Citations

Abstract Heterogeneous treatment effects (HTE) are key to precision medicine, but most real-world studies lack the scale and diversity needed to detect them. While multi-site analyses offer a potential solution, data-sharing constraints often prevent access to patient-level information across institutions. We introduce POLARIS, a federated framework for time-to-event target trial emulation. POLARIS converts each site’s weighted Cox risk function into a compact tensor shared once with the coordinating center, enabling lossless reproduction of pooled estimates without sharing patient-level data. We applied POLARIS across five U.S. health systems to study risk of gastrointestinal outcomes after GLP-1 receptor agonist (GLP-1RAs) initiation versus sodium-glucose cotransporter 2 inhibitors (SGLT2is) and dipeptidyl peptidase 4 inhibitors (DPP4is). Results showed that GLP-1RAs were consistently associated with higher risks of nausea and vomiting, particularly among men, individuals with higher baseline HbA1c (≥ 8.5%), and lipid therapy. POLARIS provides a scalable solution for distributed target trial emulation and fine-grained assessment of HTE across diverse health systems.

ABSTRACT Macrophages play a central role in determining the outcomes of healing, coordinating regeneration in some injuries and scar formation in others. In both cases, this coordination involves the cross-talk between macrophages and surrounding cells. But what drives the different cross-communication pathways to determine healing outcomes is not well known. In this study, we make use of the mouse digit tip amputation model, in which an amputation through the third phalangeal element (P3) is able to completely regenerate whereas an amputation through the second phalangeal element (P2) forms a scar. We identify a population of macrophages that is specific to the P3 regenerating digit. By integrating single-cell RNAseq, spatial transcriptomics, and metabolomic analyses, we show that this population localizes specifically to the growing bone front, express BMP ligands that drive downstream BMP activation in neighboring osteoblasts and is governed by a two-part metabolic switch involving increased fatty acid oxidation coupled with reduced glycolytic activity. This spatially restricted, BMP-expressing macrophage population is entirely absent in the scar-forming P2 injury, and our data indicate that environmental conditions unique to the regenerating digit are responsible for its emergence. Together these findings identify a regeneration-specific macrophage signaling center for patterned bone formation and suggest that targeting the metabolic conditions that drive this population could improve the efficacy of regenerative therapies.

Abstract Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of B-cell malignancies. Despite the clinical success of CD19-targeted approaches, relapsed/refractory (R/R) disease—often driven by antigen loss or immune escape—remains a major challenge. To overcome this, we developed a novel CAR-T cell therapy targeting BAFF-R, a receptor consistently expressed in chronic lymphocytic leukemia (CLL) and other B-cell malignancies. We developed a new anti-BAFF-R monoclonal antibody and engineered the MC10029 CAR construct using its scFv. Antigen-specific cytotoxicity was validated in both in vitro and in vivo models, including Nalm-6 (leukemia) and Z138 (lymphoma), and CD19-knockout tumor cells to model (R/R) disease. MC10029 CAR-T cells were generated from healthy donors and CLL patients, and their cytotoxicity was evaluated against MEC-1, a CLL cell line, as well as autologous CLL patient tumor samples. These studies demonstrated potent and specific antitumor efficacy. We transitioned to clinical-grade production, using GMP-compliant lentivirus to manufacture MC10029 CAR-T cells under regulatory guidelines. With FDA IND approval, we initiated a Phase 1 clinical trial to evaluate safety and dosing. Our first patient, treated with a low-dose, single infusion, achieved a complete metabolic response within 2 months. The therapy is well tolerated, with no severe advent events such as cytokine release syndrome or neurotoxicity, supporting both the safety and efficacy of the therapy. Conclusion: This study represents Mayo Clinic’s first homegrown CAR-T cell therapy, developed entirely in-house—from antibody discovery and CAR design to GMP manufacturing and clinical trial—marking a significant step toward personalized, next-generation immunotherapy for B-cell malignancies. Citation Format: Yan Luo, Shaohua Guo, Yaqing Qie, Martha E. Gadd, Tanya Hundal, Hemant S. Murthy, Mohamed A. Kharfan-Dabaja, Hong Qin. From gene to bedside: Mayo Clinic’s first-in-house BAFF-R CAR-T [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3719.

