Kettering University

Dietary fat composition modulates host physiology and the gut microbiome, but the long-term effects of specific fat sources and the extent to which these changes resolve after dietary reversal remain incompletely defined. Here, we present a longitudinal multi-omic resource of mice maintained for one year on a purified control diet, seven high-fat diets differing in predominant fat source, or reversal regimens in which animals were switched from high-fat to control diet after 4 or 9 months. We further incorporated two cohorts with distinct pre-existing microbiome configurations to determine how baseline community structure shapes diet-induced remodeling of the gut microbiome ecosystem. By integrating longitudinal phenotyping, fecal metagenomics, fecal metabolomics, plasma metabolomics and lipidomics, and intestinal single-cell RNA sequencing, we defined the shared and dietary fat-specific responses across host and microbiome compartments. Baseline microbiome composition strongly influenced microbial responses to diet, indicating that pre-existing community structure is a major determinant of dietary ecosystem remodeling. Although many altered features shifted toward baseline after dietary reversal, only approximately half of diet-associated microbial changes recovered within the study window. A subset of taxa exhibited persistent alterations, including sustained depletion of Lactobacillus johnsonii and Bifidobacterium pseudolongum and sustained enrichment of Alistipes finegoldii, consistent with a "microbiome memory" of prior high-fat diet exposure. This memory effect is mirrored in the host, by sustained suppression of major histocompatibility complex class II (MHC-II) gene expression in intestinal epithelial cells after dietary reversal. These findings indicate that dietary fats leave a lasting imprint on the host-microbiome interactome that survives dietary intervention. Together, these data establish a resource for defining how dietary fat source, baseline microbiome composition, and dietary history shape host-microbiome states. The entire resource is available online as an RShiny app.

Chimeric antigen receptor (CAR)-T cell therapy has shown limited success in the treatment of solid tumors, reinforcing the need to elucidate the invivo biodistribution of these engineered T cells. Here, we integrate the anti-DOTA huC825 reporter ("Thor") platform into newly developed human anti-interleukin-13 receptor α-2 (IL13Rα2)-single-cell fragment variable (scFv)-derived CAR-T cells and investigate its utility for mapping CAR-T cell distribution in a xenograft mouse model of melanoma. Methods: We engineered anti-IL13Rα2-scFv-derived CAR-T cells expressing huC825 (KLG3BBz-huC825), evaluated detection sensitivity, and monitored CAR-T cell biodistribution via weekly [86Y]Y-aminobenzyl-DOTA PET/CT and therapeutic efficacy. Results: KLG3BBz-huC825 T cells demonstrated potent antigen-specific cytotoxicity and cytokine release invitro. The Thor radiohapten capture platform offered exquisite detection sensitivity of only 3,000 engineered T cells and enabled prolonged spatiotemporal assessment of CAR-T cell kinetics up to 7 wk after infusion, corroborated by histopathology. Treatment with KLG3BBz-huC825 resulted in an overall survival benefit. Conclusion: The Thor platform offers a versatile and highly sensitive approach to study the real-time kinetics of CAR-T cells invivo.

Predicting genetic perturbation responses at a single-cell level is central to building models for cell state and disease. However, existing approaches are limited on predicting phenotypic outcomes beyond expression changes and generalizing predictions across genome-scale perturbations in biologically relevant contexts. Here we introduce pertTF, a transformer-based single-cell genetic perturbation model. pertTF was trained from a unique dataset capturing single cell expressions profiles of 30 full gene knockouts across 14 relevant cell types during human pancreatic development and beta-cell differentiation. pertTF outperforms current methods in predicting expression changes of perturbing unseen genes in unseen cellular contexts. In addition, pertTF infers perturbation-induced shifts in cell identity and population composition, an important phenotypic outcome of perturbation in many physiology and disease settings. Through transfer learning, pertTF operates in physiologically relevant systems, including primary human islets, where large-scale perturbation experiments are challenging. The generalizability of pertTF is further demonstrated by in silico pooled and single-cell CRISPR screens, capturing critical regulators of stem cells and early pancreatic cell development. These results establish pertTF as a framework for integrating large-scale single-cell perturbation data with AI models to predict genetic perturbation effects across cellular systems and disease contexts.

