Negative Cells Research Articles

Abstract 1333: Preclinical data supporting the Phase 1 trial design of SENTI-202, a next generation allogeneic logic-gated selective CAR NK cell therapy, engineered to overcome key limitations of first generation cell therapies in AML

Abstract First generation natural killer (NK) and chimeric antigen receptor (CAR) NK cell products are well-tolerated and have clinical activity (~20-60% CR) in patients with AML. CRs generally increase with the inclusion of CARs or cytokines, increased cell doses, and when Flu/Ara-C lymphodepletion (LD) is used for conditioning. Key clinical limitations have been low PK of the infused NK cells, short durability of the observed responses potentially due to immune evasion of leukemic stem cells (LSCs), and manufacturing challenges precluding higher or multiple doses. SENTI-202 is a next generation allogeneic logic-gated selective CAR NK cell therapy specifically designed to address these limitations and to augment endogenous NK anti-AML activity. SENTI-202 expresses a bivalent activating CAR (aCAR) that targets CD33 and FLT3 on bulk AML blasts and LSCs. As FLT3 is also expressed on healthy hematopoietic stem cells (HSCs), SENTI-202 is further engineered to express an inhibitory CAR that recognizes endomucin expressed on HSCs, thereby conferring protection from aCAR-mediated cytotoxicity even in the presence of FLT3/CD33. SENTI-202 expresses a calibrated-release IL15 that provides both auto- and paracrine cytokine support, mediating expansion, activation, and persistence of SENTI-202 and host immune cells. SENTI-202 demonstrated robust and specific killing of primary AML blasts, LSCs, and AML cell lines in vitro. SENTI-202 protected HSCs from CAR-mediated cytotoxicity while preserving their function. In vivo, SENTI-202 revealed robust efficacy that increased with higher E:T ratio and with 3 weekly vs 1 dose. Preclinically, 3 doses of SENTI-202 that were > 60-fold the planned starting clinical trial dose were well tolerated in acute and chronic toxicology studies with no CAR NK-related body weight, laboratory, or histopathology findings. SENTI-202 non-clinical PK revealed greater than dose proportional exposure, which was ~2-fold greater compared to non-engineered NK cells. Pretreatment of CD33/FLT3 negative AML cell lines with Ara-C resulted in upregulation of CD33 and FLT3 expression, sensitizing cells to robust SENTI-202-mediated killing, providing additional rationale for the use of Ara-C-based LD. In the presence of exogenous IL2, persistence, cytotoxicity, and serial killing activity of SENTI-202 were increased, supporting the use of low dose IL2 to further augment SENTI-202 clinical activity. Taken together, these results support the Phase 1 trial design of SENTI-202-101 in patients with R/R CD33 and/or FLT3 positive malignancies including AML, which uses Flu/Ara-C as LD followed by 3 weekly doses of SENTI-202 and includes the option of enrolling patients into cohorts that additionally receive low dose IL2 following SENTI-202 administration. Citation Format: Deepika Kaveri, Pearley Chinta, Elizabeth Leitner, Lawrence Naitmazi, Gozde Yucel, Mengxi Tian, Niran Almudhfar, Han Deng, Yongshuai Li, Alice Lam, Chen-Ting Lee, Enping Hong, Rochelle Emery, Brian Garrison, Aaron N. Nguyen, Kanya Rajangam. Preclinical data supporting the Phase 1 trial design of SENTI-202, a next generation allogeneic logic-gated selective CAR NK cell therapy, engineered to overcome key limitations of first generation cell therapies in AML [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 1333.

Abstract 5805: A novel platform of diversified payloads to drive ADC innovation

Abstract First generation of antibody drug conjugates (ADCs) for anticancer therapy has been described more than two decades ago with gemtuzumab-ozogamicin receiving the first approval in 2000. Over the last 4 years, 8 new ADCs got FDA approval essentially due to the innovation on the linker improving the disposition in patients and the manufacturability together with the technical advantages in the engineering of biologics and the introduction of new cytotoxic payloads such as deruxtecan. In the future, next generation ADCs will be developed exploring payloads beyond the currently used cytotoxins to improve safety, efficacy and durability of responses and therefore the short- and long-term therapeutic benefit to patients. Nerviano Medical Sciences (NMS) generated a portfolio of novel and diversified payload linkers leveraging on its proprietary chemical collection that includes cytotoxic and targeted payloads. A rigorous screening funnel has been applied considering diversification in terms of MoAs, pharmacokinetic properties, mechanism of resistance and combinability with other therapies to identify the optimal match with specific tumor types and targets, generating a new class of ADCs with improved selectivity and efficacy for hard-to-treat cancers even in chemoresistant settings. NMS ADC platform incorporates 1) our patent protected payload linker NMS-P945, a duocarmycin like DNA minor groove binder and alkylating agent inducing DNA damage, acting in chemoresistant cells with bystander effect and immunogenic cell death properties and generating ADCs highly efficacious in mouse xenograft models and safe when administered in NHP, 2) novel anthracyclines (PNU-682 derivatives) with an improved stability and safety profile and 3) targeted molecules with different mechanism of action including payloads active in synthetic lethality contexts. Payload linkers with different functionalization generated from each payload have been evaluated for chemical stability, sensitivity to lysosomal protease cleavage, and conjugability to monoclonal antibodies. The ADCs, generated with proof-of-concept antibodies (e.g. Trastuzumab), were characterized in terms of Drug to Antibody Ratio (DAR), aggregation level, plasma stability, antigen binding and differential antiproliferative activity in target positive vs target negative tumor cells. The best products resulting from the screening funnel have been subjected to a multidimensional analysis taking into consideration payload properties, tumor features and antigen expression in selected populations with the aim of identifying tumor types particularly sensitive to the payload. Our multidimensional approach coupling new payload linker with innovative MoA to careful selection of patients will allow to reach unprecedented efficacy and safety profile. Citation Format: Paolo Orsini, Matteo Salsa, Simona Rizzi, Ulisse Cucchi, Daniela Faiardi, Alessia Burocchi, Antonella Ciavolella, Rosita Lupi, Fabio Gasparri, Barbara Valsasina. A novel platform of diversified payloads to drive ADC innovation [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 5805.

