Korleski, Jack

Abstract Background Preparing tumor board patient summaries is time intensive. Large-language-model based systems may automate summarization but require real-world evaluation prior to clinical use. We performed an exploratory retrospective evaluation of the Microsoft Healthcare Agent Orchestrator (HAO), deployed in a Mayo Clinic–controlled staged environment, to generate tumor board–style patient summaries from retrospective Electronic Health Record (EHR) notes. Methods HAO generated summaries for breast, hepatobiliary, and neuro-oncology tumor board cases using up to the most recent 1,000 clinical notes. Clinician reviewers evaluated outputs via REDCap surveys across perceived factuality, completeness, clarity/conciseness, temporal cohesion, comparative performance, safety, and clinical utility (0–4 Likert scale). Reviewers were permitted to query the HAO chat interface to address missing details. Automated factuality was assessed using TBFact (bidirectional entailment), reporting precision and recall against available reference summaries. Results Among 57 survey responses from 5 different physicians, mean scores exceeded 2.8 across domains, with medians of 3 for most axes. In an exploratory comparison, oncology fellows required less time to review HAO-generated summaries than to manually generate patient summaries (mean difference 13.57 minutes per patient, p<0.001), although this difference may be influenced by prior familiarity with the same cases; 96% of survey responses indicated that HAO would save time. TBFact evaluations showed higher recall than precision across domains, consistent with broad capture of reference content alongside additional content that was not present in gold-standard summaries. Attribution was viewed favorably but showed issues with primary-source specificity and link reliability. Conclusions In a controlled Mayo environment, HAO demonstrated moderate performance and was associated with reduced review time for tumor board preparation. These findings are promising but preliminary and do not establish clinical safety, noninferiority to manual review, or readiness for routine clinical use. Limitations, including verbosity, specialty-specific content gaps, and inconsistent attribution, highlight the need for iterative refinement and further evaluation. Graphical Abstract Created using ChatGPT

Abstract A hallmark of GBM is its highly-immunosuppressive tumor microenvironment (TME). GBM stem-like cells (GSCs) can evade immune surveillance to allow for continued tumor growth and propagation. GSCs regulate the immune TME through recruitment and production of immunosuppressive cells and factors via tumor cell-intrinsic mechanisms. Blocking these cell-intrinsic immunosuppressive mechanisms is widely viewed as a way to enhance the effects of current immune therapies. We recently showed that TGFβ type II receptor (TGFBR2) promotes a mesenchymal transition in GBM cells characterized by high expression levels of immunosuppressive genes NT5E (CD73), ENTPD1 (CD73), LGALS1 (galectin-1), CD274 (PD-L1), PDCD1LG2 (PD-L2), and TGFB1. miRNA network analysis identified miR-16-5p and miR-124-3p as high-confidence candidates to block the expression of these immunosuppressive signaling axis in GBM cells. Transgenic miR-16-5p and miR-124-3p simultaneously reduced expression of CD274, ENTPD1, NT5E, TGFB1, and LGALS1 and increased tumor cell killing of PBMC-derived CD4+ and CD8+ T cells. We also measured an increase in drivers of the CD8+ effector state and a decrease in expression of exhaustion marker PD1 in PBMC-derived CD8+ T cells. In addition to the effects on CD4+ and CD8+ T cell activation and function, miRNA-mediated knock-down of this immunosuppressive gene signature in tumor cells also resulted in a decrease in CD206 expression and reduced release of anti-inflammatory chemokines in THP1 cells. These findings demonstrate that miRNA-based gene inhibition can block cell-intrinsic immunosuppressive pathways to enhance anti-tumor immune cell function in vitro and predicts that delivering these miRNAs using advanced nanocarriers will cooperate with current immunotherapies in vivo. Citation Format: TC Taylor, Amanda Johnson, Harmon Khela, Jack Korleski, Sophie Sall, Bachchu Lal, Karen Smith-Connor, Hernando Lopez-Bertoni. miR-16-5p and miR-124-3p block the immunosuppressive phenotype of GBM cells in vitro. [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 1357.

