Significant Reduction In Tumor Volume Research Articles

Endovascular embolization as a stand-alone treatment of head and neck paragangliomas with long-term tumor control.

The impact of therapeutic embolization as a stand-alone treatment of head and neck paragangliomas considered surgically high-risk remains insufficiently understood. The aim of this study was to investigate the procedural risks and long-term volumetric development in head and neck paragangliomas with high surgical risk following therapeutic endovascular embolization as stand-alone treatment. A retrospective database review of patients who underwent endovascular embolization as primary treatment for head and neck paragangliomas lacking appropriate curative treatment options at our institution (from January 2000 to February 2023) was conducted. Tumor volumetric analyses were performed before embolization and during follow-up. To assess the changes in tumor volume over time, the measurements were performed after embolization, first at 6 months and then on a yearly basis up to 6 years (mean follow-up time was: 33.7 ± 24.4 months). Subgroup analyses were conducted for vagal and jugular/jugulotympanic paragangliomas. A total of 32 head and neck paragangliomas in 28 patients (mean age, 56.1 years ± 16.5 [standard deviation]; 18 female) with therapeutic embolization as stand-alone treatment were evaluated, of which 11 were vagal paragangliomas, 15 jugular/jugulotympanic paragangliomas and 6 carotid body tumors. After a mean follow-up duration of 33.7 ± 24.4 months tumor control was achieved in 75%, with significant median tumor volume reduction at 6 months (p = .02, n = 21). Vagal paragangliomas responded the most to embolization with a significantly decreased median volume from 22.32 cm3; to 19.09 cm3 (p = .008, n = 8). Transient complications occurred in 3.4%. Therapeutic embolization as a stand-alone treatment offers a low-risk control of tumor growth in surgically high-risk lesions, with a significant reduction in tumor volume after treatment. Among the different subtypes, vagal paragangliomas exhibited the strongest and longest regression of the tumor volume.ABBREVIATIONS: HNPGL = head and neck paragangliomas; IQR = interquartile range; JTP = jugular/jugulotympanic paragangliomas; PVA = polyvinyl alcohol; VP = vagal paragangliomas.

Abstract PO1-16-04: Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression

Abstract Purpose: Low molecular weight isoforms of cyclin E (LMW-E) have been implicated in various human cancers, including triple negative breast cancer (TNBC), and are associated with a poor prognosis. However, targeted therapies for TNBC based on biomarkers are currently lacking. This study aims to investigate LMW-E as a potential therapeutic target in TNBC and evaluate the efficacy of RP-6306, a selective inhibitor of the Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1), in LMW-E-positive breast tumors. Experimental Design: Immunohistochemical (IHC) analysis was performed on pre-treatment tumor specimens from TNBC patients (n=36) to assess the correlation between LMW-E expression, CDK1 phosphorylation at Threonine 14 (pT14), and pathologic complete response to neoadjuvant chemotherapy. LMW-E inducible human mammary epithelial cells (hMEC) and breast cancer cell lines were used to investigate the regulatory effect of LMW-E on PKMYT1, the kinase responsible for CDK1 phosphorylation at T14, and the response to RP-6306, a first in-class and selective inhibitor of PKMYT1. Patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models of TNBC expressing human LMW-E (hLMW-E) were also utilized to assess LMW-E as a biomarker for predicting response to RP-6306. Results. Analysis of TNBC tumor biopsies revealed a significant positive correlation between LMW-E expression and CDK1 pT14, and both biomarkers were associated with a lack of pathological complete response to neoadjuvant chemotherapy. In vitro results using LMW-E inducible hMECs and breast cancer cell lines demonstrated that LMW-E up-regulates PKMYT1 and CDK1 pT14, acting as a PKMYT1 binding protein and enhancing PKMYT1 protein stability. High LMW-E protein levels predicted a favorable response to RP-6306, resulting in the accumulation of sub-G1 and polyploid cells, decreased tolerance to replication stress, increased DNA damage, chromosomal breakage, and apoptosis. In vivo treatment of TNBC PDX models and hLMW-E transgenic tumors with RP-6306 resulted in a significant reduction in tumor volume only in mice harboring high LMW-E tumors, while low cyclin E models showed no response. Immunohistochemical analysis confirmed increased γ-H2AX and decreased CDK1-pT14 and Ki67 levels, indicating the efficacy of RP-6306 in both PDX and transgenic models. Conclusion: This study highlights the regulatory axis from LMW-E to PKMYT1 and its predictive value for pathological complete response in TNBC patients receiving neoadjuvant chemotherapy. The selective PKMYT1 kinase inhibitor RP-6306 consistently induced DNA damage and inhibited tumor growth in in vitro and in vivo pre-clinical breast tumor models. Co-expression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306, emphasizing its therapeutic significance. Citation Format: Mi Li, Amriti Lulla, Cansu Karakas, Spiridon Tsavaschidis, Yan Wang, Tuyen Nguyun, Tuyen Bui, Gary Marshall, Kelly Hunt, Khandan Keyomarsi. Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-04.

Baseline parenchymal blood volume is a potential prognostic imaging biomarker in patients with malignant liver tumors treated with transarterial chemoembolization.

To assess the prognostic value of Parenchymal Blood Volume (PBV) in predicting survival, tumor response, and PBV response after transarterial chemoembolization (TACE). A total of 137 patients with malignant liver tumors who were treated with TACE between 07/2016 and 07/2018 were evaluated. Computed tomography illustrations were reworked at a dedicated workstation to create a PBV map which was overlapped with the associated magnetic resonance image to determine tumor diameter and PBV. Patients were divided into two groups according to their initial PBV value: PBV < 50 or ≥ 50ml/l. Retrospectively, for patients with at least 2 TACE and initial PBV < 50ml/l (n = 27), the tumor volume, regardless of the primary tumor type, decreased by 13.26%, and PBV showed a decrease of 23.11%. For 84 patients with PBV ≥ 50ml/l, the tumor volume decreased by 24.01%, and PBV showed a more substantial decrease of 44.69% (both p < 0.001). In the overall study population (n = 137), patients with an initial PBV ≥ 50ml/l (n = 101) survived for an average of 15.05months, while patients with an initial PBV < 50ml/l (n = 36) survived for 10.01months (p < 0.002). Subgroup analysis indicated that median survival in the HCC group was longer at PBV ≥ 50ml/l. For CRC and other primary tumors, the survival time for high and low initial PBV was almost identical. Our study reveals a noteworthy correlation between high initial PBV values and a significant reduction in both relative and absolute tumor volume. This association suggests a potential prognostic indicator, indicating that elevated PBV may signify a more favorable response to transarterial chemoembolization (TACE). Additionally, patients with high initial PBV values experienced an extended overall survival time. Notably, the subgroup analysis highlighted a prolonged survival time in the HCC group with elevated initial PBV values. These findings underscore the potential significance of assessing PBV as a predictive factor in the context of TACE, especially in specific tumor entities such as HCC. Further investigations are essential to validate and extrapolate these observations to optimize patient outcomes.

