bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3863: Flow cytometry using stabilized whole blood to assess innate and adaptive immune cells (Monocytes, NK, T, and B cells) in immunotherapies
Authors: Benko Jackie , Splitt Samantha , Bisconte Angelina , Lozza Laura , Owen Rachel , Phippard Deborah 
Published: March 2024

Abstract
Flow cytometry assays using fresh whole blood which are interrogating either an extremely rare cell subset or a depleting immunotherapy, may require samples to be processed from global clinical sites over a wide geographical area, which can pose a major challenge for clinical operations. Precision for Medicine (PfM) has addressed these operational challenges using commercial stabilizers which are added to the blood allowing samples to be frozen at -80°C and subsequently batch processed for sample testing. We have performed a fit for purpose assay qualification of a multiparameter flow cytometry panel allowing the immunophenotypic analysis of monocyte, T cell, B cell and NK immune populations using Cytodelics™ fixed/stabilized healthy donor whole blood. Additionally, the assay was verified in whole blood samples from multiple solid tumor disease states including melanoma, colorectal cancer, head & neck, non-small cell lung cancer to demonstrate clinical application. In these oncology samples, we were able to assess both immune cell frequencies and their activation status in both fresh whole blood as well as fixed whole blood over a short-term sample stability study, where the samples had been stored at -80°C for several months. This data provides a representative example of the use of commercial stabilizing fixatives for clinical studies to extend the stability window of whole blood for subsequent assessment by flow cytometry. Fixed whole blood can be stored at -80°C, shipped on dry ice allowing multiple timepoints in a cohort to be assessed together, thus minimizing assay variability as compared to fresh whole blood analysis, and mitigating the impact of shipping delays and temperature excursions on the samples. With sufficient time for assay development there are several commercial stabilizers that can be tested to determine if the markers in the flow assay are compatible with these reagents, providing a valuable option to assess the impact of immunotherapies on the immune cells of interest and reducing operational challenges in global studies.
Citation Format: Jackie Benko, Samantha Splitt, Angelina Bisconte, Laura Lozza, Rachel Owen, Deborah Phippard. Flow cytometry using stabilized whole blood to assess innate and adaptive immune cells (Monocytes, NK, T, and B cells) in immunotherapies [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 3863.
Abstract 395: An alternating magnetic field suppresses tumor growth
Authors: Umemura Masanari , Akimoto Taisuke , Islam Rafikul , Kishi Kazuhito , Nagasako Akane , Nakakaji Rina , Yamaguchi Takashi , Mizuno Yuto , Ishikawa Soichiro , Yamamoto Tetsuya , Ishikawa Yoshihiro 
Published: March 2024

Abstract
Application of physical forces, ranging from ultrasound to electric field, is recommended in various clinical practice guidelines, including those for bone fracture or cancer treatment. However, mechanistic information for such treatments has been poorly provided, due to the lack of comprehensive study model. Here, we show an alternating magnetic field (AMF) per se exhibits direct anti-cancer effect through enhancing oxidative phosphorylation (OXPOHS) and thus metabolic reprograming. Proliferation of human glioblastoma cells (U87 and LN229), but not normal human astrocytes (NHA), was inhibited upon exposure to AMF within the narrow frequency range. Extracellular acidification rate was decreased while oxygen consumption was increased. These data indicated that AMF induced metabolic reprograming. This was accompanied by increased reactive oxygen species (ROS) production. Mouse models grafted with human glioblastoma cells showed that AMF exposure, 30 minutes per day for two weeks, suppressed the tumor growth and prolonged the overall survival. Our results suggest that the AMF exposure may be a simple strategy to inhibit cancer cell growth by utilizing oxidative stress via metabolic reprograming.
Citation Format: Masanari Umemura, Taisuke Akimoto, Rafikul Islam, Kazuhito Kishi, Akane Nagasako, Rina Nakakaji, Takashi Yamaguchi, Yuto Mizuno, Soichiro Ishikawa, Tetsuya Yamamoto, Yoshihiro Ishikawa. An alternating magnetic field suppresses tumor growth [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 395.
Abstract 3951: Timing the development of chemoresistance in relapsed pediatric cancer
Authors: Blay Sasha , Layeghifard Mehdi , Davidson Scott , Chen David , Schwertschkow Astra , Ramaswamy Vijay , Taylor Michael , Papaemmanuil Elli , Villani Anita , Malkin David , Alexandrov Ludmil , Cowley Mark , Shlien Adam 
Published: March 2024