Abstract 1329: Development of the first optimised “off the shelf” MAGE-A4 targeting TCR-NK cells for advancement into the clinic for the treatment of solid tumors

Abstract T cell receptor (TCR)-T based therapies, such as MAGE-A4 TCR-T cells, have shown compelling data demonstrating effective infiltration into and targeting of solid tumors with clinical responses across various solid cancers. However, heterogeneity and down-regulation of target antigen and HLA expression limit the durability and curative potential of these types of treatments. On the other hand, owing to their clinical potency, favorable safety profile and applicability as “off the shelf” therapy, natural killer (NK) cells have emerged as promising modalities in recent years. Unguided NK cells have shown limited potential against solid cancers, due to lacking infiltration into tumors. With our proprietary TCR-NK platform, we combine the solid tumor targeting capabilities of TCRs with the pan-cancer sensing and cytotoxic potential of highly potent killer cells, redirecting and arming NK cells with a fully functional TCR-CD3 complex. Here we present our lead product, an optimized TCR-NK cell product, ZI-MA4-1a, expressing an affinity enhanced TCR (KVL-a) directed to the HLA class I-restricted clinically validated cancer-testis antigen MAGE-A4, that is broadly expressed across solid tumors. The KVL-a TCR was successfully engineered from a naturally occurring wild-type TCR, increasing the affinity to MAGE-A4-peptide-HLA complex and resulting in enhanced recognition of tumor cells expressing the target antigen. ZI-MA4-1a is produced by transducing healthy peripheral blood derived NK cells with the full CD3 complex, the KVL-a TCR and a CD8 coreceptor. The process has been optimized to enrich TCR-NK cells, increasing process robustness, diminishing the impact of donor variability, and producing a higher performing TCR-NK cell product. We show the dual functionality of ZI-MA4-1a: TCR driven cytotoxicity as well as innate NK sensing of antigen knockout or HLA negative cancers. We demonstrate killing of heterogenous tumor cell models in vitro, as well as the multifunctionality of ZI-MA4-1a with killing, degranulation and multicytokine secretion. Interestingly, we observe enhanced proliferation of ZI-MA4-1a cells after activation by MAGE-A4 expressing tumor cells, indicating that TCR-NK cells acquire further proliferative capacity when the TCR is triggered. Finally, we demonstrate higher activity of ZI-MA4-1a compared to KVL-a and benchmark TCR transduced T cells against antigen positive targets, and activity against antigen negative tumor cells escaping TCR-T cell recognition. Citation Format: Sarah Vollmers, Artur Cieslar-Pobuda, Justyna Kmiecik, Giulia Malachin, Michelle L. Sætersmoen, Frida Høsøien Haugen, Margherita Boieri, Maja Sandve, Dennis Clement, Ines Cardoso, Pimthanya Wanichawan, Martha E. Haugstøyl, Anja Oldenburg, Liliane Christ, Pranav Oberoi, Luz Mora-Velandia, Anders Holm, Namir J. Hassan, Emilie Gauthy, Julia Ino, Sylvie Pollmann, Luise U. Weigand. Development of the first optimised “off the shelf” MAGE-A4 targeting TCR-NK cells for advancement into the clinic for the treatment of solid tumors [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 1329.

Abstract 3035: Bridging the gap: Exploring the roles of ETS- coactivator interactions in promoting prostate cancer

Abstract The ETS family of transcription factors is comprised of 28 members that all share a common C- terminal DNA-binding domain. ETS play essential roles in developmental processes, such as stem cell maintenance, blood vessel formation, and cellular fate. While absent in the normal prostate, four members of the ETS family become expressed in prostate tumors: ETV1, ETV4, ETV5 and ERG. Our lab has previously classified these proteins as oncogenic ETS for their ability to promote cellular migration in normal prostate epithelial RWPE1 cells. Transcription factors may need to interact with additional proteins to serve oncogenic roles. My research explores the interactions between 1) ETV1 and EWS, as well as 2) ERG and p53. Our lab has established that ETV1, ETV4 and ETV5 form essential interactions with EWS, a transcriptional coactivator that promotes ETS activity. In prostate tumors, chromosomal rearrangements result in the N-terminal truncations of ETV1, ETV4 and ETV5. I hypothesize that the EWS interaction domain is retained and that these truncated ETVs will remain oncogenic. Pulldown assays using purified full-length and truncated ETV1 suggests EWS interacts with both N-terminal and C-terminal portions ETV1. I am attempting to express truncated ETV1, ETV4 and ETV5 in RWPE1 cells. Upon stable expression, I will evaluate their interactions with EWS and their ability to promote migration in vitro. To identify putative proteins that bridge EWS to ETV1, immunoprecipitation mass spectrometry will be used. Mutations in p53, a tumor suppressor protein, promote oncogenesis and occur in nearly half of all cancer cases. We hypothesize that mutant p53 interacts with ETS to promote its recruitment to new genomic sites. Previous studies demonstrate that mutant p53 binds ETS2 at a higher affinity than wildtype p53 and we have expanded this list of interacting ETS to include ERG. We seek to explore whether ERG and mutant p53 cooperate to promote oncogenic phenotypes. I successfully cloned wildtype p53, mutant p53 (R248W) and double-mutant p53 (R248W, T81A) into a retroviral backbone. I then transduced RWPE1-Vector and RWPE1-ERG expressing cells to express these constructs. Although additional replicates are needed, it appears that mutant p53 promotes colony formation in both cell types relative to wildtype p53. I plan to validate these observations and generate an appropriate negative control cell line before extending into a mouse model. Through the continuation of this work, I will provide critical insight into the molecular mechanisms by which a subset of ETS factors achieve oncogenic function. We address the contributions of DNA-binding specificity and coactivator interactions in promoting the phenotypes associated with prostate cancer. Citation Format: Kaitlyn M. Mills, Nicholas Downing, Stephanie Metcalf, Peter Hollenhorst. Bridging the gap: Exploring the roles of ETS- coactivator interactions in promoting prostate cancer [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 3035.

