Klinge, Skyeler

Abstract Gastrointestinal (GI) cancers are rising in people < 50 and remain major causes of cancer mortality despite therapeutic advances. Chimeric antigen receptor T (CART) cell therapy has transformed treatment of hematological cancers. However, its efficacy in GI cancers has been hindered by antigen heterogeneity and immunosuppressive cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME.) Here we aimed to 1) study CART-CAF interactions and 2) engineer CART cells capable of eliminating CAFs to overcome their inhibitory role and enhance their antitumor activity in GI cancers. We first analyzed primary CAFs from 20 GI cancer patients. CAFs showed high fibroblast activation protein (FAP) expression and significantly suppressed T cells proliferation (26-77%, p≤0.0001). We next generated CART cells capable of safely eliminating both GI cancer cells and CAFs. To target cancer cells, we developed a novel nanobody (VHH)-based CAR against Claudin18.2, a GI cancer specific target. To avoid off-tumor toxicity in healthy FAP+ tissues, we incorporated an inducible FAP-CAR using the synNotch platform, enabling conditional targeting upon engagement with Claudin18.2+ cells, restricting FAP targeting within the TME. VHH Claudin18.2 CART cells showed superior specificity and efficacy vs scFv-based CART cells (hu8E85 or zolbetuximab). In Claudin18.2+ SNU-C2B xenografts, tumors were 92mm3 (VHH) vs 48mm3 (huE85) vs 211mm3 (zolbetuximab) on day 23 post-CART, median survival undefined vs 17 days vs 23 days respectively; p=0005. In Claudin18.2+ LS411N xenografts, tumors were 315mm3 vs 106mm3 vs 382 mm3 respectively at 23 days post-CART, median survival undefined vs 19 days vs undefined respectively; p=0004. VHH Claudin18.2 CART cells showed no off-tumor effects, while hu8E85 CART induced significant toxicity. We next established a co-engraftment model. NSG mice received 1x106 SNU-C2B and 6x106 CAFs from a GI cancer patient. At 500 mm3 tumors, mice received 5x106 VHH Claudin18.2 or inducible Claudin18.2/FAP CART. Dual-targeting CART cells showed greater antitumor response (728mm3 vs 320mm3, p=0.0011 at day 20 post-CART), expansion (61 vs 2,350 CART/µl of blood, p=0.0037), and tumor infiltration (8.71 x 105 vs 1.03 x 106 CART/g of tumor, p<0.0001). This was also observed in gastric, colon, and pancreatic PDX models, with no observable off-tumor cytotoxic effects with inducible dual-targeting Claudin18.2/FAP CART. Our findings demonstrate that our novel VHH-based Claudin18.2 CART cells are highly specific and effective against GI malignancies, and that inducible Claudin18.2/FAP dual-targeting CART cells safely remodel the TME into an immune-permissive state. This approach represents a mechanistically innovative and translationally relevant strategy to overcome CAF-mediated immunosuppression, enhance CART cell efficacy, and improve clinical outcomes in refractory solid tumors. Citation Format: Omar L. Gutierrez Ruiz, Jennifer Feigin, Brooke Kimball, R. Leo Sakemura, Elizabeth L. Siegler, Dominic A. Skeele, Ateka Saleh, Claudia Manriquez Roman, Ismail Can, Olivia Sirpilla, Kun Yun, Carli M. Stewart, Mehrdad Hefazi, Yoh Yamaguchi, Lionel Aurelien Kankeu Fonkoua, Long K. Mai, Truc Huynh, Sophia Y. Goldberg, Grace E. DeFranco, Sara Foote, Ekene Johnkennedy Newton Ogbodo, Hong Xia, Michael Redig, Matthew L. Matthew, Tamiel Nichole Turley, Skyeler Klinge, John Alton Copland, Saad J. Kenderian. Optimized CART cell therapy to remodel the tumor microenvironment and eliminate GI malignancies [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 1525.

