bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 7063: Monitoring metabolic plasticity in the tumor microenvironment in vivo by mitophagy generated extracellular vesicles
Authors: Gade Purva V. , Howard Marissa , Rojas Angela , Hasanzadah Layla , Liotta Lance , Kashanchi Fatah
Published: March 2024

Tumor metabolic reprogramming is a hallmark of cancer progression, survival, and therapeutic resistance. A targetable class of cancer metabolic adaptation exploits mitophagy known to be linked to the cancer phenotype. Mitophagy selectively eliminates dysfunctional mitochondria by targeting them, via autophagosome shuttling, to the lysosome for degradation. Cancer cell mitophagy is triggered by elevated oxidative stress and mitochondrial DNA damage caused by hypoxia, radiotherapy, and immunotherapy. A high mitophagy demand can overwhelm the lysosome capacity resulting in the accumulation of damaged mitochondria and can suppress biogenesis of healthy mitochondria. We hypothesize that the newly discovered secretory mitophagy mechanism exports damaged mitochondrial fission-released segments to reduce the overload pressure on the lysosomal system, and thereby sustains cancer cell survival in the face of therapeutic mitochondrial stress. We have discovered a form of secretory mitophagy occurring in vivo in a growing solid tumor. Our molecular analysis of the full repertoire of extracellular vesicles, EVs, shed into the resident tumor interstitial fluid, IF, in vivo yielded a rich set of information about the functional state of mitochondria within the tumor cells, and the host cells. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81 positive IF EVs, including PINK1, DRP1 and FIS1. The export of cellular mitochondria proteins to CD81 positive EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti-CD81 immunoprecipitation, Mito Tracker export into CD81 positive EVs, ultrastructural characterization and Exoview analyzer. Further, we stimulated mitochondrial oxidative stress and blocked the fusion of the mitophagosome with the lysosome, which markedly stimulated the export of the secretory mitophagy unit. The outcome is new understanding of the importance of secretory mitophagy that can constitute a therapeutic target, and a new clinically relevant means of monitoring the in vivo state of mitophagic flux within the tumor microenvironment by means of mitophagy derived EVs.

Citation Format: Purva V. Gade, Marissa Howard, Angela Rojas, Layla Hasanzadah, Lance Liotta, Fatah Kashanchi. Monitoring metabolic plasticity in the tumor microenvironment in vivo by mitophagy generated extracellular vesicles [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 7063.
Abstract 7178: Neuronal cells derived from iPSCs cell to evaluate neurotoxicity after 48 or 72 hours in high-through put screening format
Authors: Sabatier Fabienne , Chaigne Justine , Martin Maryse , Barbeau Rodolphe , Houssin Luisa , Bonnin Stephane , King Alastair , Jahnke Vanessa
Published: March 2024

Neurotoxicity is a major concern in central nervous system drug discovery and a frequent cause of attrition in clinical trials or approved drug withdrawal. Neuropathy is a side effect defined by the injury of peripheral nerves causing a loss of sensation/motion. Drug development pipelines typically involve testing in cell lines followed by animal investigations with translation to humans. The induced pluripotent stem cells (iPSCs) have widened new model systems to study adverse toxicities. With evolution in regulatory guidance the use of human- iPSC-derived tissue provides high-throughput access and a more relevant testing environment. Neuropathy is one of the most chemotherapy adverse effects. In this study we have screened 8 compounds. Bortezomib (proteasome inhibitor), gemcitabine (approved ovarian cancer treatment), ivermectin (parasitic diseases treatment), flavopiridol (approved CDKs inhibitors), SNS-032 (approved CDKs inhibitor), mefloquine (anti-malaria drug), digitonin (nonionic detergent) and tamoxifen (hormone receptor-positive breast cancer treatment). These molecule toxicities were tested on neuronal-derived iPSCs Dopaneurone, GABAneurone, Glutaneurone, Motorneurone, Microglya and Astrocytes in high throughput screening. For the project, cells were plated in 384 well/plates. DMSO tolerance was performed (up to 10%). Drugs were added, then, after 48h or 72h, total protease and protease activities were measured using CytoTox-GloTM. Compounds were tested at 7 concentrations (from 10−4 to 10−9M).In this study we have validated cell culture and the assay conditions. The signal window range was good after 48h and 72h of treatment. No toxicity was found until 10% DMSO. A specific neurotoxicity signature was measurable for each drug tested. Digitonin, our positive control, induced toxicity in all IPSCs after 48h and 72h of incubation. On the contrary, Tamoxifen, our negative control, did not induce any neurotoxicity. The other molecules generated a signature of severe to mild neurotoxicity. These results are in agreement with their known adverse side-effects. In conclusion, this panel is a good tool to anticipate possible neurotoxicity within the 3Rs respect. It can be used in early drug de-risking or neuroprotection screening, with the aim of preventing/reducing/curing neuropathy in at-risk populations.