Astragaloside IV-PESV inhibits prostate cancer tumor growth by restoring gut microbiota and microbial metabolic homeostasis via the AGE-RAGE pathway.

Prostate cancer (PCa) is becoming the most common malignancy in men worldwide. We investigated the effect of astragaloside IV combined with PESV on the gut microbiota and metabolite of PCa mice and the process of treating PCa. Nude mice were genetically modified to develop tumors characteristic of PCa. The treatment of PCa mice involved the administration of a combination of astragaloside IV and peptides derived from scorpion venom (PESV). Feces were collected for both 16S rDNA and metabolic analysis. Fecal supernatant was extracted and used for fecal transplantation in PCa mice. Tumor development was observed in both PCa mice and nude mice. Tumor histopathology was examined, and the expression of inflammatory factors and the AGE-RAGE axis in PCa tissues were analyzed. PCa mice treated with Astragaloside IV in combination with PESV showed a significant reduction in tumor volume and weight, and stabilization of gut microbiota and metabolites. At the Genus level, significant differences were observed in Porphyromonas, Corynebacterium, Arthromitus and Blautia, and the differential metabolites were PA16_016_0, Astragaloside+, Vitamin A acid, Nardosinone, a-Nortestoster, D-Pantethine, Hypoxanthine, Pregnenolone, cinnamic acid, Pyridoxa, Cirtruline and Xanthurenate. There was a correlation between gut microbiota and metabolites. After the fecal transplantation, tumor growth was effectively suppressed in the PCa mice. Notably, both the mRNA and protein levels of the receptor for advanced glycation end products (RAGE) were significantly decreased. Furthermore, the expression of inflammatory factors, namely NF-κB, TNF-α, and IL-6, in the tumor tissues was significantly attenuated. Conversely, upregulation of RAGE led to increased inflammation and reversed tumor growth in the mice. Astragaloside IV combined with PESV could treat PCa by intervening in gut microbiota composition and metabolite by targeting RAGE.

Abstract LB338: Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma

Abstract Background: In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. Methods: To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. Results: CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244-/- bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Conclusion: Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients. Citation Format: Jeongsoo Kim, Tae-Jin Kim, Sehyun Chae, Hyojeong Ha, Yejin Park, Seon Ah Lim, Hyemin Lee, Younji Sim, Seyoung Oh, Geon Mo Koo, Minkyung Jo, Daehee Hwang, Kyung-Mi Lee. Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB338.

Therapeutic targeting at genome mutations of liver cancer by the insertion of HSV1 thymidine kinase through Cas9-mediated editing.

Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease. In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1- thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers. We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups. Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.

Effect of Preoperative Chemotherapy in Reduction of Tumor Volume in Wilms Tumor in Relation to Histology

Background: Wilms tumor (WT) is the most common renal tumor in children under5 years. The survival rates of children with Wilms tumor have improved dramatically over a few decades. Objective: To find out the effect of preoperative chemotherapy in the reduction of tumor volume in Wilms tumor and its correlation with tumor histology. Methodology: A total of 53 patients diagnosed with WT under 15 years were enrolled. After a complete history and physical examination, all information including demographics, metastatic status, pre and post-chemotherapy volume, histology, postop stage and surgical complication was collected and entered on Performa. All patients received neo-adjuvant chemotherapy according to the SIOP protocol. Data was entered and analyzed in SPSS 23. Results: Fifty-three patients were included. There were 33 (62.3%) male and the mean age was 4.45 +2.2 years. Thirty-two (60.4%) children had localized disease and13 (24.5%) children had lung metastasis. There was a significant reduction of tumor volume of &gt;500ml in 8(15.1%) patients after preoperative chemotherapy and the majority 18(34 %) patients had 100-200 ml decrease in tumor volume. Intermediate risk histology 40 (75%) was most prevalent. During surgery, 35 (66%) children had no complications .Thirty-nine (73.5%) patients were cured, 4 (7.5%) patients were expired and 5 (9.4%) got left against medical advice (LAMA). Conclusion: Our study infers that pre-operative chemotherapy reduces the tumor volume remarkably in WT making resection feasible

