Citations

Abstract Introduction: Heme-binding biguanides inhibit cytochrome P450 monooxygenase-mediated biosynthesis of the epoxy fatty acid (EpFA) (±)14,15-EET, which promotes progression of ER+HER2- breast cancer as well as regulatory T cell (Treg) proliferation. However, there are no known strategies to exploit this vulnerability. Metformin has shown promise but lacks sufficient potency and clinical utility. Through structure-based design, heme binding hexyl cubane derivatives of metformin were identified, which potently inhibited (±)14,15-EET biosynthesis. Fluorination of the hexyl moiety improved pharmacokinetics (PK) and reduced toxicity. Among the fluorinated derivatives, C5F2-HCB potently inhibited (±)14,15-EET biosynthesis (IC50=3.1 uM for C5F2-HCB vs. 5 mM for metformin). C5F2-HCB and metformin are candidate agents for TME activation from “cold” to “hot” due to their inhibition of EETs biosynthesis and EET-driven oxidative phosphorylation (OXPHOS) which promotes tumor hypoxia. We then tested whether C5F2-HCB or metformin could inhibit immune exclusion in the immunologically “cold” ovarian dependent STAT1 KO SSM2ucd mammary carcinoma model, in part, by reducing Tregs and/or increasing CD8 or CD4 tumor infiltrating T cells (TIL). We hypothesized that biguanides, by reducing EETs, would suppress Tregs and promote effector T cells in the TME. We also hypothesized that C5F2-HCB would be more effective than metformin in modulating the TME, based on greater potency for inhibition of (±)14,15-EET biosynthesis. Both biguanides were tested at their maximum tolerated dose (C5F2-HCB; 18 mg/kg daily and metformin; 50 mg/kg every other day). Results: In the SSM2ucd allograft, all combination treatment groups with biguanides reduced tumor growth relative to the IgG control at the endpoint (day 63) : metformin + IgG (51.5±9.8% of control), C5F2-HCB + IgG (53.3±12.8%), metformin + anti-PD-1 (50.4±11.5%), and C5F2-HCB + anti-PD-1 (49.6±12.8%) [(P < 0.05) for all comparisons]. In contrast, anti-PD-1 antibody alone failed to reduce tumor growth at the endpoint. Flow cytometry analysis of the TME revealed that only C5F2-HCB + IgG treatment increased CD8+ tumor-infiltrating lymphocytes (TILs) compared to all other treatment groups (>2-fold; P < 0.05). Additionally, only C5F2-HCB + anti-PD-1 reduced intratumoral Tregs (>3-fold; P < 0.05). C5F2-HCB in combination with IgG or anti-PD-1 reduced intratumoral macrophages by >50% (P < 0.001) and reduced the M2/M1 macrophage ratio by 32-61% (P < 0.05) relative to other groups. No effects on CD4+ T cells were observed. Furthermore, C5F2-HCB and IgG or anti-PD-1 increased intratumoral neutrophils by 75- 120% compared to the anti-PD-1 or IgG (P < 0.05). (Multiple comparisons tested by ANOVA). Conclusion: Relative to IgG alone, C5F2-HCB and metformin inhibited growth of the SSM2ucd allograft in combination with either anti-PD1 or IgG. In contrast to metformin, only C5F2-HCB modulated the TME, including increased CD8+ TILs with IgG, reduced Tregs with anti-PD-1, and decreased M2/M1 macrophage ratio with IgG and anti-PD-1. C5F2-HCB, in contrast tometformin, was effective in modulating an immune excluded TME and may contribute to a strategy for clinical translation. Citation Format: Zhijun Guo, Jianxun Lei, Joshua McCarra, Qing Cao, Michael J. Pryzbilla, Brenda Koniar, Beverly Norris, Robert J. Schumacher, Swaathi Jayaraman, John R. Hawse, Antonino B. D'Assoro, Ilia G. Denisov, Stephen G. Sligar, Kathryn L. Schwertfeger, Michael A. Farrar, Dipak Panigrahy, Hammock D. Bruce, Elizabeth A. Ambrose, Gunda I. Georg, Goetz P. Matthew, David A. Potter. C5F2-hexyl-(cuban-1-yl-methyl)-biguanide (C5F2-HCB) overcomes an immune excluded tumor microenvironment (TME) and suppresses tumor growth in the ovarian dependent ER+HER2- SSM2ucd mammary carcinoma allograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB010.