Citation Format: Fabienne Sabatier, Justine Chaigne, Maryse Martin, Rodolphe Barbeau, Luisa Houssin, Stephane Bonnin, Alastair King, Vanessa Jahnke. Neuronal cells derived from iPSCs cell to evaluate neurotoxicity after 48 or 72 hours in high-through put screening format [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 7178.
Abstract 7180: Tumor growth modeling to support dose justification of Dato-DXd in hormone receptor positive, HER2 negative breast cancer (HR+HER2-BC) subjects
Authors: Tang Zoey , Jiang Yu , Yang Haitao , Dai David , Gong Chang , Irons Linda , Pan Yuzhuo , Hong Ying , Liang Shuang , Ren Song , Phipps Alex , Zhou Diansong
Published: March 2024

BACKGROUND: Dato-DXd, a TROP2-directed antibody-drug conjugate, has demonstrated efficacy and a manageable safety profile in multiple tumor types. A dosing regimen of 6 mg/kg every three weeks (Q3W) has been proposed as the optimal schedule based on phase I data in patients with non-small cell lung cancer (NSCLC)1. Dato-DXd demonstrated statistically significant and clinically meaningful improvement in PFS compared with ICC in patients with HR+HER2-BC in TROPION-Breast012. A tumor growth inhibition (TGI) model was developed to support dose justification at 6 mg/kg in HR+HER2-BC subjects.

METHODS: A tumor growth inhibition (TGI) model was developed based on tumor size data from HR+HER2-BC patients received 6 mg/kg Dato-DXd Q3W in a Phase I study (N = 39, Data cutoff July 2022). The TGI model described the tumor size as the result of an exponential tumor growth, a tumor shrinkage that was driven by Dato-DXd cycle-specific AUC, and a resistance term suggesting that the shrinkage effect by Dato-DXd decreases over time. Cycle-specific Dato-DXd AUC was derived based on the AUC and time at each cycle for each individual. Simulations were conducted using the TGI model to compare tumor size change from baseline at 4, 6, and 8 mg/kg in HR+HER2-BC patients, with consideration of dropouts.

RESULTS: The TGI model well-described tumor size data with reasonable parameter estimations. Simulated time-courses of tumor sizes at Dato-DXd 4, 6, and 8 mg/kg suggested that Dato-DXd at 6 mg/kg may lead to greater tumor shrinkage effect than that at 4 mg/kg in HR+/HER2-BC patients. At approximately 180 days post first treatment, tumor shrinkage at 6 mg/kg was almost 2-times greater than at 4 mg/kg (-51% vs. -25%, respectively).

CONCLUSION: Simulations based on the TGI model suggested greater tumor size reduction at Dato-DXd 6 mg/kg compared to 4 mg/kg in HR+HER2-BC patient population.

Reference: 1 ACoP13 2022 2 ESMO 2023.

Citation Format: Zoey Tang, Yu Jiang, Haitao Yang, David Dai, Chang Gong, Linda Irons, Yuzhuo Pan, Ying Hong, Shuang Liang, Song Ren, Alex Phipps, Diansong Zhou. Tumor growth modeling to support dose justification of Dato-DXd in hormone receptor positive, HER2 negative breast cancer (HR+HER2-BC) subjects [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 7180.
Abstract 7198: A small molecule Notch1 inhibitor (ASR490) restores chemosensitivity in triple-negative breast cancer
Authors: Tyagi Neha , Chandrasekaran Balaji , Tyagi Ashish , Singh Balpreet , Chandran Megha , Singh Amandeep , Sharma Arun , Damodaran Chendil
Published: March 2024

Metastatic triple-negative Breast cancer (mTNBC) is an aggressive and often a chemo resistant disease for which no effective druggable target has yet been identified. We and others reported that the aberrant expression of neurogenic locus Notch homolog protein 1 (Notch1) serves as a molecular signature for TNBC. Hence, we postulated that pharmacologically targeting Notch1 in conjunction with a standard chemotherapeutic agent (Doxorubicin: DXR) may completely eradicate TNBC growth and overcome systemic and debilitating toxicity. Hence, this study aims to elucidate the role of our recently discovered notch1 inhibitor ASR490 and its ability to chemosensitize TNBC without significant systemic toxicity.

In our results, pretreatment of 1/10 th of the inhibitory concentration of ASR490 combined with 1/10 th or 1/20th of DXR significantly inhibited the growth of TNBC cells (MDA-MB-231 and BT549). Notably, DXR treatment induced Notch1 activation which led to chemoresistance in TNBC; however, co-treatment with the Notch1 inhibitor ASR490, led to the downregulation of Notch1 activation and eradication of the cell viability and clonogenicity of the TNBC cells. Further, molecular analysis suggested that pretreatment of 1/10th of ASR490 facilitates DXR-mediated sensitivity, by inhibiting the DNA repair pathway and upregulating (cleaved) caspase 3, (cleaved) caspase-9 and poly (ADP-Ribose) polymerase (PARP)-mediated apoptosis. Similarly, the combination effectively blocked the epithelial and mesenchymal transition (EMT) regulators and suppressed metastatic properties of MDA-MB231 and BT-549 cells. Moreover, combining ASR490(1/10th) and DXR (1/20th) significantly eradicated xenotransplanted TNBC tumors compared to ASR490 or DXR alone. In addition to being effective at inhibiting tumor growth, the significantly lower dose requirements of DXR and ASR490 in combination makes it a highly safe therapeutic modality. These findings suggest that aberrant activation of Notch1 is responsible for TNBC chemoresistance and concomitantly inhibiting Notch1 activation is an effective therapeutic strategy to restore chemosensitivity in TNBC.