Abstract 1423: Harnessing the gut microbiome to modulate immune checkpoint blockade response in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. The discovery that this subtype has the highest levels of tumor-infiltrating immune cells and programmed death ligand 1 (PD-L1) expression in the tumor microenvironment (TME) has propelled the use of anti-PD-1 immune checkpoint blockade (ICB) therapy, in combination with chemotherapy, as a treatment for TNBC patients. This has advantageously impacted TNBC patient outcomes such as survival, yet there remains a need to improve patient response to ICB therapies. The gut microbiome, in particular, increased abundance of Akkermansia muciniphila was associated with ICB therapy response in other cancer types. A. muciniphila can degrade mucins and produce short-chain fatty acid (SCFA) metabolites. Therefore, we investigated the interplay between A. muciniphila, SCFAs, and ICB therapy efficacy in TNBC models. As diet is the main modifier of the gut microbiome, we first investigated diet-gut microbiome interactions on ICB efficacy. Using the EO771 syngeneic murine model of TNBC, female C57BL/6 tumor-bearing mice consuming a low-fat control, or high-fat Western, or Mediterranean diet, were treated with 3 doses of 200µg of IgG or anti-PD-L1 antibodies and tumor progression was monitored. To assess changes in tumoral immune cell populations, end-of-study tumor tissue was used for immunohistochemistry (IHC). To evaluate modulation on the gut microbiome, metagenomic sequencing was performed on DNA isolated from fecal samples collected at the end-of-study, and targeted SCFA metabolomic analysis was performed on plasma collected at the study endpoint. Results show mice consuming a Mediterranean diet treated with anti-PD-L1 had significant increases in the abundance of gut A. muciniphila and plasma SCFAs, as well as significantly reduced primary tumor volume and increased immune cell activity within the TME. To then investigate if direct supplementation of A. muciniphila or SCFAs could enhance ICB efficacy, EO771 tumor-bearing mice were treated with 3 doses of 200µg of IgG, anti-PD-L1 or anti-PD-1 antibodies with a subset of mice stratified to also receive A. muciniphila or exogenoussupplementation of SCFAs. Tumor progression was measured and changes in tumoral immune cell populations were assessed with end-of-study tumor tissue prepared for flow cytometry and IHC. Results show mice supplemented with A. muciniphila and treated with anti-PD-L1 therapy demonstrated response to treatment, with a significant reduction in tumor volume as well as increased levels of immune cell populations in the TME. Mice supplemented with SCFAs and treated with a combination of anti-PD-1 and chemotherapy demonstrated enhanced response to treatment, with a significant reduction in tumor volume. In conclusion, data from our lab suggest that increased levels of gut A. muciniphila and SCFAs drive ICB efficacy in TNBC. Citation Format: Kenysha YJ. Clear, Adam S. Wilson, Elizabeth R. Stirling, Yu-Ting Tsai, Valerie Payne, David R. Soto-Pantoja, Katherine L. Cook. Harnessing the gut microbiome to modulate immune checkpoint blockade response in triple-negative 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 1423.

Abstract 5232: Placental circulating T cells expressing CD16 in combination with trastuzumab demonstrate robust anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against gastric cancer

Abstract Introduction: Celularity is developing PT-CD16VS as a novel platform for use in combination with approved monoclonal antibodies for the potential treatment of multiple cancers. PT-CD16VS is an allogeneic cell therapy derived from human postpartum placental circulating T (P-T) cells that are genetically modified to express a proprietary CD16 variant and endogenous T cell receptor (TCR) knockout. Here we report the characterization and preclinical evaluation of PT-CD16VS against HER2+ gastric cancer cells in combination with Trastuzumab. In vitro safety studies evaluating the potential for on-target/off-tumor activity of PT-CD16VS against HER2 low expressing lung epithelial cells (LEC) and human dermal fibroblasts (HDF) are also highlighted. Methods: P-T cells were activated and transduced with a lentiviral vector containing a CD16 construct expressing a high affinity CD16 variant, CD16VS. Following transduction, P-T cells were transfected to knock out the TCR. In vitro, the functional activity of PT-CD16VS cells in combination with Trastuzumab was assessed against the HER2+ gastric cancer NCI-N87 cell line in cytotoxicity, cytokine release, and proliferation assays. For on-target/off-tumor studies, similar assays were utilized to assess activity against the HER2 low expressing LEC and HDF cell lines. In vivo, PT-CD16VS with Trastuzumab was evaluated in a subcutaneous NCI-N87 xenograft model in NSG mice. Tumor volume was measured and tumor samples were evaluated for PT-CD16VS infiltration. Results: High CD16 transduction efficiency (&amp;gt;60%) was achieved across donors. In vitro, PT-CD16VS plus Trastuzumab demonstrated potent cytotoxicity against NCI-N87 cells at low E:T ratios (0.625:1) and at 12 hours of co-culture with moderate cytokine secretion at 24-hour of co-culture. PT-CD16VS proliferated following 48-hour incubation with NCI-N87 cells and Trastuzumab, as compared to an IgG1 control. PT-CD16VS in combination with Trastuzumab did not elicit on-target/off-tumor cytotoxicity or cytokine release in response to low HER2 expression on normal LEC or HDF cells. In vivo, PT-CD16VS with Trastuzumab demonstrated significant reduction in tumor volume compared to vehicle, Trastuzumab alone, and the positive control Enhertu groups. In addition, PT-CD16VS infiltration into the tumor was shown to be Trastuzumab dependent, with infiltrating cells expressing high levels of CD16 and Ki67. Conclusions: Our results show that PT-CD16VS has robust in vitro and in vivo ADCC activity against gastric cancer tumor cells when combined with Trastuzumab. Furthermore, PT-CD16VS activity is specific to tumor HER2 antigen expression and does not target low HER2 expression on normal healthy cells. PT-CD16VS is being developed in combination with various monoclonal antibodies for the treatment of multiple cancers. Citation Format: Kathy Karasiewicz-Mendez, Joseph Gleason, Kristina Tess, Mansour Djedaini, Shengchen Lin, Chenfei Huang, Shuyang He, Robert Hariri, Adrian Kilcoyne. Placental circulating T cells expressing CD16 in combination with trastuzumab demonstrate robust anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against gastric 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 5232.

Abstract 1346: Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in Osimertinib resistant H1975 lung cancer with EGFR mutations