ImportanceUnderstanding the global barriers to otolaryngology–head and neck surgery (OHNS) care is crucial in addressing disparities in access to care, particularly in low-income and middle-income countries (LMICs).ObjectiveTo characterize barriers to comprehensive otolaryngology care across World Bank income groups.Design, Setting, and ParticipantsUsing an online cross-sectional survey that was administered by the Global OHNS Initiative via international and national professional societies, personal contacts, and social media, this study captured the perceptions of otolaryngologists regarding barriers to OHNS care at the levels of the country, health sector, clinician, and patient. Participants included otolaryngologists from the 194 World Health Organization member states and Taiwan. Eligibility criteria included a medical degree and specialized training in managing conditions of the ear, nose, and throat. The nonresponse rate was not recorded. The data collection period spanned from October 2022 to June 2023.ExposuresExposures included World Bank income group classification.Main Outcomes and MeasuresThe primary outcomes were Likert scale responses regarding frequency of barriers to OHNS care. The a priori hypothesis was that LMICs would report more frequent barriers to OHNS care.ResultsThe study involved 146 otolaryngologists (47 female individuals [32%]), with 69 (47%) from high-income countries and 77 (53%) from LMICs. Male individuals represented 45 high-income country respondents (65%) and 54 LMIC respondents (70%). Barriers were reported across income groups at all levels of the health system, with a higher frequency in LMICs. Several barriers were reported to be more substantial in LMICs, such as the national volume and distribution of otolaryngologists, financial compensation for clinicians, and patient stigma, and others were common across income settings, such as the availability of operating rooms and inflexible working hours. Common concerns included national and health sector barriers: OHNS workforce, referral networks, government support; clinician barriers: excessive clinician workloads, understaffing, poor administration support; patient barriers: distance to health care facilities, financial burdens such as medical costs and foregone wages, stigma, and health literacy.Conclusions and RelevanceThe results of this cross-sectional study suggest that there are barriers to otolaryngology care globally that affected all levels of the health care system. This work may inform prioritization of otolaryngology within research and policy, and it emphasizes the need for effective strategies to expand access to otolaryngology care, particularly in lower-income settings.

Background: Although it is well established that the type of organism can be a risk factor for failure in patients with periprosthetic joint infection (PJI), no study to date has examined the impact of the number of positive cultures on treatment outcomes in patients undergoing 2-stage exchange. The purpose of this multicenter study was to determine the prognostic utility of multiple positive cultures at resection as a predictor of failure following reimplantation. Methods: This retrospective multicenter study identified 437 patients with chronic knee PJI who had undergone 2-stage exchange arthroplasty with a minimum of 1 year of follow-up following reimplantation. PJI was defined with use of the 2013 Musculoskeletal Infection Society (MSIS) criteria. Patients with culture-negative PJI were excluded (n = 138). Treatment failure was defined as either any reoperation for infection or PJI-related mortality. Multivariable regression controlling for risk factors for failure after a 2-stage arthroplasty was performed to determine whether ≥2 positive intraoperative cultures at resection can predict outcomes following reimplantation when compared with a single positive culture. Results: Two hundred and ninety-nine patients were included. At a mean follow-up of 6.2 ± 2.6 years, 48 patients (16.1%) experienced failure. Patients who had a failure were more likely to have had a longer interstage interval (p = 0.038) and were also more likely to have had ≥2 positive cultures at the time of resection arthroplasty (95.8% versus 75.3%; p = 0.001). On regression analysis, ≥2 positive cultures at resection was the only variable that was identified as a risk factor for failure following reimplantation in both the univariate (odds ratio [OR], 7.55 [95% CI, 2.24 to 47.0]; p = 0.006) and multivariable models (OR, 8.12 [95% CI, 2.31 to 51.9]; p = 0.005). Conclusions: This is the first study to examine the impact of the number of positive cultures on outcomes in patients with PJI. We found that the presence of ≥2 positive cultures at resection was an indicator of a poor prognosis and resulted in a greater than eightfold increase in the risk of treatment failure in patients undergoing a 2-stage exchange. Level of Evidence: Prognostic Level III . See Instructions for Authors for a complete description of levels of evidence.