Citation Format: Neha Tyagi, Balaji Chandrasekaran, Ashish Tyagi, Balpreet Singh, Megha Chandran, Amandeep Singh, Arun Sharma, Chendil Damodaran. A small molecule Notch1 inhibitor (ASR490) restores chemosensitivity 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 7198.
Abstract 7202: Strategy of targeting PKCβΙ protein to overcoming the acquired gemcitabine resistant pancreatic cancer cells
Authors: Bae EunSeo , Lim Yijae , Hong Junhwa , Byun Woong Sub , Chun Simin , Hong Suckchang , Lee Sang Kook
Published: March 2024

Pancreatic cancer (PC) is one of the most malignant cancers with limitation of treatment options such as resection surgery, radiation therapy and gemcitabine-based chemotherapy. To treating PC patients, gemcitabine-based chemotherapy is common and effective for PC, acquired gemcitabine resistance is one of the major reasons for treatment failure. Therefore, there is a need to suggest effective strategies and novel therapeutic approach for gemcitabine-resistant PC. Protein Kinase C β1 (PKC β1) is known as involved in multiple signal transduction related to oncogenic functions in cancer. We suggested that PKC β as one of the novel biomarker of drug resistance and further investigated the molecular mechanism. Among the natural products as small molecule inhibitor, we found that evodiamine has potent antitumor activity and various biological activities. Based on various biological activities of evodiamine, the scaffold was synthesized and investigated for antiproliferative and antitumor activities against gemcitabine resistant PANC-1 (PANC-GR) cells. Also, target validation experiment was employed to elucidate the molecular mechanism and verifying the target to overcoming gemcitabine resistance. Mechanistically, evodiamine derivatives have shown that inhibition of PKC β1 can modulate the proliferation of PANC-GR cells and exhibited tumor growth inhibitory activity in xenograft mouse model.

Keywords: Pancreatic cancer cell, Gemcitabine resistance, Protein Kinase C β1 (PKC β1), Molecular mechanism

Acknowledgements: This research was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (RS-2023-00273240).

Citation Format: EunSeo Bae, Yijae Lim, Junhwa Hong, Woong Sub Byun, Simin Chun, Suckchang Hong, Sang Kook Lee. Strategy of targeting PKCβΙ protein to overcoming the acquired gemcitabine resistant pancreatic cancer 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 7202.
Abstract 723: Reduction of immunosuppressive function of regulatory T cells enhances antigen-specific T cell mediated cytotoxicity against tumor cells
Authors: Khan Arif Azam , Park Jaehyung , LaVoy Nate , Tompkins Chris , Bellinder Jeff , Miguel Tisha San , Lawrence Neal , Schoen Brianna
Published: March 2024

Regulatory T cells (Tregs) maintain immune homeostasis by suppressing abnormal immune response to self and non-self-antigens. Tregs are also involved in tumor development and progression by inhibiting anti-tumor immunity. Infiltration of Tregs in the tumor microenvironment (TME) inhibit anti-tumor immunity by suppressing tumor antigen-specific T cell responses. This results in tumor progression and poor prognosis in patients with various types of cancers. Therefore, depletion of Tregs could be a promising immunotherapy target to augment the anti-tumor immune response. Molecules highly expressed by Tregs such as CD25, immune checkpoint molecules, and chemokine receptors have been targeted by antibodies or small molecules to deplete Tregs and its immunosuppressive function has been tested in the clinic. However, a thin line exists between augmenting effective immunity and inducing autoimmunity, posing difficulties in harnessing Tregs modulation in cancer treatment. Thus, additional strategies to selectively control Tregs are urgently needed for the effective treatment of cancer. In this study we evaluated the role of Tregs in suppressing the effector function of antigen-specific T cells and targeted killing of cancer cells in vitro. We observed Tregs mediated suppression of antigen-specific cytotoxicity against cancer cells. When Tregs were treated with cyclophosphamide, CD25 depleting antibody and anti-CTLA4 antibody alone or in combination, we observed enhanced killing of cancer cells in vitro as a result of potentiated effector function of antigen-specific T cells. This study provides a path forward to fine-tune and optimize strategies to target various molecules that are highly expressed on Tregs and augment anti-tumor immunity by antigen-specific T cells to achieve therapeutic outcome.