Abstract Extracellular vesicles (EVs) derived from immune cells (e.g., NK cells, dendritic cells) may be able to treat malignancies that are resistant to other treatments (e.g., chemotherapy) relying on the hypothesis that EVs carry almost the same cargo of the parent cells. In this study, we evaluated IL-15 stimulated natural killer cell-derived EVs (NK-EVs) as therapeutic agents in vitro and in vivo in Osimertinib resistant lung cancer (H1975R) with EGFR mutations(L858R). NK-EVs were isolated by ultracentrifugation and nanoparticle tracking analysis revealed a size distribution of 89.5 ± 3.4 nm, zeta potential of -31.38 ± 0.25 mV. Atomic force microscopy imaging revealed vesicles with a spherical form and comparable sizes, meeting the criteria of exosomal EVs. Further, western blot studies demonstrated the presence of regular EV markers along with specific NK markers (perforin and granzyme). EVs were also characterized by using proteomic analysis which demonstrated that EVs had proteins for natural killer cell mediated cytotoxicity (Granzyme B) and T cell activation (Perforin and Plastin-2). Gene oncology (GO) analysis also showed that these are differentially expressed proteins (DEPs) that are involved in programmed cell death and positive regulation of cell death. Further, isolated NK-EVs (1*1011 particles/mL) were cytotoxic to H1975R cells in vitro with 55% and 68% cell viability in 2D and 3D cell cultures respectively against control. Carboplatin’s IC50 was reduced by approximately 1.9 and 1.7-fold (p less than 0.001) in 2D and 3D cell culture respectively when combined with NK-EVs. The EVs were then combined with carboplatin (25 mg/kg) and administered by i.p. route to H1975R tumor xenografts and significant reduction in tumor volume in vivo (≈1000 mm3 compared to ≈2000 mm3 of control group, p less than 0.001) was observed after 10 days. Our findings show for the first time that NK-EVs target the PD-L1/PD-1 immunological checkpoint and induce apoptosis and anti-inflammatory response by downregulation of SOD2, PARP, BCL2, SET, NF-κB and TGF-ß. MicroRNAs regulating cytotoxic proteins like perforin, granzyme and plastin were also identified using sequencing and the miRNAs: hsa-miR-5193/149-5P/3133/193-5p are picked for further studies. These miRNAs have been encapsulated in NK-EVs by electroporation and will be evaluated against Lung PDX xenografts along with carboplatin loaded NK-EVs. These miRNAs are directly involved in T cell or NK cell mediated cytotoxicity and are being currently investigated. The ability to isolate functional NK-EVs on a large scale and using them with platinum-based drugs may lead to new clinical applications. The results of the present study suggest the possibility of the combination of NK-cell-derived EVs and carboplatin as a viable immunochemotherapeutic strategy for resistant cancers. Citation Format: Aakash Nathani, Li Sun, Islauddin Khan, Mounika Aare, Arvind Bagde, Yan Li, Mandip Sachdeva. Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in Osimertinib resistant H1975 lung cancer with EGFR mutations [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 1346.

Abstract 3065: Targeting of metabolic reprogramming in lung cancer by utilizing patient-derived organoid tissue for precision medicine

Abstract In a preliminary pilot study, we reported elevated activity of indoleamine 2,3-dioxygenase 1 (IDO1) in serum from cisplatin-resistant (CR) lung cancer patients compared to cisplatin-sensitive (CS) patients. This heightened IDO1 activity was associated with increased tryptophan (TRP) catabolism, resulting in the upregulation of kynurenine (KYN) production. The detection of KYN can now be readily achieved through immunohistochemistry (IHC). We demonstrated that this upregulation contributed to increased Treg (regulatory T cell) and MDSC (myeloid-derived suppressor cell) populations resulting in an immunosuppressive tumor microenvironment. To investigate this effect, we used dual inhibition of IDO1/TDO2 (tryptophan2,3-dioxygenase 2) in an orthotopic mouse model of CR-Lewis lung carcinoma. This resulted in a significant reduction in tumor volume compared to LLC-CS (n=7, p&amp;lt;0.01), confirming the role of TRP catabolism in CR tumorigenesis. To determine the relevance of the data derived from this mouse model to clinical settings, we established 50 patient-derived organoid tissues (PDOT) from patients with NSCLC. PDOT is an experimental ex vivo tissue model developed to recapitulate the intricate features of lung architecture, including the lung acinus. Upon optimizing our collection and growth protocols, each PDOT successfully developed into a spherical body within 14 days. Furthermore, through bulk RNA-seq analysis, we showed that PDOT closely mirrors the expression of key molecular markers (PD-L1 and mutations), as observed in the original tumor tissues. PDOT from patients possessing high levels of PD-L1 and KYN were exquisitely sensitive to dual inhibition with 2-3-fold greater sensitivity compared to patients with undetectable KYN expression in growth inhibition assays (GI50=20±5.5μM vs. 65±7.5μM). Increased lipid peroxidation was detected via flow cytometry by accumulation of the C11-BODIPY reporter dye in the dual inhibition treatment group. Moreover, we integrated PDOT with a novel 3D microfluidic culture that can be supplied with patient-matched peripheral blood mononuclear cells (PBMC) to assess immune response. Using CyTOF, we showed that dual inhibitors with Atezolizumab (anti-PD-L1) enhanced immune effector (CD8+, NK) and suppressed immunosuppressive (Treg, MDSC) populations. We believe that our swift progress in PDOT development may bridge the divide between cancer biomarker expression and patient trials by timely complementing conventional drug studies based on cell lines and xenografts to facilitate the design of personalized therapies. As a result, the extensive reporting that we have contributed for KYN expression profiling in CR has the potential to serve as a valuable companion diagnostic tool in the future. This can assist in applying precision oncology by finding the most appropriate patients for immunotherapies. Citation Format: Chunjing Wu, Jonathan D. Nguyen, George Theodore, Estelamari Rodriguez, Sydney Spector, Emily Kim, Taranatee Khan, Pablo Puente, Yujie Wang, Dao M. Nguyen, Cheng-Bang Chen, Diane Lim, Lynn G. Feun, Niramol Savaraj, Medhi Wangpaichitr. Targeting of metabolic reprogramming in lung cancer by utilizing patient-derived organoid tissue for precision medicine [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 3065.