Abstract The range of Babesia microti (Franca, 1910)-infected ticks is expanding, resulting in locally acquired human babesiosis cases occurring in new areas: Maryland (2009), the District of Columbia (2013), Virginia (2016), and West Virginia (2017). We collected host-seeking ticks from old fields, ecotones, forested habitats and animal hosts in Delaware, Maryland, and Virginia, 2010 to 2024. Ixodes scapularis Say, the tick vector of babesiosis, was captured in all 3 states. PCR revealed B. microti in 2.7% (36/1310) of I. scapularis, with site prevalence ranging from <1% to 12.5% infected. The first B. microti-positive I. scapularis was collected in Northampton County, Virginia, 2012. Of the B. microti-infected ticks, 50% (18/36) were coinfected with Borrelia burgdorferi and one was triple-infected with B. microti, B. burgdorferi, and Anaplasma phagocytophilum. We collected Ixodes keiransi Beati, Nava, Venzal, and Guglielmone ticks from Delaware and Virginia. We found B. microti and B. burgdorferi in those from Virginia, and B. burgdorferi in ticks from a shrew in Delaware. To our knowledge, this is the first report of B. microti and B. burgdorferi-positive I. keiransi from Virginia, and the first report of B. burgdorferi-positive I. keiransi from Delaware. Ixodes keiransi ticks rarely bite humans but are involved in the maintenance and spread of pathogens when sympatric with I. scapularis. We tested a subset of both tick species for Babesia duncani; none were positive. Jurisdictions in the southern mid-Atlantic region should expect babesiosis cases, and Lyme disease and anaplasmosis coinfections, and healthcare providers should consider these tick-borne infections as part of the differential diagnosis.

Background. MicroRNAs are small, single-stranded non-coding RNA molecules that play major role in gene regulation. miRNAs have been reported as both tumor suppressive and oncogenic and are promising candidates for cancer prognosis and treatment. miR-494 is known to promote cell migration and invasion in CRC. Our study aimed to further explore the role of miR-494 modulation (inhibition and overexpression) in CRC. Methods. We evaluated the impact of tumor gene expression of MIR494 on outcomes (overall survival: OS; progression-free survival: PFS) in data for 433 metastatic CRC (mCRC) patients (pts) (bevacizumab [bev] (N = 226) vs cetuximab [cet] (N = 207)) enrolled in randomized phase III CALGB/SWOG 80405 trial obtained from Alliance for Clinical Trials in Oncology. RNA extracted from FFPE tumor samples was sequenced using the HiSeq 2500 (Illumina). Gene expression was divided into high, medium and low. Associations were assessed by log-rank test and multivariable Cox proportional hazards regression model. Treatment-gene interactions were analyzed using the likelihood ratio test. CRC cell lines HCT116 and HT29 were transfected with miR-494 inhibitors and mimics for 48 hours and total RNA was collected, followed by RT-qPCR (miR-494 and target genes BMAL1, PAK1, CDH1, CDH2, SNAI1, TWIST) and clonogenic assays. Results. High miR-494 was significantly associated with worse PFS in overall (median survival: high vs low: 9.3 vs 11.3 months, P<0.025) and cet-treated (median survival high vs low: 9.2 vs 9.6 months, P<0.025) mCRC cohorts, while no significance was observed in bev-treated mCRC. Multivariable Cox model showed significant association between high miR-494 and worse PFS and OS in the cet-treated cohort; however, treatment-gene interaction was not statistically significant. Expression analysis revealed miR-494 was significantly reduced and increased following inhibitor and mimic transfection, respectively. Differential gene expression of the target genes analyzed was observed between the miR-494 inhibitor and mimic-treated HCT116 and HT29 samples. In HCT116 cells, inhibition of miR-494 significantly (P < 0.01) reduced expression of PAK1, while increased the expression of CDH1. The number of colonies formed relatively increased after transfection with miR-494 inhibitor compared to miR-494 mimic. Conclusion. Our findings showed that high miR-494 expression correlated with worse PFS overall and in cet-treated mCRC pts. It would be interesting to check for potential associations with EGFR signaling (direct or indirect). Further studies are warranted for efficient targeting and to gain better understanding of the associated signaling involved in miR-494 modulation in CRC. Support: U10CA180821, U10CA180882; U10CA180888 (SWOG): Genentech. https://acknowledgments.alliancefound.org; P30CA014089, UG1CA180830 [HJL]. Clinicaltrials.gov Identifier: NCT00265850 Citation Format: Pooja Mittal, Joshua Millstein, Shivani Soni, Francesca Battaglin, Karam Ashouri, Sandra Algaze, Priya Jayachandran, Yan Yang, Lesly T. Gonzalez, Unnati H. Shah, Wu Zhang, Fang-Shu Ou, Alan P. Venook, Indrakant K. Singh, Heinz J. Lenz. Effect of miR-494 modulation in colorectal cancer (CRC): Clinical outcome, target expression and cell characteristics [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 2787.