Abstract 2636: Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck squamous cell carcinoma

Abstract Background Head and neck cancer is the 8th most common cancer worldwide. Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) overall survival rates remain low (25-40%) despite advances in treatments. CD8+ T cell differentiation into activated/cytotoxic (CTL), exhausted (TEX) and tissue-resident memory (TRM) phenotypes is a major determinant of successful cancer immunotherapy, which relies on the ability of CD8+ T cells to detect and eliminate cancer cells. Recent evidence suggests the importance of epigenetic regulation in the development of CD8+ T cell exhaustion and tissue residency; yet the epigenetic mechanisms that govern the transition between CTL, TEX, and TRM CD8+ T cell states remain unclear. This study investigates the epigenetic modifications associated with CTL, TEX, and TRM states using CD8+ T cells from healthy donors and HPV-negative HNSCC tumors. Methods An in vitro human CD8+ T cell stimulation assay using healthy donor cells was utilized to generate CTL, TEX and TRM phenotypes. Naïve T cells underwent CD3/28, TCR-independent stimulation for 9 days to promote CTL (3 days) and TEX (9 days) phenotypes, and TGF-β exposure (days 3-9) occurred to induce a TRM phenotype. Flow cytometry to confirm expression of cell-surface and intracellular markers for each phenotype is ongoing. Genome-wide mapping for activating (H3K4me1, H3K4me3) and repressive (H3K9me3, H3K27me3, H4K20me3) histone marks with CUT&Tag assays will be pursued for each of the in vitro CTL, TEX and TRM states, along with RNA-seq. CUT&Tag and RNA-seq will also characterize the epigenetic profiles of CD8+ T cells from single-cell suspensions generated from HPV-negative HNSCC tumors (n=5). Findings will be correlated with results from our in vitro T cell states. Results Preliminary flow cytometry confirmed increased expression of CD25, PD-1, Tim3 after 9 days of in vitro stimulation, supporting the induction of a CD8+ TEX phenotype. CD103 was also highly expressed in CD8+ T cells exposed to TGF-β, supporting the induction of the CD8+ TRM phenotype. Validation of the CTL state is ongoing. Phenotypic validation of cell states with flow cytometry (18 cell surface and intracellular markers) is ongoing. Feasibility of genome-wide mapping for activating and repressive histone marks with CUT&Tag on naïve CD8+ T cells has been confirmed. Conclusion This study investigates epigenetic changes that characterize the transition between CTL, TEX, and TRM states of CD8+ T cells using healthy donor cells through an in vitro assay and CD8+ T cells isolated from HPV-negative HNSCC tumors. Understanding the epigenetic profile of T cell states can help determine the major epigenetic regulators that lead to exhaustion and tissue residency, paving the way for improved T cell-based immunotherapies. Future experiments aim to validate our findings using TCR-specific in vitro and in vivo models. Citation Format: Marie K. Luff, Marco Craviero, Mohd Saleem Dar, Katherine McKinnon, Amir Kaskas, Clint T. Allen, Vassiliki Saloura. Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck 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 2636.

Abstract 6788: Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models

Abstract Robust techniques are needed to screen the anti-tumor activity of antibody-drug conjugates (ADCs) in vitro. Traditional monolayer models provide a cost-effective and scalable option but lack many features of the tumor microenvironment, such as complex cell-cell and cell-extracellular matrix interactions. Tumor spheroids afford a more translational model for quantification of ADCs. Here, spheroids are used to compare the mechanism of action (MoA) of anti-HER2 ADCs. Single spheroids were formed from mono- or co-cultures of HER2 over-expressing cancer cells and HER2 negative control cells. Incucyte® Nuclight Green Lentivirus was used to label HER2 positive BT474 cells, whilst HER2 negative MDA-MB-231 cells were left unlabeled. Antibodies were added after 72 hours of spheroid formation in 96-well ultra-low attachment plates. Brightfield and fluorescence images were captured every 3 hours using the Incucyte® Live-Cell Analysis System and quantified, for metrics such as spheroid size and fluorescence intensity, to give an indication of ADC cytotoxicity. Co-culture spheroids were dissociated at assay endpoint and the remaining proportion of green and unlabeled cells was measured using the iQue® Advanced Flow Cytometry Platform. The area of the BT474 mono-culture spheroids decreased in a concentration-dependent manner in the presence of both ADCs tested (trastuzumab emtansine and trastuzumab deruxtecan), indicating increased cytotoxicity. In contrast, only the highest concentration of trastuzumab, which is the monoclonal antibody backbone on which they are based, resulted in a reduction in spheroid size. This displays the increased anti-tumor activity due to the addition of the ADC payloads. In the co-culture model, only the trastuzumab deruxtecan induced a reduction in spheroid area, with the size of the spheroids in the presence of trastuzumab emtansine and trastuzumab remaining comparable to control levels. Analysis of the spheroid composition using flow cytometry showed that for the BT474 cells, again, percentage cell death compared to the IgG control was greater for the two ADCs, at 96.4% and 97.2%, respectively. This death was greater than with trastuzumab, which induced 46.4% cell death. Unlike in the mono-culture assays, a high level of death of the HER2 low expressing cells was also seen in the co-culture in the presence of trastuzumab deruxtecan (94.1% death compared to control). This displays the unique bystander activity of trastuzumab deruxtecan, which has led to its approval as a treatment for both HER2 high and HER2 low breast cancers. These data display how the Incucyte® Live-Cell Analysis System combined with iQue® Advanced Flow Cytometry can be leveraged to provide comprehensive assessment of the in vitro function of ADCs and how it can enable differences in their MoAs to be distinguished in 3D spheroid models. Citation Format: Kirsty McBain, Kalpana Barnes, Nicola Bevan. Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models [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 6788.

Abstract 5157: Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells

Abstract Cell surface proteins are increasingly considered in the development of novel anticancer agents, as demonstrated by recent clinical successes in the field of immuno-oncology and antibody-drug conjugates (ADCs). These conjugates specifically target antigens present on the surface of tumor cells, enabling the delivery of cytotoxic agents to specific tumor sites. During the preclinical development of such compounds, for example, when they are tested on a wide panel of cell lines, it is essential to have a rapid and reliable method for monitoring target expression and distribution. Complementing our 160 Cell line panel ProLiFiler™, we report here the development of a novel method named oncoFLOW-Profiler™ which includes steps of cell fixation, micronic storage, staining, and measurement by flow cytometry. This method is applied in a 96-well format, enabling screening of all cell lines in parallel and analysis of the expression of different targets in just one day. In this proof-of-concept study, we investigated the level of ERBB2 expression across the ProLiFiler panel of 160-cell lines in order to validate this methodology. We first compared the impact of fixation methods and sample storage on knowingly positive and negative control cell lines over a period of several months. ERBB2 measurements from the oncoFLOW-Profiler™ will be analyzed and validated by comparison with various OMICs datasets from the Cancer DataMiner platform (4HF Biotec), including RNA expression, mutational status, and protein expression levels. To determine the expression cutoff of ERBB2 amplified models, results will be analyzed according to gene copy number variation. Finally, the flow cytometry data generated will be investigated for use as a predictor of response to clinically approved ADCs targeting ERBB2: Kadcyla and Enhertu. Here we present a method that we have demonstrated is feasible for investigating cell surface receptor/antigens during preclinical phase of drug development. Our method has enabled us to successfully assess ERBB2 in biological samples and to identify models sensitive to ADCs targeting ERBB2. We will also discuss the results of ongoing experiments analyzing the applicability of this screen to intracellular targets and carbohydrate-based surface antigens, which cannot be analyzed either by transcriptomics or proteomics. Citation Format: Kaja Holtorf, Anne-Lise Peille, Daniel Feger, Vincent Vuaroqueaux, Jan Erik Ehlert, Nadine Obier. Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells [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 5157.

