Eliminate Cancer Cells Research Articles

Abstract 33: Revitalizing CAR-T cells for TNBC: Targeting phosphorylation of TCF7 to overcome T cell exhaustion

Abstract Breast cancer stands as the second leading cause of cancer-related deaths among women in the United States, with triple-negative breast cancer (TNBC) representing the most aggressive subtype. Although immune checkpoint inhibitors (ICI) demonstrated durable response in some TNBC patients, the added benefit of current FDA-approved ICI in combination with chemotherapy is estimated to be only around 10%. Fortunately, TNBC displays the highest number of tumor-infiltrating lymphocytes (TILs), compared to other BC subtypes, indicating an optimal context for the survival of T cells. Therefore, CAR-T therapy emerges as a promising therapeutic option for TNBC patients. Expanding the application of CAR-T therapies to solid tumors, including breast cancer, remains challenging. One of the key hurdles lies in the immunosuppressive tumor microenvironment, where the T cell receptor experiences long-term exposure to tumor antigens, leading to T cell exhaustion. This state impedes the T cells' capacity to efficiently target and eliminate cancer cells. Identifying key regulatory mechanisms downstream of the T cell receptor holds the potential to revolutionize existing immunotherapeutic strategies. In our study, we discovered that the presence of the master transcriptional factor of T cell stemness, TCF1, in CD8+ T cells, is essential for controlling TNBC tumors. TCF1 undergoes phosphorylation by LCK, a crucial component of initiating T cell receptor signaling, upon T cell activation. We found this phosphorylation site is indispensable for TCF1's transcriptional activities, inhibiting the downstream program of T-cell persistence and causing T-cell exhaustion. Reversing this process, either through LCK inhibition or introducing a non-phosphorylated form of TCF1, results in a more sustained CAR-T cell-mediated anti-tumor response. Our findings reveal the significance of TCF1 post-translational modification in the development and longevity of stem-like CD8+ T cells, bridging the gap between TCR signaling and transcriptional and epigenetic programs of stem-like CD8+ T cells. This insight contributes to a potential cure for TNBC by maintaining T cell persistence. Citation Format: Zhou Jiang, Yu-Yi Chu, Heng-Huan Lee, Mien-Chie Hung, Liuqing Yang, Chunru Lin. Revitalizing CAR-T cells for TNBC: Targeting phosphorylation of TCF7 to overcome T cell exhaustion [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 33.

Abstract 4123: Attenuating breast cancer metastasis with BCL-XL-targeting PROTACs

Abstract B-cell lymphoma X-large (BCL-XL) emerges as a significant player in cancer progression. In our recent findings, we unveil the efficacy of proteolysis-targeting chimeras (PROTACs) directed against BCL-XL, demonstrating their capacity to not only eliminate cancer cells but also target tumor-induced regulatory T cells (TI-Tregs). Notably, BCL-XL's role in promoting breast cancer metastasis extends beyond its anti-apoptotic function. We posit that BCL-XL-targeting PROTACs (BCL-XL-Ps) hold promise for treating metastatic breast cancer. Their potential lies in directly eliminating circulating tumor cells during vulnerable stages and/or triggering anti-cancer immunity through Treg cell depletion. Our experiments underscore the potency of IAP-based PROTACs in facilitating BCL-XL degradation via the ubiquitin-proteasome system (UPS) while mitigating on-target platelet toxicity. Treatment of breast cancer-bearing mice with these compounds demonstrates a tangible reduction in breast cancer metastasis to the lungs. Additionally, our studies reveal a significant depletion of tumor-infiltrating Tregs in mice treated with BCL-XL-Ps. While affirming BCL-XL as a viable therapeutic target for breast cancer metastasis, further exploration into the mechanisms behind BCL-XL-P targeting is imperative. Our future endeavors will be devoted to unraveling whether our BCL-XL-Ps effectively target both tumor-intrinsic BCL-XL, potentially inducing tumor cell death, and tumor-infiltrating Tregs, thereby fostering an adaptive immune response against the tumor cells. Citation Format: Madison E. Carelock, Peiyi Zhang, Mo Jiao, Umasankar De, Zeng Jin, Rachel M. Stump, Sarah G. Williams, Guangrong Zheng, Weizhou Zhang. Attenuating breast cancer metastasis with BCL-XL-targeting PROTACs [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 4123.