Abstract Background The first-line standard of care (SOC) for patients (pts) with estrogen receptor-positive, HER2-negative advanced breast cancer (ER+, HER2- aBC) is CDK4/6 inhibitor (i) + endocrine therapy (ET) but effective post-CDK4/6i options remain limited. Giredestrant (GIRE) targets the ER pathway while everolimus (E) targets the PI3K/AKT/mTOR pathway; both of which are implicated in driving resistance in the post-CDK4/6i setting. evERA BC (NCT05306340) is the first Phase III trial to demonstrate statistically significant and clinically meaningful improvement in investigator-assessed progression-free survival (INV-PFS) with an all-oral selective ER antagonist and degrader combination of GIRE + E vs SOC ET + E in pts with ER+, HER2- aBC post-CDK4/6i + ET, both in pts whose tumors had a detectable ESR1 mutation (m) and in the intent-to-treat (ITT) population (Mayer ESMO 2025). The safety profile of GIRE + E was manageable with no unexpected findings (Mayer ESMO 2025). We report results from prespecified exploratory subgroup analyses. Methods Pts who had ER+, HER2- aBC with disease progression (PD) post-CDK4/6i + ET in the aBC setting, or relapse during/after CDK4/6i + ET in the adjuvant setting, were randomized 1:1 to once-daily oral 30 mg GIRE + 10 mg E or SOC ET (exemestane, fulvestrant, or tamoxifen) + E until PD or unacceptable toxicity. Mutational status was determined using circulating tumor DNA at baseline. The co-primary endpoints were INV-PFS per Response Evaluation Criteria in Solid Tumors v1.1 in pts whose tumors had detectable ESR1m and in the ITT population. INV-PFS was assessed by subgroup analyses. Results Three-hundred-and-seventy-three pts were randomized; 183 pts were randomized to GIRE + E and 190 to SOC ET + E. A total of 207 pts (55%) had tumors with ESR1m, 115 (31%) had PIK3CAm, and 137 (37%) had alterations (alt) in the PI3K pathway genes (PIK3CA/AKT1/PTEN). Sixty-four pts (17%) had both ESR1m and PIK3CAm; 76 (20%) had both ESR1m and PIK3CA/AKT1/PTEN alt. Ninety-eight percent of pts received a CDK4/6i in the metastatic setting. INV-PFS benefit was observed for GIRE + E vs SOC ET + E regardless of PIK3CAm status or PIK3CA/AKT1/PTEN alt in both the ESR1m and ITT populations (Table). Consistent benefit was observed regardless of duration of prior CDK4/6i (Table). Data for additional subgroup analyses for prior therapy will be presented. Conclusions GIRE + E led to clinically meaningful improvements in INV-PFS vs SOC ET + E irrespective of PIK3CAm and PIK3CA/AKT1/PTEN alt, and other key relevant subgroups, regardless of ESR1m status. These data support the use of GIRE + E vs SOC ET + E in pts with ER+, HER2- aBC post-CDK4/6i + ET. Citation Format: H. S. Rugo, S. M. Tolaney, K. L. Jhaveri, M. Martin, G. A. Vidal, L. Moscetti, A. Brufsky, W. J. Gradishar, A. Schneeweiss, N. Niikura, A. Favret, M. Alfie, K. S. Lee, S. Khan, M. Feldman, B. M. Day, L. H. Lam, W. C. Darbonne, T. M. Fernando, P. Perez-Moreno, E. L. Mayer.Clinical and biomarker subgroup analysis of evERA Breast Cancer: A Phase III trial of giredestrant plus everolimus in patients with estrogen receptor-positive, HER2-negative advanced breast cancer previously treated with a CDK4/6 inhibitor [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr GS3-09.