Abstract 3039: Pan cancer therapeutic inhibition of oncogenic c-MYC by the engineered destabilized 3’UTR of c-MYC

Abstract c-MYC is a master transcription factor that is over expressed in more than 70% of human cancers. There is no clinically approved direct inhibitor of c-MYC. Recently, we reported the development of the engineered destabilized 3’UTR of MYC constructs which is a novel mRNA overwriting technology through which we directly destabilized and degraded c-MYC. Here we report that we have extended this technology in directly targeting and degrading c-MYC in multiple ethnically diverse triple negative breast cancer cell lines, ovarian cancers, neuroendocrine prostate cancers and pancreatic cancers. The constructs are specific, titratable, and directly engaged the c-MYC target validated by RNA transcript technologies. We have performed head-to-head IC50 dose dependent titration comparison with the engineered destabilized 3’UTR of c-MYC constructs and chemotherapies (DNA damaging agents, microtubules inhibiting drugs, alkylating agents) as well as immune checkpoint inhibitor. We found evidence that suggests that the engineered destabilized 3’UTR of c-MYC is likely to be superior to these therapies in pan cancers driven by c-MYC. Citation Format: Chidiebere U. Awah. Pan cancer therapeutic inhibition of oncogenic c-MYC by the engineered destabilized 3’UTR of c-MYC [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 3039.

Abstract 1530: Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice

Abstract The relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2−/−, IL2Rγ−/−, SIRPαNOD and Flt3−/−) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models overexpressing human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and test novel therapeutics. Effect of Flt3L-induced boost on myeloid compartment is seen in a non-small cell lung cancer (A549) tumor model. A single boost prior to tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the tumor microenvironment (TME). Human triple negative cancer cell (TNBC) MDA-MB-231 and human pancreatic adenocarcinoma HPAFII cell lines are widely used in cancer research and drug development. We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft (CDX) is CD34+ donor independent and the TME composition varies according to cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231 bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII bearing mice is mainly composed of T cells enabling T cells-based therapy. Treatment of BRGSF-HIS mice bearing HPAFII tumor cells with combotherapy based on CD3xTAA + CD28xTAA efficiently reduces tumor growth in vivo compared to vehicle-treated mice or mice injected with CD3xTAA alone. Induced systemic immunomodulation is observed in the TME where increased numbers of both CD4 and CD8 T cells are observed upon combotherapy. Remarkably, while no CD34+ donor effect is observed in CDX growth kinetic, response to treatment appears to be donor dependent with apparition of “weak” and “good” responders. This heterogeneity of response mimics what is observed in clinic and enables further investigations of treatment mode of action and immune escape mechanisms. Citation Format: Perrine Martin-Jeantet, Siham Hedir, Fabiane Sonego, Gaëlle Martin, Yacine Cherifi, Kader Thiam. Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice [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 1530.

Abstract 4006: IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells

Abstract Introduction: EGFRvIII is a commonly studied tumor-specific antigen in glioblastoma (GBM) and poses an enticing target for immunotherapy. Its varied expression, however, complicates its effectiveness as a Chimeric Antigen Receptor (CAR) T cell target. Given the increasing prominence of Interleukin-12 (IL12) in treating solid tumors, its synergy with EGFRvIII specific CARs in treating preclinical models of GBM is explored. Methods: To simulate tumor heterogeneity, mice were implanted with equal ratios of CT2A and CT2A EGFRvIII+ cells. We developed EGFRvIII-specific third-generation CAR T cells, both with and without IL12 using a MSGV retrovirus backbone and transduced them into murine T cells. Their antitumor effectiveness was gauged by evaluating survival rates, and their capability to enhance and modify the tumor environment was determined using single-cell sequencing. Results: Standalone EGFRvIII CARs were unsuccessful in curing mice with heterogeneous tumors. However, 60% of mice treated with EGFRvIII CARs combined with IL12 were long term survivors. IL12 secreting CARs exhibited prolonged persistence in vivo, and there was no need for prior lymphodepletion before ALT introduction. Our studies reveal that EGFRvIII CARs secreting IL12 can eliminate EGFRvIII negative tumor cells by drawing on the inherent CD8 T cell compartment. Moreover, IL12 prominently impacted microglia among all Antigen Presenting Cells in the tumor environment, amplifying antigen presentation processes and broadening epitope exposure. Conclusion: By leveraging IL12, EGFRvIII CARs can counteract antigen heterogeneity in tumors by recruiting endogenous CD8 T cells and polarizing microglia. These IL12-secreting CARs outlast their traditional counterparts without requiring lymphodepletion. Harnessing CAR-T cells that secrete IL12 represents a novel strategy for overcoming tumor heterogeneity and treating GBM in a clinical context. Citation Format: Aditya Mohan, Steven Shen, Kelly Hotchkiss, Sarah Quackenbush, John Sampson, Peter Fecci, Anoop Patel. IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells [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 4006.