Abstract 6663: Innovative CPMV immunotherapy: A canine model for poor-prognosis breast cancer treatment

Abstract Cowpea Mosaic Virus (CPMV) is a plant comovirus non-infectious to humans or animals. In mice models, intratumor immunotherapy (ITI) with CPMV devoid of nucleic acids (empty CPMV, eCPMV) and full CPMV results in a highly potent, systemic, antitumor immune response, with full CPMV being much more effective. Dogs with spontaneous mammary (breast) cancer (MC) serve as a valuable model for studying breast cancer (BC), bridging the gap between studies in rodents and human clinical trials. Canine MC develops in the presence of an intact immune system and has a unique advantage of shorter endpoint periods compared to women BC clinical trials. New immunotherapeutic approaches are necessary for poor-prognosis BC, such as triple-negative BC, metastatic BC, and the very aggressive and lethal inflammatory BC (IBC). Previous studies by our group using eCPMV ITI in dogs with MC revealed a substantial reduction in tumor size in both injected and non-injected tumors (abscopal effect), extended survival, and improved quality of life without any adverse effects or toxicity. In this clinical trial, we employed full CPMV nanoparticles ITI in dogs with poor-prognosis (inoperable) MC, comparing them with untreated controls. Regular tumor volume assessments and clinical evaluations have been conducted. Histopathology and immunohistochemistry of immune cell populations were performed to study tumor microenvironment (TME) modifications. Thus far, 5 dogs have been treated: four cases of inflammatory mammary cancer (IMC) and one case of metastatic MC. After at least two weekly CPMV ITI sessions, a significant reduction in tumor volumes was observed. In IMC cases, marked clinical improvement of typical signs (warmth, edema, erythema, and cutaneous pain) was observed in 3 out of 4 cases, and tumor reduction was so remarkable in one dog that surgery was recommended and successfully carried out. In IMC CPMV ITI cases, the survival of 2 dogs exceeded historical IMC records; the dog with metastatic MC maintains stable disease after one year, which is also superior to controls. TME studies showed that eCPMV immunotherapy significantly increased intratumor inflammatory infiltrates, neutrophils, T lymphocytes, and macrophages. Additionally, Treg cells (FOXP3+)/T lymphocytes (CD3+) ratio was significantly reduced. Our results demonstrate that immunotherapy with eCPMV induces a potent immunostimulatory effect on dogs with poor-prognosis MC by recruiting and activating neutrophils as the main drivers of the inflammatory response. This process reduces the immunosuppressive action of regulatory T lymphocytes and produces a breakdown of tumor immune suppression. These findings underscore the therapeutic potential of CPMV nanoparticles ITI in the management of canine IMC and metastatic MC. From a translational point of view, this study lays the groundwork for advancing the treatment of human breast cancer. Citation Format: Guillermo Valdivia, Dolores Pérez-Alenza, Lucía Barreno, Ángela Alonso-Diez, Jessica Fernanda Affonso de Oliveira, María Suárez-Redondo, Steven F. Fiering, Nicole F. Steinmetz, Laura Peña. Innovative CPMV immunotherapy: A canine model for poor-prognosis breast cancer treatment [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 6663.

Abstract 5282: Sulindac modulates the response of triple negative breast cancer to anti-PD-L1 immunotherapy

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous tumor, and there is a lack of effective therapies. Immune checkpoint inhibitors (ICIs) therapy has been widely used to treat a variety of human cancers including TNBC. Despite the FDA's approval of therapies like Atezolizumab (anti-PD-L1) and Pembrolizumab (anti-PD-1) for certain TNBC cases, more than half of all TNBC patients, especially those with low PD-L1 levels and in advanced stages, remain unresponsive. Therefore, there is urgent clinical need to find other innovative combination therapy strategy for these TNBC patients. In this study, we examined the efficacy of sulindac to enhance the response of TNBC to anti-PD-L1 immunotherapy. We utilized a 4T1 syngeneic mouse tumor model to compare the inhibitory effects of PD-L1 antibody, sulindac, and their combination on 4T1 tumor growth. We found that mice treated with combination therapy showed a significant reduction in tumor volume, along with increased infiltration of activated T lymphocytes (Granzyme B+/CD8+ T cells) in the tumor tissues. We also established a PBMC humanized mouse model to further confirmed that combination therapy could significantly reduce the tumor size of TNBC patient-derived xenografts (56S) and the combination group were found to have the most infiltrating activated human T lymphocytes (Granzyme B+/CD8+ T cells) in the tumor tissues. Immunofluorescent staining of organoids also confirmed that organoids from combination group have more CD8+ T cell and the results of organoid cell viability assay in vitro indicated that combination of sulindac with PD-L1 antibody together with activated human PBMC significantly reduce 56S organoid cell viability, which is consistent with in vivo study. When investigating the mechanism of action, first we found sulindac could downregulate exosomal PD-L1 by decreasing expression of nsMase2 which is a major regulator in production of exosomal PD-L1. Second, we demonstrated that sulindac could downregulate PD-L1 by blocking Stat3 signaling and enhancing the expression of miR-570-3p which can potentially target PD-L1, which in turn led to a further decrease in exosomal PD-L1. Previous study showed that PD-L1 antibody could be bound and consumed by exosomal PD-L1 in the blood circulation. Therefore, in combination therapy, sulindac downregulating exosomal PD-L1 leads to increased availability of PD-L1 Ab, which potentially improves the overall efficacy of anti-PD-L1 therapy. In conclusion, our findings provide unique insights into the mechanism of action and efficacy for sulindac as an immunomodulatory agent in combination with anti-PD-L1 therapy for the treatment of TNBC. Citation Format: Bin Yi, Ruixia Ma, Yaguang Xi. Sulindac modulates the response of triple negative breast cancer to anti-PD-L1 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 5282.

Abstract 62: Intra-tumor persistence of activated anti-mesothelin hYP218 CAR T cells is associated with increased efficacy in gastric and colorectal cancers

