ITGB6, the gene encoding the β6 subunit of integrin αvβ6, is a potent prognostic marker across multiple cancer types. As a major activator of latent TGFβ and a potent modulator of the tumor immune environment, αvβ6, and consequently, ITGB6, has considerable therapeutic implications. ITGB6 is highly upregulated in squamous cell carcinomas and pancreatic adenocarcinomas, where it disrupts tumor-immune cell signaling. We identify ITGB6 as a potent clinical prognostic marker of anti-tumor immune response and were able to recapitulate the immune-mediated anti-tumor effect of ITGB6 in pre-clinical mouse models. Genetic knockout of ITGB6 in heterotopically-injected head and neck squamous cell carcinoma and pancreatic adenocarcinoma cell lines shows markedly reduced tumor progression and immunogenic cytokine profiles in immunocompetent mice. Additionally, co-cultures of human head and neck squamous cell carcinoma and pancreatic adenocarcinoma with human T-cells show increased T-cell killing upon cancer cell ITGB6 inhibition. Colony formation experiments give further evidence that the reduced tumor growth observed upon ITGB6 inhibition in vivo is through immunological clearance of cancer cells and not merely through intrinsic factors. Analysis of The Cancer Genome Atlas (TCGA) reveals the high prognostic value of ITGB6 on overall survival and that high ITGB6 expression in patients is associated with an inferior response to α-PD-1 and α-PD-L1 immune checkpoint blockade. The potent anti-tumor immune response observed both in vitro and in vivo upon ITGB6 inhibition, combined with analysis of RNA-seq data from immune checkpoint blockade-treated patients, encourages the development of ITGB6 blockade and immunotherapy combination regimens. Further pre-clinical studies should facilitate translation of our findings into therapeutic clinical trials for treating immunotherapy-resistant cancers.

Abstract Introduction: Androgen Deprivation Therapy (ADT) is a highly effective treatment for prostate cancer. However, resistance to ADT develops in most patients, leading to a lethal state of castration-resistant prostate cancer (CRPC). Senescent prostate cancer (SPC) cells can arise after ADT and contribute to therapy resistance. SPCs can restore replicative function and alter the tumor microenvironment through pro-tumorigenic senescence-associated secretory phenotype (SASP). We aimed to characterize changes in SASP in prostate cancer cell lines and serum samples from patients receiving ADT. Methods: SASP factors (Luminex 100/200 System, xMAP Instruments) were analyzed in patients undergoing ADT. To correlate SASP with the sensitivity to ADT, we sought to evaluate the regulation of SASP in vitro using PCa cell lines. The PC3 (AR resistant) and LNCaP (AR sensitive) cell lines were used in the experiments. We performed an analysis of archival tumor tissue (paired) from 18 patients with or without alterations in DNA repair genes (BRCA1, BRCA2, and CDK12). We performed immunohistochemical (IHC) staining of known SASP markers, such as P16, uPAR, gamma-H2AX, and Lamin B1. Results: In a cohort of patients (n=8) with pre and post-ADT matched status, we demonstrate increased levels of SASP factors such as IL-6 (pre-ADT: 1.54±0.24 pg/ml, post-ADT: 5.23±1.3 pg/ml p=0.001), IL-7 (pre-ADT: 3.11±0.6 pg/ml, post-ADT: 13.5±2.7 pg/ml p=0.01), IL-1 alpha (pre-ADT: 1.06±0.3 pg/ml, post-ADT: 15.3±3.5 pg/ml p=0.001), IL-15 (pre-ADT: 15.6±3.1 pg/ml, post-ADT: 37.1±4.8 pg/ml p=0.002), MIF (pre-ADT: 1036±67.6pg/ml, post-ADT: 2362±55.5 pg/ml p=0.003), and IL-15 (pre-ADT: 5±2.1 pg/ml, post-ADT: 37.5±5.1 pg/ml p=0.002). In vitro, the AR-responsive LNCaP cell line, exposed to AR blockade, showed similar findings in upregulating IL-6, IL-7, and IL-15 but not the PC3 cell line. We also demonstrate measurable differences between the expression of u-PAR, gamma-2AX, p16, and loss of Lamin B1 expression in tumor specimens from patients with advanced castration-resistant prostate cancer (n=18). The expression of p16 was significantly upregulated post-treatment irrespective of DDR status (p=0.0417). Other SASP factors showed a trend in upregulation but were insignificant (u-PAR p=0.056, gamma-H2AX p=0.62, Lamin B1 p=0.72) Conclusion: These results provide evidence of systemic increases in SASP-related cytokines following treatment with ADT. The in vitro results suggest that secretion of SASP is promoted by an AR-mediated mechanism. Preliminary results from tumor specimens suggest increased senescence marker p16 expression induced by therapy. Ongoing experiments are further characterizing the senescence markers in prostate tumor specimens and correlated to treatment effect. Citation Format: Maximilian Schwermann, Matthew Hadfield, Shaolei Lu, Praveen Srinivasan, Vida Tajiknia, William MacDonald, Andrew George, Shengliang Zhang, Leiqing Zhang, Kimberly Meza, Andre De Souza, Dragan Golijanin, Elias Hyams, Galina Lagos, Sheldon Holder, Anthony Mega, John Sedivy, Howard Safran, Wafik El-Deiry, Benedito Carneiro. Androgen deprivation therapy (ADT) and senescence-associated secretory phenotype (SASP) in vitro: Correlation with SASP in tumor specimens as well as in the serum of patients after ADT [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 2954.