Abstract 6025: Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display

Abstract Radioligand therapeutics (RLT) aim to selectively deliver radioisotopes to cancer tissues to eradicate tumor cells while limiting the damage to surrounding tissues. They are composed of a tumor-targeting ligand and a radioactive payload. The therapeutic potential of RLT strongly depends on their biostability and residence time in tumor and healthy tissue after injection. Macrocyclic peptides are effective tumor-targeting ligands because their size and molecular characteristics confer optimal pharmacologic properties that can be fine-tuned during the development process. mRNA display is a powerful platform for macrocyclic peptide drug discovery that identifies high affinity peptide ligands through in vitro selection. Here, we describe the selection and initial characterization of macrocyclic peptides discovered by mRNA display that bind to the extracellular domain of a tumor target. Enrichment of the library for peptides that bound to the recombinant target protein was observed after four rounds of selection with no further enrichment in round five. The library of round 5 was sequenced by Illumina sequencing and the top 1,000 sequences (highest counts) were used to perform cluster analysis. We tested the top sequences of 43 different clusters in in vitro translation binding assays to validate the selection output and identify sequences for hit confirmation. Most of the sequences tested showed clear binding to the target protein and only a few peptides showed binding to a closely related protein from the same protein family. No non-specific binding of the discovered peptides to an irrelevant protein was observed showing the high specificity of the generated library. From this confirmatory selection data, we chose a subset of peptides for chemical synthesis. Binding affinity measurements were determined by surface plasmon resonance (SPR) and cellular binding of selected candidates to target-positive and -negative tumor cells was analyzed by flow cytometry. From this hit confirmation data, in vitro internalization and in vitro anti-tumor efficacy of radiolabeled peptide candidates for RLT are assessed. Our data highlights the potential of mRNA display platforms for fast and efficient discovery of highly specific macrocyclic peptides with optimal binding properties suitable for RLT. Citation Format: Anika Jaekel, Kathryn Sherry, Prachi Desai, David Fox, Tang Guo-Qing, Manuel Sturzbecher-Hoehne, Dennis Mewis, Kaori Noridomi, Liam Rayman, Ping Ye. Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display [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 6025.

Abstract 1691: p300 catalytic inhibition selectively targets IRF4 oncogenic activity in multiple myeloma

Abstract Multiple myeloma (MM), an incurable plasma cell malignancy, is addicted to oncogenic transcription factor (TF) IRF4 signaling. Lineage TFs like IRF4 are difficult to drug but represent high value oncology targets owing to the depth and context selectivity of their genetic dependency. Using a transcription regulatory network (TRN) based approach to integrate a high dimensional multi-platform dataset, we defined the key regulatory activity of IRF4 and its druggable co-factors. This unbiased analysis identified p300 lysine acetyl transferase (KAT) function as the nearest selective neighbor for therapeutic intervention, with more potent and selective effects than historically observed with p300/CBP bromodomain inhibitors. To further probe this biology, we developed a highly selective inhibitor of the KAT domain of p300 and its close homolog CBP. p300 KAT inhibition led to selective downregulation of IRF4 at the mRNA and protein level as well as reduction in acetylation at highly specific sites co-localized with IRF4, in contrast to more general agents like BET inhibitors. Gene expression reduction was most pronounced at IRF4/p300 co-bound genes, in particular IRF4 itself and MYC. IRF4 and p300 also directly bind to each other and other key MM nodes regulating them at the chromatin, mRNA, and protein levels. p300 KAT inhibition exhibits strong antiproliferative activity across in vitro MM models regardless of cytogenetics or resistance to other therapies. Moreover, this relationship is mediated by the KAT domain and not its acetyl-lysine recognizing bromodomain. Comparative pharmacology studies found that a p300 KAT inhibitor downregulated the IRF4 TRN and induced apoptosis in MM cells more effectively than existing standard of care agents, including IMiDs and glucocorticoid receptor agonists, or p300/CBP bromodomain inhibitors. Combinations of a p300 KAT inhibitor with clinically active molecules such as IMiDs and glucocorticoid agonists showed synergistic impacts on reducing key MM TFs and cell line viability. Ex vivo treatment of primary patient samples demonstrated broad anti-proliferative activity with loss of IRF4 and induction of apoptosis in relapsed-refractory MM patient samples, independent of prior lines of therapy. Importantly, the induction of MM cell apoptosis occurs in the milieu of CD138 negative bone marrow stromal cells while also sparing them from toxicity, supporting a potential therapeutic index. Finally, p300 KAT inhibition demonstrated activity in in vivo MM models with robust tumor growth inhibition at tolerated doses and exposures correlated to reduced p300-dependent substrate acetylation. IRF4 dependency is a hallmark of MM that has been challenging to target with existing therapies, motivating further development of p300 KAT targeting therapeutics. Citation Format: Michael R. McKeown, Giulia Giorgetti, Walter F. Lenoir, Marek J. Kobylarz, Tamara D. Hopkins, Wayne L. Glore, Michelle G. Shum, Yare Calderon, Kameron R. Mori, Hua Gao, Luis A. Carvajal, Nikolaus D. Obholzer, Benjamin W. Trotter, Christopher D. Dinsmore, Nikhil C. Munshi, Peter B. Rahl, Mariateresa Fulciniti, Charles Y. Lin. p300 catalytic inhibition selectively targets IRF4 oncogenic activity in multiple myeloma [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 1691.

Abstract 7486: Development and analytic validation of an ARv7/PSMA dual biomarker circulating tumor cell assay

Abstract Background Prostate cancer drug targets and biomarkers may be investigated non-invasively on circulating tumor cells (CTCs) obtained via liquid biopsies. PSMA is an important drug target in prostate cancer, and ARv7 is a splice variant that confers resistance to second-generation androgen receptor inhibitors. In this study, we describe the development and analytic validation of a novel assay to quantify ARv7 and PSMA protein expression on CTCs. The assay was validated with blood samples from patients with Stage IV prostate cancer to demonstrate clinical feasibility. To our knowledge, a PSMA/ARv7 dual protein assay for CTCs has not previously been reported. Methods LNCaP (PSMA positive), PC3 (PSMA negative), 22Rv1 (ARv7 positive), and BT474 (ARv7 negative) cells were spiked into healthy donor blood to generate model CTCs. Nucleated cells were collected from these spike-in samples using the AccuCyte® sample preparation kit and spread onto microscope slides. Slides were fixed and stained with antibodies to cytokeratin, EpCAM, and CD45 to detect CTCs and exclude white blood cells (WBCs), and with PSMA and ARv7 to evaluate protein expression on identified CTCs. The slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope that identifies CTCs for confirmation by trained reviewers based on machine learning algorithms and quantitatively measures biomarker signal mean fluorescence intensity (MFI). Results For ARv7/PSMA expression classification of the model CTCs, the dual biomarker assay showed analytic sensitivity of 83%/97% and specificity 98%/99%, yielding accuracy of 90.2%/97.9%, respectively. The dynamic range of MFI for both biomarkers was approximately three orders of magnitude. Inter-run reproducibility CV was 13.6% (ARv7) and 6.9% (PSMA). Slide repeatability mean CV was 11.1% (ARv7) and 12.0% (PSMA). Clinical feasibility of the assay was investigated in 9 progressing Stage IV prostate cancer patient blood samples. CTCs were identified in all clinical samples (median 4, range 1 - 384). Thresholds for biomarker positivity were established based on MFI measurements of positive and negative control cell lines. ARv7 positivity in clinical samples ranged from 33% to 100%, and PSMA positivity ranged from 0% to 75%. Conclusions We have developed a dual ARv7/PSMA assay that simultaneously measures protein expression of both biomarkers on individual CTCs. The assay is accurate and reproducible for the assessment of ARv7 and PSMA expression by liquid biopsy in clinical studies. Citation Format: Jon Ladd, Erin Bayer, Brock Bartels, Eric Kaldjian, Arturo Ramirez. Development and analytic validation of an ARv7/PSMA dual biomarker circulating tumor cell assay [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 7486.