Citation Format: Arif Azam Khan, Jaehyung Park, Nate LaVoy, Chris Tompkins, Jeff Bellinder, Tisha San Miguel, Neal Lawrence, Brianna Schoen. Reduction of immunosuppressive function of regulatory T cells enhances antigen-specific T cell mediated cytotoxicity against tumor 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 723.
Abstract 7238: Exploring the potential of targeted DNA Nanobot delivery systems for gene delivery
Authors: Shahhoessini Melika , Spitzner Jeffrey R. , Lucas Christopher R. , Castro Carlos E. , Halley Patrick D.
Published: March 2024

Gene therapy is a cornerstone of modern medicine's quest for precision treatment to cure human disease including cancer. The current approach to gene delivery technology employs viral vector delivery systems. While effective, these vectors present significant challenges including immunogenicity, safety concerns due to off-target non-tissue specific delivery, payload limitations, and significant cost ultimately diluting their therapeutic potential. Thus, novel approaches to gene delivery are necessary. One such approach to address these critical limitations involves DNA Nanobots, a DNA-based nanotechnology that allows for a tissue-/target-cell specific gene delivery system. Utilizing DNA origami molecular assembly techniques, our DNA Nanobots offer a customizable and highly precise delivery platform, free from viral vector constraints. This innovative approach has the potential to transform the landscape of gene therapy by providing a safer, more efficient, and targeted alternative. Key technical advancements with our DNA Nanobots Gene Delivery Platform include: •CRISPR Cas-9 Integration: By incorporating CRISPR Cas-9 functional proteins, our nanobots were shown to enhance gene editing precision in primary human T cells, broadening therapeutic applications across various genetic conditions. •Targeted mRNA Delivery: Our nanobots revealed promising results in the targeted delivery of mRNA, vital for vaccine development and personalized treatments. •Efficient antisense/siRNA Delivery: Prior studies have overcome traditional challenges including endosomal escape and cytoplasmic entry, demonstrating effective siRNA delivery critical for gene silencing therapeutic development. In summary, DNA Nanobots mark a paradigm shift in gene therapy. Their ability to deliver various nucleic acids with high specificity and efficiency addresses many of the current methodological limitations especially off-target safety concerns and high cost. This advancement not only enhances the therapeutic index of existing treatments but also opens avenues for novel therapeutic strategies, potentially reshaping the future of molecular medicine and gene therapy.

Citation Format: Melika Shahhoessini, Jeffrey R. Spitzner, Christopher R. Lucas, Carlos E. Castro, Patrick D. Halley. Exploring the potential of targeted DNA Nanobot delivery systems for gene delivery [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 7238.
Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy
Authors: Wei Han
Published: March 2024

Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions.

Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay.

Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models.

Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels.

Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [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 7248.
Abstract 7251: Surface-engineered extracellular vesicles to modulate antigen-specific T cell expansion for cancer immunotherapy
Authors: Lyu Xiabing , Yamano Tomoyoshi , Imai Shota , Van Le Toan , Hanayama Rikinari
Published: March 2024

Tumor immunotherapy, including immune checkpoint therapy, CAR T cell therapy, and TCR-T cell therapy, has made significant progress in the treatment of cancer. Recently, newly developed nanomaterials have also been applied to anti-tumor immunotherapy. Among them, extracellular vesicles (EVs) have been well-engineered in recent years due to their low immunogenicity, high stability, and wide distribution in biological fluids. Despite the recent development of several EVs-based cancer immunotherapies, their clinical efficacy remains limited. In this study, we have developed engineered EVs designed to mimic the function of antigen-presenting cells, which can co-deliver multiple immune-activation signals to tumor-associated T cells. We named these vesicles Antigen-presenting Extracellular Vesicles (AP-EVs). AP-EVs express a high level of MHC I-peptide complexes, costimulatory molecules, and surface-bound cytokines, allowing for the simultaneous presentation of multiple immune modulators to antigen-specific CD8+ T cells. AP-EVs promote the clonal expansion and differentiation of antigen-specific cytotoxic T lymphocytes, resulting in potent anticancer immune responses. Notably, our findings indicate that combination therapy involving AP-EVs and anti-PD-1 yields superior anticancer immunity against established tumors compared to anti-PD-1 monotherapy. This synergistic effect underscores the potential of our engineered EVs as a novel and highly effective strategy for cancer immunotherapy.

Citation Format: Xiabing Lyu, Tomoyoshi Yamano, Shota Imai, Toan Van Le, Rikinari Hanayama. Surface-engineered extracellular vesicles to modulate antigen-specific T cell expansion for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7251.
Abstract 7259: Effectiveness and comparison of CAR-T cell therapies for synovial sarcoma; HER2-targeted CAR-T and NKG2D-based CAR-T
Authors: Miyazaki Tomohiro , Imai Chihaya , Oike Naoki , Murayama Yudai , Ariizumi Takashi , Baba Minori , Kasahara Yasushi , Kawashima Hiroyuki
Published: March 2024

Introduction: In Japan since 2019, CAR-T cell therapy has been used to treat patients with relapsed or refractory B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma and has shown excellent clinical results. Clinical studies of CAR-T cell therapies targeting various antigens, such as HER2, have been conducted, but the therapeutic outcome in sarcoma is still limited. We have previously reported that HER2-targeted CAR-T cells show antitumor effects for synovial sarcoma in vitro. The purpose of this study is to compare HER2-targeted CAR-T cells and NKG2D-based CAR-T cells in antitumor effect and specific responses for synovial sarcoma in vitro.