e14033 Background: Glioblastoma (GBM) is the most common primary brain malignancy with dismal prognosis. Tumor Treating Fields (TTFields) therapy is available for the treatment of both newly diagnosed and recurrent glioblastoma and represents a new category of treatment modalities in oncologic therapy. Despite decades of study, few FDA approved modalities are available for GBM and provide limited survival improvements for patients. AKT functions as a key serine/threonine kinase in the RTK/PTEN/PI3K pathway, and extensive genomic analysis of GBM has revealed that this pathway is mutated in a significant majority of GBM cases. Activation of this pathway ultimately leads to the activation of AKT, and phospho-AKT levels are elevated in the majority of GBM tumor samples and cell lines. Studies have shown that increased p-AKT levels contribute to uncontrolled growth, resistance to apoptosis, and enhanced invasive capabilities of glioma cells, which are characteristics of tumor progression in GBM. AKT serves as a critical hub in this pathway, enabling the amplification of growth signals, thereby making the inhibition of AKT an appealing and promising therapeutic target for treating GBM. Imipridone ONC206 is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). Here we show inhibition of p-AKT as well as upregulation of cleaved PARP following cotreatment of ONC206 and TTFields. Methods: We investigated the effect of ONC206 at IC50s plus TTFields in LN229, U138, U251 and T98G human GBM cell lines at different time points. Effect of treatment on cPARP, BCL-2, CHOP, caspase-10, and p-AKT were investigated using western blot. Results: Treatment with ONC206 at the IC50 in T98G cells with TTFields at 24h showed synergistic upregulation of cPARP as well as inhibition of BCL-2. At 96hour time point, inhibition of p-AKT was observed following all treatments (ONC206, TTFields, and co-application compared to control. In U251 cells, following treatment of ONC206 and TTFields, inhibition of BCL-2 was observed at 24h time point. Upregulation of CHOP and Casapse-10 at 96h time point was seen, as well as inhibition of p-AKT. Spheroid models showed structure disruption and growth arrest following combination treatment. At 72 h time point co treatment with ONC206 and TTFields resulted in synergistic upregulation of cleaved PARP. Conclusions: Here we showed cotreatment with TTFields and ONC206 results in inhibition of p-AKT, the key node in GBM growth pathway. This is the first report of cleaved PARP upregulation following TTFields and ONC206 in Human GBM cell line. Further studies to investigate the in vivo efficacy of TTField treatment in GBM with ONC206 are warranted. More studies are needed to understand the exact mechanism of synergy.

Abstract Metastatic colorectal cancer (mCRC) is a deadly disease with a five-year survival rate of 14%. Most patients receive 5-FU (F) and folinic acid (FOL) combined with oxaliplatin (OX), irinotecan (IRI), or both (OXIRI). Immune checkpoint inhibitors (ICIs) are effective against microsatellite instable (MSI) tumors, but 95% of mCRC tumors are microsatellite stable (MSS) and ICI-resistant. Though the primary targets of 5-FU, irinotecan, and oxaliplatin are well-understood, their effects on the tumor microenvironment (TME) remain incompletely characterized. Chemotherapy-mediated immune stimulation in MSS CRC has been observed in preclinical models and clinical trials. We hypothesize that combination chemotherapy treatment modulates the immune microenvironment of CRC -/+ ICI, with differences across subtype and treatment regimen. Chemotherapy-dependent changes in cancer cell gene expression, PD-L1 levels, cytokine secretion, and T cell activation were measured using in vitro models of MSS and MSI CRC. A preliminary evaluation of spleen and tumor T cells and dendritic cells (DCs) in murine models of MSS and MSI CRC after immunotherapy -/+ chemotherapy was conducted. CD8+ T cells, DCs, PD-L1, CD69, and GM-CSF expression were measured in clinical specimens of chemotherapy-treated MSS mCRC patients. In MSS CRC, FOX increased GM-CSF and activated CD8+ T cells in vitro and activated splenic CD8+ and CD4+ T cells in vivo. FOX and FIRI suppressed an anti-PD-1-mediated enhancement of type 1 cDCs in the tumor in vivo. FOLFOX was associated with increased tumor CD8+ T cells and decreased GM-CSF in MSS mCRC biopsies. FIRI treatment increased PD-L1 in vitro. FOLFIRI was not associated with increased tumor CD8+ T cells in MSS mCRC biopsies. In MSI CRC, FOX increased tumor CD8+ T cells in vivo. Here, we elucidate novel effects of clinically relevant chemotherapy combinations on the TME in MSS and MSI CRC. These data point to a FOX-specific mechanism by which CD8+ T cell infiltration into MSS mCRC tumors is enhanced, but their activation is halted potentially by GM-CSF suppression and/or type 1 cDC depletion in the TME. These findings contribute to our understanding of the mechanisms of chemotherapy-dependent immune modulation and bring the field closer to harnessing these effects for therapeutic gain. Citation Format: Lindsey Carlsen, Maximilian Pinho-Schwermann, Leiqing Zhang, Andrew Elliott, Kelsey E. Huntington, William J. MacDonald, Brooke Verschleiser, Laura Jinxuan Wu, Wafik S. El-Deiry. Modulation of the MSS and MSI colorectal cancer immune microenvironment with FOLFOX and FOLFIRI -/+ anti-PD-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 1189.

OBJECTIVES:Prostate cancer (PCa) is a leading cause of cancer death in men worldwide. Approximately 30% of castrate-resistant PCa becomes refractory to therapy due to neuroendocrine differentiation (NED) that is present in <1% of de-novo tumors. First-in-class imipridone ONC201/TIC10 therapy has shown clinical activity against midline gliomas, neuroendocrine tumors, and PCa. We explored whether NED promotes sensitivity to imipridones ONC201 and ONC206 by inducibly overexpressing SOX2 and BRN2, well-known neuroendocrine drivers. METHODS:Inducible SOX2 or BRN2 systems were cloned into human PCa cell lines LNCaP and DU145. Inducible cell lines were characterized based on protein expression, morphology, and migration. The sensitivity of the inducible cell lines to imipridone therapy was determined by viability, cell growth, or clonogenic assays. RESULTS:Slight protection from ONC201 or ONC206 with SOX2 and BRN2 overexpression was observed in the inducible LNCaP cells but not in the DU145 cells. At 2 months, there was an apparent increase in CLpP expression in LNCaP SOX2-overexpressing cells, though this did not confer enhanced sensitivity to ONC201. DU145 SOX2-overexpressing cells had a significantly reduced ONC201 sensitivity than DU145 control cells. CONCLUSIONS:The results suggest that treatment of castrate-resistant prostate cancer by imipridones may not be substantially affected by neuroendocrine differentiation as a therapy-resistance mechanism. The results support further testing of imipridones across subtypes of androgen-sensitive and castrate-resistant prostate cancer.