Abstract 1375: Abundance of KLRB1+ (CD161) T cells in anti-PD1 non responders coupled with enhanced tumor cytotoxicity of anti-CD161 (IMT-009) with anti-PD1 makes it a rational target for combination with anti-PD-(L)1 immunotherapy

Abstract Background: Intra-tumoral CD4+ and CD8+ T cells expressing inhibitory receptor CD161 have an effector memory phenotype with high cytotoxic potential. CD161 inhibits T and NK cell function in the presence of its ligand, CLEC2D. This suppression can be recovered by treatment with IMT-009, a first in class anti-CD161 antibody. Single cell transcriptomic analysis of anti-PD1 refractory microsatellite stable colorectal tumors identified presence of CD161+ CD4+ cytotoxic T cells and CD161+ PD1+ exhausted CD8 T cells suggesting that anti-CD161 treatment may provide benefit in these tumors both as a monotherapy and in combination with anti-PD1. It was also observed that CD161+ CD4/CD8T cells are more abundant in anti-PD1/L1 non responders compared with responders in hepatocellular carcinoma, cutaneous squamous cell carcinoma, clear cell renal carcinoma and breast cancer, providing a rationale for targeting CD161. Using engineered ex-vivo tumor models and dissociated human tumors, we have demonstrated that IMT-009 reverses CLEC2D mediated inhibition and restores primary T cell function. Additionally, co-treatment of IMT-009 with anti-PD1 further enhanced T cell mediated cytotoxicity, providing rationale for studies combining IMT-009 and anti PD1 in the clinic. Methods: Using the Snakemake pipeline developed in house, we processed publicly available scRNAseq data. Cells were clustered by Louvian method followed by cell type annotation by Seurat, and gene expression and gene signature levels per cluster were plotted. For ex-vivo assays, antigen specific CD8 T cells expanded from HLA-A2:01 donors were co-cultured with HLA-A2:01 expressing MDA-MB 231 triple negative breast cancer cell line or Raji Burkitt Lymphoma model with HLA-A2:01 allelic overexpression. Target tumor cells were engineered to express the ligands CLEC2D or PDL1. These cell lines were made to overexpress luciferase for monitoring target cell death using a luminescence readout. Additionally, dissociated tumor cells (DTCs) were cultured in presence of the treatment molecules. Intracellular staining of cytotoxic T cells along with detection of IFNγ, IL-2, TNFα and Granzyme B levels in culture supernatants by MSD/ELISA were used to assess activation profile of tumor infiltrated T cells. Results: Combining anti-PD1 and IMT-009 significantly enhanced cytotoxic benefit compared to individual monotherapy in both Raji and MDA-MB-231 target cell line models. IFNγ levels in supernatants of DTCs treated with this combination were elevated as compared to individual monotherapy. Together with scRNAseq data of increased abundance of KLRB1 (CD161) expressing T cells in anti PD1 non-responder patients, our data provide rationale for testing combination of IMT-009 with anti-PD1 in patients that may not respond to anti PD1 alone. Citation Format: Adwitiya Kar, Tim Nieuwenhuis, Meixue Duan, Siamak Tabrizi, Matthew Huggins, Elizabeth Scanlon, Cynthia Hession, Matthew Bernstein, Michael Ross, Ming Tang, Shruti Malu. Abundance of KLRB1+ (CD161) T cells in anti-PD1 non responders coupled with enhanced tumor cytotoxicity of anti-CD161 (IMT-009) with anti-PD1 makes it a rational target for combination with anti-PD-(L)1 immunotherapy [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 1375.

Abstract 1779: De novo purine pathway enzyme ATIC promotes tumor progression and modulates metabolic reprogramming in breast cancer

Abstract Breast cancer (BC), a complex and heterogeneous disease, poses a significant global health challenge with a pressing need for enhanced prognostic markers and therapeutic targets. This study focuses on 5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), an enzyme essential in de novo purine biosynthesis and previously implicated in myeloma and hepatocellular carcinoma progression. Leveraging publicly available datasets, including TCGA, METABRIC, and GENT2, we identified elevated ATIC expression in BC tissues compared to normal tissues. Notably, patients with elevated ATIC expression displayed significantly diminished overall survival rates, underscoring ATIC's potential as a prognostic marker. Functional assays involving ATIC downregulation in BC cell lines revealed suppressed cell growth, migration, and increased sensitivity to chemotherapeutic drug, Doxorubicin, implicating ATIC in BC progression. Further, RNA-sequencing analysis of ATIC knockdown (KD) BC cell lines revealed a positive correlation between ATIC expression and the activation of MYC and E2F-regulated pathways. The marked attenuation in cell growth and various tumor properties upon ATIC KD provides mechanistic insights into the role of ATIC in fueling BC progression, potentially through the modulation of MYC and E2F target gene expression. Gene Set Enrichment Analysis (GSEA) on differentially expressed genes identified between tumors with high ATIC expression and low ATIC expression in BC patients from the TCGA cohort revealed a notable enrichment of genes associated with MYC and E2F targets, providing additional support for the observed molecular associations in our experimental model. Intriguingly, ATIC KD in Triple Negative BC cell line unexpectedly led to an increase in fatty acid β-oxidation (FAO), uncovering a metabolic vulnerability in these cells. In conclusion, this study unravels the multifaceted role of ATIC in BC, demonstrating its influence on BC tumor progression and metabolism. The observed increase in FAO upon ATIC KD presents a novel metabolic vulnerability that could be explored for targeted therapeutic interventions in BC. This comprehensive analysis contributes valuable insights toward understanding ATIC as a potential therapeutic target and prognostic indicator in BC. Citation Format: Divya Murthy, Kuldeep S. Attri, Jun Hyoung Park, Benny A. Kaipparettu. De novo purine pathway enzyme ATIC promotes tumor progression and modulates metabolic reprogramming in breast cancer [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 1779.