Abstract Several allogeneic CAR T-cell therapies are in clinical trials to evaluate safety, many of which rely on CRISPR/Cas-based genome editing. However, the Cas enzyme’s random repair mechanism elevates the risk of undesired, off-target effects, posing a challenge for safe allogeneic CAR T-cell generation. To mitigate the risk of therapy-related oncogenesis, we sought to pursue an alternative safe strategy to manufacture allogeneic CAR T-cells distinct from the preexisting ones in the clinic. We have developed a CRISPR RNA (crRNA) targeting T-cell receptor beta constant (TRBC) gene, which in conjunction with AsCas12a Ultra enzyme, causes site-specific editing by eliciting a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, which mitigates off-targeting risk. Targeted amplicon sequencing was employed to evaluate off-targeting and repair mechanisms. Graft versus host alloreactivity as well as CAR T persistence was evaluated using immunocompromised mice. In vivo tumor challenge study was conducted for over 3 months. Engraftment confirmation was done using immunohistochemistry and flow cytometry. Interestingly while evaluating the edited T-cells, we sequestered a unique T-cell population that showed TCR disruption yet remained CD3-positive and did not cause in vivo alloreactivity. These CD3-Retained, Allogeneically Functioning T-cells (CRAFT-cells) showed a similar growth rate as unedited T-cells and were then used as a platform to produce CD19-targeted CAR T-cells and BAFF-R-targeted CAR T-cells. When compared to the standard CD3-disrupted allogeneic CAR T-cells, CRAFT CAR T-cells displayed an equally robust cytotoxicity while possessing a safer genomic profile. CRAFT CAR T-cells could function as effector cells for bispecific T-cell engager (BiTE) and induce CD3-dependent cytotoxicity against tumor cells. Pertinently, CRAFT CAR T-cells as opposed to CD3-disrupted CAR T-cells, were present in mouse xenografts without alloreactivity. Having the onset of MMEJ-directed repair and minimal off-targeting, positions this CRAFT crRNA to generate safer allogeneic CAR T-cells with an overall reduced genotoxic profile in a clinical setting. This unique CRAFT CAR T-cell population displays in vivo persistence. Additionally, CRAFT CAR T can be combined with BiTE therapy, offering a promising and potentially more durable alternative to preexisting ex vivo allogeneic CAR T-cell therapies. Citation Format: Tanya Hundal, Yan Luo, Yaqing Qie, Martha E. Gadd, Shaohua Guo, Mohamed A. Kharfan-Dabaja, Hong Qin. CD3-positive allogeneic CAR T-cells: A novel platform with persistence, BiTE-capture, and no alloreactivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4283.

ABSTRACT Tumorigenesis and metastasis are frequently attributed to the intricate interplay between cancer cells and the tumor microenvironment (TME). Comprehending the mechanisms and key regulators of cancer‐immune crosstalk in the TME is imperative for developing efficacious immunotherapy. Through a series of in vivo CRISPR screens, we identified tumor‐intrinsic ITGB1 as a critical regulator of triple‐negative breast cancer (TNBC) development and deciphered its underlying mechanisms. Tumoral ITGB1 facilitated the establishment of pro‐tumorigenic TME by orchestrating tumor‐associated myeloid populations. Suppressing ITGB1 favored the enrichment of anti‐tumorigenic myeloid cells and enhanced infiltration of CD4 and CD8 T cells, culminating in superior antitumor effects. CRISPR scanning pinpointed a previously unrecognized functional domain essential for ITGB1's pro‐tumorigenic activity. This domain is distinct from all known ligand‐binding sites in ITGB1. An antibody capable of sterically blocking this domain significantly impaired TNBC progression. These findings position tumoral ITGB1 as a promising therapeutic target for reprogramming the TME from a pro‐ to an anti‐tumorigenic state, thereby effectively inhibiting TNBC development. Our study uncovers a novel mechanism of TNBC development and provides a unique therapeutic strategy for targeting ITGB1 in TNBC treatment.

