)0.07 (0.12)0.10 (0.05)0.02 (0.02)0.06 (0.10)HR, hormone receptor; MAF, mutant allelic frequency; SD, standard deviation; TNBC, triple-negative breast cancer. Table 2.TP53 MAF correlation with tumor volume decrease by ultrasound and MRIOutcomeScreeningCycle 1 Day 28 Cycle 2 Day 28 Pre-Surgeryn11997TP53 MAF, mean, (SD)0.09 (0.11)000.01 (0.02)n11112Percent change in tumor volume from baseline by MRI, mean (SD)-N/A*−72.6 (22.5)N/A*Percent change in tumor volume from baseline by ultrasound, mean (SD)-−60.8 (24.0)−80.6 (21.8)−95.9 (4.3)*MRI was only performed at Cycle 2 Day 28. MAF, mutant allelic frequency; MRI, magnetic resonance imaging; N/A, not applicable; SD, standard deviation. Citation Format: Ming Shan, Laura Spring, Minetta C Liu, Erika Hamilton, Hanna Irie, Cesar A Santa-Maria, Steven J Isakoff, James Reeves, Leif W Ellisen, Lee P Lim, Kavita Garg, Caterina Bertucci, Bin Feng, Hailei Zhang, Kaiming Sun, Julie R Graham, Erin Hofstatter, Hyo Han. Relationship between circulating tumor DNA and response to neoadjuvant niraparib in HER2-negative, BRCA-mutated breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-21.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Although combined treatment with gemcitabine and albumin-bound paclitaxel has improved the prognosis of PDAC, both intrinsic and acquired chemoresistance cloud the outcomes. Hence, there is an urgent need to explore new therapeutic targets and formulate innovative strategies for the more effective treatment of PDAC.Methods: We transduced pancreatic cancer cells with a protein kinase library, and transfected cells were selected by puromycin treatment, followed by continuous exposure to vehicle control or gemcitabine/paclitaxel chemotherapy for 21 days. Next-generation sequencing as well as MAGeCK-VISPR analysis were performed to the identify candidate genes. Finally, we used the CRISPR-Cas9 to knockout the target gene, confirming that it is a viable target for overcoming chemoresistance in vivo and in vitro.Results: We identified several cell cycle checkpoint kinases and DNA damage-related kinases as targets for overcoming chemoresistance. Among them, cyclin-dependent kinase 7 (CDK7) ranked highly in both screenings. We demonstrated that both gene knockout and pharmacological inhibition of CDK7 by THZ1 result in cell cycle arrest, apoptosis induction. Furthermore, THZ1 synergized with gemcitabine and paclitaxel in vitro and in vivo, resulting in enhanced antitumor effects.Conclusions: Our findings support the application of CRISPR-Cas9 screening in identifying novel therapeutic targets and provide new strategies for overcoming chemoresistance in pancreatic cancer.

518 Background: Cisplatin (C) based neoadjuvant chemotherapy (NAC) has been the mainstay treatment for muscle invasive bladder cancer (MIBC). However, the optimal choice of NAC is not well-established. We therefore conducted a network-meta-analysis (NMA) to assess comparative efficacy of different treatment options. Methods: Several electronic databases (MEDLINE, and EMBASE) and conference proceedings were systematically used to identify randomized trials evaluating first-line treatments in neoadjuvant MIBC. Outcomes of interest included overall survival (OS), progression-free survival (PFS), objective response rate (ORR) and pathologic complete response (pCR). DerSimonian-Laird random-effects meta-analysis was used to compute direct comparisons between NAC and local therapy (LT) only. Fixed effect NMA was conducted within the frequentist framework for mixed treatment comparisons. P-scores were used to assess relative treatment rankings. All statistical analyses were conducted in R (v 4.1.1). Results: This systematic review included 23 trials (25 references) with a total of 5313 patients with 10 unique treatments. Direct comparisons showed significant benefit in PFS (HR: 0.82; 95% CI: 0.71-0.94), and in OS (HR: 0.85; 95% CI: 0.78-0.92) with NAC when compared to LT