Abstract Purpose: Patients with gastric and colorectal cancers that have progressed after standard therapies have poor prognosis. Given the high expression of mesothelin in these tumor types, we examined the potential utility of the anti-mesothelin hYP218 CAR T cells to treat these cancers. Experimental Design: Cell surface mesothelin expression in a panel of gastric and colorectal cancer cell-lines was analyzed using flow cytometry. In vitro efficacy of hYP218 CAR T cells against these cell lines was evaluated using cytotoxicity and cytokine release assays. In vivo efficacy of hYP218 CAR T cells was studied using gastric (HGC27) and colorectal (SW48) tumor xenografts in NSG mice. NSG mice were injected with 1 × 106 cancer cells and the tumors were allowed to grow until they reached a volume of 80-100mm³, after which the mice were treated with either 5 × 106 hYP218 CAR T cells, 5 × 106 untransduced T cells, or saline and monitored over time. Persistence of CAR T cells in the tumor was evaluated over time and expression of activation and exhaustion markers were studied. Results: hYP218 CAR T cells demonstrated strong cytotoxic activity against mesothelin positive gastric and colorectal cancer cell lines and exhibited increased pro-inflammatory cytokine production. In both the HGC27 and SW48 tumor models hYP218 CAR T cells demonstrated significant reduction in tumor volume and overall survival compared to mice treated with untransduced T cells or saline. CAR T cells isolated from tumors of both the xenograft models, at day 40 after treatment, displayed higher expression levels of activation markers CD39 and CD69 when compared to the infused product. Furthermore, a concomitant elevation in the levels of effector cytokines, TNF-α and IFN-γ, was detected in the blood of treated mice demonstrating the functional capacity of the CAR T cells to not only persist but also to maintain an activated state. Conclusions: hYP218 CAR T cells show anti-tumor efficacy against gastric and colorectal tumor xenografts due to increased accumulation of activated CAR T cells in the tumor and warrants further investigation for treatment of patients with these cancers. Citation Format: Sameer A. Mir, Abhilash Venugopalan, Jingli Zhang, Mana Kamana Khanal, Chaido Stathopoulou, Manjistha Sengupta, Qun Jiang, Raffit Hassan. Intra-tumor persistence of activated anti-mesothelin hYP218 CAR T cells is associated with increased efficacy in gastric and colorectal cancers [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 62.

Abstract 7548: Role of bromodomain extra terminal inhibitor (BETi) in combination with radiotherapy for the treatment of NF1-associated malignant peripheral nerve sheath tumor

Abstract Introduction: Neurofibromatosis Type 1 (NF-1) associated malignant peripheral nerve sheath tumors (MPNST) harbor mutations in the polycomb repressive complex 2 (PRC2) in 70% of tumors. PRC2 loss results in epigenetic changes, which increase histone acetylation and make the DNA vulnerable to binding by bromodomain proteins. This interaction could be targeted to uncouple histone acetylation from transcription using bromodomain extra terminal inhibitors (BETi). As radiation therapy (RT) is a cornerstone of MPNST treatment, we analyzed the effects of BETi with and without RT. Methods: Mice were implanted with patient-derived xenografts (PDX) of PRC2-wildtype model developed in the Torres laboratory, MPNST007. Mice were segregated into vehicle and I-BET 762 treatment groups and treated with either vehicle or drug for one week before the RT was initiated. Half of the mice in each treatment group (n = 10) received radiation (2 Gy per fraction for 5 days for a total of 10 Gy). The study endpoints were (1) tumor growth rates, (2) tumor size and weight at study termination, (3) immunohistochemical (IHC) staining for biomarkers of proliferation (Ki67), apoptosis (cleaved caspase 3, CC3), and DNA damage (γH2AX), and (4) RNA sequencing of PDXs from each experimental arm. Results: Significant tumor regression was observed in PRC2-mutant PDX tumors treated with I-BET 762 based on tumor growth kinetics over 45 days of treatment (p &amp;lt; 0.001). Tumor volume was significantly reduced by I-BET 762 compared to vehicle (p &amp;lt; 0.01). Combining I-BET 762 and RT resulted in significant tumor volume reduction (p &amp;lt;0.001). RNA sequencing and intersection of differentially expressed genes related to “DNA damage repair” and those repressed by I-BET 762 was performed for the MPNST007 PDXs. The expression of 259 genes related to DNA damage repair decreased after I-BET 762 treatment, including XRCC1, ERCC4, RAD50, RAD51D, and LIG4 (data not shown). IHC analysis prompted by the RNA sequencing results of the I-BET 762-treated PDXs revealed higher levels of γH2AX. An increase in necrotic and fibrotic tissue with increased CC3 in the BETi plus radiation group was observed. Conclusions: Genetic mutations in NF-1-associated MPNST tumors result in susceptibility to BETi treatment. In a PDX model of PRC2 wild-type tumors, BETi treatment resulted in tumor regression and sensitized MPNST xenografts to RT. Combining BETi and RT enhanced tumor death and increased expression of genes associated with DNA damage and apoptosis. Citation Format: Alcia A. Gingrich, Sharon Landers, Angela Bhalla, Cristian Rodriguez-Aguayo, Rossana Lazcano, Suresh Satpati, Alines Lebron-Torres, Emily Z. Keung, Christopher P. Scally, Christina L. Roland, Kelly K. Hunt, Heather Lyu, Heather Lillemoe, Ashleigh Guadagnolo, Alexander Lazar, John Slopis, Ian McCutcheon, John A. Livingston, Ahsan Farooqi, Kunal Rai, Keila E. Torres. Role of bromodomain extra terminal inhibitor (BETi) in combination with radiotherapy for the treatment of NF1-associated malignant peripheral nerve sheath tumor [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 7548.

Abstract 4987: TREM2 is a radiation-induced target for immunomodulation of tumor-associated macrophages in the prostate cancer tumor microenvironment