Abstract
Survivors of pediatric cancer face lifelong battles with severe morbidities, which includes a significant risk of recurrence. Pre-existing genetic variation within primary tumors may offer certain cell populations an evolutionary advantage, increasing the likelihood that some will be resistant to treatment. But the inherent complexity of the tumor means finding these markers of resistance has yet to be fully explored across childhood cancer. To address this, we developed a pipeline that combines clonal evolution reconstruction with mutational signature extraction at the subclonal level to elucidate changes in mutational processes. Using this pipeline to analyze 1,743 pediatric tumor genomes we found a common pattern of parallel evolution in most pediatric cancers, irrespective of their tissue of origin. Further, we detected therapy-associated mutational signatures linked to specific treatments such as temozolomide, platinum, fluorouracil, and thiopurine in post-treatment tumor subpopulations. Of 235 samples with known treatment exposure, 37.9% displayed one or more treatment-associated signatures. In the context of the phylogenetic tree, we identified specific subpopulations with therapy signatures. In multi-sample cases, resistant subpopulations had ancestors present in primary tumors at the time of diagnosis, suggesting certain subpopulations possessed the ability to withstand chemotherapy-induced pressures from an early stage and then expanded following treatment. Finally, we observed differences in the timing of mutational signatures between antimetabolite and alkylating agent treatments, reflecting the mechanisms of these drugs. Antimetabolite agents showed subclonal mutational signatures occurring in later branches, likely due to dependence on cell cycling. In contrast, alkylating agents displayed signatures in both early and late subpopulations, as they are unaffected by cell cycle state. Thus, investigating mutational signatures at the subclonal level unveils new insights into the clonal dynamics of pediatric cancers and the development of chemoresistance. This research offers valuable insights for identifying pediatric cancer patients at greater risk of recurrence, enabling the development of more effective treatment plans to enhance their quality of life and overall survival rates.
Citation Format: Sasha Blay, Mehdi Layeghifard, Scott Davidson, David Chen, Astra Schwertschkow, Vijay Ramaswamy, Michael Taylor, Elli Papaemmanuil, Anita Villani, David Malkin, Ludmil Alexandrov, Mark Cowley, Adam Shlien. Timing the development of chemoresistance in relapsed pediatric 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 3951.
Abstract 3967: ISM9182A, a novel HPK1 inhibitor, exhibits immune modulatory activity and robust monotherapy anti-tumor effects in preclinical studies
Authors: Chen Celia X.-J. , Pu Congying , Peng Jingjing , Ding Xiaoyu , Lu Hongfu , Ding Xiao , Zhang Man , Bavadekar Supriya , Rao Sujata , Ren Feng , Zhavoronkov Alex 
Published: March 2024

Abstract
The hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a member of the Ste20 serine/threonine kinase super family that negatively regulates immune cells, including T cells, B cells and dendritic cells (DCs). Thus HPK1 is an attractive immuno-oncology target in many tumor types. Here, we report ISM9182A as a novel, potent, and selective inhibitor of HPK1.ISM9182A demonstrated nanomolar enzymatic potency against HPK1 with &gt;100-fold selectivity against other MAP4K family kinases. ISM9182A inhibited the phosphorylation of SLP-76 at Ser376 dose-dependently with an IC50 of 30nM in human peripheral blood mononuclear cells. Moreover, ISM9182A enhanced IL-2 and IFNγ secretion upon CD3/CD28 co-stimulation in a dose-dependent manner without affecting T cell proliferation. Treatment of human pan T cells with ISM9182A also significantly reversed PGE2-mediated immunosuppression of human pan T cell activation and further boosted IL-2 and IFNγ secretion compared to CD3/CD28 co-stimulation. In monocyte-derived DCs stimulated with TNF-α/IL-1β/PGE-2, ISM9182A significantly upregulated CD80, CD86, and CD83 expression, demonstrating its potency to promote DC maturation. ISM9182A showed no obvious toxicity towards human T cell, B cells, and monocytes without CD3/CD28 co-stimulation. In vivo PK data revealed favorable ADME properties and good oral bioavailability of ISM9182A across different species. In a CT-26 murine syngeneic mouse model, oral administration of ISM9182A exhibited potent antitumor effects with p-SLP76 inhibition in both murine spleen and tumor, without body weight loss. In summary, ISM9182A is a potent and selective HPK1 inhibitor and a promising immuno-oncology agent.
Citation Format: Celia X.-J. Chen, Congying Pu, Jingjing Peng, Xiaoyu Ding, Hongfu Lu, Xiao Ding, Man Zhang, Supriya Bavadekar, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM9182A, a novel HPK1 inhibitor, exhibits immune modulatory activity and robust monotherapy anti-tumor effects in preclinical studies [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 3967.
Abstract 3978: B-cell activating factor (BAFF) enhances antitumor immunity
Authors: Aziz Khaled , Munjal Kabeer , Howe Kathryn , Yarchoan Mark 
Published: March 2024

