Citations

ABSTRACT Knowledge of Treponema pallidum subspecies pallidum ( TPA ) outer membrane protein (OMP) sequence variability is essential for understanding spirochete proliferation within endemic populations as well as design of a globally effective syphilis vaccine. Our group has identified extracellular loops (ECLs) of TPA BamA (TP0326) and members of the FadL family (TP0548, TP0856, TP0858, TP0859, and TP0865) as potential components of a multivalent vaccine cocktail. As part of a consortium to explore TPA strain diversity, we mapped the variability of BamA and FadL orthologs in 186 TPA strains from Malawi, China and Colombia onto predicted 3D structures. The 186 genomes fell into eight subclades (five Nichols-, three SS14-lineage) with substantial geographic restriction. Single nucleotide variants accounted for the large majority of proteoforms, with variability notably higher within the Nichols-lineage strains. Most mutations were in regions of the proteins predicted to be extracellular and harboring B cell epitopes. We observed a striking difference in the degree of variability between the six OMPs, suggesting that these proteins are following divergent evolutionary paths. Concatenation of OMP sequences recapitulated the phylogenetic structure of the TPA strains, effectively segregating within clades and largely clustering by subclades. Lastly, we noted that BamA and FadL candidate ECL vaccinogens previously shown to elicit antibodies that kill treponemes during in vitro cultivation are well conserved. Taken as a whole, our study establishes a structural-phylogenetic approach for analyzing the forces shaping the host-pathogen interface in syphilis within endemic populations while informing selection of vaccine targets. IMPORTANCE Syphilis remains a major global health concern, reinforcing the need for a safe and effective vaccine. Understanding the variability of TPA OMPs is essential for tracking pathogen evolution and informing vaccine design. Here, we analyzed the variability of six TPA OMPs in 186 strains from Malawi, China, and Colombia, identifying protein-specific evolutionary patterns. Most mutations were localized in extracellular regions and, notably, appeared to correlate with the phylogenetic structure of TPA . Despite OMP heterogeneity, several candidate vaccinogens remained highly conserved, reinforcing their potential as globally effective vaccine targets. Our study establishes a structural-phylogenetic framework for dissecting the forces shaping the host-spirochete interface within endemic populations and provides a foundation for designing a globally effective syphilis vaccine.

Abstract Poly ADP ribose polymerase inhibitors (PARPi) are being investigated in combination with photon and proton radiotherapy in clinical trials of pediatric and adult malignancies. The precise mechanisms of PARPi radiosensitization, and whether differences in photon and proton-mediated relative efficacy exist, have not been fully elucidated. To study mechanisms of PARPi radiosensitization we investigated the effects of PARPi in combination with photons and two different ionizing densities of protons delivered at the entrance (ENT) or the Bragg peak (BP) of the profile (dose-averaged LET [LETd] 2.2 and 7.0 keV/µm, respectively) of a 76.8 MeV proton beam after observing an extreme tumor and normal tissue response in an 18-year-old female patient with high grade glioma treated veliparib and proton therapy in a Children’s Oncology Group clinical trial. BRCA1/2 wild-type hTERT RPE-1, U2OS, and MDA-MB-231 cells were hypersensitive to the combination of veliparib and proton BP, with enhanced relative biological effectiveness (RBE) at 2 Gy values of 1.7, 1.4, and 1.3, respectively. Similar findings were observed with talazoparib and olaparib, suggesting that PARP trapping efficiency was not a major influence on the proton BP RBE. Further, we did not observe differences in transcription replication conflict between the different proton ionizing densities. Unexpectedly, DNA fiber assays revealed more elongated replication fork tracks in PARPi treated cells 60 minutes after proton BP compared with proton ENT, indicative of accelerated replication fork progression. To assess gap formation in the vicinity of nascent DNA we treated cells with the ssDNA-specific S1 nuclease and found that the lengths of CldU-labeled tracks were significantly reduced in cells treated with the PARPi/proton BP combination, suggestive of greater gap formation at replication forks. We postulated that more ssDNA gaps were being converted to DSBs during replication as we further observed that PARPi/proton BP resulted in increased γH2AX foci at 4- and 24-hours after treatment. Consistent with increased DSB formation, a higher proportion of cells arrested in the G2/M phase of the cell cycle at 24 hours following PARPi/proton BP. Despite being arrested in G2/M when homologous recombination is active, there was a significant increase in 53BP1 and a decrease in RAD51 recruitment, indicative of upregulated repair by more error-prone non-homologous end-joining (NHEJ) repair pathway. The enhanced proton BP RBE in PARPi treated cells was abrogated upon knockdown of XRCC4, a critical component of NHEJ repair pathway, suggesting that toxic NHEJ may be contributing to the enhanced proton BP RBE. In conclusion, PARPi renders cells hypersensitive to high LET BP proton irradiation. Enhancing the LET in combination with PARPi warrants further investigation to overcome therapeutic resistance. Citation Format: Yiqun Han, Qin Zhou, Anna Olson, Cameron M. Callaghan, Bin Chen, Nicholas B. Remmes, Meng Xu Welliver, Zheming Wu, Daniel K. Ebner, Jake A. Kloeber, Zhenkun Lou, Robert W. Mutter. Poly ADP ribose polymerase inhibitors potentiate proton therapy end-of-range relative biological effective dose by increasing replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4689.