Abstract Background: The prostate cancer (PCa) tumor microenvironment (TME) is characterized by a predominance of myeloid cells that may have a key role in immunosuppression and disease progression. Stereotactic Body Radiation Therapy (SBRT) is utilized for treatment of localized and metastatic PCa and promotes inflammatory remodeling of the TME. We analyzed human PCa specimens at acute post-SBRT timepoints to identify radiation-induced targets in the PCa TME and used in vitro and in vivo models to test and validate our findings. Methods: We performed 10X single-cell RNA sequencing (scRNA-seq) on prostatectomy specimens from men with high-risk localized PCa; 12,988 cells from 4 patients that underwent pre-operative SBRT and 47,314 cells from 6 patients that underwent prostatectomy alone. THP-1 monocytes polarized into macrophage (Mɸ) states [M0, M1, M2 and tumor-associated macrophages (TAM), were used to characterize TREM2 expression and immune responses to radiation or conditioned media (CM) derived from irradiated PC3 cells. In vivo studies with C57BL/6 mice inoculated with MC38 flank tumors or PTEN-/- SPOPmut CHD1del mouse prostate organoid line were used to analyze intratumoral myeloid populations in response to SBRT (37.5Gy in 5 fractions). Results: scRNA-seq analysis (Cell Ranger 7.1.0 pipelines; Seurat v5 R package) revealed enrichment of a cluster of myeloid cells defined by a damage-associated Mɸ gene signature (TREM2, APOE, SPP1, LGALS3, CD9) in response to SBRT compared with non-irradiated specimens (35.6% vs 5.7%). TREM2 expression was significantly upregulated in THP-1 cells polarized in M2 (p&amp;lt;0.001) and TAM (p&amp;lt;0.001) conditions, compared to M1 conditions. Irradiation (20Gy x1) of THP-1 cells significantly increased TREM2 expression in M0 (p&amp;lt;0.01), M1 (p&amp;lt;0.001), and TAM (p&amp;lt;0.01) conditions. Proliferation of PC3 cells exposed to CM from irradiated TAMs was significantly increased (p&amp;lt;0.0001) compared to CM from non-irradiated TAM. Alternatively, CM derived from irradiated PC3 cells significantly increased (p&amp;lt;0.05) TREM2 expression in M0-polarized Mɸ. In vivo studies, in MC38 and organoid models, noted a significant reduction (p&amp;lt;0.0001) in tumor volume at day 7 post-SBRT. Multi-parametric flow cytometry demonstrated robust myeloid infiltration in response to SBRT dominated by CD11b+F4/80+ TAMs in irradiated tumors. Among irradiated tumors, TREM2 expression was markedly elevated in TAMs (organoid and MC38, p&amp;lt;0.0001). Conclusion: Preliminary data in human irradiated PCa and validation studies in preclinical models suggest TREM2 expression on myeloid cells is a radiation-induced target. Further studies are underway to understand the functional and immunological implications of SBRT-induced TREM2 expression on TAMs and whether this can be therapeutically targeted to enhance anti-tumor immunity in PCa. Citation Format: Janny A. Villa-Pulgarin, Un In Chan, Francesca Khani, Fabio Socciarelli, Jiansheng Wu, Ryan Carelli, Jeffrey Kraynak, Silvia C. Formenti, Luigi Marchionni, Massimo Loda, Himanshu Nagar, Ariel E. Marciscano, Christopher E. Barbieri. TREM2 is a radiation-induced target for immunomodulation of tumor-associated macrophages in the prostate cancer tumor microenvironment [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 4987.

Abstract 4558: A triterpenoid derivative has strong antitumor activities in gemcitabine-resistant advanced bladder cancer models and promotes immune cell infiltration in a high grade murine syngeneic model

Abstract Bladder cancer (BC) presents a formidable challenge in treatment and patient survival, requiring particular attention to advanced stages, such as muscle-invasive stage (MIBC), due to its potential for rapid progression and substantial impact on patient morbidity and mortality. The five-year survival rate for MIBC is a meager 30%, which drops further down to 8% once tumors metastasize. Advanced bladder cancers (High grade T1) are treated with intravesical therapy with Gemcitabine and Cisplatin (G+C) combination and Stage T2 are treated neoadjuvant with G+C and cystectomy, thereby decreasing the quality of life for patients with BC. Furthermore, tumors progress beyond responding to other chemotherapy regimens such as dose-dense treatment with Methotrexate +Vinblastine + Adriamycin and Cis-platin (MVAC), all of which will further decrease the quality of life with persistent adverse toxicity. Our laboratory has developed a novel compound derived from the pentacyclic triterpenoid, Ursolic acid, termed N-methyl piperazine conjugated-Ursolic acid, or UA4, and have demonstrated its antitumor activities against both Gem-Sensitive (Gem-S) and Gem-Resistant (Gem-R) human advanced BC cell lines, T24 and 5637. We tested the combination of Gem, UA4 and Gem + UA4 in a murine non-MIBC model (MB49) to investigate the role of immune cell infiltration during tumor development and treatment response. UA4 treatment was equally effective in chemo-sensitive and chemo-resistant cell lines at dosages between 4-5µM. Previous in vitro mechanistic studies on UA4 and UA4+Gem combination treatments revealed cell death occurred through apoptosis and autophagy, along with a highly selective form of mitochondrial-mediated autophagy (mitophagy). These findings were confirmed with transmission electron microscopy ultrastructural analysis, cell cycle phase analyses, excess, changes in mitochondrial membrane potential and reactive oxygen species measurements. Gem-S and Gem-R xenografts in athymic mice further supported UA4’s efficacy in vivo, demonstrating a significant reduction in both tumor weight and volume following oral gavage (100mg/kg), with mice exhibiting no signs of systemic toxicity. Combination treatment with UA4 and Gem synergistically increased antiproliferative activity. MB49 syngeneic mice model further supported UA4 as an effective therapy, and immunofluorescence assays with immune-related markers, suggested an anti-inflammatory effect in vivo. Overall, we demonstrate UA4 has a potential to be used as an efficacious, non-toxic compound for further adjuvant use in clinical settings. Citation Format: Juliette Rose Seremak, Kunj Bihari Gupta, Siva S. Panda, Bal L. Lokeshwar. A triterpenoid derivative has strong antitumor activities in gemcitabine-resistant advanced bladder cancer models and promotes immune cell infiltration in a high grade murine syngeneic model [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 4558.

Abstract 6963: Cysteinyl leukotriene receptors promote melanoma progression and metastasis