Abstract 3437: Stromal tumor-infiltrating lymphocytes in association with body size and composition among Black and White women with breast cancer

Abstract Background: Obesity is a major risk factor for breast cancer and negatively affects disease prognosis. Tumor-infiltrating lymphocytes (TILs) in the stroma of breast tissue play a major role in eliminating cancer cells and modulating immunotherapy efficacy. We aim to study the association between body size and composition with stromal TILs in breast tumor tissue by race. Methods: The study included 920 Black and 395 White women with newly diagnosed invasive breast cancer who have stromal TIL scores from the Women’s Circle of Health Study (WCHS). Body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) were measured during the home interview. Pre-treatment H&E-stained tumor tissue sections were reviewed by our study pathologist and stromal TILs were scored on a scale of 0-100% at 10% increment according to the recommendations of the International TILs Working Group. Linear regressions adjusting for age at diagnosis, tumor grade, stage, and molecular subtype were used to test the differences in TILs across the categories of body size and composition indicators separately for Black and White women. Results: A higher stromal TILs score was associated with more aggressive breast cancer features, including higher grade, larger tumor size, more advanced stage, hormone receptor (HR) negativity, and human epidermal growth factor receptor 2 (HER2) positivity in both Black and White women (all P<0.05). Among Black women, WHR >0.90 vs ≤0.85 was associated with lower TILs overall (β = -3.5%, P = 0.045) and in triple-negative tumors (β = -12.3%, P = 0.003). Among White women, BMI 25.0 - 29.9 vs <25 was associated with higher TILs in HR-/HER2+ tumors (β = 28.1%, P = 0.031). A pattern suggested that BMI ≥30 vs <25 was associated with lower TILs in both HR-/HER2+ (β = -10.2, P=0.52) and triple-negative (β = -12.8, P=0.14) tumors, although the associations were not significant. No association was observed between TILs and any of the body composition parameters in women with HR+ tumors. Conclusions and Relevance: Body size and composition correlate with stromal TILs mainly in patients with HR- tumors, and this relation may be race-dependent. These findings suggest that body size and composition potentially modulate breast cancer treatment outcomes at least in part through TILs-dependent mechanisms. Funding: This work was in part supported by grants from the US National Institutes of Health (P01CA151135, R01CA100598, R01CA185623, P30CA016056, P30CA072720, K07CA201334, and R37CA248371), US Army Medical Research and Material Command (DAMD-17-01-1-0334), the Breast Cancer Research Foundation (CBA), and the Philip L. Hubbell family. Citation Format: Rand T. Akasheh, Thaer Khoury, Song Yao, Angela Omilian, Chi-Chen Hong, Elisa V. Bandera, Bonnie Qin, Nur Zeinomar, Christine Ambrosone, Ting-Yuan David Cheng. Stromal tumor-infiltrating lymphocytes in association with body size and composition among Black and White women with 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 3437.

Abstract 5371: Multimodal CRISPR activation screens reveal new mechanisms to sensitize cancer cells to targeted T cell mediated elimination

Abstract Cell autonomous resistance mechanisms can protect cancer cells from targeted T cell elimination, resulting in resistance to a broad range of immunotherapies. High-throughput gene knockout and gene inhibition screens have been instrumental in identifying key drivers of such mechanisms, yet often uncovering gene activity that is essential but not sufficient for response. Here we set out to leverage gene activation as an orthogonal approach to identify novel regulators of the cancer-immune interface. First, we performed CRISPR activation screens in melanoma cells co-cultured with antigen-specific primary CD8+ T cells. Our findings revealed a collection of novel genes that substantially sensitize melanoma cells to targeted T cell-driven elimination when activated, including DNA damage and unfolded protein response genes (e.g., ETS1, TSPYL2), Wnt ligands (e.g., WNT3A, WNT1), and others that are deleterious only in combination with targeting T cells. Among the resistance genes we found regulators of insulin signaling (e.g., HGF, TCF7L2) and numerous glycoproteins (e.g., PDPN, CD44). Second, we validated top novel hits in 2D and in 3D spheroid models, using optical readouts to investigate their impact on cancer cell elimination and cancer-T-cell spatial dynamics. Third, we performed a Perturb-seq screen to stratify dozens of hits into functional groups based on the transcriptional readouts in the cancer cells. Lastly, we developed an in situ Perturb-Seq method, where we combine high-plex spatial transcriptomic readouts at subcellular resolution with in situ detection of genetically modified cells, and applied it to track the impact of the (de)sensitizing perturbations not only on the cancer cells themselves, but also on T cell recruitment and suppression. Taken together, our study provides a catalog of immune evasion mechanisms and an array of novel targets, demonstrating that synthetic gene activation can diminish immune evasion and open novel avenues for development of agonist-based and RNA-based interventions. Citation Format: Reece V. Akana, Chang Sun, YoungMin Kim, Yuxin Cai, Subin Kim, Jeehyun Yoe, Olivia Laveroni, Livnat Jerby-Arnon. Multimodal CRISPR activation screens reveal new mechanisms to sensitize cancer cells to targeted T cell mediated elimination [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 5371.