Abstract Background: The cellular balance between saturated and monounsaturated fatty acids is a defining feature of tumor lipid metabolism, with SCD1 functioning as a key modulator through the production of MUFAs. Elevated SCD1 activity and MUFA accumulation have been documented in hepatocellular carcinomas, although the consequences of restricting MUFA synthesis have not been fully clarified. Methods: A selective SCD1 inhibitor, SSI-4, was evaluated across established human HCC cell lines and xenograft models. Measurements included cell viability, lipid compositional analyses, and characterization of stress response pathways at both molecular and biochemical levels. The effects of MUFA depletion were studied in vitro and in vivo, with attention to ER stress, apoptosis, ferroptosis markers, and autophagy induction. Results: SSI-4 treatment led to marked reduction in cellular MUFA content and an increase in saturated fatty acid pools. The resulting metabolic stress correlated with activation of unfolded protein response components, caspase-mediated cell death, and the upregulation of ferroptosis-associated genes. Triglyceride and MUFA levels declined precipitously, as confirmed by mass spectrometry-based lipidomics. Tumor growth was suppressed in PDX models and evidence of autophagic flux was apparent in sensitive cell lines. Variability in response highlights metabolic heterogeneity in HCC. Conclusions: Targeted disruption of MUFA biosynthesis inflicts metabolic vulnerability on HCC cells, precipitating membrane alterations and activation of multiple cell death programs. These findings recommend further investigation into metabolic interventions exploiting MUFA depletion, with consideration for stratifying patients according to lipid metabolic phenotypes. Funding: This work is supported by Mayo Clinic Comprehensive Cancer Center and the Jay and Deanie Stein Career Development Award for Cancer Research at Mayo Clinic Jacksonville and the Mayo Clinic Translational Hepatobiliary Cancer SPORE P50CA210964 Career Enhancement Program (CEP) and Florida Department of Health/Florida Cancer Innovation Fund C25C23. Citation Format: Justyna J. Gleba, Victoria N. Rios, Brian M. Necela, Skyeler M. Klinge, Matt L. Pawlush, Mike J. Redig, Aylin Alasonyalilar-Demirer, Han W. Tun, John A. Copland III, . Disrupting tumor lipid homeostasis by MUFA depletion drives cell death in HCC [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 7333.

Abstract Chimeric antigen receptor T (CART) cell therapy has shown unprecedented success in the treatment of hematological malignancies. However, the immunosuppressive tumor microenvironment of both solid and liquid tumors is a major barrier for CART efficacy. Immunosuppressive myeloid cells inhibit CART efficacy. Colony-stimulating factor 1 receptor (CSF1R) promotes macrophage proliferation, survival, and anti-inflammatory response and is upregulated on myeloid cells in many cancers, making CSF1R an appealing therapeutic target. We aimed to test the interactions between CSF1R, myeloid cells, and CART response, and to study CSF1R targeting in the context of CART cell therapy. We utilized CD19-targeted CART cell therapy (CART19) in lymphoma preclinical models and thyroid-stimulating hormone receptor (TSHR)-targeting CART cell therapy (TSHR-CART) in thyroid cancer models. We first measured CSF1R expression on myeloid cells in vitro. Suppressive myeloid cells, polarized from healthy donor monocytes using an anaplastic thyroid cancer (ATC) cell line, 8505c, or mantle cell lymphoma cell line, JeKo-1, significantly upregulate CSF1R (p=0.0002) compared to media control. After polarization, myeloid cells significantly inhibit TSHR-CART and CART19 antigen-specific proliferation compared to freshly isolated monocytes (p=0.012, p<0.01, respectively) indicating that suppressive myeloid cells contribute to CART failure in multiple tumor types. We then performed single cell RNA sequencing on peripheral blood taken at baseline from patients with mantle cell lymphoma treated with CART19. We found a significant increase of CSF1R on monocyte subsets in CART19 non-responders vs responders (padj=0.0037). Next, we determined the impact of CSF1R blockade on CART cell therapy in vivo. Using lymphoma models, B6 mice were engrafted with the luciferase+ CD19+ murine lymphoma cell line TBL-12 (1x106 cells i.v. 1 day after 250 mg/kg cyclophosphamide). Tumor burden was significantly reduced after 7 days of treatment with low dose CART19 (0.5x106 i.v.) + anti-mouse CSF1R antibody (16mg/kg i.p. twice weekly) vs CART19 + IgG control (p=0.0146). We then tested the impact of CSF1R blockade in mice experiencing tumor relapse. After TBL-12 engraftment, mice were treated with anti-mouse CSF1R or IgG control antibody followed two days later with high dose CART19 (1x106 i.v.) to induce remission. Mice were re-challenged with TBL-12 14 days later. Mice treated with anti-mouse CSF1R antibody showed increased tumor control and significantly higher survival (p=0.0416). Using thyroid cancer models, first we identified high macrophage infiltration in biopsy specimens of patients with ATC compared to normal thyroid tissue and benign thyroid tumors through immunohistochemistry (IHC) of patient samples (p<0.01, p<0.0001, respectively). To test if macrophages are associated with CART failure in ATC, NSG mice were subcutaneously engrafted with 1x106 TSHR+ THJ529 tumors, an ATC cell line derived from patient biopsy samples from the Mayo Clinic thyroid cancer biobank, followed by i.v. treatment with 1x107 TSHR-CART or untransduced T cell control (UTD). IHC staining of tumor tissue collected at experimental endpoints revealed a dose-dependent, significant infiltration of human CD3+ T cells into the tumors which inversely correlated with murine F4/80+ myeloid cell and murine YM-1+ M2 macrophage infiltration. This led us to hypothesize that myeloid cell targeting with CSF1R blockade can improve TSHR-CART efficacy. We utilized the same ATC model described above to test CSF1R targeting. When TSHR+ THJ529 tumors reached approximately 100 mm3, mice were randomized by tumor volume and treated with CSF1R inhibitor PLX5622 (600mg/kg) or vehicle control, followed by either UTD or TSHR-CART cells 5x106 i.v. Mice treated with TSHR-CART + PLX5622 showed significantly improved tumor control vs TSHR-CART + vehicle control (p<0.0001) or UTD + PLX5622 (p<0.0001). Together, our results highlight immunosuppressive myeloid cells which express CSF1R as a major cell type limiting CART efficacy in both solid and liquid tumors. We also identified a strategy to overcome myeloid-mediated suppression of CART cells by targeting CSF1R with antibody or small molecule inhibitors.