e16334 Background: Immunotherapy has experienced limited success in PDAC, with αPD-1 therapy being approved only in a subset that makes up < 2% of all PDAC cancers. TGFβ is a contributor to T-cell suppression in the PDAC tumor microenvironment. However, the success of blocking TGFβ in preclinical models has failed to translate into the clinic due to the off-target effects of systemic TGFβ blockade. Integrin ITGB6 is an activator of the inert latent TGFβ into the active, immunosuppressive form. Consequently, inhibition of ITGB6 is a potent and targeted therapeutic approach for disrupting TGFβ to overcome T-cell suppression in PDAC. Methods: Analysis of TCGA molecular and survival data was performed to determine the specificity and prognostic value of ITGB6 in PDAC. Subsequently, candidate cell lines were screened for ITGB6 expression using flow cytometry to facilitate an in vitro model. Using a lentiviral system, PDAC cells were transduced with a doxycycline-inducible short hairpin RNA (shRNA) knockdown (KD) of ITGB6. Both KD and control shRNA cell lines were induced with doxycycline and subsequently co-cultured with TALL-104 T-cells. Furthermore, conditioned media was analyzed for secreted active TGFβ levels using a luciferase-based TGFβ reporter cell line. Finally, a clonogenic assay was performed to determine the autocrine effects of TGFβ activation in PDAC. Results: ITGB6 is highly specific to PDAC and also shows upregulation in comparison to normal pancreatic tissue (FC 3.38, p < 0.0001). Capan-2 cells were chosen as a model for high integrin PDAC (Capan-2 99.73% cells ITGB6+, negative control: HCT116WT 0.96% cells ITGB6+). Fluorescent microscopy of the co-culture revealed that the KD of ITGB6 increased T-cell killing over 24 hours (Capan-2 CTRL shRNA 7.71%±3.63 dead vs. Capan-2 ITGB6 shRNA 21.68%±4.57 dead, p < 0.05). Media from ITGB6 KD cells contained significantly decreased TGFβ (+TGFβ: 673000 RLU vs. CTRL: 175500 RLU, p = 0.015). A clonogenic assay of Capan-2 cells treated with active-TGFβ demonstrates that PDAC cells resist the autocrine growth inhibitory effects of TGFβ through loss of SMAD4. TCGA survival data revealed ITGB6 as prognosis marker in PDAC (high ITGB6 median survival 16.79 mos, vs. low ITGB6 21.88 mos, HR = 2.730, p < 0.004). Conclusions: Survival data that clearly demonstrates the poor prognosis of ITGB6 expression, as well as the presently proposed mechanism of immunosuppression, provide ample justification for deploying anti-ITGB6 combination therapies to in vivo studies that facilitate translation of our findings into therapeutic clinical trials.

Abstract Neuroendocrine prostate cancer (NEPC) is an aggressive histologic subtype associated with poor prognosis that commonly arises in later stages as a mechanism of treatment resistance. Activation of oncogenic drivers, in combination with epigenetic changes (such as EZH2 overexpression and DNA methylation) further promotes tumor proliferation and expression of downstream neuroendocrine lineage pathways (in part controlled by transcription factors, including SOX2, ASCL1, and BRN2). Importantly, EZH2 inhibitors (EZH2i) can restore AR expression in CRPC (Ku et al., 2017). Imipridones, including Dordaviprone (ONC201), show promise in treating neuroendocrine tumors by interacting with DRD2 and CLpP (Anderson et al., 2022). These results warrant investigation into the potential for synergism in imipridone/EZH2i combination therapies. In the PCa cell lines LNCap, PC3, 22Rv1, and DU145 the combination of ONC201 and EZH2i GSK126 was assessed. Cell viability data after treatment with ONC201 and GSK126 reveals several dose ranges with potential synergistic activity after 72h. Preliminary western blots after 72 hours of GSK126 treatment indicate modulation of proteins relevant to the mechanism of action of imipridones, specifically DR5 and CLpP, in LNCap, PC3, and 22Rv1. Notably, the decrease in DR5 expression warrants investigation into the effects of EZH2i on TRAIL sensitivity and whether imipridones may rescue DR5 expression to maintain TRAIL sensitivity. Subsequent experiments will investigate dosing and time point-based effects on viability and protein expression, in addition to assessing the synergy of other imipridones and EZH2i’s. Further, while the cell lines PC3, 22Rv1, and DU145 are castration-resistant, the neuroendocrine prostate cell line NCI-H660 will be used as a positive control as it expresses higher neuroendocrine features, including DRD2. Additionally, the modulation in DR5 expression warrants inquiry into the effects of combination therapy on tumor-immune cell interactions. Further, we will manipulate the expression of neuroendocrine transcription factors to assess changes in therapy sensitivity. Citation Format: Connor Purcell, Praveen Srinivasan, Maximilian Pinho-Schwermann, William MacDonald, Elizabeth C. Ding, Vida Tajiknia, Wafik El-Deiry. In-vitro efficacy of Dordaviprone/GSK126 combination therapy on castration resistant and neuroendocrine 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 7228.