Abstract Background One in five adults aged 40 will develop heart failure (HF) during their lifetime. Risk factors (e.g., hypertension, diabetes mellitus, coronary artery disease, etc.) lead to structural and functional changes in cardiomyocytes, resulting in HF. At the cellular level, these changes consist of mis/over‐expression of genes that regulate cardiac identity (e.g., CamK2δ, PKC, Stat3, etc.). The current paradigm for treating HF is pharmacological or device‐based intervention; however, with few exceptions, the condition worsens with time. We are proposing to implement a change in HF treatment, shifting from a drug‐centric system to a cardiac target‐specific molecular approach that would reverse hypertrophy and adverse remodelling of affected cardiomyocytes. Methods A cardiomyocyte targeting peptide (CTP) was reversibly linked to miRNA106a for delivery to a mouse model of HF. Reversal of morphological, signalling, and physiological HF parameters was measured. Additionally, CTP‐miRNA106a was introduced into a human cardiomyocyte cell line to identify mechanism(s) at play for reversing HF characteristics (e.g., hypertrophy). Results Bio‐distribution studies showed that intravenously injected CTP‐miRNA106 delivered its cargo specifically to the heart within 30 min, followed by clearance of CTP from the heart to the kidneys, and to a lesser extent, the liver by 35 h with persistence of miRNA106a in cardiomyocytes until day 7 (the latest tested time‐point). CTP‐miRNA106a reversed angiotensin2/isoproterenol‐induced hypertrophy in 90% of the treated mice. We also identified two potential HF intracellular signalling pathways/mechanisms (PLCβ1/PKC/IP3 and NF‐κB) targeted by CTP‐miRNA106a that could benefit many pathophysiologies underlying HF, including inflammation. Conclusions CTP‐miRNA106a, a first‐of‐a‐kind cardiac‐specific drug, downregulates genes involved in cardiac hypertrophy and inflammation through the PLCβ1 and CamKIIδ kinase pathways. CTP delivery of miRNA106a cargo is specific to cardiomyocytes both in vitro and in vivo, and once delivered, many HF parameters, including hypertrophy, are reversed. Key points Cardiac Targeting Peptide (CTP) delivers, specifically to the heart, reversibly linked miRNA106a. MiRNA106a targets genes in various pathways known to cause heart failure when they are over/hyper activated returning their expression to normal levels. In vivo analyses using a mouse heart failure model resulted in reversal of heart failure parameters in 19/20 mice.

5076 Background: C and Lu are both life-prolonging therapies for pts with mCRPC after progression on androgen receptor pathway inhibitor (ARPI) and docetaxel (D). Herein, our objective was to assess the comparative effectiveness of C vs. Lu in pts with mCRPC with prior progression on D and ARPI in real-world pts in the USA. Methods: A de-identified nationwide Flatiron Health electronic health record (EHR)-derived database was used to extract pt-level data. Eligibility: pts with mCRPC who progressed on D and ARPI and received for the first-time single-agent C or Lu. Pts who received alternative Lu or C in a later line of therapy (LOT) were excluded. Endpoints: real-world time to next therapy (rwTTNT) and real-world overall survival (rwOS). These were summarized via Kaplan-Meier survival estimates with a 95% confidence interval (CI) and compared in the context of propensity score (PS) matching weighted analysis using the Cox proportional hazard model. PS model included the following covariates: age, race-ethnicity, socioeconomic status, treatment year, Gleason score, ECOG performance status, log2PSA, alkaline phosphatase, hemoglobin, creatinine, LOT, insurance, and practice type. All the covariates achieved balance after PS matching weighting. Results: Among 24,105 pts with metastatic prostate cancer in the dataset, 1,445 met the eligibility criteria and were included (1,227 treated with C, 218 treated with Lu). In C cohort: median age was 73 (IQR 67 – 78), 66.9% had Gleason score ≥ 8, and median LOT was 4 (IQR 3-4). In Lu cohort: median age was 75 (IQR 67.25 – 80), 68.1% had Gleason score ≥ 8, and median LOT was 4 (IQR 3-5). In PS matching weighting analysis, there was evidence that pts receiving Lu had a significantly improved rwTTNT (median 8.3 months [mo], 95% CI 6.3 – 9.8) compared to those receiving C (median 4.7 mo, 95% CI 4.2 – 5.4) (HR 0.49, 95% CI 0.37 – 0.63, p < 0.001), which persisted after adjusting for covariates (HR 0.43, 95% CI 0.32 – 0.57, p < 0.001). Additionally, there was evidence that rwOS was longer in pts receiving Lu (median 10.3, 95% CI 8.9 – 12.8) compared to those receiving C (median 8.8 mo, 95% CI 6.6 – 10.8) (HR 0.69, 95% CI 0.51 – 0.92, p = 0.01). This improvement in rwOS with Lu compared to C persisted after adjusting for covariates (HR 0.61, 95% CI 0.44 – 0.84, p < 0.001). Conclusions: This is the largest real-world data to date assessing the comparative effectiveness of Lu vs. C, and it showed significantly longer rwTTNT and rwOS with Lu compared to C in pts with mCRPC pretreated with ARPI and D. Limitations: retrospective nature, selection bias, missingness, lack of randomization, etc. and residual confounding in real-world datasets. Upon external validation, these findings could guide treatment selection in the clinic and design of clinical trials.