Attempts to activate an anti-tumor immune response in glioblastoma (GBM) have been met with many challenges due to its inherently immunosuppressive tumor microenvironment. The degree and mechanisms by which molecularly and phenotypically diverse tumor-propagating glioma stem cells (GSCs) contribute to this state are poorly defined. In this study, our multifaceted approach combining bioinformatics analyses of clinical and experimental datasets, single-cell sequencing, and the molecular and pharmacologic manipulation of patient-derived cells identified GSCs expressing immunosuppressive effectors mimicking regulatory T cells (Tregs). We showed that this immunosuppressive Treg-like (ITL) GSC state is specific to the mesenchymal GSC subset and is associated with and driven specifically by TGFβ type II receptor (TGFBR2) in contrast to TGFBR1. Transgenic TGFBR2 expression in patient-derived GBM neurospheres promoted a mesenchymal transition and induced a six-gene ITL signature consisting of CD274 (PD-L1), NT5E (CD73), ENTPD1 (CD39), LGALS1 (galectin-1), PDCD1LG2 (PD-L2), and TGFB1. This TGFBR2-driven ITL signature was identified in clinical GBM specimens, patient-derived GSCs, and systemic mesenchymal malignancies. TGFBR2high GSCs inhibited CD4+ and CD8+ T cell viability and their capacity to kill GBM cells, effects reversed by pharmacologic and shRNA-based TGFBR2 inhibition. Collectively, our data identify an immunosuppressive GSC state that is TGFBR2-dependent and susceptible to TGFBR2-targeted therapeutics.

Abstract Addressing the limited responsiveness to immune checkpoint blockade (ICB) in human cancers remains a critical challenge. Efforts over the past two decades have primarily focused on enhancing CD8 T cell function and preventing their exhaustion. However, less attention has been given to understanding the molecular mechanisms that contribute to the immunosuppressive tumor microenvironment (TME), which impedes CD8 T cell infiltration and fosters ICB resistance. The TME in ICB-resistant tumors is characterized by Transforming Growth Factor beta (TGF-β) cytokine signaling, leading to a fibrotic extracellular matrix (ECM) and the accumulation of immunosuppressive fibroblasts and myeloid cells. These features hinder CD8 T cell penetration, thereby promoting immune escape. The immune checkpoint molecule B7-H3, overexpressed in ICB-resistant cancers, has been linked to impaired CD8 T cell infiltration and poor prognosis. However, the molecular mechanisms governing B7-H3 function remain poorly understood. We investigated the relationship between B7-H3 gene expression and ICB response using transcriptomic data from two randomized phase 3 trials (IMvigor010 and IMvigor210). Bulk RNAseq data, tissue staining, and spectral flow cytometry were used to characterize the TME composition of B7-H3 high and low tumors. Multi-omic profiling of tumor cell lines with manipulated B7-H3 expression was performed to identify downstream targets of B7-H3. Syngeneic mouse tumor models were used to assess the impact of B7-H3 on tumor growth and antitumor immunity. High B7-H3 expression was associated with shorter overall survival in both IMvigor010 (HR=0.48, 95% CI: 0.33-0.70, p&lt;0.0001) and IMvigor210 trials (HR=0.68, 95% CI: 0.51-0.92, p=0.01). In ICB-refractory cancers, B7-H3 expression correlated with high levels of fibroblasts, collagen deposits, and M2 macrophages. Gene set enrichment analysis revealed a positive association between B7-H3 and TGF-β signaling. Genetic deletion of B7-H3 in tumor cells reduced the surface expression of MMP14, a metalloprotease that activates TGF-β. Pan-cancer analysis identified MMP14 as the gene most correlated with B7-H3 (Pearson r=0.69, p&lt;0.0001). TGF-β activation was reduced by 70% in B7-H3 knockout cells compared to wild-type. Loss of B7-H3 led to MMP14 accumulation in lysosomes, resulting in its degradation. We generated a B7-H3 inhibitor, 5D10, which promotes MMP14 degradation and inhibits TGF-β activation. In humanized B7-H3 mouse models, 5D10 significantly reduced TGF-β activation, enhancing CD8 T cell infiltration and impairing tumor growth. Our research identifies B7-H3 as a driver of immune exclusion and ICB resistance. We describe a novel, receptor-independent mechanism where B7-H3 promotes TGF-β activation via stabilization of MMP14. Targeting B7-H3 represents a strategy to modulate the TME, transforming immune-excluded tumors into immune-inflamed states that may respond better to ICB. Citation Format: Fabrice Lucien, Roxane Lavoie, Jack Korleski, Yohan Kim, Bharath Wootla, Liguo Wang, Ava Farrell, Kelly Harper, Martine Charbonneau, Claire Dubois, Igor Frank, Sounak Gupta, John Cheville, Jacob Orme, Stephen Boorjian, Parash Shah, Eugene Kwon, Sean Park, Haidong Dong. The immune checkpoint molecule B7-H3 is a driver of tumor immune exclusion by mediating TGF-β activation [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B034.