Materials and Methods: Surface expressions of HER2 and NKG2D ligands on three synovial sarcoma cell lines were detected by using recombinant human HER2 Fc chimeric protein and that of NKG2D, followed by staining with PE-conjugated anti-human IgG secondary antibody. We generated 4-1BB-type second generation CAR-T cells targeting NKG2D ligands and examined their antitumor effects and specific responses against synovial sarcoma cells. In addition, we compared the differences between HER2-targeted CAR-T cells and NKG2D-based CAR-T cells by co-culturing with synovial sarcoma cells using short-term assay (WST-8 assay), long-term assay (Real-time Cell Analysis) and CD107a assay.

Results: HER2 and NKG2D ligands were found to be expressed by flow cytometry in three synovial sarcoma cell lines. In vitro, NKG2D-based CAR-T cells showed antitumor effects against synovial sarcoma cells, including degranulation and cytokine production. Furthermore, NKG2D-based CAR-T cells showed stronger antitumor effects than HER2-targeted CAR-T cells in both short- and long-term assays and expressed higher levels of CD107a.

Conclusions: In this study, NKG2D-based CAR-T cells showed more antitumor effects than HER2-targeted CAR-T cells in vitro. Although the reason for this is not clear, we hypothesize that NKG2D ligands may be induced in synovial sarcoma cells by co-culture with CAR-T cells because HER2 is a cancer-specific antigen, whereas NKG2D ligands are ligands for activating receptors of natural killer cells. Further studies, including in vivo studies, will be conducted to verify the difference in anti-tumor effects between these CAR-T cells.

Citation Format: Tomohiro Miyazaki, Chihaya Imai, Naoki Oike, Yudai Murayama, Takashi Ariizumi, Minori Baba, Yasushi Kasahara, Hiroyuki Kawashima. Effectiveness and comparison of CAR-T cell therapies for synovial sarcoma; HER2-targeted CAR-T and NKG2D-based CAR-T [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 7259.
Abstract 7265: ETS-005, a highly selective TEAD4 palmitoylation inhibitor with potent anti-tumor activity and brain penetrating capability
Authors: Lu Jiajun , Du Wenpei , Gao Ming , Fan Xiaofei , Li Jinping , Feng Lijian , Wang Mengya , Li Yingjie , Zhong Xiaofeng , Cui Wenqi , Zheng Qiangang , Zhu Jidong
Published: March 2024

The Hippo pathway is a highly conserved network that regulates cell proliferation, tissue regeneration, and organ size by orchestrating YAP/TAZ-TEADs, the core effectors of signaling. Aberrant activation of YAP/TAZ has been implicated in tumorigenesis, tumor progression, metastasis and the resistance to targeted therapeutics, including MAPK and RTK inhibitors. Among the four TEAD paralogs, TEAD4 is found widely overexpressed in various solid tumors and potentially less associated with YAP/TEAD-related renal toxicity. Here we reported the identification of ETS-005, a highly selective TEAD4 small molecule inhibitor that shows preference for binding to the palmitoylation pocket of TEAD4 than other TEAD paralogs over 15 folds. ETS-005 displayed low nanomolar inhibition of cell proliferation in NF2 or LATS1/2 mutant mesothelioma in vitro, and robust anti-tumor efficacy in vivo. ETS-005 showed favorable oral availability, high brain penetration capability and broad safety window. Moreover, ETS-005 had relative long projected T1/2 in human, supporting a more convenient clinical QD dosing schedule. Our study demonstrated that selective TEAD4 palmitoylation inhibitor ETS-005 exhibited potent anti-tumor activity as much as pan-TEAD palmitoylation inhibitors while may gain broader therapeutic index. Additionally, ETS-005 also exhibited profound synergistic effects in drug combination studies, offering broad opportunities combined with MAPK and RTK therapies. ETS-005 is currently in IND-enabling study and expected to enter Phase 1 in 2024.

Citation Format: Jiajun Lu, Wenpei Du, Ming Gao, Xiaofei Fan, Jinping Li, Lijian Feng, Mengya Wang, Yingjie Li, Xiaofeng Zhong, Wenqi Cui, Qiangang Zheng, Jidong Zhu. ETS-005, a highly selective TEAD4 palmitoylation inhibitor with potent anti-tumor activity and brain penetrating capability [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 7265.
Abstract 7351: Delineating the prostate cancer tumor microenvironment through scRNA-seq profiling at different timepoints
Authors: Valencia Itzel , Pakula Hubert , Omar Mohamed , Loda Massimo , Marchionni Luigi
Published: March 2024