Abstract 3586: A Hedgehog-dependent signaling axis drives neural plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine tumors

Abstract Background: Neuroendocrine tumors (NETs) represent heterogenous malignancies whose origins and etiology remain poorly understood. Men1-driven reprogramming of neural crest-derived glial cells was recently implicated in NET development. In these studies, aberrant Sonic hedgehog (SHH) pathway activation known to pattern neural cell fate coincided with the neuroendocrine phenotype in mice. Here, we investigated the hypothesis that loss of menin encoded by the MEN1 gene drives SHH-dependent oncogenic reprogramming of NETs by modulating the neural crest cell fate. Methods: Glial cell specific Men1 deletion was accomplished by expressing Cre recombinase downstream of the human glial fibrillary acidic protein promoter (GFAPΔMen1). Hedgehog (HH) activation of Men1-deficient glial cells was blocked by deleting the gene encoding primary ciliary protein Kif3a required for transducing SHH signaling. The resulting GFAPΔMen1 mice were evaluated for NET development and dysregulated hormone profiles. To identify aberrant SHH activation leading to neuroendocrine reprogramming, we performed single cell RNA- and Assay for Transposase Accessible Chromatin sequencing on GFAPΔMen1 NETs and primary glial cultures. Hyperactivation of SHH in human gastroenteropancreatic (GEP)-NETs was confirmed by immunofluorescent staining and western blot. Lastly, primary mouse and human NET organoids were treated with an agonist and inhibitors of HH signaling and evaluated for ERK/AKT activation, proliferation, and transcript fluctuations indicative of neural crest cell reprogramming. Results: GFAPΔMen1 mice developed NETs in the pancreas, pituitary, and small intestine. Astonishingly, impaired SHH activation in GFAP+/Men1−/− cells abolished the development of NETs and restored hormone levels to that of wild type mice. GFAPΔMen1 NETs and glial cultures showed increased SHH signaling and chromatin accessibility of HH pathway genes, whereas additive Kif3a deletion blocked HH activation. Moreover, menin negative glial cells demonstrated a phenotypic shift in neuroglial lineages that suggested SHH-mediated reprogramming of neural crest-derived cells following the loss of menin. Consistent with hyperactive SHH activation in the GFAPΔMen1 mice, human GEP-NETs overexpressed SHH and downstream signaling targets. Functionally, SHH induced ERK/AKT activation, cell proliferation, and neuroendocrine transcript expression in human and mouse NET organoids, whereas pharmacological inhibition of HH signaling reversed these effects. Conclusions: Our observations implicate neural crest-derived glial cells as potential neuroendocrine cell precursors that are susceptible to transformation through increased HH signaling. These studies warrant future investigation into the delivery of Hedgehog inhibitors in the adjuvant setting for NET treatment. Citation Format: Suzann Duan, Ricky A. Sontz, Martin Deymier, Juanita L. Merchant. A Hedgehog-dependent signaling axis drives neural plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine tumors [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 3586.

Abstract 138: UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma

Abstract PAX3-FOXO1 positive rhabdomyosarcoma (RMS) is an aggressive pediatric sarcoma with a terrible prognosis. The resultant fusion acts as a chimeric transcription factor, in part remodeling chromatin to inhibit terminal differentiation and maintain cancer cell proliferation. As this oncoprotein is not directly druggable, we have used genetic screens to identify dependencies downstream of PAX3-FOXO1 to improve our understanding of pathogenesis and develop new therapeutic targets. We performed pooled CRISPRi screening in an isogenic system of a patient-derived RMS cell line with or without knockdown of PAX3-FOXO1. Loss of the ubiquitin hydroxylase UCHL5 led to selective depletion of PAX3-FOXO1 positive Rh30 cells compared to PAX3-FOXO1 knockdown cells, suggesting that the fusion creates UCHL5 dependence. We validated this finding in competition growth assays in additional fusion positive (FP) and fusion negative (FN) RMS cell lines, and observed UCHL5 dependency only in the former. By contrast, UCHL5 was dispensable for cell growth and survival in FN RMS cell lines. These data were recapitulated in the large DepMap dataset, which also identified the top genetic co-dependencies of UCHL5 as INO80-complex (INO80c) members. We therefore investigated whether the fusion selective effects of UCHL5 were mediated by the INO80c, of which UCHL5 is an N-terminal member. We observed that knockdown of another N-terminal component of INO80c, NFRKB, phenocopies the fusion-selective effects of UCHL5 loss. Conversely, the catalytic ATPase INO80 was pan-essential, suggesting that only N-terminal subunits of INO80c regulate PAX3-FOXO1 specific functions. INO80c plays an essential role in mitigating DNA replication stress. Indeed, loss of UCHL5 led to DNA damage as measured by γH2AX and slowing of replication forks as measured by fiber assays in FP, but not FN RMS. Unexpectedly, acute degradation of PAX3-FOXO1 also slowed DNA replication fork speed, suggesting a genetic model in which UCHL5 and PAX3-FOXO1 cooperate to resolve replication stress in FP RMS. Finally, in vivo experiments using cell line xenografts confirms that loss of UCHL5 slows the growth of FP RMS, but not FN RMS. These findings suggest that loss of UCHL5 induces replication stress in a fusion-specific manner, and offers both an opportunity to study the mechanisms by which the PAX3-FOXO1 oncoprotein mitigates replication stress, and to develop UCHL5 inhibitors as precision therapies for FP RMS patients. Citation Format: Amit J. Sabnis, Pushpendra K. Sahu, Yue Pan. UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma [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 138.