Abstract Attempts to activate an anti-tumor immune response in glioblastoma (GBM) have been met with many challenges due to its inherently immunosuppressive tumor microenvironment. The degree and mechanisms by which molecularly and phenotypically diverse tumor-propagating glioma stem cells (GSCs) contribute to this state are poorly defined. In this study, our multifaceted approach combining bioinformatics analyses of clinical and experimental datasets, single-cell sequencing, and molecular and pharmacologic manipulation of patient-derived cells identified GSCs expressing immunosuppressive effectors mimicking regulatory T cells (Tregs). We show that this I mmunosuppressive T reg- L ike (ITL) GSC state is specific to the mesenchymal GSC subset and is associated with and driven specifically by TGF-β type II receptor (TGFBR2) in contrast to TGFBR1. Transgenic TGFBR2 expression in patient-derived GBM neurospheres promoted a mesenchymal transition and induced a 6-gene ITL signature consisting of CD274 (PD-L1), NT5E (CD73), ENTPD1 (CD39), LGALS1 (galectin-1), PDCD1LG2 (PD-L2), and TGFB1. This TGFBR2-driven ITL signature was identified in clinical GBM specimens, patient-derived GSCs and systemic mesenchymal malignancies. TGFBR2 High GSCs inhibited CD4+ and CD8+ T cell viability and their capacity to kill GBM cells, effects reversed by pharmacologic and shRNA-based TGFBR2 inhibition. Collectively, our data identify an immunosuppressive GSC state that is TGFBR2-dependent and susceptible to TGFBR2-targeted therapeutics.

Background/Objectives: CSCs are critical drivers of the tumor and stem cell phenotypes of glioblastoma (GBM) cells. Chromatin modifications play a fundamental role in driving a GBM CSC phenotype. The goal of this study is to further our understanding of how stem cell-driving events control changes in chromatin architecture that contribute to the tumor-propagating phenotype of GBM. Methods: We utilized computational analyses to identify a subset of clinically relevant genes that were predicted to be repressed in a Polycomb repressive complex 2 (PRC2)-dependent manner in GBM upon induction of stem cell-driving events. These associations were validated in patient-derived GBM neurosphere models using state-of-the-art molecular techniques to express, silence, and measure microRNA (miRNA) and gene expression changes. Advanced Poly(β-amino ester) nanoparticle formulations (PBAEs) were used to deliver miRNAs in vivo to orthotopic human GBM tumor models. Results: We show that glioma stem cell (GSC) formation and tumor propagation involve the crosstalk between multiple epigenetic mechanisms, resulting in the repression of the miRNAs that regulate PRC2 function and histone H3 lysine 27 tri-methylation (H3K27me3). We also identified miR-217-5p as an EZH2 regulator repressed in GSCs and showed that miR-217-5p reconstitution using advanced nanoparticle formulations re-activates the PRC2-repressed genes, inhibits GSC formation, impairs tumor growth, and enhances the effects of ionizing radiation in an orthotopic model of GBM. Conclusions: These findings suggest that inhibiting PRC2 function by targeting EZH2 with miR-217-5p advanced nanoparticle formulations could have a therapeutic benefit in GBM.