Abstract
Immunotherapies that modulate T cell function have been firmly established in cancer immunotherapy, whereas the potential for B cells in the antitumor immune response is not as well understood. B cell-activating factor (BAFF) is a cytokine belonging to the TNF ligand family that activates B cells and is linked to autoimmunity. Our preliminary research has identified BAFF as a target for enhancing antitumor immunity through enhancing presentation of tumor neoantigens and inducing tertiary lymphoid structures which can orchestrate tumor-specific T cell immune responses and correlate with improved clinical response. Since broad activation of B cell response could be detrimental and possibly induce autoimmune side effects, identification of specific B cell receptors that coordinate anti-tumor immunity is prudent. BAFF has three known receptors (BAFF-R, TACI and BCMA), and the effects of these specific BAFF ligand-receptor interactions on antitumor immunity are not known. Here we first generated BAFF-overexpressing cell lines using genomic editing [KPC-BAFF (pancreas), B16F10-BAFF (melanoma), and PancO2-BAFF (pancreas)] and examined their tumor growth in syngeneic immunocompetent host. Secondly, we investigated the effects of BAFF-overexpression on tumor response when individual receptors are knocked out using BAFF-R-KO, TACI-KO and BCMA-KO mice. Our results show significantly hindered tumor growth when the BAFF expressing stable cell lines are introduced to immunocompetent host, compared to parental cell lines. Tumor response experiments in receptor knockout mice is currently in progress. Successful identification of the BAFF receptor candidate that drives anti-tumor response is an exciting prospect for clinical translation. Development of agonists for the specific receptor may allow selective modulation of this particular B cell function in context of cancer therapy without perturbation of broad systemic humoral immunity.
Citation Format: Khaled Aziz, Kabeer Munjal, Kathryn Howe, Mark Yarchoan. B-cell activating factor (BAFF) enhances antitumor immunity [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 3978.
Abstract 4004: Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma
Authors: Yuan Stephen Hsien-Chi , Wang Yufei , Abbas Rabia , Mathenge Peterson , Chien Charles , Coherd Christian D. , Bajoua Branson , Chang Matthew R. , Murugan Nithyassree , Kastrunes Gabriella M. , Wang Guilin , Hoang Song-My , Marasco Wayne A. 
Published: March 2024

Abstract
On-target off-tumor toxicity (OTOT) is one of the major hurdles preventing tumor-associated antigen (TAA) targeted therapy from effectively treating patients with solid tumors due to the shared presence of the same TAA on normal tissues. We have previously demonstrated high expression of carbonic anhydrase IX (CAIX) in ccRCC patient samples and low-density expression on healthy bile duct epithelium. In 2006, a clinical trial was stopped due to OTOT bile duct toxicity observed in patients infused with anti-CAIX CAR T cell therapy. To address the issue of OTOT in the bile duct, we performed affinity/avidity fine-tuning of the anti-CAIX CAR G36 to specifically target ccRCC tumor cells with high CAIX expression while sparing cholangiocytes with low CAIX expression. We generated a complementarity-determining region (CDR) single variant mutagenesis library of G36 and displayed the library on yeast for screening. Using flow cytometry, we were able to identify six populations based on antigen-binding affinity: high, med-high, med, med-low, low, and loss of binding. These libraries were sequenced via PacBio, a next generation sequencing (NGS) platform that excels in long-read sequencing, providing a powerful tool to detect single mutations in the scFv. The results were analyzed to identify enriched mutations compared to the wild-type G36, and inspected using in silico modeling and docking. Furthermore, enriched mutants from med-low and low populations were tested in CAR-T format for their cytotoxic capacity on tumor cells and bile duct cholangiocytes, as well as their avidity. We demonstrate that mutations in CDR2 and CDR3 of the heavy chain successfully mitigate OTOT toxicity on cholangiocytes with physiological levels of CAIX expression while maintaining tumor-killing capacity, shedding light on CDR mutagenesis as a useful tool to mitigate OTOT toxicity.
Citation Format: Stephen Hsien-Chi Yuan, Yufei Wang, Rabia Abbas, Peterson Mathenge, Charles Chien, Christian D. Coherd, Branson Bajoua, Matthew R. Chang, Nithyassree Murugan, Gabriella M. Kastrunes, Guilin Wang, Song-My Hoang, Wayne A. Marasco. Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4004.
Abstract 4006: IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells
Authors: Mohan Aditya , Shen Steven , Hotchkiss Kelly , Quackenbush Sarah , Sampson John , Fecci Peter , Patel Anoop 
Published: March 2024