Abstract Purpose: Black women are disproportionately affected by aggressive breast cancers compared to white women, suggesting underlying biological differences in normal breast tissue. This study aims to identify molecular disparities in normal breast tissue between premenopausal Black and white women that may contribute to increased breast cancer risk. Experimental Procedures: Visium spatial transcriptomic analysis was performed on a pilot cohort of normal breast tissue samples from premenopausal Black and white women. Tissue sections were spatially profiled, and capture spots were classified based on predominant cell types: epithelial, stromal, or endothelial. Differential gene expression analysis between racial groups was conducted for each cell type classification. Multiplex immunofluorescence (mIF) was used to validate selected markers in the pilot cohort. Meta-analysis compared differentially expressed genes with published gene sets associated with breast cancer subtypes. Results: Significant racial differences in gene expression profiles were identified in both epithelial and stromal spots. In epithelial spots, genes upregulated in Black women significantly overlapped with over 20 published gene sets associated with estrogen receptor-negative (ER−) breast cancer, including key genes involved in Wnt signaling inhibition (e.g., WIF1) and basal epithelial markers (e.g., KRT14, KRT15). This suggests alterations in pathways linked to more aggressive breast cancer phenotypes. In stromal spots, differentially expressed genes related to extracellular matrix remodeling and fibroblast activation (e.g., PI16, DPT, EFEMP1) were observed, indicating potential differences in stromal-epithelial interactions. Ongoing deconvolution and spatial analyses are being performed to further elucidate these findings. Preliminary mIF validation confirmed increased expression of PI16 in samples from Black women. Validation of additional markers is underway in a larger cohort of premenopausal Black and white women. Conclusions: Spatial transcriptomic profiling of normal breast tissue reveals distinct molecular differences between Black and white women, particularly in pathways associated with aggressive breast cancer subtypes and stromal interactions. These findings suggest that inherent variations in the normal breast microenvironment may contribute to the increased risk and aggressiveness of breast cancer in Black women. Further validation and analysis may identify potential biomarkers for risk assessment and inform targeted prevention strategies. Citation Format: Camila Pacheco Erak, Nicole Cruz-Reyes, Pria Westerman, Joshua Ogony, Jean R. Clemenceau, Tae Hyun Hwang, Sarah McLaughlin, Derek Radisky. Spatial transcriptomic profiling of normal breast tissue reveals potential mechanisms underlying increased breast cancer risk in Black women [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 765.

Abstract Background: Postpartum breast cancer (PPBC) is associated with increased morbidity and mortality among women within 5 to 10 years following childbirth. The postpartum breast undergoes involution, involving tissue remodeling and immune cell infiltration, potentially creating a pro-tumorigenic microenvironment. The molecular mechanisms underlying this elevated risk remain poorly understood. In this study, we compared normal breast tissue samples and human mammary epithelial cells (HMECs) from postpartum women to those from age- and BMI-matched nulliparous women to elucidate potential molecular contributors to PPBC risk. Methods: Transcriptomic profiling was performed on RNA extracted from FFPE normal breast tissues using the NanoString IO 360 and BC 360 platforms to examine gene expression in nulliparous and postpartum women under 45 years of age. HMECs were isolated from organoids derived from breast tissue samples and used for next-generation sequencing and knockdown experiments targeting STAT1 and GBP1. Gene set enrichment analysis (GSEA) was applied to NanoString and RNA-seq datasets to identify differentially enriched genes between postpartum and nulliparous women. Results: Transcriptomic analysis revealed significant upregulation of interferon-stimulated genes, including STAT1 and GBP1, in postpartum breast tissues, consistent with increased immune responses during involution. NanoString data showed that markers of cell proliferation, such as Ki67, STAT1, and GBP1, were significantly higher in samples from parous compared to nulliparous women (p < 0.05). GSEA revealed that cell cycle-promoting genes (GBP1, cyclins, CDKs) were upregulated, while cyclin-dependent kinase inhibitors (CDKNs) were downregulated in postpartum breast tissues. Immunohistochemistry confirmed significantly higher expression of Ki67, STAT1, and GBP1 in postpartum versus nulliparous breast tissues (p < 0.0001). Silencing STAT1 and GBP1 in HMECs resulted in decreased cell proliferation, G1 cell cycle arrest, and increased expression of CDKN1A and CDKN1C. Conclusion: These findings suggest that STAT1 and GBP1 promote proliferation in postpartum breast epithelial cells by downregulating cyclin-dependent kinase inhibitors, potentially contributing to the increased risk of PPBC. Targeting these pathways may offer novel strategies for mitigating postpartum breast cancer risk. Citation Format: Joshua Ogony, Melody Stallings-Mann, Sarah McLaughlin, Mark Sherman, Derek C. Radisky. STAT1 and GBP1 promote proliferation in postpartum breast epithelium by downregulating cyclin-dependent kinase inhibitors: implications for increased postpartum breast cancer risk [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 187.