Abstract 4893: Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas

Abstract Introduction: Colorectal cancer (CRC) is second most common cause of cancer-related mortality in the USA and most patients with advanced tumors lack effective therapeutic options. Epigenetic silencing resulting in reduced expression of O6-methylguanine-DNA methyltransferase (MGMT) has been described in diverse tumor types including CRC and it is linked with reduced DNA repair capability and increased sensitivity to alkylating agents such as temozolomide. However, the potential role and clinical significance of MGMT overexpression in cancer cells has not been studied. Methods: We used spatially resolved multiplex quantitative immunofluorescence (mQIF) analysis to concurrently measure the levels of MGMT protein, γH2AX and CD8+ tumor infiltrating lymphocytes (TILs) in a cohort of 111 CRCs and paired non-tumor colorectal mucosa from the same patient represented in tissue microarray format. To assess the genomic and immunologic context of MGMT expression, we studied the methylome, mutation profiling and mRNA expression of 526 CRCs from TCGA. The immunologic landscape of tumors was studied using GSEA and multiple transcriptomic-based tumor immune infiltration scores such as TIMER, CIBERSORT, etc. Finally, the genomic alterations and immunogenicity of SW620 human CRC cells (with baseline MGMT hypermethylation) with or without MGMT transfection were studied using in vitro co-culture preparations coupled to ultra-deep DNAseq and flow cytometry analysis. Results: On mQIF analysis, cancer-cell MGMT protein upregulation was identified in 73.9% of CRCs and was associated with reduced levels of γH2Ax in malignant cells and lower CD8+ TILs. 26.1% of cases showed MGMT protein downregulation. The genomic analysis of the TCGA cohort revealed that the mean nonsynonymous mutational load was significantly lower in MGMT-high than low tumors with marked differences in the type of variants and frequency of oncogenic mutations. The transcriptomic analysis revealed that MGMT overexpressing tumors showed lower immune infiltration than the MGMT-low group and reduced representation of immune associated signatures including interferon gamma response and TNF-alpha signaling. In contrast, DNA repair, TP53 pathways, were enriched. The exogenous expression of MGMT in SW620 cells reduced the number of nonsynonymous mutations by 15.6% (141 vs 119 mean mutations) and was also associated with low T-cell activation (CD69+/CD8+ T-cells) and cancer-cell elimination on allogenic co-cultures with human PBMCs. Conclusion: MGMT protein upregulation is common in human CRCs and is associated with reduced DNA damage response, distinct genomic features, and adaptive immune evasion. To our knowledge, this study is the first to report the biological and clinical characteristics of CRCs with MGMT overexpression that could support diagnostic and therapeutic developments. Citation Format: Barani Kumar Rajendran, Janie Zhang, Shruti Desai, Joanna Gibson, Jassim DiPalermo, Patricia LoRusso, Yong Kong, Hongyu Zhao, Michael Cecchini, Kurt Alex Schalper. Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas [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 4893.

Abstract 4: Identifying novel patient-derived T cell receptors targeting TP53 public neoantigens