Abstract Despite aggressive therapy consisting of surgery followed by radio/chemotherapy Glioblastoma (GBM) recurs in almost all patients and, currently, there are no proven therapies to treat recurrent GBM (rGBM). Recent developments in nanomedicine provide new and promising opportunities to develop new targeted therapeutics to treat brain tumors. In this study we combine bioinformatics, forward-thinking understanding of miRNA biology and cutting-edge nucleic acid delivery vehicles to advance targeted therapeutics for rGBM. Bioinformatic analysis of RNA sequencing from GSCs and clinical rGBM specimens identified TGF-beta receptor II (TGFBR2) signaling as a targetable pathway in rGBM. Mechanistically, we show that alterations in chromatin state driven by stem-cell driving events are conducive to a therapy-resistant state induced by TGFBR2. We show that blocking TGFBR2 via molecular and pharmacological approaches decreases the stem cell capacity, cell viability and re-sensitizes clinical rGBM isolates to temozolomide (TMZ) in vitro. Network analysis of SMAD2/3 targets enriched in rGBM predicted a subset of miRNAs that can target multiple genes within this set. Notably, miR-590-3p was predicted to inhibit 37 SMAD 2/3 gene targets that are also enriched in rGBMs. Bioinformatics analysis also showed that expression of 24 of those 37 miR-590-3p targets are positively correlated with TGFBR2 expression in rGBM clinical specimens. Consistent with these predictions, miR-590-3p inhibited expression of 25 of the 37 targets across 3 distinct therapy-resistant GSC isolates. To translate these in vitro finding, we developed novel bioreducible Lipophilic poly(β-amino ester) nanoparticles (LiPBAEs) for in vivo miRNA delivery. Following direct intratumoral infusion, these nanomiRs efficiently distribute through the tumors and spatial transcriptomic analysis (STA) found a decrease in 34 out of the 37 miR- 590-3p targets in animals that received the active nanomiR compared to control miRNA. Furthermore, STA identified 3 cell populations with distinct transcriptomic profiles reflecting a gradient pharmacodynamic response within tumors that received miR-590-3p nanomiRs with decreasing target expression towards the core of the tumor closest to catheter tip placement. To test the effect of 590-3p nanomiR therapy on survival, animals bearing pre-established intracranial rGBM xenografts received three infusions of either control or miR-590-3p nanomiRs every other day beginning on post-implantation day 7. All 10 animals treated with control nanomiRs were either dead or premorbid requiring euthanasia by post-implantation day 23. In contrast, 3 of 10 animals treated with miR-590-3p nanomiRs remained alive and healthy by post-implantation day 45, at which time the experiment was terminated. These results show that miRNA-based targeted therapeutics provide new opportunities to treat rGBM and bypass the resistance that is developed to standard of care. Citation Format: Hernando Lopez-Bertoni, Jack Korleski, Sophie Sall, Kathryn Luly, Maya K Johnson, Amanda L Johnson, Harmon Khela, Bachchu Lal, TC Taylor, Jean Micheal Ashby, Hector Alonso, Alice Li, Stephany Tzeng, John Laterra, Jordan J Green. Multipronged SMAD Pathway Targeting by LiPBAE miR-590-3p NanomiRs Inhibits Recurrent Glioblastoma Growth and Prolongs Survival [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR011.