Distant metastasis remains the leading cause of death in patients with prostate cancer (PCa), however, the underlying mechanisms of this process remain unclear. Using samples from genetically-engineered mouse models (GEMMs) of PCa, we have identified eight different stromal cell states linked to epithelial mutations that may impact PCa progression to metastatic stages. To further investigate the unique intercellular interactions in the tumor microenvironment (TME) involved in mediating disease progression, we use single cell RNA sequencing (scRNA-seq) to profile primary PCa samples from different GEMMs including TRAMP, Hi-MYC, and FVBN, at different timepoints. Analysis of the scRNA-seq data revealed a significant enrichment of three clusters, designated as stromal c6 (s6), epithelial c7 (e7), and epithelial c10 (e10), exclusively in TRAMP, each expressing proliferative markers such as Mki67 and Top2a. To investigate the intercellular interactions among these clusters, we conducted ligand-receptor (LR) interaction analysis using CellChat. Notably, s6 is expressing the genes encoding ligands Bmp4, Fn1, and Igf1, which interact with the receptors Bmpr1b/Bmpr2, Itga3/Itgb1, and Igf1r, respectively expressed in e7. Subsequently, the transcriptomic profiles of these cells were used to build and validate a robust gene signature that can predict metastasis and survival in patients with PCa using a large cohort of 1239 samples, 930 of which were used for training while the remaining 309 samples were used for testing the model’s performance. In conclusion, these results underscore the role of the TME mediating PCa progression and highlight the potential of leveraging the TME to extract predictive and prognostic features which can be later employed to build predictive and prognostic models for patients with PCa.

Citation Format: Itzel Valencia, Hubert Pakula, Mohamed Omar, Massimo Loda, Luigi Marchionni. Delineating the prostate cancer tumor microenvironment through scRNA-seq profiling at different timepoints [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 7351.
Abstract 7358: Role of noncoding genome architecture in esophageal cancer
Authors: Erkizan Hayriye Verda , Wadleigh Robert
Published: March 2024

Esophageal Cancer is a deadly disease with a tendency to be diagnosed at the later stages. Considered a rare disease, accounting for 1.1% of all cancers, EC comprises 2.6% of all cancer deaths. The survival rate for all stages is 21.7%. However, the survival rate decreases to 5.6 % for advanced-stage cancers. EC is more common in men and associated with older age and heavy alcohol and tobacco use. In the last 25 years, the death rate of EC has been decreasing slightly from 4.3 to 3.7. The death rates for lung, prostate, and colon cancers have decreased by half in the same time frame. Therefore, novel therapies and biomarkers are urgently needed to accelerate survival and reduce the death rates by at least half for patients with EC.We previously completed exome sequencing in a small sample size and observed that the mutations in the noncoding regions of the EC genome are highly affected. In this study, we perform exome sequencing in 110 paired esophageal tumors and normal tissues using an Agilent SSELXT HS Exon V8+UTR library enrichment kit. Twelve paired EC and normal tissues are sequenced using the PacBio Hifi system.In WES, we detected almost half of the noncoding region mutations in the 3UTR and 2kb downstream regions of the genome, significantly affecting 51 oncogenic or tumor suppressor genes. In addition, the involved genes are over-represented in critical signaling pathways and transcriptional regulation of pluripotent stem cells. The number of changes in the 5UTR and 2kb upstream are similar to the number of variations in the intronic regions. Apoptosis, stress response, and DNA double-strand break repair pathways are affected by 5UTR mutations. Mutations in the intronic regions frequently occurred in pro-tumorigenic immune regulation genes, such as IL-33 and IL-6, and oncogene-induced senescence.The combined effect of these genetic and coding region mutations on oncogenes, significant signaling pathways, cancer cell stemness, DNA damage repair pathways, and immune system regulation may significantly impact poor prognosis in EC patients. Further studies will verify the effectiveness of targeting these genes for developing candidate therapeutics.

Citation Format: Hayriye Verda Erkizan, Robert Wadleigh. Role of noncoding genome architecture in esophageal 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 7358.
Abstract 7385: Explainable AI model incorporating uncertainty estimation for enhanced MSI-H prediction and immunotherapy response in gastrointestinal cancer
Authors: Park Sunho , Kim Minj , Sung Ji-Youn , Park Jeong Hwan , Lee Sung Hak , Wang Sam C. , Cheong Jaeho , Hwang Tae Hyun
Published: March 2024

Background: Microsatellite instability-high (MSI-H) tumors respond well to immune checkpoint blockade and oftentimes not to chemotherapeutics. Despite value of determining MSI status to guide therapy, many patients’ samples are not untested. Deep learning models may be able to predict MSI status by analyzing hematoxylin and eosin (H&E) stained whole-slide images (WSIs). Validation of this tool will provide another tool to increase MSI testing. However, to be deployed into clinical routine care, prediction models need be externally validated in large and diverse patient cohorts. In addition, these models should provide uncertainty of prediction to help clinicians make informed decisions.