Abstract Adoptive cell transfer (ACT) is a promising immunotherapeutic strategy to bolster reactivity against immunologically cold tumors through infusion of T cells that can recognize and eliminate cancer cells bearing mutation-derived neoantigens (NeoAgs). T cell receptors (TCRs) that confer recognition of public NeoAgs–an elite subset of Human leukocyte antigen (HLA)-restricted epitopes arising from recurrent hotspot mutations in driver genes that enhance cancer cell fitness and are clonally conserved–are highly desirable and would be effective against many tumors sharing the common hotspot mutation. Tumor suppressor TP53 is a supreme target for this approach: it is the most frequently mutated gene in cancer yet remains untargetable with FDA-approved therapies. The most common TP53 alteration (R175H) generates a public NeoAg in the context of HLA-A*02, an allele expressed by ~40% of the North American and Western European populations. Earlier work established the immunogenicity of this public NeoAg by isolating a TP53(R175H)-specific clone from a colorectal cancer patient. ACT of this TCR in a single patient produced an objective anti-tumor response, but also resulted in multisystem organ failure indicating this receptor might possess off-target reactivity to unrelated proteins in healthy tissue. To discover safe and effective TP53(R175H)-specific TCRs, we assembled a unique biorepository of peripheral blood mononuclear cells (PBMCs) from n=26 cancer patients with diverse tumors that co-express HLA-A*02/TP53(R175H) and applied a novel high-throughput screening technique to retrieve candidate TCRs. These efforts resulted in a panel of n=9 genetically distinct and functionally validated receptors, each with NeoAg-specific anti-tumor activity against a panel of cancer cell lines. To define the cross-reactivity profile of these novel TCRs and the benchmark TCR that caused toxicity, we performed amino acid scanning mutagenesis (X-Scan) to identify potential off-targets. Importantly, we identified significant off-target reactivity to normal human proteins for the benchmark TCR, which may explain the lung and kidney toxicities observed in the trial patient. By contrast, a subset of the novel TCRs exhibited exquisite specificity for the TP53 public NeoAg with no predicted off-targets. Finally, we performed ACT of all TCRs in a mouse xenograft model and identified several novel TCRs that potently eliminated TP53(R175H)+/HLA*A02+ tumors in vivo. Based on these data, we are now proceeding with the clinical development of a best-in-class TCR. This work is poised to bring an important new class of precision immunotherapeutics to patients with TP53-mutant tumors that presently lack effective molecularly targeted agents. Citation Format: Michael V. Gormally, Smita S. Chandran, Inaki Etxebarria, Christopher Klebanoff. Identifying novel patient-derived T cell receptors targeting TP53 public neoantigens [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 4.

Abstract 4598: Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers

Abstract Homozygous deletions of the p16/CDKN2a (cyclin dependent kinase inhibitor 2A) locus, which is responsible for regulating the cell cycle, are commonly found in cancer and often involve the deletion of adjacent genes. One such adjacent gene is methylthioadenosine phosphorylase (MTAP), involved in metabolism, located on chromosome 9p21 in close proximity to the p16/CDKN2A tumor-suppressor locus. Co-deletion of MTAP is observed in approximately 80-90% of tumors with homozygous deletion of CDKN2A, representing 10-15% of all human tumors. These tumor types, including non-small cell lung cancer, pancreatic adenocarcinoma, glioblastoma, and mesothelioma, have a poor prognosis, highlighting the significant unmet medical need in this area. Deletion of MTAP leads to a significant accumulation of methylthioadenosine (MTA) in cells. MTA, at high concentrations, selectively inhibits the PRMT5 methyltransferase enzyme, competing with the substrate S-adenosylmethionine (SAM) required for methylation reactions. As a result, the overall level of symmetric arginine dimethylation throughout the proteome is reduced. This heightened sensitivity to modulation of methylosome activity makes cells with MTAP deletion more susceptible to therapeutic targeting of PRMT5. Hence, selective targeting of PRMT5 in cancers with homozygous MTAP deletion represents a promising strategy for specifically eliminating cancer cells with this genetic alteration. Ryvu has developed MTA-cooperative PRMT5 inhibitors characterized by good drug-like physicochemical properties and inhibition of methyltransferase activity with IC50 values in the low nanomolar range. A structure-based lead optimization delivered compounds coming from two independent series with high selective potency in MTAP-deleted cell lines and DMPK profiles allowing an oral administration. The antitumor activities were compared in vitro and in vivo to MRTX1719 and AMG193 in MTAP null tumors such as HCT116 MTAP KO, DoHH-2 and Lu99. The correlation between compound exposure and on-target effect was confirmed in PK/PD and efficacy studies. Taken together, these studies confirm that MTA cooperative PRMT5 inhibitors exert strong synthetic lethal phenotype in MTAP deleted cancers and offer an exciting therapeutic opportunity for a large patient population. Citation Format: Adam Radzimierski, Aneta Bobowska, Agata Stachowicz, Kamil Kuś, Kamila Kozłowska-Tomczyk, Agnieszka Ludwig-Słomczyńska, Paulina Podkalicka-Gołda, Aniela Gołas, Monika Żukowska, Pavlo Lebed, Przemysław Wyrębek, Daria Szukiel, Matylda Stefaniak, Oleksandr Popika, Julia Krzywik, Sujit Sasmal, Paulina Niedziejko-Ćwiertnia, Klara Korta-Piątek, Karolina Fijołkowska, Aleksandra Więckowska, Marta Olszak-Płachta, Swapnil Nipunge, Mateusz Świrski, Marek Wronowski, Henryk Pawlik, Quỳnh Vũ, Katarzyna Łagosz-Ćwik, Mateusz Stoszko, Szymon Woroszyło, Igor Tomczyk, Ewelina Gabor-Worwa, Nilesh Gaud, Anna Kowal-Chwast, Dawid Gogola, Magdalena Miodek, Róża Starczak, Agata Dudek, Jacek Faber, Bożena Winnik, Sanja Novak-Ratajczak, Agnieszka Świrska, Karolina Gluza, Paweł Guzik, Katarzyna Banaszak, Nicolas Boutard, Grzegorz Ćwiertnia, Krzysztof Brzózka, Mateusz Nowak, Anna Bartosik, Didier Pez. Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted 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 4598.