Calcific aortic valve disease (CAVD) is a highly active process, and upregulation of osteogenic signaling is thought to be a major driver to initiation and progression of disease. While previous reports by our group and others demonstrated dysregulation of antioxidants and pro-oxidants in calcified aortic valves, it remains unclear if loss of antioxidant is a causal event driving valvular calcification and dysfunction. We hypothesized that MnSOD deficiency—an essential mitochondrial antioxidant—would result in accelerated reductions of cusp separation distance, increased osteogenic signaling, and augmented calcium deposition in a hypercholesterolemic mouse model of CAVD. We used ldlr -/- / ApoB 100/100 (LA) mice that were either wild-type (LA-MnSOD +/+ ) or MnSOD-deficient (LA-MnSOD +/- ), fed a Western Diet for 3 or 6 months. We used qRT-PCR and immunohistochemistry to measure changes in pro-osteogenic signaling, histopathological analyses of calcium burden (Alizarin Red), and echocardiography to assess aortic valve function. BMP2—a pro-osteogenic marker—increased over time with Western diet feeding, though MnSOD-deficiency did not exacerbate these increases. mRNA levels of pro-osteogenic markers (Runx2, Msx2) were also unchanged with either time on diet or with experimental reduction of MnSOD. Surprisingly, protein levels of late- stage osteoblast marker, osterix, were modestly decreased at the three month time point in in LA-MnSOD +/- mice compared to age-matched wild-type littermates. Consistent with our hypothesis, however, loss of mitochondrial antioxidant capacity substantially increased osterix protein levels and calcium burden at the 6 month time point compared to age-matched wild-type littermates. Functionally, loss of mitochondrial antioxidant capacity did not alter aortic valve cusp separation distance at both 3 and 6 month time points. Collectively, these data suggest that loss of MnSOD does not alter disease initiation but instead augments osteogenic signaling and valvular calcification in established aortic valve disease. Future studies aimed at targeting mitochondrial oxidative stress could elucidate novel strategies to slow progression of established CAVD.

Marfan Syndrome (MFS) is often associated with progressive aortic root dilation, ultimately leading to aortic aneurysm and dissection. While recent work showed increased transforming growth factor beta (TGF-β) signaling contributes to aneurysm formation in aorta, efficacious therapeutic targets remain elusive. Consistent with prior reports of progeroid phenotypes in a subset of Marfan patients we previously demonstrated increased cellular senescence in a MFS mouse model. Here, we sought to determine whether administration of senolytic drugs reduces cellular senescent markers, TGF-β signaling, and aortic root dilation in aged MFS mice by assessing aortic root dimensions by echocardiography and gene expression levels by quantitative real time PCR at 15 months of age following treatment with vehicle (VEH) or the senolytic compound Fisetin for one month. As expected, aortic sinus dimensions were increased dramatically in fibrillin-1 mutant mice compared to wild-type controls and were associated with increased expression of TGF-β1, the senescence marker CDKN2A, the matrix remodeling enzyme MMP2, and the osteogenic and proliferative gene RUNX2. Consistent with previous data from our group, Fisetin treatment dramatically reduced CDKN2A expression in marfanoid mice compared to VEH, which was associated with reductions in TGF-β1, MMP2, and RUNX2. Phenotypically, we observed modest reductions in aortic sinus dimensions compared to VEH, but no substantive change in ascending aortic dilatation. Collectively, these data suggest that senescent cells are significant contributors to molecular changes occurring in MFS and clearance of senescent cells with Fisetin slows the progression aortic root-dilation in fibrillin-1 mutant mice.