Medical imaging and nuclear medicine are fundamental techniques used in today’s health care for diagnosing and treating diseases, with a measurable impact on saving patients’ lives. However, the global availability of nuclear medicine is markedly different between high-income and low- and middle

Identifying appropriate clinical trials for cancer patients with specific gene mutations remains a significant challenge, largely due to limitations in current search tools like ClinicalTrials.gov, which at times return irrelevant or misleading results. This diagnostic accuracy study investigates the efficacy of 2 large language models (LLMs), GPT-4.0 and Gemini 2.0, in evaluating the eligibility of patients with specific cancer-related gene mutations for clinical trials. The study prompts GPT 4.0 and Gemini 2.0 with trial details from ClinicalTrials.gov and a particular cancer mutation. We then assess model performance against physician-curated benchmarks across 6 gene mutations (ALK, BRAF, EGFR, ERBB2, KIT, and KRAS). The results demonstrate good F1-scores for both LLMs-averaging 64% for GPT-4.0 and 70% for Gemini 2.0-highlighting their potential to streamline clinical trial matching. Furthermore, decision trees provided interpretability by identifying key textual indicators that LLMs use. This work demonstrates the feasibility of using proprietary LLMs such as GPT 4.0 and Gemini 2.0 "off the shelf" with both limited LLM fine-tuning and limited patient information to evaluate clinical trial eligibility.

The Mycobacteriales are an order of diverse bacteria that thrive in many environmental and host-associated niches. Because the most notorious member of this clade, Mycobacterium tuberculosis, is a major human pathogen, research on Mycobacteriales has focused on pathogenesis, and, as a consequence, many fundamental aspects of Mycobacterial biology remain understudied. Here, we address this gap by performing a genome-wide CRISPRi chemical genomics screen using a diverse set of >35 antibiotics, detergents, and other anti-microbials predominantly targeting the cell envelope of Mycobacterium smegmatis, a saprophytic model Mycobacterium. We highlight new information derived from this screen, including the identification of novel functions for previously uncharacterized conserved and essential genes (in mycolic acid and arabinogalactan synthesis), the discovery of a new drug scaffold/protein target pair, and insights into the mechanism of action of two commonly used antibiotics. These data are also a valuable resource for the mycobacterial research community, as they provide thousands of novel phenotypes for uncharacterized genes and meaningful phenotypic correlations between annotated and uncharacterized genes.

Abstract Stem cell-like castration resistant prostate cancer (SCL-CRPC) is a major subtype of double-negative prostate cancer (DNPC) that is resistant to conventional and next-generation of androgen signaling inhibition. SCL-CRPC is characterized by a distinct chromatin accessibility signature enriched with motifs for the AP-1 family of transcription factors. Here, we seek to identify the members of the AP-1 family that are critical to the growth and survival of SCL-CRPC. To inhibit AP-1 activity, we induced the expression of the dominant negative AFOS (constructs kindly provided by Jude Owiredu in Chris Barbieri lab at Weill Cornell) in SCL models and observed growth suppression, suggesting a dependency of this disease subtype on AP-1. Using in-house developed SCL prostate organoids (MSKPCa series) and conventional cell lines, we knocked down AP-1 factors individually or in combination via siRNAs and found that FOS proto-oncogenes FOSL1 and cFOS are required for the rapid growth of these models. Given that there is known crosstalk between MAPK signaling and these FOS proto-oncogenes, we hypothesize that SCL-CPRC also depends on MAPK signaling for growth. Pharmacological inhibition of MEK via trametinib in SCL models resulted in rapid downregulation of cFOS, FOSL1 and TAZ transcripts in as early as 1hrs in our RNA-Seq, suggesting that MAPK signaling positively relates to the core SCL-CRPC transcriptional program. MEK inhibition also reduces proteins levels in 4hrs and 24hrs. Surprisingly, MEK inhibition also led to G1 arrest in DU145 which has RB loss, leading to new investigations in the cell cycle status of our SCL model in response to MAPK inhibition as well as FOS proto-oncogene knock-down. To identify upstream regulators of FOSL1 and cFOS, we will perform a CRISPR sgRNA screen for E3 ligases targeting these FOS proto-oncogenes to the proteosome. In summary, we have determined the role of the FOS proto-oncogenes FOSL1 and cFOS as critical drivers of SCL-CRPC. We will investigate its interaction with MAPK signaling as well as regulators of its protein stability and assess the impact of its inhibition via AFOS on cell cycle in vitro and in vivo. With these mechanisms clearly studied, we would be able to give strategic insights for drug targeting. Citation Format: Hongsu Wang, Chen Khuan Wong, Dana Schoeps, Yu Chen. Dissecting the mechanisms of dependency of stem-cell-like castration resistant prostate cancer on FOS proto-oncogenes and MAPK signaling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr A072.