Abstract 115: Developing uPAR CAR T Cells for High-Grade Serous Ovarian Cancer

Abstract Chimeric Antigen Receptor (CAR) T cells are a modality of immunotherapy that act to eliminate cancer cells by redirecting the cytolytic activity through targeting a protein overexpressed on the surface of the cancer cell. In contrast to move conventional cancer therapies, their anti-cancer activity does not depend on a cancer-specific molecular vulnerability but rather the differential expression of the target antigen on tumor cells compared to normal tissues. We have developed CAR T cells targeting the urokinase plasminogen activator receptor (uPAR), which is overexpressed on senescent cells but not expressed highly in vital organs, to selectively target senescent cells in a range of tissue damage pathologies where they are known to be pathogenic. uPAR is also highly overexpressed in a wide range of cancer types, including pancreatic, kidney, bladder, brain and ovarian carcinomas, respectively, raising the possibility that CAR T cells targeting uPAR may also be effective against cancer. Indeed, leveraging an electroporation-based genetically-engineered mouse model of ovarian cancer, we demonstrate the robust anti-tumor efficacy of murine uPAR CAR T cells in immunocompetent syngeneic mice. Moreover, through an exhaustive functional screening of 37 distinct human uPAR single-chain fragment variants (scFVs), we have successfully identified lead scFVs exhibiting subnanomolar affinity to membrane-anchored uPAR. Significantly, these human uPAR CAR T cells exhibit the capability to eliminate both orthotopic and metastatic human HGSOC xenograft tumors without inducing severe adverse effects. Ongoing efforts encompass the assessment of the long-term safety and toxicity profiles of uPAR CAR T cells utilizing a humanized mouse model platform. Our results provide a strong rationale for developing uPAR CAR T cells as an anticancer strategy relevant to a broad range of tumor types, and provide an avenue for safety profiling of uPAR CAR T cells prior to clinical testing in non-cancer patients harboring senescence-related pathologies. Citation Format: Zeda Zhang, Xin Fang, Yu-jui Ho, Sascha Haubner, Friederike Kogel, Clemens Hinterleitner, Stella Paffenholz, Kevin Chen, Wei Luan, Amanda Kulick, Gertrude Gunset, Andreina Garcia Angus, Jing Zhang, Zijian Xu, Adam Wang, Qingwen Jiang, Elisa de Stanchina, Britta Weigelt, Dmitriy Zamarin, Aveline Filliol, Judith Feucht, Jorge Mansilla-Soto, Corina Amor, Michel Sadelain, Scott Lowe. Developing uPAR CAR T Cells for High-Grade Serous Ovarian 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 115.

Abstract 3968: Class I HLA-independent lysis of cancer cells by tumor-infiltrating CD8 T cells

Abstract T cell-based therapies have transformed cancer treatment, although intrinsic or acquired resistance develops in nearly half of the patients. Tumor-infiltrating CD8+ T lymphocytes (TILs) are key determinants of anti-tumor immunity by secretion of cytotoxic granules, such as granzymes and perforin, and effector cytokines, such as TNFa and IFNg. A deep understanding of how CD8+ TILs eliminate cancer cells will help identify resistance mechanisms and develop new therapeutic strategies. Here, we demonstrate that TILs eliminated matched melanoma cells in a time and dose-dependent manner but in a class I HLA-independent fashion. Specifically, blocking class I HLA-TCR interaction either by the deletion of B2M (resulting in loss of class I HLA surface expression) or TCR genes did not alter the activity of these TILs. We further confirmed that TIL-mediated killing is contact-dependent. After excluding the potential roles of CD1d and MR1-restrictive T cells, gamma delta T cells, and NKG2D in TIL-mediated killing, a whole-genome CRISPR/Cas9 screen was performed in cancer cells. We identified that genes involved in JAK-STAT and cell-extrinsic apoptosis pathways are necessary for TIL-mediated killing. Together, these findings demonstrate that expanded CD8 TILs from melanoma patients are capable of eliminating melanoma cells via a novel, class I MHC-independent mechanism by coactivation of both JAK-STAT and cell-extrinsic apoptosis pathways. Citation Format: Hongyan Xie, Aiping Jiang, Jonathan Perera, Aonkon Dey, Neal Smith, Moshe Sade-Feldman, Robert T. Manguso, Nir Hacohen, Russell W. Jenkins. Class I HLA-independent lysis of cancer cells by tumor-infiltrating CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3968.