Abstract Head and neck squamous cell carcinoma (HNSCC) encompasses malignancies of the mucosal epithelium in the oral cavity, pharynx, and larynx. The lack of effective screening strategies and the typically late stage of detection are contributors to a considerable annual death toll of HNSCC. Additionally, the anatomical proximity of the malignancy to delicate structures of the head and neck leads to tremendous treatment-related morbidity. Therefore, there is great interest in developing life-saving therapies and strategies for treatment de-escalation. The immune checkpoint inhibitor pembrolizumab (αPD-1) has been approved for recurrent or metastatic HNSCC and as a frontline therapy for unresectable disease. However, only about a quarter of patients respond to αPD-1 therapy. The immunosuppressive effect of TGFβ has been widely described as relevant to the HNSCC tumor microenvironment. However, attempts to blockade TGFβ have failed mainly due to the widespread side effects of disrupting a highly promiscuous cytokine. Integrin αvβ6 is a major activator of the inert latent TGFβ into the active, immunosuppressive form. With its high relative specificity to HNSCC tissue, inhibition of αvβ6 could offer a safer approach for disrupting TGFβ to overcome resistance to immune checkpoint blockade. After screening ɑvβ6 expression levels of HNSCC cell lines via flow cytometry, cell lines FaDu and CAL27 were selected for further investigation due to high endogenous ITGB6 expression (FaDu 35.57% cells ITGB6+, CAL27 95.69% cells ITGB6+). The cell lines were transduced with a doxycycline-inducible short hairpin RNA (shRNA) knockdown of αvβ6 using a lentiviral system. Both knockdown and control shRNA cell lines were pretreated with 1 μg/mL of doxycycline for five days to induce the shRNA and subsequently co-cultured with TALL-104 T-cells. Quantitative fluorescence microscopy of the co-culture revealed that the knockdown of αvβ6 substantially increased T-cell killing over 24hrs (FaDu CTRL shRNA 6.72%±2.49 dead vs. FaDu ITGB6 shRNA 12.80%±3.16 dead, p<0.001). However, treatment of FaDu and CAL27 cell lines with active-TGFβ showed no change in PD-L1 expression via flow cytometric analysis. To determine whether the T cell evasive effect of αvβ6 is driven by other immune checkpoints on the cancer cells or by the direct effect that TGFβ has on immune cells, we show the generation of relevant mouse αvβ6 shRNA knockdown cells. These syngeneic in vivo studies will facilitate cytokine profiling to uncover the dynamics of tumor-immune cell interactions in response to αvβ6 inhibition. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Maximilian Pinho-Schwermann, Shengliang Zhang, Vida Tajiknia, Connor Purcell, Jillian Strandberg, Nolan Stubbs, Wafik S. El-Deiry. Integrin ɑvβ6 upregulation as a mechanism of T-cell evasion in 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 6528.

Abstract Radiation pneumonitis is one of the most common but challenging toxicities seen in patients that are receiving ionizing radiotherapy for thoracic malignancies. While radiation effectively eliminates cancer cells, the damage it causes to surrounding healthy lung tissue leads to an immune response that can impact treatment regimen and lead to long term fibrotic tissue formation. The mechanism of radiation pneumonitis and the inflammatory response in the lungs is poorly understood. However, it is known that the TRAIL pathway plays an important role in inflammatory and fibrotic response. Through our previous work, it was discovered that modulating the TRAIL pathway via pegylated recombinant long-acting TRAIL (TLY012) in both WT and TRAIL-/- C57Bl/6 could suppress radiation pneumonitis in mice that were exposed to 20 Gy of thoracic radiation with shielding of other organs as early two weeks post exposure. In addition to the TRAIL pathway, transforming growth factor-beta (TGF-β) is a cytokine that is known to play a key role in fibrosis. Broad spectrum integrin inhibitor GLPG0187 is known to inhibit the activation of TGF-β via binding to specific integrin receptors. In order to determine if the inhibition of TGF-β could suppress radiation pneumonitis without stimulation of the TRAIL pathway, DR5 null mice were utilized for the experiment. Male DR5-/- C57Bl/6 mice received a single 20 Gy thoracic radiation dose with shielding of other organs and were treated with 100 mg/kg of GLPG-0187 or control twice a week via IP injection with the first dose being administered 1 hour before radiation (n = 4/treatment/group). 13 days post-irradiation, lungs, sternal bone marrow, and peripheral serum were collected. Upon histological analysis, it was observed that there were thinner alveolar borders and lessened inflammation compared to the control mice. Mice were weighed every three days, and it was discovered that mice treated with GLPG-0187 maintained steady weight compared to the control group. Additional analysis including immunohistochemistry, cytokine profiling, and quantification needs to be conducted. As the TRAIL pathway could not be modulated in the DR5 null mice, these findings suggest that GLPG-0187 was able to suppress radiation pneumonitis via inhibition of TGF-β. While some rescue from radiation pneumonitis was observed via GLPG0187, it was to a lesser extent compared to treatment of WT and TRAIL-/- C57Bl/6 mice with TLY012. Future directions include investigating the synergistic effects between TLY012 and GLPG0187 in suppressing radiation pneumonitis and decreasing fibrotic response to radiation. Citation Format: Jillian Strandberg, Anna Louie, William MacDonald, Praveen Srinivasan, Leiqing Zhang, Lanlan Zhou, Seulki Lee, Wafik El-Deiry. Post-exposure suppression of radiation pneumonitis suggests non-redundant independent effects of TGF-beta and TRAIL/DR5 [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 710.

Abstract Our previous results demonstrate an integrin-mediated mechanism of immune evasion in colorectal cancer. Increased levels of latent TGFβ in the experimental tumor environment led to the upregulation of the immune checkpoint marker PD-L1 in HCT116WT cells, facilitating escape from T-cell killing. This effect was rescued in immune co-cultures by pretreating cancer cells with the broad-spectrum integrin inhibitor GLPG-0187. To further elucidate this system, we investigated the TGFβ-integrin axis. Integrin αvβ6 is a major activator of the inert latent TGFβ into the active, immunosuppressive form. With its high specificity within malignant tissue, inhibition of αvβ6 poses an attractive therapeutic target for disrupting TGFβ signaling while avoiding the toxicities of systemic blockade. To identify an endogenous in vitro model of integrin αvβ6-expressing cancer, computational analysis of publicly available mRNA expression data was performed to determine cancer types with high αvβ6 upregulation. This investigation revealed a strong presence of αvβ6 in pancreatic, bladder, head and neck, and cervical cancer tissues. Further in-silico work identified the head and neck squamous cell carcinoma cell lines CAL-27 and FaDu, as well as the pancreatic adenocarcinoma line Capan-2, as suitable models for in vitro study. Flow cytometry of these cell lines was performed to determine the surface expression of integrin αvβ6, revealing that protein expression correlated with the mRNA data from the previous computational studies. Capan-2 and CAL-27 exhibited high levels αvβ6 of expression, while FaDu expressed moderate levels, and HCT116WT exhibited low αvβ6 expression. These cell lines were pretreated with latent TGFβ, simulating sequestered latent TGFβ in the extracellular matrix, and subsequently co-cultured with TALL-104 T-cells. Quantitative fluorescence microscopy revealed that resistance to killing by T-cells correlated with αvβ6 expression across cell lines. These findings are consistent with the hypothesis that upregulated expression of integrin αvβ6 functions as an immunosuppressive resistance mechanism by increasing tumor microenvironment levels of active TGFβ. Analysis of mRNA sequencing data from the TCGA Pancreatic Adenocarcinoma Firehose database revealed that integrin αvβ6 is a strong marker of poor prognosis in pancreatic adenocarcinoma. Future co-culture experiments will investigate the dynamics of TGFβ activation in the tumor microenvironment in relation to αvβ6 expression. Further in vivo studies can facilitate translation of our findings to therapeutic clinical trials. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Vida Tajiknia, Maximilian Pinho-Schwermann, Connor Purcell, Brooke Verschleiser, Wafik S. El-Deiry. A gradient expression of integrin αvβ6 expression in human cancer cells associates with TGFβ mediated immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B022.