Abstract Background: Sequential Tyrosine Kinase Inhibitor (TKI) therapy is a well-established strategy to overcome on-target resistance in ALK, ROS1, TRK, and RET kinase fusion-positive (+) tumors. However, this approach does not address off-target resistance that occurs when tumors acquire oncogenic alterations that activate bypass signaling pathways. A hallmark of off-target resistance is the sustained activation of ERK, a process driven by the hyperactivation of Receptor Tyrosine Kinases (RTKs) and/or acquired alterations within the downstream RAS-MAPK signaling pathway. Hypothesis: We hypothesize that inhibition of the MAPK/ERK pathway (using SHP2i or ERKi) can overcome off-target resistance by resensitizing tumors to their original TKIs. Methods: We generated multiple transduced and patient-derived kinase fusion+ cell lines and PDXs with off-target resistance to TKIs. These included ALK, TRK, and RET fusion+ models carrying MET or IGF1R amplification as well as ALK, TRK, and ROS1 fusion+ models carrying a KRAS mutation. The activity and safety of next-generation ERKi (ulixertinib, temuterkib, ERAS-007) or SHP2i (TNO-155, BBP-398, ERAS-601) as monotherapies or combined with TKIs was tested using viability assays, synergy tests, downstream signaling profiling, and in vivo tolerability and efficacy studies. Results: ERK and SHP2 inhibition was able to resensitize all models to the original TKIs in vitro. Among the SHP2is tested, TNO-155 and ERAS-601 were equally active, and both synergized (BLISS score >10) with TKIs (e.g., BLISS scores of 23.38 and 33.88 for TNO+TKI and 601+TKI, respectively in an ALK+, IGF1R amplified model). Among ERKis, only ERAS-007 resensitized these cell lines to TKIs, with BLISS scores up to 25.36. Notably, ERAS-007 also demonstrated potent single-agent activity at nanomolar concentrations in most models (e.g., 90.6% cell growth inhibition at 25 nM in an ALK+, KRAS mutant cell line; P=.000018). Mechanistically, dual inhibition using SHP2i or ERAS-007 in combination with TKIs completely suppressed phosphorylation of RSK1, a direct downstream substrate of ERK. To determine the optimal drugs’ doses for the in vivo testing, we performed a 12-day tolerability study in healthy mice and found that standard doses of TKIs combined with 30mg/kg QD of SHP2i or ERKi did not cause toxicity (no mouse weight loss or impaired liver/kidney function). Importantly, these combinatorial regimens were significantly more effective than each of the single agents in inhibiting tumor growth in xenografts derived from an ALK+, IGF1R amplified NSCLC cell line (P=.006584 for SHP2i+TKI and P=.013847 for ERKi+TKI) and in a PDX derived from a TRK+, KRAS G12D mutant tumor (P<.000001 for SHP2i+TKI and P=.003215 for ERKi+TKI). Conclusions: ERKi or SHP2i synergize with TKIs in kinase fusion+ tumors with off-target resistance to therapy. Defining the biochemical properties of clinically available ERKis and SHP2is is crucial to design effective and tolerable combinatorial therapies to treat patients with TKI-resistant kinase fusion+ tumors. Citation Format: Shaza M. Sayed Ahmed, Valentina Fogglizo, Zane Mamman, Courtney Lynn. Binaco, Sherifah Kemigisha Muzungu, Paola Roa, Amanda Kulick, Elisa de Stanchina, Matteo Repetto, Yonina R. Murciano-Goroff, Ezra Rosen, Sandra Misale, Eneda Toska, Robert Shoemaker, Alexander Drilon, Emiliano Cocco. Overcoming off-target resistance to therapy in tumors driven by kinase fusions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B030.