Abstract 710: Post-exposure suppression of radiation pneumonitis suggests non-redundant independent effects of TGF-beta and TRAIL/DR5

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 6724: Mechanistic validation of MAIT cell engagers in HUB patient-derived organoids (PDO) co-culture model

Abstract In the field of cancer immunotherapy, bispecific antibodies have emerged as a promising approach for redirecting T-cells to target and combat tumors. However, their efficacy is limited to only a subset of patients. A major challenge that hinders the development of bispecific antibody targets is the lack of preclinical models that preserve patient-specific tumor antigens and recapitulate the complex interaction between the human immune system and tumor cells. HUB Organoids technology presents a valuable platform for novel immunotherapies testing and validation as they are directly derived from patient tumor tissues, accurately reflecting patient-specific tumor antigens. BIOMUNEX Pharmaceuticals has developed the BiXAb® technology platform, a computational modeling approach that rapidly generates bispecific antibodies from various monospecific monoclonal antibodies, eliminating the need for extensive engineering. This platform includes a bivalent T cell engager specifically targeting MAIT cells and human epithelial growth factor receptor (HER2), a tumor antigen highly expressed in some colorectal cancer (CRC) patients. This innovative approach forms an efficient immunological synapse, allowing exclusive redirection of MAIT cells to directly eliminate cancer cells. In a collaborative research initiative between HUB Organoids® and BIOMUNEX Pharmaceuticals, the mechanisms by which BiXAb® induce tumor cell killing were confirmed using CRC patient-derived organoids (HUB Organoids® or PDOs) co-cultured with allogenic MAIT cells in an in vitro model. The study results depicted BiXAb® induced MAIT-cell activation in a dose-dependent manner, as detected by FACS and ELISA. Furthermore, BiXAb®-mediated MAIT-cell-specific killing of CRC PDOs was observed in a HER2-dependent manner, determined using an image-based viability assay. In conclusion, the co-culture platform of CRC PDOs-MAIT cells described here effectively captured the activation and targeted killing of MAIT cells mediated by BiXAb®, underscoring the added value of this PDO-based co-culture platform in developing novel immunotherapies. Citation Format: Yasmine Abouleila, Julie Prigent, Mayke Doorn, Claire Germain, Sylvia Boj, Simon Plyte, Carla Verissimo. Mechanistic validation of MAIT cell engagers in HUB patient-derived organoids (PDO) co-culture 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 6724.

Abstract 5366: ATR promotes extracellular matrix stiffness mediated cytoskeleton remodeling and immunosuppression

Abstract The stiffening of the extracellular matrix (ECM), a phenomenon that often promotes metastasis and immunosuppression in many cancers, is a process that is not yet fully understood. Our study reveals that ATR, a molecule primarily recognized for its role in maintaining the genome, plays a crucial role in ECM stiffness-induced immune evasion and metastasis in triple negative breast cancer (TNBC) through DNA repair independent manner. In response to ECM stiffness, ATR activates SUN2, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. This activation enhances the interaction of SUN2 with outer nuclear membrane proteins KASH1 and KASH2. The interaction between SUN2 and KASH1/2 facilitates the nuclear localization of β-catenin, which in turn promotes epithelial mesenchymal transition (EMT) and reduces the expression of E-cadherin. E-cadherin is essential for attracting CD103+ immune cells that perform anti-tumor immunity. Interestingly, inhibiting ATR reduces EMT in tumor cells, leading to an increase in CD103+ dendritic cell infiltration while decreasing neutrophil and polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) infiltration. Further, we found that ATR levels are inversely correlated with immunotherapy response rates in patients. The alteration in immune cell composition sensitizes tumors to immunotherapy, thereby suppressing tumor growth and metastasis. In conclusion, the role of ATR in remodeling the immune environment could be crucial for optimizing the clinical use of ATR inhibitors. This makes ATR a promising target for overcoming immunotherapy resistance. We suggest early or prolonged treatment with an ATR inhibitor to potentially reduce metastasis rates and improve the prognosis of TNBC patients. Furthermore, we suggest using ATR levels as a marker to predict response rates and prognosis in TNBC patients. For patients with higher ATR expression levels, combination treatment with ATR inhibitors and immunotherapy may yield better outcomes. Enhanced anti-tumor immunity could increase the chances for patients to eliminate cancer cells and help end breast cancer. Finally, our research suggests the combined treatment of ATR inhibition and immunotherapy for patients without necessarily applying DNA damage stimulation. This approach could potentially reduce toxicity while still benefiting patients. Citation Format: Xinyi Tu, Zhenkun Lou, Robert Mutter. ATR promotes extracellular matrix stiffness mediated cytoskeleton remodeling and immunosuppression [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 5366.