Abstract Pancreatic and colorectal cancer are leading causes of cancer deaths worldwide. Resistance to current therapeutics is a significant challenge in treating these cancers. Ferroptosis, a non-apoptotic iron-dependent form of cell death characterized by overwhelming lipid peroxidation, has emerged as a potential strategy to overcome drug resistance. Our lab previously identified the small-molecule NSC59984 as a p53 pathway restoring compound that induces reactive oxygen species, requires p73, and targets mutant p53 for ubiquitin-mediated proteolysis involving MDM2 and HSP90. In pre-clinical models of pancreatic and colorectal cancer, we now show that the small-molecule NSC59984 induces lipid peroxidation and causes reactive oxygen species-dependent apoptosis as monotherapy. However, when combined with ferroptosis inducers or cystine deprivation, NSC59984 potently induces ferroptosis in a mitochondrial complex III-dependent manner. Using CRISPR/Cas9 in pancreatic cancer cells, we further show that HRI, an EIF2-alpha kinase responsible for the cellular response to mitochondrial stress, induces the integrated stress response (ISR) upon treatment with NSC59984 or ferroptosis inducers. The ISR serves as a mechanism for resistance to cell death induced by NSC59984 or ferroptosis inducers, causing upregulation of key anti-oxidative stress and anti-ferroptosis proteins, including the cystine importer SLC7A11 and the GPX4-stabilizing protein HSPA5/BIP/GRP78. The combination therapy overcomes the ISR to induce potent cell death. Our work demonstrates the importance of the interplay between mitochondria and the ISR during ferroptosis induction in pancreatic and colorectal cancer cells. Citation Format: Praveen R. Srinivasan, Arielle J. De La Cruz, Maximilian Pinho-Schwermann, Andrew George, William J. MacDonald, Shengliang Zhang, Wafik S. El-Deiry. Small molecule NSC59984 stimulates mitochondria-dependent ferroptosis and overcomes integrated stress response pro-survival signaling in pre-clinical pancreatic and colorectal cancer 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 672.

Abstract Background: Both colorectal and pancreatic cancers are major global health issues and among deadliest cancers with great disease burden. The most commonly mutated oncogenic alteration in human cancers is KRAS, which is prevalent in pancreatic malignancies. The KRAS G12D mutation subtype is present in more than 40% of pancreatic ductal adenocarcinoma (PDAC). To this date there is no available targeted therapy options for patients with KRAS subtype mutations. MRTX1133 has been identified as a non-covalent, potent, and selective inhibitor of KRASG12D. This small molecule inhibitor has been shown to suppress KRASG12D signaling in cells and in vivo. Tumor Treating Fields (TTFields) therapy is a novel approach to treating cancer, the electric fields alter the behavior of cancer cells and prevent them from growing and dividing. We hypothesize that the co-application of MRTX1133 with TTFields will enhance the effects of MRTX1133 in PDAC and colorectal cancer (CRC). Martials and Methods: PDAC cell line CFPAC1 with KRAS G12 V mutation and LS513 CRC cell line with KRAS G12D Mutation were treated with same dose of MRTX1133 (500 nM) and were co-treated with 150kHz TTFields. After 48 hours western blot was used to probe for cleaved PARP, cleaved C3, phosphor-ERK and DUSP6. Results: In both KRAS G12D & KRAS G12V cell lines, synergistic upregulation of cPARP was observed following 48-hour co-treatment. Synergistic inhibition of pERK happened in KRAS G12D cell line but no change in pERK level following TTFields or MRTX1133 treatment was seen. Upregulation of cC3 in KRAS G12D happened in only co-treatment conditions. DUSP6 levels increased following MRTX1133 treatment in KRAS G12V cell line. Conclusions: The surprising synergistic upregulation of cPARP in both KRAS G12V and G12D cells following co-treatment of KRAS G12D inhibitor MRTX1133 and TTFields can bring hope for other KRAS mutation subtypes as well as G12D. the increase in cPARP in KRAS G12V with no effect on pERK is particularly interesting and demonstrates the need of more studies to investigate the mechanisms of this synergy. Citation Format: Vida Tajiknia, Praveen Srinivasan, Maximilian PInho-Schwermann, William MacDonald, Connor Purcell, Wafik El-Deiry. Co-treatment with KRAS G12D inhibitor MRTX1133 plus TTFields against human pancreatic and Colorectal cancer cell lines results in synergistic up-regulation of cleaved PARP in KRAS G12D & unexpectedly in KRAS G12V as well [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 2093.