Perivascular adipose tissue releases inflammatory paracrine factors that accelerate development of vascular dysfunction, atherosclerosis, and large vessel diseases such as abdominal aortic aneurysm. Our group has shown that chronic hypercholesterolemia and chronological aging both significantly increase senescent cell burden in vascular and endothelial cell populations of large arteries, but the effects of chronic hypercholesterolemia on senescent cell burden in perivascular adipose tissue—as well as the biological impact of reducing those senescent cells using pharmacological or genetic interventions—is poorly understood. Here, we sought to investigate the effects of chronic, intermittent senescent cell clearance by treating mice with Fisetin—a flavenoid shown to be a potent senolytic in multiple tissue types—and its potential synergies with risk factor mitigation. In brief, “Reversa” mice (ldlr-deficient, apoB100-only mice in which lipids can be lowered with a genetic switch) were fed a Western diet for 6 months, at which point they were randomized to one of three groups for 2 additional months: 1) continued progression on Western Diet (WD), 2) risk factor mitigation/lipid lowering with a genetic switch (LL) or 3) weekly, intermittent treatment with Fisetin + lipid lowering (FIS+LL). Compared to WD mice, LL mice had modest reductions in perivascular adipose inflammatory markers such as IL-6, CCL2, and CD-68, as well as the pro-oxidant Nox2; however, LL mice experienced no impact on expression of the pro-inflammatory cytokine TNFalpha. Interestingly, while intermittent Fisetin treatment and lipid lowering in the FIS+LL group had a significant additive effect on reducing IL-6, Nox2, and CD68, these changes were associated with modest reductions in expression of p16 ink4a or p21 (both of which are markers of senescent cell burden). Collectively, these data suggest that Fisetin is likely to modestly reduce senescent cell burden in perivascular fat, while effectively synergizing with lipid lowering to reduce a subset of inflammatory markers in this tissue, which may ultimately have implications for management of multiple age-associated vascular diseases.

Activation of soluble guanylate cyclase (sGC), a key downstream target of nitric oxide (NO) signaling, is reduced in valve tissue from patients with fibrocalcific aortic valve stenosis (FCAVS). We hypothesized that restoring NO signaling pathway using Ataciguat (ATA)—an activator of oxidized sGC—would slow progression of aortic valve calcification. To determine whether Ataciguat attenuates bone morphogenetic protein (BMP2) signaling in vitro, we treated mouse aortic valve interstitial cells with exogenous BMP2(200ng/ml) with or without ATA(10nm), and demonstrated that ATA significantly reduced canonical BMP2 signaling by more than 50% (p< 0.01). To determine whether Ataciguat slows progression of FCAVS in vivo , ldlr -/- /apoB 100/100 mice were assigned to one of two groups:1) 9 months of western diet feeding (WD), or 2) 6 months of WD followed by 3 months of WD + ATA (5mg/kg ATA). In this model, Ataciguat attenuated BMP signaling in aortic valve tissue (p < 0.05), reduced aortic valve calcification (p < 0.05), and prevented progression of aortic valve dysfunction (p < 0.05). Finally, to determine whether ATA slows progression of calcification in humans, we treated patients with mild-to-moderate FCAVS with placebo or ATA (200 mg/day) for 6 months. In this Phase II clinical trial, ATA significantly slowed progression of aortic valve calcification (computed tomography imaging; p = 0.05) and also tended to slow progressive reductions in aortic valve area and valvular stenosis. Collectively, we believe this is the first study to demonstrate the pre-clinical and clinical utility of pharmacologically stimulating sGC to attenuate osteogenic signaling and slow progression of aortic valve disease in mice and humans, and creates a unique opportunity for larger trials focused on the effectiveness of this strategy to delay or prevent surgery in FCAVS.

While the strong connection between oxidative stress and atherosclerosis has many to hypothesize that antioxidant therapy may be viable therapeutic target in combating progression of atherosclerosis, numerous clinical trials of antioxidant interventions that scavenge multiple reactive oxygen species have been negative. Here, we test the hypothesis that overexpression of catalase—an enzyme which selectively degrades H 2 O 2 —plays a protective role against the initiation and expansion of calcific lesions in advanced atherosclerotic plaques. In vitro, we used aortic smooth muscle cells (SMC) from Ldlr -/- ApoB 100/100 (LA) mice that were either wild-type for human catalase (LA-Cat 0/0 ) or overexpressing human catalase (LA-Cat Tg/0 ). After treatment with BMP2 or TNFα for 24 hours, we found that catalase-overexpressing VSMCs had modest reductions in TNFα-induced inflammatory markers but paradoxical increases in mRNA levels of BMP2-induced osterix and the SMAD-specific target ID1. To determine if these effects were conserved in vivo, we analyzed aortic tissue from LA mice fed a Western diet for 3 or 12 months. Surprisingly—and unlike our cell-based experiments—aorta from LA-Cat Tg/0 tended to have higher expression of inflammatory markers compared to their LA-Cat 0/0 littermates. Similarly, expression of osterix was increased in LA-Cat Tg/0 mice compare to their LA-Cat 0/0 littermates, which was also associated with an increase in intimal plaque calcification. Collectively, these data strongly suggest that systemically overexpression of catalase (a close parallel to systemic administration of a selective H 2 O 2 -scavenging antioxidant) does not result in molecular, histopathological, or functional benefits in large arteries from hypercholesterolemic mice. Future studies aimed at characterizing the complex interplay between reactive oxygen species and specific intracellular signaling pathways are an essential next step to understanding the role of endogenous reactive oxygen species in the pathogenesis of atherosclerotic vascular disease.