Abstract 1335: Generating functional CAR-NK cells for cancer immunotherapy

Abstract Chimeric Antigen Receptor-Natural Killer (CAR-NK) cells are rapidly emerging as a promising cellular therapeutic overcoming the potential life-threatening adverse events that may result from CAR-T therapy, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). Due to their intrinsic characteristics, NK cells can kill tumor cells without the requirement for recognition of antigen in the context of MHC. This removes the constraint of HLA matching and supports antigen independent cytotoxicity through NK cell intrinsic mechanisms. As such CAR-NK cells kill via both CAR-dependent and -independent mechanisms. For these reasons, they offer the advantage of being an “off-the-shelf” product; satisfying the requirement for large-scale production of cancer immunotherapies, with the potential to reach more patients at a reduced cost. The frequency of NK cells in the blood is low (~ 2-5%), therefore improving their in vitro expansion is key to generating sufficient material to offer a viable CAR-NK cell therapy. Here we outline an in-vitro method to generate, expand, and test primary human CAR-NK cells using engineered feeder cells, and go on to demonstrate the efficacy of the CAR-NK against target cancer cells. Primary human NK cells were cultured and expanded in the presence of irradiated K562 cells (iK562) modified to express 4-1BBL and membrane bound IL-21. Following initial expansion, NK cells were transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV; as an empty vector transduced control. Following transduction, CD19 CAR-NK and GFP-NK were enriched by FACS sorting to reach >80% purity and further expanded in the presence of 4-1BBL/mbIL21 iK562 feeder cells. Following sufficient expansion, cytotoxicity of fresh or cryopreserved CD19 CAR-NK cells was tested in vitro in a Tumor Killing Assay (TKA) against CD19+ or CD19- target cells. As expected, CD19 CAR-NK cells showed increased on-target cytotoxicity, increased degranulation and IFNꝩ secretion compared to controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting CD19 CAR engagement of the target antigen to mediate killing. Taken together these data show the generation, expansion, and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This approach provides a platform for in vitro characterization of novel CAR-constructs, allied with in-vitro safety assessment and in vivo pharmacology testing. Together, this offers the potential to further develop new and more widely available cellular therapeutics to treat cancer. Citation Format: Karina Di Gregoli, Henry Leonard, Dan Rocca, Sabrina de Munnik, Gemma Moiset, Robert Nunan, Lauren Schewitz-Bowers, Louise Brackenbury. Generating functional CAR-NK cells for cancer immunotherapy [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 1335.

Abstract 430: Precision diet and lipid modulating agents to induce lipid oxidation in breast cancer

Abstract Metastasis is the primary cause of breast cancer-related mortality, and understanding the intricate relationship between the tumor microenvironment, lipid metabolism, and metastasis is crucial for preventing cancer progression. Previous work from our group demonstrated that high exogenous levels of oleic acid, a monounsaturated fatty acid (MUFA), in the lymph node environment protects melanoma cells from ferroptosis. Ferroptosis is an iron-dependent form of cell death induced by accumulation of lipid peroxides, which are commonly formed from polyunsaturated fatty acids (PUFAs), but not MUFAs. Here, we hypothesized that increasing dietary PUFAs would alter the exogeneous lipid content of the lymph node environment, thus rendering breast cancer cells more sensitive to ferroptosis. To test this, we determined the in vitro sensitivity of 4T1 triple negative breast cancer cells to ferroptosis when treated with various PUFAs and MUFAs. We also assessed the impact of various PUFA and MUFA fatty acid-enriched diets on breast cancer metastasis and ferroptosis resistance in the lymph nodes in vivo. In concordance with previously published work, our results demonstrated that pharmacological induction of ferroptosis in vitro increased lipid oxidation and was mitigated by fatty acid treatments with higher MUFA compared to PUFA content. In vivo, cancer cells in lymph nodes of mice on palm oil diets had lower lipid oxidation compared to cancer cells from tumors in the mammary fat pad. These findings support the hypothesis that dietary lipids can alter ferroptosis response and the extent of lipid oxidation in breast cancer cells within lymph nodes. Our research has the potential to further reveal mechanistic connections between dietary lipids, the lymphatic microenvironment, and breast cancer progression. Citation Format: Mahsa Yavari, Jordan Torpey, Marie Sabatier, Alanis Carmona, Krystina Szylo, Midori Flores, Mario Palma, Cameron Fraser, Yanshan Liang, Sheng Tony Hui, Oana Zeleznik, Heather Eliassen, Jessalyn Ubellacker. Precision diet and lipid modulating agents to induce lipid oxidation in breast cancer [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 430.

Abstract 740: CNTN4 as a novel target for solid cancer with antibody-drug conjugate

Abstract As a novel immune checkpoint, we previously confirmed that contactin 4 (CNTN4) regulates T cell activity negatively by binding to amyloid precursor protein (APP) on T cells. CNTN4 is highly expressed in various types of tumors, including gallbladder, pancreas, stomach, endometrium, liver, prostate, bladder cancer, melanoma, and other tumors, with positive rates over 70% through immunohistochemistry analysis in contrast to low-level expression in normal tissues. Based on the tumor-specific expression profile of CNTN4, we evaluated the potential of CNTN4 as a novel target for antibody-drug conjugate (ADC). First, the internalization of the anti-CNTN4 antibody in the IND submission stage, GENA-104A16, into CNTN4-positive cancer cells was confirmed. For investigation of the potential of targeting CNTN4 using ADC, clinically validated various linker-payloads (MMAE, MMAF, SN-38, and exatecan derivate) were conjugated to GENA-104A16 using thiol maleimide conjugation with average drug-to-antibody ratio (DAR) in the range of 4.0 to 4.8. After preparing ADCs, we confirmed the binding affinity for CNTN4 was maintained as high even after the payload conjugation through an ELISA. In vitro studies have demonstrated the highly cytotoxic effect of MMAF conjugate among the conjugates of GENA-104A16 on CNTN4-positive cancer cells compared to negative cancer cells. And there was no cytotoxic effect on normal cells. Furthermore, treating MMAF conjugate of GENA-104A16 showed promising efficacy in the Pan02 pancreatic orthotopic mouse model and the patient-derived xenograft model. These results suggest that CNTN4 could be a highly potential ADC target that could expand the options for cancer treatment strategies. Citation Format: Mi Young Cha, Hyunuk Kim, Seungmin Byun, Youngeun Ha, Kitae Park, Hyunkyung Yu, Bu-Nam Jeon, Mira Kim, Soojung Moon, Kyung Mi Park, Hansoo Park. CNTN4 as a novel target for solid cancer with antibody-drug conjugate [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 740.