e15196 Background: CXCL8, serves as a crucial multifunctional cytokine that plays a significant role in regulating tumor growth, invasion, and migration. Although in some cancers the critical role of CXCL8 in patient prognosis and disease progression is stablished but its role in CRC & PDAC patients is under investigation. High levels of CXCL8 in tumor microenvironment (TME) and association with cancer stem cell survival, immune surveillance escape & metastasis make it an interesting therapeutic target. Both CRC & PDAC are among the deadliest cancers with dismal prognosis. The need for novel therapeutics with a focus on TME is emerging. MRTX1133 was identified as an inhibitor of KRASG12D. ONC212 is a fluorinated imipridone with strong anti-cancer activity in nM range and preclinical efficacy against pancreatic and other malignancies. Here we report the synergistic inhibition of CXCL8 in both KRAS G12D & KRAS G12V cell lines with use of KRAS G12D inhibitor in combination with 5-FU or ONC212. Methods: Different CRC & PDAC cells were treated with MRTX1133 with 5-FU or ONC212 cytokine profiling at 48h time point was done. Human protein atlas was used for protein and RNA data &TCGA data for survival was used. Results: We previously have shown the synergistic inhibition of pERK and cytokine alteration between MRTX1133 KRAS G12D inhibitor with 5FU or ONC212 in both cell lines with KRAS G12D & KRAS G12V mutation (Tajiknia V et al. AACR 2023 annual meeting). In LS513 KRAS G12D CRC cell line, treatment of ONC212, MRTX1133 and combination of both showed a decrease of 52.36%,58.83% and 71.65% respectively in CXCL8 levels compared to control at 48h time point. In HPAF-II KRAS G12V PDAC cell line, treatment of ONC212, MRTX1133 and combination of both resulted in 38.69%, 64.44%, 57% inhibition of CXCL8 levels respectively compared to control. In SW480 KRAS G12V CRC cell line, treatment of ONC212, MRTX1133 and combination of both demonstrated 25.7%,47.3% and 66.5% inhibition in CXCL8 level respectively compared to control at 48h time point. Single treatment of MRTX1133 caused a decline in CXCL8/IL-8 levels by 30% while single treatment of 5-FU showed a 36% decrease, the combination of both resulted in more than 55% decrease compared to control in LS513 KRAS G12D cell line. In SW480 KRAS G12V cell line, combination of 5-FU & MRTX1133 showed 30% inhibition while in Capan-2 PDAC KRAS G12V cells the same combination showed a 48.2% reduction in CXCL8 compared to control at 48h time point. TCGA data for RNA shows low specificity of CXCL8 for cancer type but it is significantly high in CRC and PDAC. Protein concentration in the Pan-cancer cohort shows significant high level of CXCL8 in CRC. Conclusions: Considering the important role of KRAS mutations and CXCL8 in pathogenesis of CRC & PDAC, the effective inhibition of CXCL8 with the use of targeted therapy in combination could enhance treatment response and patient survival.

Abstract Background: Glioblastoma (GBM) is the most common primary brain malignancy with dismal prognosis. Tumor Treating Fields (TTFields) therapy is available for the treatment of both newly diagnosed and recurrent glioblastoma and represents a new category of treatment modalities in oncologic therapy. Despite decades of study, few FDA approved modalities are available for GBM and provide limited survival improvements for patients. AKT functions as a key serine/threonine kinase in the RTK/PTEN/PI3K pathway, and extensive genomic analysis of GBM has revealed that this pathway is mutated in a significant majority of GBM cases. Activation of this pathway ultimately leads to the activation of AKT, and p-AKT levels are elevated in the majority of GBM tumor samples and cell lines. Studies have shown that increased p-AKT levels contribute to uncontrolled growth, resistance to apoptosis, and enhanced invasive capabilities of glioma cells, which are characteristics of tumor progression in GBM. AKT serves as a critical hub in this pathway, enabling the amplification of growth signals, thereby making the inhibition of AKT an appealing and promising therapeutic target for treating GBM. Imipridone ONC206 is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). Here we show inhibition of p-AKT as well as upregulation of CHOP and caspase-10 following cotreatment of ONC206 and TTFields. Materials & Methods: We investigated the effect of ONC206 (at IC50s) plus TTFields in U251 and T98G human GBM cell lines at different time points. Effect of treatment on cPARP, BCL-2, CHOP, caspase-10, and p-AKT were investigated using western blot. Neurospheres were generated using mentioned cell lines and used for investigating effect of co-treatment on 3D structure. Results: Treatment with ONC206 at the IC50 in T98G cells with TTFields at 24h showed synergistic upregulation of cPARP as well as inhibition of BCL-2. At 96hour time point, inhibition of p-AKT was observed following all treatments (ONC206, TTFields, and co-application compared to control. In U251 cells, following treatment of ONC206 and TTFields, inhibition of BCL-2 was observed at 24h time point at all treatment conditions. Upregulation of CHOP and Casapse-10 at 96h time point was seen, as well as inhibition of p-AKT. Spheroid models showed structure disruption and growth arrest following combination treatment. Conclusion: Here we showed cotreatment results in inhibition of p-AKT, the key node in GBM growth pathway. Also, the upregulation of CHOP and caspase-10 that has not been discussed before can be the key to focus on novel therapeutic targets for GBM More studies are needed to elaborate the exact mechanism of synergy but these new findings can be used to develop novel treatment combination Citation Format: Vida Tajiknia, Praveen Srinivasan, Maximilian Pinho-Schwermann, William MacDonald, Connor Purcell, Wafik El-Deiry. (TTFields) and imipridone ONC206 co-treatment inhibits p-AKT and spheroid growth and upregulates caspase-10 in human GBM cell lines [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 7596.