Results: In this study, we introduce a novel Bayesian prediction model using whole-slide images (WSIs) and deep Gaussian processes (DGPs) in a weakly supervised learning context. Extensively tested on large datasets (n=3,146) from multiple centers, our model not only demonstrated superior performance but also showed that incorporating uncertainty into predictions significantly improves MSI prediction accuracy, achieving AUROC of >0.91 in colon cancer and >0.94 in gastric cancer. Additionally, an in-depth analysis within the gastric cancer cohort indicated a significant correlation between predicted MSI-H regions and immunotherapy response. Patients who responded to immune checkpoint inhibitors had a higher proportion of MSI-H regions (P-value < 0.05). This correlation was also observed in patients categorized as MSS but responsive to ICIs, highlighting the potential of our model to identify MSI-H patterns not detected through standard laboratory tests. This advancement could lead to more precise patient selection for ICI therapy, enhancing personalized treatment strategies in gastrointestinal cancer.

Citation Format: Sunho Park, Minj Kim, Ji-Youn Sung, Jeong Hwan Park, Sung Hak Lee, Sam C. Wang, Jaeho Cheong, Tae Hyun Hwang. Explainable AI model incorporating uncertainty estimation for enhanced MSI-H prediction and immunotherapy response in gastrointestinal 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 7385.
Abstract 7473: Flotillin-2 ablation in T cells enhances antigen sensitivity and functionality
Authors: Moon Sookjin , Karmaus Peer W. , Fessler Michael B.
Published: March 2024

The membrane scaffolding protein flotillin-2 (Flot2) has been proposed to support receptor signaling, but its involvement in T cell receptor (TCR) signaling and T cell responses remains unclear. Here, we show that the deletion of Flot2 increases antigen sensitivity along with changes in surface TCR clustering, resulting in enhanced TCR signaling and effector function to weak TCR stimulation. T cell-specific Flot2-deficient mice showed enhanced proliferation and effector cytokine production in both CD4+ and CD8+ T cells, resulting in better control of tumor growth and infection. However, in vitro TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 showed increased TCR signaling and early activation of CD4+, but not CD8+ Flot2-deficient T cells upon weak stimulation. In addition, Flot2-deficient CD4+ T cells showed better in vitro polarization toward T helper 1 cells upon weak TCR stimulation, likely due to increased sensitivity of the TCR-triggering threshold. Changes in TCR clustering on the membrane have been suggested to influence the early detection of foreign stimuli and the formation of immune synapses. Flot2 ablation indeed increased the number of membrane TCR nanoclusters on naïve CD4+ T cells. Collectively, this study suggests a mechanism for increasing T cell functionality to weak TCR stimulation by modulating the membrane protein Flot2, which affects surface TCR clustering. These results may have important implications for improving T cell reactivity in diseases with poor antigenicity, such as anti-tumor immune responses.

Citation Format: Sookjin Moon, Peer W. Karmaus, Michael B. Fessler. Flotillin-2 ablation in T cells enhances antigen sensitivity and functionality [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 7473.
Abstract 7514: Tumor intrinsic CDC42BPB confers resistance to anti-PD-1 immune checkpoint blockade in breast cancer
Authors: Deshpande Ravindra Pramod , Wu Kerui , Wu Shih-Ying , Tyagi Abhishek , Hunting John , Ruiz Jimmy , Li Wencheng , Watabe Kounosuke
Published: March 2024

Immune checkpoint blockade has been used to treat breast cancer, but the clinical responses remain relatively poor, especially for the triple-negative subtype. The weak responsiveness is attributed to tumor intrinsic as well as tumor extrinsic factors, suggesting a need for novel approaches. We have used the CRISPR Cas9 kinome knockout library consisting of 763 kinase genes to identify tumor intrinsic kinase conferring resistance to anti-PD-1 immune checkpoint blockade and identified the CDC42BPB kinase as a potential target to overcome the resistance to anti-PD-1 immunotherapy. We found that CDC42BPB is highly expressed in breast cancer patients who are non-responsive to immunotherapy. Importantly, a small molecule pharmacological inhibitor, BDP5290, which targets CDC42BPB synergized with anti-PD-1 and enhanced tumor cell killing by promoting T-cell proliferation in both in vitro and in vivo assays. Moreover, anti-PD-1 resistant breast cancer cells showed higher expression of CDC42BPB, and its inhibition rendered the resistant cells more susceptible to T cell killing in the presence of anti-PD-1. We also found that CDC42BPB phosphorylated AURKA which in turn upregulated PD-L1 through cMYC. Our results have revealed a robust link between tumor intrinsic kinase and immunotherapy resistance and provided a rationale for a novel combination therapy of CDC42BPB inhibition and anti-PD-1 immunotherapy for breast cancer.