Abstract Lung cancer is the leading cause of cancer deaths in the US. Non-small cell lung cancer (NSCLC) accounts for ~80% of lung cancer cases and is further classified into adenocarcinoma (LUAD), squamous cell carcinoma (LUSC), and large cell carcinoma. New approaches targeting pathways in NSCLC have emerged, resulting in encouraging new treatments that benefit LUAD patients, but very few advances have been made in the treatment options for LUSC which accounts for ~30% of lung cancer cases. NSCLCs, including LUSCs, contain a sub-population of cells that exhibit properties of cancer stem cells (CSCs) and are responsible for tumor initiation, maintenance, therapeutic resistance, and relapse. Therefore, CSCs must be effectively targeted to elicit long lasting therapeutic results in LUSC patients. Our studies have shown that: 1) PKCι is an oncogene and prognostic marker in LUSC; 2) copy number gains (CNGs) of the PKCι gene (PRKCI) drives overexpression of PKCι in 90% of LUSCs; 3) PKCι phosphorylates SOX2, and controls SOX2-mediated transcriptional activation of Hedgehog Acyl Transferase, resulting in Hedgehog (Hh) pathway activation that drives a CSC phenotype in LUSC cells; and 4) the FDA approved compound Auranofin (ANF) is a potent inhibitor of oncogenic PKCι. In this study, we assessed the effect of ANF in combination with the Hh pathway inhibitor, LDE225, on LUSC CSCs and tumor growth. Cell viability, sphere formation and soft agar assays were used to assess the effects of ANF and LDE225 alone or in combination on the growth of LUSC CSCs with PRKCI CNGs or LUAD and LUSC CSCs without PRKCI CNG to determine if PRKCI CNG can be used to predict response to ANF and LDE225 and test the selectivity of response for LUSC. The effects of ANF and LDE225 on the levels of PKCι-Hh signaling components was assessed by qPCR and western blotting. Finally, the efficacy of ANF and LDE225 in combination was evaluated in human primary LUSC patient-derived xenograft (PDX) models that harbor PRKCI CNGs and the ability of PKCι-Hh signaling intermediates to serve as biomarkers of PKCι-Hh pathway inhibition was validated in vivo. We observed a dose-dependent decrease in cell proliferation, clonal expansion and transformed growth in CSCs treated with ANF and LDE225 alone. ANF and LDE225 combined treatment of LUSC CSCs with PRKCI CNGs (but not LUAD or LUSC CSCs without PRKCI CNG) synergistically inhibited CSC phenotypes based on results of combination index analysis. Furthermore, treatment of LUSC PDX models with ANF and LDE225 in combination had a synergistic effect on tumor growth inhibition. Lastly, ANF and LDE225 treatment led to inhibition of PKCι-Hh signaling intermediates demonstrating the on-target effects of treatment. Our data indicate that a PKCι-Hh vertical blockade strategy of combined ANF and LDE225 effectively targets LUSC CSCs and blocks tumor growth in vivo and provides compelling rationale to develop this novel combination strategy further in clinical trials. Citation Format: Dania Alqasrawi, Ryan Argo, Christine Ratliff Rang, Prita Pandya, Skyeler Klinge, Jennifer Lindemann, Nyla Searl, Alan Fields, Verline Justilien. A novel combination therapy for lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5434.