Abstract 1917: HER2-FGFR1 interaction induced by bispecific molecules: A novel strategy for treating breast cancer

Abstract Breast cancer poses a significant global health challenge, demanding effective therapies targeting specific receptors. In our prior study, we engineered molecules capable of binding to both HER2 and FGFR1, pivotal receptors in breast cancer progression. By combining the high-affinity Affibody ZHER2:2891 for HER2 with a modified, stable form of FGF2 binding to FGFR1, we created the bispecific protein (AfHER2-lFGF2). This innovative molecule could simultaneously recognize HER2 and FGFR1, exhibiting enhanced cell penetration. When coupled with the potent cytotoxic agent monomethyl auristatin E (MMAE), it demonstrated exceptional efficacy in eliminating cancer cells expressing HER2, FGFR, or both receptors. Our current investigation focused on quantifying the interaction between HER2 and FGFR1 receptors on the cell surface upon treatment with these bispecific molecules. Leveraging fully quantified spectral imaging FRET (FSI-FRET), a technique utilizing spectrally resolved two-photon microscopy. We directly monitor the association of HER2 and FGFR1 in the plasma membrane. Our findings revealed that the bispecific AfHER2-lFGF2 molecule forces interaction between HER2 and FGFR1 on the cell surface. Furthermore, we analyzed the activation of the HER2-FGFR1 complex and subsequent downstream signaling pathways using western blotting. In summary, our research uncovers the ability of the AfHER2-lFGF2 bispecific protein to foster direct interaction between HER2 and FGFR receptors on the cell membrane. By employing state-of-the-art techniques like FSI-FRET, we elucidated the mechanisms governing this interaction and, by western blotting, downstream signaling pathways. These insights emphasize the potential of AfHER2-lFGF2 as a promising therapeutic agent for targeting HER2, FGFRs, and HER2/FGFRs-positive cancers. This approach paves the way for targeted therapies in addressing the prevalent global health challenge of breast cancer. Citation Format: Mateusz Adam Krzyscik, Kalina Hristova. HER2-FGFR1 interaction induced by bispecific molecules: A novel strategy for treating 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 1917.

Global analysis of T-cell groups reveals immunological features and common antigen targets of digestive tract tumors

BackgroundT cells are key players in the tumor immune microenvironment (TIME), as they can recognize and eliminate cancer cells that express neoantigens derived from somatic mutations. However, the diversity and specificity of T-cell receptors (TCRs) that recognize neoantigens are largely unknown, due to the high variability of TCR sequences among individuals.MethodsTo address this challenge, we applied GLIPH2, a novel algorithm that groups TCRs based on their predicted antigen specificity and HLA restriction, to cluster the TCR repertoire of 1,702 patients with digestive tract cancer. The patients were divided into five groups based on whether they carried tumor-infiltrating or clonal-expanded TCRs and calculated their TCR diversity. The prognosis, tumor subtype, gene mutation, gene expression, and immune microenvironment of these groups were compared. Viral specificity inference and immunotherapy relevance analysis performed for the TCR groups.ResultsThis approach reduced the complexity of TCR sequences to 249 clonally expanded and 150 tumor-infiltrating TCR groups, which revealed distinct patterns of TRBV usage, HLA association, and TCR diversity. In gastric adenocarcinoma (STAD), patients with tumor-infiltrating TCRs (Patients-TI) had significantly worse prognosis than other patients (Patients-nonTI). Patients-TI had richer CD8+ T cells in the immune microenvironment, and their gene expression features were positively correlated with immunotherapy response. We also found that tumor-infiltrating TCR groups were associated with four distinct tumor subtypes, 26 common gene mutations, and 39 gene expression signatures. We discovered that tumor-infiltrating TCRs had cross-reactivity with viral antigens, indicating a possible link between viral infections and tumor immunity.ConclusionBy applying GLIPH2 to TCR sequences from digestive tract tumors, we uncovered novel insights into the tumor immune landscape and identified potential candidates for shared TCRs and neoantigens.