Sirtuin-6 (SIRT6) is a protein deacetylase that functions in several pathways related to aging, as complete loss of SIRT6 in mice elicits multiple age-associated phenotypes and premature death. While increasing age is a major risk factor for development of calcific aortic valve disease (CAVD), it is unclear whether loss of SIRT6 accelerates aortic valve calcification. Here, we report the role of SIRT6 in a subset of aortic valve interstitial cell progenitors (neural crest-derived VICs) by crossing mice carrying Cre recombinase under the control of a PAX3 promoter with mice harboring a SIRT6 gene that can be conditionally inactivated (SIRT6 FL/+ ). We hypothesized that tissue-specific reductions of SIRT6 would increase calcification, increase pro-osteogenic signaling, and impair aortic valve function. We examined mice of the genotypes Cre-/SIRT6 FL/+ or Cre+/ SIRT6 FL/+ at two different time points: young (3 months) and old (14-24 months). First, we used qRT-PCR to demonstrate that SIRT6 was effectively reduced in the aortic valve from PAX3-cre/SIRT6 FL/FL mice. Although reductions in SIRT6 did not change aortic valve calcium (Alizarin Red) in young mice, reducing SIRT6 increased valvular calcium levels in old mice compared to their WT littermates. Interestingly, protein levels of osterix were substantially increased in young, SIRT6-deficient mice compared to WT littermates but were paradoxically decreased in old SIRT6-deficient mice. Regardless of age, histopathological, or molecular changes, we did not observe detectable changes in cusp separation distance at any time point. Collectively, these data suggest that, contrary to our hypothesis, depletion of SIRT6 in neural crest cells—which are key progenitors of valve interstitial cells—is not sufficient to impair aortic valve function. Importantly, this finding suggests that age-associated reductions in SIRT6 may be a permissive context for development of CAVD but relies heavily on the presence of additional risk factors.

ABSTRACTBackgroundThe treatment landscape for multiple myeloma continues to evolve. Recently, the addition of anti‐CD38 monoclonal antibodies (mAbs) to the triplet regimen, comprising a proteasome inhibitor, an immunomodulatory agent, and a steroid, for transplant‐eligible newly diagnosed multiple myeloma (TENDMM) has shown promising results.AimsTo evaluate the overall efficacy and safety of quadruplet therapy with an anti‐CD38 mAb compared to a triplet regimen.MethodsA systematic search of Medline, Scopus, and EMBASE databases from inception to July 2024 identified relevant randomized controlled trials (RCTs). Efficacy and safety outcomes were derived using random‐effects meta‐analysis. Summarized outcomes include hazard ratios (HR) for progression‐free survival (PFS) and overall survival (OS), odds ratios (OR) for response rates, measurable residual disease (MRD) negativity rate, and grade 3 or higher adverse events (G ≥ 3 AEs).ResultsFive RCTs involving 2963 patients were included. A statistically significant PFS was observed for quadruplet therapy when compared to the triplet regimen (HR 0.44; 95% Confidence Interval [CI] 0.35–0.56). PFS benefit was consistent for the standard risk (SR) group (HR 0.38; 95% CI 0.27–0.52) and high risk (HiR) group (HR 0.62; 95% CI 0.41–0.92). No statistically significant benefit was observed for OS (HR 0.55; 95% CI 0.28–1.08). A statistically significant benefit was observed for the overall response rate (OR 1.77; 95% CI 1.02–3.06) and MRD negativity rate (OR 2.67; 95% CI 1.79–3.99). No significant differences were observed for G ≥ 3 AE (OR 1.21; 95% CI 0.92–1.58), lymphopenia (OR 1.09; 95% CI 0.62–1.89), and anemia (OR 1.06; 95% CI 0.83–1.37). However, a significantly increased risk was observed for all‐grade thrombocytopenia (OR 1.64; 95% CI 1.37–1.97), neutropenia (OR 2.24; 95% CI 1.67–3.02) and infections (OR 1.88; 95% CI 1.07–3.31).ConclusionQuadruplet therapy demonstrated a favorable efficacy and safety profile, with consistent benefit across subgroups. The findings support its potential as the new standard of care for TENDMM.