Abstract Introduction: Cholangiocarcinoma (CCA) is an aggressive biliary malignancy with poor survival outcomes and multiple mechanisms of resistance to targeted therapies. We previously validated NXP800, a novel GCN2 small-molecule activator, as an effective anti-cancer drug in six CCA cell lines. In vitro testing identified upregulation of phosphorylated eIF2a, as well as upregulation of ATF4, in CCA cells treated with of NXP800 compared to vehicle, suggesting that treated cells led to stimulation of stress-induced genes and subsequently, apoptosis. Furthermore, patient derived xenografts (PDX) models treated with NXP800 were found to have decreased tumor size, particularly in models containing ARID1A, FGFR1, and ATM mutations. Here, we aimed to predict response to NXP800 using sensitivity and resistance signatures from our profiled PDX models. Methods: GCN2 activation in multiple previously profiled PDX models was evaluated. Whole Exome Sequencing (WES) was done on multiple PDX models to characterize the mutational signature and activity in CCA. Two additional CCA xenografts were expanded into the flank of NOD/SCID mice. Tumor bearing mice were treated with NXP800 (35 mg/kg) or vehicle via oral gavage for 28 days once tumors averaged 120 mm3. Tumor volume and animal weight were collected twice weekly. Median Efficacy Index (MEI) scores were generated as drug response rates based on tumor growth. Predicted sensitivity and resistance kinase signatures to predict drug response were produced using multi-omics. Results: Treatment with NXP800 was associated with a statistically significant decrease in tumor size in two additional PDX models, which included ARID1A and BRAF mutants. Preliminary multi-omics on the PDX models revealed that sensitivity mechanisms were upregulated most in EGFR signaling, while resistance signatures were focused around CSNK2A1 signaling. Conclusion: Multi-omics analysis revealed a sensitivity mechanism structured around EGFR signaling, providing insights into which patients should be selected for treatment with NXP800. A Phase 1b clinical trial investigating the effects of NXP800 use in patients with advanced CCA is open and currently accepting patients. Citation Format: Danielle Marie Carlson, Hendrien Kuipers, Amro Abdelrahman, Erik Jessen, Joshua Roldan Kalill, Jennifer Tomlinson, Jack Sample, Akhilesh Pandey, Mark Truty, Rory Smoot. Modeling response prediction to a novel GCN2 kinase activator (NXP800) in patient-derived xenograft cholangiocarcinoma models using a multi-omics approach [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 5592.

Abstract Text While the importance of estrogen in driving ER+ breast cancer progression is clear, data from pre-clinical studies exploring the role of tumoral progesterone (PR) and androgen (AR) receptors are conflicting, likely due in part to the promiscuity of steroid receptor ligands, as well as relative levels of steroid hormone receptor expression and of steroid hormones within the tumor milieu. While bone is usually the first site of metastases in ER+ metastatic breast cancer, the roles of tumoral PR and AR (present in 58 percent and 86 percent of clinical ER+ bone metastases [BMET], respectively) in ER+ breast cancer progression at this site have not been well studied. Studies were undertaken to examine the effects of ovariectomy (OVX), a model of surgical menopause that significantly reduces endogenous progesterone and androgen levels, in a pre-clinical model of human ER+ bone metastases that is dependent on exogenous 17ß-estradiol (E2) supplementation. Effects of OVX (at 9-weeks of age) were ascertained using skeletally mature female nude mice supplemented with a modest dose of E2 (0.36 mg 60 day pellets) prior to intracardiac inoculation (at 10 weeks of age) with bone metastasis-derived MCF7 cells (43.4M cells) that form robust E2- and TGFß-dependent osteolytic metastasis in vivo, and express PR and AR in vitro. Osteolytic bone metastatic lesions in E2-supplemented mice were reduced in both incidence and size in OVX (vs ovary intact) mice. Anti-tumoral effects of OVX were not limited to bone; E2-driven orthotopic tumor progression was similarly reduced. Histologic assessment of cytokeratin-positive breast cancer tumor cells within bone revealed a small but statistically significant reduction in Ki67-positivity in OVX (vs ovary -intact) mice that correlated with osteolytic lesion size. In addition, PR and AR expression in BMET lesions, which was readily detected by IHC in ovary-intact mice, was markedly reduced in OVX mice. While additional experiments are underway to query possible roles of specific reproductive hormones altered in OVX mice, these results are consistent with the postulate that tumoral AR and PR signaling may promote ER+ bone metastases progression in this model. In addition, while ovariectomy is a common pre-clinical means of reducing estrogen to model natural menopause in women, resultant changes in steroid receptor signaling are clearly much more complex. Funding: NIH 5R25CA275753, 5P30CA023074 Date of Presentation October 17, 2024