Abstract Background: Cysteinyl leukotrienes (cys-LTs; LTC4, LTD4, LTE4) are pro-inflammatory mediators mainly produced by hematopoietic cells, which enhance inflammation through their receptors, CysLT1R and CysLT2R. They play a pivotal role in the development of chronic asthma in humans. Recent association of inflammation with cancer has led to immense interest on the role of cysteinyl leukotrienes in cancer progression and metastasis. However, the exact molecular mechanisms regarding how these inflammatory mediators regulate the tumor proliferation and metastasis remain unexplored. A thorough investigation of these precise molecular mechanisms and identification of the specific receptor/s responsible can aid in understanding the therapeutic potential of these receptors in various cancers like melanoma. Since the inhibitors of these receptors are already FDA approved for the treatment of asthma, our goal is to repurpose these drugs to treat melanoma progression. Objective: To determine the mechanistic aspects of how CysLTRs regulate melanoma tumor initiation, progression, and metastasis. Methods: Protein expression by western blotting and ELISA, transcript expression by qPCR, viability, and proliferation by XTT and BrDU, migration using trans-well assay, tumor growth and metastasis were confirmed through in vivo experiments. Results &amp; Conclusion: B16F10 melanoma cells express high CysLT1R compared to CysLT2R and produce endogenous cys-LTs that function in an autocrine signaling pattern. Further, cys-LTs mediated the activation of several signaling proteins such as ERK, p38, AKT, and CREB that are important for melanoma survival and proliferation. Moreover, treatment with CysLTR antagonists significantly reduced melanoma cell proliferation, survival, and migration. Accordingly, we observed a significant reduction in the melanoma tumor volume in vivo in both Cysltr1−/- and Cysltr2−/− mice compared to the WT mice. Interestingly, angiogenesis was significantly reduced in Cysltr2−/- mice but not in Cysltr1−/−.Therefore, we speculate that while both receptors play a crucial role in tumor proliferation in vivo, CysLT2R is the main driver of angiogenesis and metastasis. Therefore, targeting both these receptors using their specific antagonists can offer potential therapy for melanoma progression and metastasis. Citation Format: Emma Elizabeth Sabu Kattuman, Lakshminarayan Reddy Teegala, Somayeh Darzi, Sailaja Paruchuri. Cysteinyl leukotriene receptors promote melanoma progression and metastasis [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 6963.

Abstract 6034: PSMA targeted alpha radiopharmaceutical [211At]PSAt-3 inhibits tumor growth in a preclinical model of prostate cancer

Abstract Introduction: Prostate cancer continues to be a highly prevalent cancer diagnosis among men and a leading cause of cancer related deaths worldwide. With the introduction[177Lu]Lu-PSMA-617, targeted radionuclide therapy has proven successful in increasing survival time for patients with metastasized castration resistant prostate cancer. However, despite encouraging results, such patients tend to develop progressive disease, and thus, enhanced efficacy is warranted. To this end, the use of radiopharmaceuticals harnessing alpha-emitters is a promising venue due to the potent energy deposition and short range of alpha particles in tissue. Here, we employ the PSMA targeting vector, [211At]PSAt-3, for targeted alpha radionuclide therapy in a preclinical prostate cancer model. Methods: [211At]PSAt-3, a structural derivative of PSMA-617, in which the L-2-naphtylalanine was replaced by L-3-[211At]astato-phenylalanine, was obtained by electrophilic [211At]astatodesilylation. Evaluation of [211At]PSAt-3 was conducted in male inbred nude mice of Balb/c background xenografted subcutaneously with human prostate cancer cells (LNCaP). Prior biodistribution studies were carried out with approximately 100 kBq intravenous injections, and subsequent biodistribution 2, 6 and 24h post injection (P.I.). For establishment of therapeutic effect, a single intravenous dose of [211At]PSAt-3 was administered. To asses hematological toxicity, blood cell composition was assessed weekly. Results: Favorable tumor uptake of 15.2±6 %ID/g was observed 2h after injection of [211At]PSAt-3 in LNCaP xenografted nude mice, with subsequent 9±5.1% ID/g and 6.4±2.4% ID/g 6 and 24h P.I., respectively. [211At]PSAt-3 cleared mainly by the kidneys, in which uptake was 72, 38 and 6% ID/g at 2, 6 and 24h P.I., respectively. Thyroid/throat uptake remained low at ~0.1% ID for all timepoints investigated, indicating a low degree of de-astatination. Initial efficacy studies using a single intravenous dose of 0.5 MBq (~20 MBq/kg) demonstrated significant growth delay and tumor volume reduction compared with control mice (saline). Blood platelet count was reduced after administration of [211At]PSAt-3 and subsequently recovered. No signs of severe toxicity, and no significant weight loss between control and treated mice was observed, suggesting that the treatment is well tolerated. Conclusion: 0.5 MBq [211At]PSAt-3 was well tolerated in mice and demonstrated tumor volume reduction and significant delay of tumor growth. These findings suggest [211At]PSAt-3 as a promising candidate for clinical translation. Citation Format: Lars Hvass, Marius Müller, Anne Clausen, Matthias Herth, Andreas Kjaer. PSMA targeted alpha radiopharmaceutical [211At]PSAt-3 inhibits tumor growth in a preclinical model of 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 6034.

Enhanced Anti-tumor Effect of Flavopiridol in Combination With Gemcitabine in Pancreatic Cancer.

The efficacy of current chemotherapies for pancreatic ductal adenocarcinoma (PDAC) is still unsatisfactory. Flavopiridol inhibits multiple cyclin-dependent kinases, causing cell cycle arrest and inducing cancer cell apoptosis. This study aimed to evaluate the anti-tumor effect of flavopiridol and gemcitabine in PDAC in vitro and in vivo. PANC-1 and MIA PaCa-2 cell lines were treated with gemcitabine and flavopiridol alone, in combination, and sequentially, and cell proliferation, apoptosis, and the cell cycle were evaluated. Proteins related to cell cycle progression (cyclin A, CDK2, E2F-1, and p53) were quantified using western blotting. A xenograft mouse model was generated, and the effects of gemcitabine and flavopiridol, administered alone or in combination, were evaluated by measuring tumor volume and apoptosis degree using the TUNEL assay. Sequential administration of gemcitabine and flavopiridol suppressed PDAC cell proliferation and induced apoptosis. Flavopiridol treatment led to an increase in the number of cells in the S and a decrease in those in the G0/G1 phases. Gemcitabine increased and decreased the number of S- and G2/M-phase cells, respectively. Furthermore, flavopiridol treatment decreased cyclin A and CDK2 expression and increased E2F-1 expression. In a xenograft mouse model, the combined administration of gemcitabine and flavopiridol demonstrated the most significant reduction in tumor volume and induction of apoptosis. Flavopiridol potentiates the anti-tumor activity of gemcitabine by inducing cell cycle arrest and apoptosis. Its synergistic inhibition of PDAC cell proliferation, when combined with gemcitabine, positions flavopiridol as a promising candidate for cancer treatment.