only. However, the likelihood of achieving an objective response (RR: 1.18; 95% CI: 0.72-1.95: I2: 85%) or CR (RR: 1.51; 95% CI: 0.87-2.61; I2: 79%) was not different between the two arms. Data available in 15 trials contributed to the network for PFS outcome while only eight trials contributed to OS outcome. Mixed treatment comparisons showed significantly improved PFS with nintedanib-gemcitabine-cisplatin (NinGC; HR: 0.42; 95% CI: 0.20-0.87) and dose-dense-methotrexate-vinblastine-doxorubicin-cisplatin (ddMVAC; HR: 0.61; 95% CI: 0.43-0.88) compared to LT only. Consistent benefit was observed when NinGC (HR: 0.48; 95% CI: 0.24-0.97) and ddMVAC (HR: 0.70; 95% CI: 0.51-0.96) were compared to GC only. However, no significant differences were observed between NinGC (rank 1) and ddMVAC (rank 2) or among other mixed treatment comparisons. Similarly, OS was significantly improved with NinGC (rank 1) relative to MVAC (HR: 0.41; 95% CI: 0.17-0.97), MVC (HR: 0.37; 95% CI: 0.16-0.88), MC (HR: 0.38; 95% CI: 0.16-0.92), AC (HR: 0.36; 95% CI: 0.15-0.91), and GC (HR: 0.39; 95% CI: 0.17-0.90). While similar results were observed with ddMVAC (rank 2) when compared to MVC (HR: 0.63; 95% CI: 0.42-0.94), MC (HR: 0.64; 95% CI: 0.42-0.98), and GC (HR: 0.66; 95% CI: 0.47-0.92), no significant differences were observed with ddMVAC when compared to MVAC (HR: 0.62; 95% CI: 0.38-1.01) and NinGC (HR: 1.70; 95% CI: 0.69-4.19). Conclusions: Current evidence shows improved PFS and OS with the use of neoadjuvant ddMVAC and Nin-GC in patients with MIBC relative to other treatment options.

481 Background: EV is an anti-Nectin-4 antibody-drug conjugate that has been approved by the US FDA for mUC progressing on platinum and PD1/L1 inhibitor therapy and in cisplatin-ineligible pts following prior therapy. However, prognostic and predictive biomarkers in the setting of EV therapy are unclear. We aimed to investigate baseline and early changes in commonly available clinical and laboratory tests that could help predict survival and toxicities in patients with mUC treated with EV. Methods: Data from patients with mUC who were treated with EV at Dana Farber Cancer Institute between 2017 and 2021 were reviewed retrospectively. We assessed the association of demographic (age, sex), clinical (ECOG-performance status [PS], sites of metastasis) and laboratory variables (hemoglobin, RDW, neutrophil count, lymphocyte count, neutrophil-to-lymphocyte ratio, platelet count and serum albumin) at baseline as well as after 4-7 weeks of treatment with Overall Survival (OS) by constructing univariate and multivariable Cox proportional-hazards models. Any variables significantly associated with OS at the univariate level (using an alpha-level of 0.1 to determine statistical significance) were then entered into the multivariable model. Predictors were eliminated from the final multivariable models by a backward selection process, retaining only those with a p-value of ≤ 0.05. Results: A total of 49 patients who received EV were included in the analysis. The median age was 72 years (range 48-88) and 36 (73.5%) were male. The median OS was 13.3 months (range: 2.7-38.7) and grade 3-4 toxicities were seen in 11 pts (22.4%). In the final multivariable models, lower serum albumin at 4-7 weeks (HR = 0.18; 95% CI = 0.05-0.71; p = 0.015), and baseline ECOG-PS of 1 vs. 0 (HR = 2.56; 95% CI = 1.05-6.21; p = 0.038) were significantly associated with worse OS (Table). Patients with higher serum albumin levels at 4-7 weeks also had significantly lower odds of experiencing grade 3-4 adverse events on EV (OR = 0.05; 95% CI = 0.002-0.52; p = 0.02). Conclusions: Lower serum albumin appeared to predict worse survival and severe toxicities in mUC pts receiving EV. This information may guide therapy and assist in the prevention of toxicities. Additionally, ECOG-PS was prognostic while the location of metastatic disease was not prognostic in the setting of EV.[Table: see text]