95 Background: Most pts with prostate cancer with bone metastasis experience symptomatic skeletal events (SSEs) [PMID: 23884473], leading to increased morbidity and mortality. BMAs have been approved since 2007 and are recommended in mCRPC to prevent SSEs in pts with bone metastases or a high risk of osteoporotic fracture. Herein, we sought to assess the real-world trends of BMA administration in pts with mCRPC. Methods: We used the de-identified nationwide Flatiron Health electronic health record (EHR)-derived database to extract pt-level data. Inclusion criteria: pts with mCRPC with available date of diagnosis of mCRPC and information on receipt of BMA. Pts were categorized into 2 cohorts based on BMA receipt (e.g., bisphosphonates, denosumab, teriparatide, romosozumab) or not. Treatment trends of BMA by year of mCRPC diagnosis were summarized using frequency and percentages. Baseline characteristics at the time of mCRPC diagnosis (age, race-ethnicity, practice type, insurance) were compared between cohorts using the Wilcoxon rank-sum and Chi-squared tests. Results: Of 24,105 pts with metastatic prostate cancer in the dataset, 14,112 with mCRPC were eligible and included. 7,990 (56.6%) received BMA while 6,122 (43.4%) did not. In BMA receipt cohort: median age was 75 (IQR 67 – 81), majority were White (61%), treated in community practice (87%) and had commercial health plan (80%). In BMA non receipt cohort: median age was 75 (IQR 68 – 82), majority were White (61%), treated in a community practice (78%), and had commercial health plan (76%). The use of BMA decreased over time from 56% in 2013 to 46% in 2024 (Table). Statistically significant differences existed between cohorts in race-ethnicity, practice type, and insurance (p < 0.001) and will be presented at the meeting. Conclusions: Despite recommendations to use BMAs in pts with mCRPC and bone metastasis or high risk of osteoporotic fracture, our findings reveal a low utilization rate of BMAs in mCRPC setting with a notable decline over time. This highlights the need to incorporate the use of BMAs in clinical practice and improve access to these agents. Trends by year of mCRPC diagnosis in receipt of the first BMA and frequency and percentage of each agent. Year 2013N = 549 2014N = 1035 2015N = 1223 2016N = 1345 2017N = 1441 2018N = 1346 2019N = 1469 2020N = 1380 2021N = 1352 2022N = 1393 2023N = 1196 2024N = 383 Receipt of BMA, n (%) 316 (56) 674 (65) 792 (65) 837 (62) 886 (61) 766 (57) 816 (56) 766 (56) 692 (51) 713 (51) 556 (46) 176 (46) Denosumab, n (%) 190 (60.1) 450 (66.8) 557 (70.3) 599 (71.6) 606 (68.4) 533 (70) 526 (64.5) 488 (63.7) 410 (59.2) 414 (58.1) 290 (52.2) 94 (53) Zoledronic acid, n (%) 120 (38) 220 (32.6) 228 (28.7) 233 (27.8) 270 (30.5) 225 (29) 281 (34.4) 273 (35.6) 277 (40) 295 (41.4) 258 (46.4) 82 (47) Teriparatide, other bisphosphonates, n (%) 6 (1.9) 4 (0.6) 7 (1) 5 (0.6) 10 (1.1) 8 (1) 9 (1.1) 5 (0.7) 5 (0.7) 4 (0.5) 8 (1.4) 0