Citation Format: Ravindra Pramod Deshpande, Kerui Wu, Shih-Ying Wu, Abhishek Tyagi, John Hunting, Jimmy Ruiz, Wencheng Li, Kounosuke Watabe. Tumor intrinsic CDC42BPB confers resistance to anti-PD-1 immune checkpoint blockade in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7514.
Abstract 7569: Characterizing pathways for metastasis of retinoblastoma in forebrain organoids
Authors: Shtanko Yulia , Rodriguez Sara , Schachner Benjamin , Adis Emily , McLaughlin Samantha , Borges Sebastian , Dollar James , Zuniga Ashley , Quintana Gabriela , Lopez Sofia , Owens Dawn , Moulin Chloe , Braun Steffanie , Yenisehirli Gulum , Pelaez Daniel , Harbour J. William , Kurtenbach Stefan
Published: March 2024

Retinoblastoma (Rb) is a childhood cancer of the eye, which usually presents in children under the age of five. When detected early, Rb has favorable prognosis in developed countries, but metastasis into the central nervous system (CNS) through the optic nerve correlate with a high mortality rate. Even if detected early, the available harsh and unspecific treatments can cause serious side effects, and enucleations are still performed frequently. There is still uncertainty regarding the mechanism for metastasis into the central nervous system. We performed bioinformatic analysis of single cell sequencing data for retinoblastoma, retinal tissues, optic nerve, and brain, to identify potential pathways and cell-cell communications fostering Rb invasion into the optic nerve and brain. Our analysis revealed several potential metastatic drivers, including immune modulation involving MIF, secreted by the Rb cells, as well as cell-adhesion pathways promoting a change in cellular phenotype. Given that animal models do not faithfully recapitulate human Rb initiation and progression, we developed a novel human iPSC-derived forebrain organoid model to investigate mechanisms of Rb CNS metastasis. Microinjections of GFP-labelled Rb cells allow to track growth and spread. In this fully human model including microglial cells, we can, for the first time, study the Rb forebrain interactions and pathways involved. RNA-seq analysis of Rb cells growing in forebrain organoids shows increased activity in neuron projection development and neuron migration, overlapping with the observed phenotype switch of invading Rb. Together, we present a novel iPSC-derived human forebrain model to monitor growth and spread of metastatic Rb, and describe new pathways involved in invading and metastasizing Rb.

Citation Format: Yulia Shtanko, Sara Rodriguez, Benjamin Schachner, Emily Adis, Samantha McLaughlin, Sebastian Borges, James Dollar, Ashley Zuniga, Gabriela Quintana, Sofia Lopez, Dawn Owens, Chloe Moulin, Steffanie Braun, Gulum Yenisehirli, Daniel Pelaez, J. William Harbour, Stefan Kurtenbach. Characterizing pathways for metastasis of retinoblastoma in forebrain organoids [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 7569.
Abstract 7611: Circulating blood RNAseq detects activation of immune system and predicts response to immunotherapy in bladder cancer
Authors: Richard Corentin , Chevrier Sandy , Marabelle Aurélien , Boidot Romain
Published: March 2024

Background: Anti-PD(L)1 immunotherapies have revolutionized oncology by getting durable tumor responses in advanced cancers. Nevertheless, currently approved biomarkers (PD-L1, MSI, TMB) have suboptimal positive/negative predictive values for tumor responses and survival.

Aims & Methods: We sought to assess the value of gene expression on circulating blood to predict responses to immunotherapy. We performed total paired-end RNAseq at 20-million reads and analyzed differential gene expressions according to cancer outcomes on baseline frozen whole blood from a cohort of 186 patients prospectively enrolled in the IOPREDI study, a French ancillary cohort of the international multi-center STRONG Phase III trial testing the anti-PD-L1 durvalumab in advanced bladder cancers (NCT03084471).

Results: One hundred and sixty four out of the 186 baseline samples generated valuable blood transcriptome data. Thanks to mathematical corrections, all samples were normalized, and the analysis was possible without introducing any bias despite the sample origin from 16 enrolling sites. We first observed that patients with a complete or partial response or a stable disease overexpressed 19 genes, 5 of which are markers of immune cell activation: CXCL8, EOMES, GBP5, GZMA, and GZMH. Conversely, patients with a progressive disease overexpressed 11 genes including CD177, a marker of neutrophil cells, recently associated to immunotherapy resistance. Next, patients with a PFS > 3 months showed an overexpression of 25 genes associated with immune cells activation such as CD8A, CXCL8, EOMES, FASLG, GZM genes, whereas patients with a shorter PFS highly expressed genes of immune cell inhibition: CD177, CD300LG, CD99, and CST7. Finally, patients with an OS > 7 months had also a higher expression of the same previous genes completed with 6 other genes, still indicators of immunity activation (CD3D, CD3E, CD3G, GZMM, IL2RB, and PTGDS).

Conclusion: Total RNAseq from RNA directly extracted from whole blood can detect baseline activation of T-cells in patients who will subsequently benefit from anti-PD-L1 immunotherapy. This work shows that RNAseq can detect an immune activation state in circulating blood without specific cell selection. This technique could be used in clinical trials to select for patients prone to benefit from cancer immunotherapies.

Citation Format: Corentin Richard, Sandy Chevrier, Aurélien Marabelle, Romain Boidot. Circulating blood RNAseq detects activation of immune system and predicts response to immunotherapy in bladder 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 7611.