190 Background: The blockade of the androgen receptor (AR) pathway is an effective treatment for prostate cancer (PCa), but most patients progress to castration-resistant prostate cancer (mCRPC). AR signaling modulates CD8+ T cytotoxic function, revealing the immune-modulatory function of the AR pathway. We have described the activation of NK cells by AR inhibitors and the potential synergistic effect with anti-NKG2a combination.1 Strategies to activate NK cells can be limited by the expression of HLA-E, a ligand of the inhibitory checkpoints on NK cells NKG2A. We investigated the mechanisms of AR-dependent modulation of HLA-E on tumor cells and the ADT-enhancing effect on patient-derived NK cells. Methods: PC cell lines were treated with second-generation androgen pathway inhibitors in vitro (enzalutamide 10uM, darolutamide 15 uM), and the expression of HLA-E was evaluated by flow cytometry. To evaluate the modulation of HLA-E by AR, the AR-negative cell lines (PC3 and DU145) were stably transduced with an inducible AR system. The pan HDAC inhibitors vorinostat (0.4 uM) and panobinostat (2.5 uM) were used to evaluate the regulation of HLA-E by epigenetics. We analyzed the activation status of paired peripheral blood NK cells isolated from patients with PCa prior to and post-androgen deprivation therapy (ADT). The patients (n=6) had a median time between collection of 26.3±2.3 days. Results: The AR-responsive LNCaP cell line displayed an increase in surface expression of HLA-E upon treatment with enzalutamide (Enza) or darolutamide (Daro) ([C]: 12.1±1.3%, Enza: 22.3±3.3%, Daro: 19.1±1.1%, p=0.004). AR blockade did not change the surface expression of HLA-E of AR negative PC3 ([C]: 10.5±2.7%, Daro: 13.1±2.3%, p=0.17) or DU145 cell lines ([C]: 3.5±1.7%, Daro: 2.5±0.6%, p=0.12). Transduction of AR in PC3 and DU145 lead to upregulation of HLA-E expression with AR blockade (PC3-AR+ [C]: 13.1±3.7%, Daro: 43.1±5.3%, DU145-AR+ [C]: 4.5±1.4%, Daro: 27.2±3.6%, p=0.001). The upregulation of HLA-E upon AR blockade was suppressed if cell lines were co-treated with vorinostat or panobinostat. Patient-derived peripheral blood NK cells displayed enhanced cytotoxic activity after ADT (expression of Granzyme B pre-ADT: 12.3±2.3%, post-ADT: 36.5±5.7% p=0.0017, Perforin pre-ADT: 3±0.6%, post-ADT: 26.5±3.7% p=0.001), and upregulation of the inhibitory checkpoint NKG2a (pre-ADT: 5.2±1.2%, post-ADT: 12.3±1.4%, p=0.04). Conclusions: Androgen inhibitors upregulate the expression of HLA-E in PCa cell lines by an AR-dependent mechanism regulated by epigenetics. ADT promotes peripheral blood patient-derived NK cell activation and upregulation of inhibitory NKG2A receptor. These findings support further investigative approaches targeting the HLA-E and NKG2A in mCRPC. 1. Schwermann, AACR 2023.

Abstract Background: GBM is the most common primary brain malignancy with dismal prognosis. Current standard of care is chemoradiation&surgery. The main issue with GBM is the resistance & recurrence. GBM can have high Ferritin levels associated with disease progression & immunosuppression in the GBM TME. Ferritin light chain (FTL), a key protein in iron metabolism, is mutated in GBM leading to high ferritin levels (both light& heavy chain) & is associated with poor survival of (GBM) patients in part due to inhibition of ferroptosis. Panobinostat is a histone deacetylase inhibitor with antineoplastic & antiangiogenic effects in glioma. Imipridone ONC206 is under Phase I clinical development in treatment of adult & pediatric patients with newly diagnosed and/or recurrent primary brain tumors (NCT04732065 and NCT04541082).We investigated the novel combination of irradiation, Panobinostat and ONC206 with regard to GBM cell line sensitivity&ferritin levels. Materials & methods: We investigated cell viability and drug synergies of ONC206 plus Panobinostat with or without radiation in 4 human GBM cell lines at 72 hours. Changes in Ferritin level treatment in U87 & U251 cells were measured using cytokine profiling at 24 h time point. Information regarding recurrent cases of adult GBM from TCGA &GlioVis were used for survival data. Results: We previously have shown the synergy between Panobinostat, Irradiation and ONC206 (2023 AACR meeting). Treatment of U87 cells with Panobinostat and ONC206 at their IC50s +/- 4 Gy radiation at 24h time point led to reduction in secreted ferritin levels (Panobinostat alone: 26.3% decrease; ONC206 alone: 14.7% decrease; RT 4Gy alone 14.7% decrease; triple therapy: 32.2% decrease). In panobinostat treated U251 cells there was a 10% increase in ferritin at 24 h. ONC206 resulted in a reduction by 34.71%, 4 Gy RT caused a slight increase of 4% in ferritin amount while the triple showed 49.96% reduction in ferritin levels. mRNA expression of FTL gene (Ferritin Light Chain) is significantly higher in GBM tumors compared to normal brain tissue. From TCGA 17 cases of recurrent gliomas with FTL mutation were available with median survival of less than a year. In data from GlioVis of recurrent GBM cases 263 patients had high FTL with median survival of 12.6 months compared to 262 patients with low FTL with median survival of 14.9 months. Conclusions: GBM with high ferritin correlates with poor patient survival. FTL is associated with cell proliferation &angiogenesis of GBM cells & is protective from ferroptosis in glioma cells. ferritin may be a therapeutic target in recurrent & therapy resistant cases of GBM and ferroptosis modulating drugs &can be a novel treatment option. ONC206 & Panobinostat with irradiation is a novel combination & can be considered for temozolomide or radiation-resistant & recurrent cases. More studies are needed to investigate the mechanism & impact of ferritin in disease progression, therapy resistance& ferroptosis targeting therapeutics. We are currently exploring these directions including in vivo studies. Citation Format: Vida Tajiknia, Wafik El Deiry, Lanlan Zhou, William Macdonald, Praveen Srinivasan. Ferritin, associated with worse overall survival in recurrent GBM, is reduced by combination of HDACi, ONC206 and radiation [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A050.