A dual-mode portable platform with spatiotemporal temperature–pressure signal readouts for ultrasensitive quantitative determination of cancer cells

Early precise diagnosis of cancer is a prerequisite for cancer-related mortality and improved therapeutic outcomes. Direct quantitative cancer cell detection technologies show a great potential for early screening and diagnosis of cancer. However, the reported techniques generally involve exorbitant costs, lengthy procedures, and inevitable use of large-scale apparatus. We develop herein a spatiotemporal dual-mode sensing platform based on temperature and pressure signals for rapid, sensitive, and straightforward quantitative detection of cancer cells. Specifically, this platform is constructed by a dual-signal response probe consisting of hyaluronic acid (HA)-modified hollow copper sulfide nanoparticles (CuS@HA NPs), which not only elicits a robust and spatiotemporal photothermal effect via localized surface plasmon resonance (LSPR) but also promotes a rapid photothermal decomposition of NH4HCO3. This dual-signal response probe demonstrates a broad detection range from 10 to 105 cells/mL for sensing triple-negative breast cancer cells (MDA-MB-231) due to the specific recognition between HA and CD44 receptors. Notably, the remarkable sensitivities, with detection limits of 9 and 4 cells/mL for temperature and pressure signal readouts respectively, display about one order of magnitude lower than conventional immunoassay methods. The practicability and high reliability of the proposed strategy are further validated via analyzing clinic serum samples. Moreover, the localized photothermal therapy induced by the specific binding of CuS@HA NPs can realize targeted elimination of cancer cells. Overall, the developed dual-mode sensing platform demonstrates significant potential as a facile yet robust strategy for real-time and on-site determination of cancer cells in actual samples.

Identification and Characterization of a Small Molecule Bcl-2 Functional Converter.

We report the identification of a small molecule that exposes the Bcl-2 killer conformation and induces death in Bcl-2-expressing cancer cells. Selective targeting of Bcl-2 and elimination of cancer cells expressing Bcl-2 opens up new therapeutic avenues.

Bispecific immune cell engager enhances the anticancer activity of CD16+ NK cells and macrophages in vitro, and eliminates cancer metastasis in NK humanized NOG mice

BackgroundIn a prior report, we detailed the isolation and engineering of a bispecific killer cell engager, referred to as BiKE:E5C1. The BiKE:E5C1 exhibits high affinity/specificity for the CD16a activating receptor...

ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia

BackgroundIn recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold‑inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC).MethodsCCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot.ResultsOur results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)‑like population. Moreover, hyperthermia substantially improved the sensitivity of radiation‑resistant NPC cells and CSC‑like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti‑tumor‑killing activity of hyperthermia against NPC cells and CSC‑like cells, whereas ectopic expression of Cirbp compromised tumor‑killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC‑like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance.ConclusionTaken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.

In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. We show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

Nanomedicines: Emerging Platforms in Smart Chemotherapy Treatment-A Recent Review.

Cancer continues to pose one of the most critical challenges in global healthcare. Despite the wide array of existing cancer drugs, the primary obstacle remains in selectively targeting and eliminating cancer cells while minimizing damage to healthy ones, thereby reducing treatment side effects. The revolutionary approach of utilizing nanomaterials for delivering cancer therapeutic agents has significantly enhanced the efficacy and safety of chemotherapeutic drugs. This crucial shift is attributed to the unique properties of nanomaterials, enabling nanocarriers to transport therapeutic agents to tumor sites in both passive and active modes, while minimizing drug elimination from delivery systems. Furthermore, these nanocarriers can be designed to respond to internal or external stimuli, thus facilitating controlled drug release. However, the production of nanomedications for cancer therapy encounters various challenges that can impede progress in this field. This review aims to provide a comprehensive overview of the current state of nanomedication in cancer treatment. It explores a variety of nanomaterials, focusing on their unique properties that are crucial for overcoming the limitations of conventional chemotherapy. Additionally, the review delves into the properties and functionalities of nanocarriers, highlighting their significant impact on the evolution of nanomedicine. It also critically assesses recent advancements in drug delivery systems, covering a range of innovative delivery methodologies. Finally, the review succinctly addresses the challenges encountered in developing nanomedications, offering insightful perspectives to guide future research in this field.