Abstract Background Chimeric antigen receptor (CAR) T-cell therapy has been revolutionary in the treatment of blood cancers, but has been associated with arrhythmias such as atrial fibrillation (AF). Whether the incidence of AF has changed over time and if contributing risk factors are present remains to be defined. Methods Leveraging the Mayo Electronic Medical Record, we extracted a cohort of patients who underwent CAR T-cell therapy between 2016 and 2022. From this cohort, we identified those who had a diagnosis of AF at any point. We then defined the endpoint of interest as the first episode of new or recurrent AF during or after CAR T-cell infusion. Fine and Gray subdistribution hazard modeling was used to calculate the association of patient characteristics to the endpoint of interest. Variables with P&lt;0.20 in univariate modeling were chosen for the multivariate model. Significance was defined as P&lt;0.05. Results There were 328 patients who underwent CAR T-cell therapy between 2016 and 2022. Of this cohort, 27 (8.2%) patients had a pre-existing diagnosis of AF, of which 6 had recurrence of AF after CAR T (22.2%), while 21 (77.8%) did not. Of the 301 patients without a prior history of AF, 8 (2.7%) patients developed new-onset AF after CAR T. The majority of the 14 new or recurrent AF events (4.3% overall incidence) occurred within 30 days of receiving CAR T (N=10, 71.4%), the remainder after 180 days (Figure 1). No trend in declining incidence over time was noted. Prior AF, neurotoxicity, and inflammation (cytokine release syndrome and macrophage activating syndrome-like (MAS-L)) were strongly associated with the risk of developing new or recurrent AF (Table 1). Only presence of MAS-L remained an independent predictor in the multivariate model (HR 15.4, 95% CI 3.9-61.2, P&lt;0.001). Conclusion AF is mainly seen early following CAR T-cell therapy, and mainly in association with severe inflammatory responses. Early and aggressive treatment of CRS and MAS-L may be recommendable for patients undergoing CAR-T therapy, especially for those with a prior history of AF.

Abstract Background: KRAS is a significant driver gene for pancreatic ductal adenocarcinoma (PDAC) and is present in up to 90% of PDAC cases. KRAS mutant (mt) PDAC tends to have worse prognosis compared to KRAS wild-type (wt) PDAC. However, the clinical and biological impact of the KRAS mutation subtypes (e.g. G12D, G12R, G12V, G12C and Q61) remains unclear. We sought to explore the clinical and molecular characteristics of the KRAS mt subtypes. Methods: We performed a retrospective review of patients with KRAS mt PDAC who underwent next generation sequencing at our institution from December 1, 2018 to December 1, 2021. We collected patient demographics, disease characteristics, clinical outcomes, DNA and RNA sequencing results via chart review. Bulk RNA-seq was performed via Tempus and The Cancer Genome Atlas. Count data were processed and separated by KRAS mutation subtype using a custom python script. Differential gene expression was performed using the DifSeq2 R package. Results: A total of 158 KRAS mt patients (at diagnosis, average age 63.8 years, 57% male, 88 (56%) Stage IV) were included. 10% of patients had KRAS Q61 mutation, 14% G12R, 30% G12V and 46% G12D; only 1.1% had the potentially actionable G12C mutation. G12R had a longer overall survival (OS) compared to the others (median 24.8 vs 17.5 months (m), p=0.05). Among patients with de novo metastatic disease, G12V had the lowest OS (median 9.8 vs 12.4 m, p=0.02). Between the KRAS mt subtypes, there were no statistically significant differences in tumor mutation burden, location of PDAC or subsequent metastasis, lines of treatment (average 2.3), age at diagnosis, gender, or stage at diagnosis. One hundred thirty-seven (87%) patients had at least one pathogenic somatic variant, 14 of which had statistically significant differences between KRAS mt subtypes. 82 (52%) patients had at least one RNA expression variant and bulk RNA-seq showed that each KRAS G12 mutation subtype had unique gene expression patterns, including several oncogenes and tumor suppressor genes. Conclusions: Our data suggest that KRAS mt subtypes may confer different clinically significant phenotypes of PDAC. We found G12R to be associated with improved prognosis compared to other KRAS mt subtypes and that among patients with de novo metastatic disease, G12V had the worst prognostic implication. As there were no statistically significant differences in patient demographics between the KRAS mt subtypes, we hypothesize survival differences are driven by biological phenotype. This is supported by our RNA-seq findings, which demonstrated unique gene expression patterns for each KRAS mt subtype. Citation Format: Alexander Xiao, Jack Korleski, Ryan Carr. KRAS mutation subtypes are associated with clinical and phenotypic differences in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C095.