109 Background: C and Lu are associated with overall survival benefit and approved for pts with mCRPC after prior treatment with docetaxel and an androgen receptor pathway inhibitor (ARPI). However, in real-world settings, administering Lu requires coordination across specialties and resources [PMID: 36637862], which could delay its administration. Herein, we sought to compare the time from medication order to administering the first dose of C versus Lu. Methods: This study used pt level data from the nationwide Flatiron Health electronic health record (EHR)-derived de-identified database. Inclusion criteria: availability of the date of the first order of single-agent C or Lu and the date of first administration of the drugs. The time between the first medication order to the administration of single-agent C or Lu was measured. Wilcoxon rank-sum test was used to compare the median days between medication order to administration between these 2 agents and a stratified analysis by practice type and insurance was performed. Results: Of 2,329 orders, 2,045 orders were for C between 1/1/2013 and 1/26/2024, while 284 were for Lu between 8/1/2013 and 12/6/2023. The time from medication order to administration was significantly longer with Lu (median 22 days, IQR 12-43) compared to C (median 7 days, IQR 3-14, p < 0.001). Time from medication order to administration of C and Lu by practice type and insurance is summarized (Table). At the time of the first order, pts in Lu cohort, compared to C cohort, were significantly older (median age 74 vs. 72, p < 0.001), more likely to be treated at an academic practice (27% vs. 15%, p < 0.001), and more likely to be covered by a commercial health plan (80% vs. 73%, p < 0.001). Conclusions: Our results show that it takes significantly longer to administer Lu compared to C across different practice types and most insurance plans. These results could inform healthcare policies aimed at improving access to Lu and guide treatment selection in the clinic. Time from medication order to administration of C and Lu by practice type and insurance. Category Subtype (number of orders for C/number of orders for Lu) CN = 2045 LuN = 284 p-value Practice type, mean (range) (days) Academic (315/77) 1.3 (0 – 59) 9.2 (0 – 55) < 0.001 Community (1730/207) 13 (0 – 243) 44 (1 – 282) < 0.001 Insurance, mean (range) (days) Commercial (1494/226) 11 (0 – 163) 32 (0 – 215) < 0.001 Medicare/other government programs (266/46) 10 (0 – 120) 46 (0 – 282) < 0.001 Medicaid (32/2) 9 (0 – 76) 28 (0 – 55) 0.8 Others (72/6) 12 (0 – 42) 21 (0 – 41) 0.2 Unknown (181/4) 14 (0 – 243) 76 (39 – 91) < 0.001

104 Background: Androgen deprivation therapy (ADT) intensification [ADT + androgen receptor-pathway inhibitor (ARPIs) +/- docetaxel] has been shown to improve survival outcomes in pts with mHSPC and is recommended as standard of care by all major guidelines. However, prior studies have shown underutilization of ADT intensification [PMID: 39191549]. There are limited updated and large-scale studies on the use of ADT intensification, beyond January 2023. In this study, we aimed to assess the treatment patterns of ADT intensification in real-world pts with mHSPC in the US, including data on those treated since January 2023. Methods: This retrospective study utilized the nationwide Flatiron Health Electronic Health Record (EHR) derived de-identified database. Eligibility: Pts diagnosed with mHSPC and availability of treatment information (ADT and/or line of therapy in mHSPC). The data cut-off date was 5/31/2024. Treatment patterns were summarized using frequency and percentages. All analysis was done using R version 4.2.3. Results: Among 24,105 pts with metastatic prostate cancer in the database, a total of 14,084 were eligible and included. The median age at metastatic diagnosis was 72 years (IQR 65 – 79), and 60% were White non-Hispanic. In the overall cohort (1/1/2013–5/15/2024), 56.8% received ADT monotherapy, and 37.7% received ADT intensification. Table summarizes the treatment patterns by year, highlightinga consistent increase in adoption of intensified ADT regimens, and a decline in use of ADT monotherapy. Notably, since January 2023, 76.8% pts with mHSPC received ADT intensification while only 17.3% of pts received ADT monotherapy. Conclusions: To our knowledge, this is the first and largest study to evaluate the current use of ADT intensification. These data indicate a notable improvement in the adoption of level 1 evidence (ADT intensification) in the treatment of pts with mHSPC. These encouraging data emphasize the benefits of continued efforts to promote guideline-concordant care and provide the current treatment landscape to design clinical trials. Treatment patterns by year in pts with mHSPC. Treatment 2013–2017n = 5,263(%) 2018n = 1,313 (%) 2019n = 1,407(%) 2020n = 1,353(%) 2021n = 1,483(%) 2022n = 1,556(%) 2023n = 1,459(%) 2024n = 250(%) ADT monotherapy 4,095 (77.8) 856 (65.2) 819 (58.2) 664 (49) 698 (47.1) 569 (36.6) 291 (20) 5 (2) ADT + ARPI 155 (2.9) 238 (18) 354 (25.1) 452 (33) 555 (37.4) 695 (44.7) 842 (58) 154 (61.6) ADT + Docetaxel 709 (13.5) 156 (12) 175 (12.4) 156 (12) 134 (9.0) 117 (7.5) 78 (5.3) 19 (7.6) Triplet(ADT + ARPI + Docetaxel) 6 (0.1) 2 (0.2) 1 (0.1) 2 (0.1) 9 (0.6) 82 (5.2) 166 (11.1) 53 (21.2) Other therapies 298 (5.7) 61 (4.6) 58 (4.1) 79 (5.8) 87 (5.9) 93 (6) 82 (5.6) 19 (7.6)