Abstract Our previous work studying the effects of integrin αvβ6 knockdown in cancer cells demonstrated αvβ6 as a method of T cell suppression in a co-culture model. ITGB6, the gene encoding the β6 subunit of αvβ6, is a potent prognostic marker across multiple cancer types. As a major activator of latent TGFβ, αvβ6, and consequently, ITGB6, has considerable therapeutic implications due to the immunosuppressive effect that activated TGFβ has on the tumor microenvironment. A Human Protein Atlas search of ITGB6 expression surveyed the upregulation of ITGB6 across cancer types. Cancer ITGB6 expression was then compared to expression in paired adjacent normal tissue using the TNMplot tool (Bartha, 2021) on a concatenated set of GEO, GTex, TCGA, and TARGET RNA-seq databases. Kaplan-Meier curves were generated using TCGA mRNA data through the cBio portal, splitting the cohorts according to an ITGB6 mRNA expression threshold of one standard deviation above the mean (z-score ± 1.0). ITGB6 expression and immune checkpoint blockade (ICB) response were evaluated with the Kaplan-Meier Plotter tool (Kovacs, 2023). ITGB6 expression levels between ICB responders and non-responders were quantified using the ROC Plotter tool (Fekete, 2023), and the figures were generated using Matplotlib in Python. Lung, bladder, head and neck, pancreatic, and cervical cancer all demonstrated high expression of ITGB6. Head and neck squamous cell carcinoma (median fold change 2.70, p<0.0001), bladder urothelial carcinoma (FC 2.41, p<0.001), cholangiocarcinoma (FC 5.33, p<0.01), and esophageal carcinoma (FC 2.72, p<0.03) showed strong upregulation of ITGB6 compared to normal tissue. Interestingly, lung adenocarcinoma (FC 0.77, p<0.04) and lung squamous cell carcinoma (FC 0.55, p<0.0001) showed downregulation of ITGB6 compared to normal tissue. Survival data revealed that ITGB6 was a potent marker of a poor prognosis in pancreatic adenocarcinoma (high ITGB6 median survival 16.79 mos, vs. low ITGB6 21.88 mos, p<0.004) and head and neck squamous cell carcinoma (high ITGB6 30.06 mos vs. low ITGB6 57.42 mos, p<0.03). However, no significant prognostic difference was observed in non-small cell lung cancers. Finally, high pan-cancer expression of ITGB6 led to poorer response to αPD-1 (high ITGB6 Hazard Ratio 1.37, p<0.05) and αPD-L1 (high ITGB6 HR 1.50, p<0.001). In vitro and in vivo experiments to validate the benefit of ITGB6 as a biomarker and as a therapeutic target are underway. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Maximilian Pinho-Schwermann, Vida Tajiknia, Connor Purcell, Lindsey Carlsen, Wafik S. El-Deiry. ITGB6 as a predictive biomarker for overall prognosis and PD-(L)1 immune checkpoint blockade response in various cancer types [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 5140.

Abstract Introduction: Natural killer (NK) cell tumor infiltration and higher expression of activating receptors NKp30 and NKp46 are associated with improved clinical outcomes in prostate cancer (PC). Activation of NK cells by androgen receptor inhibitors enhanced the killing of PC cell lines (Schwermann 2023). These findings support the therapeutic potential of strategies inducing NK cell activation to treat CRPC. NK cells induce apoptosis of cancer cells via TNF-related apoptosis-inducing ligand) (TRAIL) binding of death receptor 5 (DR5), which is overexpressed in PC cells. This is the first report of adoptive therapy utilizing high expression of TRAIL as a novel therapeutic strategy. Methods: We transduced NK-92-MI and CD4+ (Jurkat) cell lines with human TRAIL (pLVX-EF1alpha-TRAIL-IRES-eGFP).Transduced cells were pre-treated with simvastatin or atorvastatin (4uM) for 36h, followed by stimulation (IL-2 100 IU/ml and IL-12 100 ng/ml) for 2h. The eGFP-expressing cells were then sorted by FACS. PC (PC3, DU145, LNCaP, and 22Rv1), fibroblast (IMR-90), and brain endothelial (hCMEC/D3) cell lines were used in co-culture experiments with non-transduced and transduced NK cells and CD4+ cells. These experiments were quantified using the ImageXpress Confocal HT instrument. Viability assays were performed using Annexin-PI and flow cytometry staining for caspases 3/7. Results: Transduced NK-92 MI cells with TRAIL displayed increased surface expression of TRAIL (control[C]: 4.38%±1; TRAIL-overexpression[NK-TRAIL]: 78.54%±3.76; p<0.001). Co-culture of PC cell lines with TRAIL-NK-92 MI cells resulted in significant tumor-induced apoptosis compared to co-culture with non-transduced NK cells after 48 hours (LNCaP + NK [C]: 16.52%±5, LNCaP + NK-TRAIL: 73.19%±6; PC [C]: 14.4%±3, PC3 + NK-TRAIL: 79%±8; 22Rv1 [C]: 16.78%±1.7, 22Rv1 + NK-TRAIL: 76%±8; DU145 [C]: 13%±4, DU145 + NK-TRAIL: 59.1%±10; p<0.001). This enhanced cytotoxic function was also observed in CD4+ cells expressing high levels of TRAIL. Annexin-PI staining as well as Caspase 3 and 7 showed increased apoptosis in PC co-cultured with NK-TRAIL. Co-culture of NK-TRAIL cells did not affect the viability of the non-malignant cells (IMR-90 and hCMEC/D3) over 48h (IMR-90 + NK: 4.6%±1, IMR-90 + NK-TRAIL: 4.47±2, p=0.9; hCMEC/D3 + NK: 2.7%±0.8, hCMEC/D3 + NK-TRAIL: 2.2±0.5, p>0.99). These results align with the lower expression of DR5 on non-malignant cells compared to PC cell lines (IMR-90: 1.46%±0.5, hCMEC/D3: 1.9%±0.8, LNCaP: 92.3%±4.5). Conclusion: Transduced NK-92 MI cells with human TRAIL-induced higher levels of apoptosis of PC cell lines compared with non-transduced NK cells in co-culture experiments. The results highlight the potential of TRAIL-expressing adoptive cell therapy for CRPC. Engaging adoptive therapies with specific targets (anti-PSMA CAR) might increase the therapeutic potential of this strategy. Citation Format: Maximilian Schwermann, Praveen Srinivasan, William MacDonald, Vida Tajiknia, Andrea Schmidt, Shengliang Zhang, Lindsey Carlsen, Andrew George, Connor Purcell, Shaolei Lu, Matthew Hadfield, Anthony Mega, Benedito Carneiro, Wafik El-Deiry. TRAIL-NK-92 MI as a novel adoptive therapy for castration-resistant prostate cancer (CRPC): Preliminary in vitro results [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 3602.