Abstract
Introduction: EGFRvIII is a commonly studied tumor-specific antigen in glioblastoma (GBM) and poses an enticing target for immunotherapy. Its varied expression, however, complicates its effectiveness as a Chimeric Antigen Receptor (CAR) T cell target. Given the increasing prominence of Interleukin-12 (IL12) in treating solid tumors, its synergy with EGFRvIII specific CARs in treating preclinical models of GBM is explored.
Methods: To simulate tumor heterogeneity, mice were implanted with equal ratios of CT2A and CT2A EGFRvIII+ cells. We developed EGFRvIII-specific third-generation CAR T cells, both with and without IL12 using a MSGV retrovirus backbone and transduced them into murine T cells. Their antitumor effectiveness was gauged by evaluating survival rates, and their capability to enhance and modify the tumor environment was determined using single-cell sequencing.
Results: Standalone EGFRvIII CARs were unsuccessful in curing mice with heterogeneous tumors. However, 60% of mice treated with EGFRvIII CARs combined with IL12 were long term survivors. IL12 secreting CARs exhibited prolonged persistence in vivo, and there was no need for prior lymphodepletion before ALT introduction. Our studies reveal that EGFRvIII CARs secreting IL12 can eliminate EGFRvIII negative tumor cells by drawing on the inherent CD8 T cell compartment. Moreover, IL12 prominently impacted microglia among all Antigen Presenting Cells in the tumor environment, amplifying antigen presentation processes and broadening epitope exposure.
Conclusion: By leveraging IL12, EGFRvIII CARs can counteract antigen heterogeneity in tumors by recruiting endogenous CD8 T cells and polarizing microglia. These IL12-secreting CARs outlast their traditional counterparts without requiring lymphodepletion. Harnessing CAR-T cells that secrete IL12 represents a novel strategy for overcoming tumor heterogeneity and treating GBM in a clinical context.
Citation Format: Aditya Mohan, Steven Shen, Kelly Hotchkiss, Sarah Quackenbush, John Sampson, Peter Fecci, Anoop Patel. IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4006.
Abstract 4013: Increasing mitochondrial respiration by silencing MCJ/DnaJC15 enhances CD8 CAR-T cell therapy efficacy
Authors: Wu Meng-Han , Giddings Emily L. , Valenca-Pereira Felipe , Pham-Danis Catherine , Cendali Francesca , Yarnell Michael C. , Novak Amanda J. , Henao-Mejia Jorge , Flavell Richard A. , D'Alessandro Angelo , Kohler Mark Eric , Rincon Mercedes 
Published: March 2024

Abstract
Chimeric antigen receptor (CAR)-T cell therapy is a promising treatment against leukemia and lymphoma. However, poor initial treatment response and post-therapy relapse have found to be common and remained to be resolved. Modulating T cell immunometabolism is considered a novel therapeutic approach to enhance CAR-T cell therapy. We have identified MCJ (DnaJC15) as an endogenous negative regulator of Complex I of the electron transport chain in mitochondria. MCJ is abundantly expressed in mouse CD8 cells relative to other immune cells. We propose that MCJ can be a therapeutic target to enhance mitochondrial respiration in CD8 CAR-T cells. We will show that loss of MCJ in mouse CD8 cells leads to superior anti-tumor response against melanoma. In addition, we will show that MCJ-deficient murine CD8 CAR-T cells have enhanced killing activity against leukemia both in vitro and in vivo. We have recently revealed that MCJ is also expressed in human CD8 cells and the levels of MCJ are heterogenous among human population. Similar to murine CD8 cells, MCJ knockdown in human CD8 cells enhances mitochondrial membrane potential and effector functions. We have designed a new CAR construct to silence MCJ in human CD8 CAR-T cells. MCJ-deficient human CD8 cells show superior cytotoxicity against leukemia with increased mitochondrial metabolism. Together, these data demonstrate that MCJ is a promising target to improve CAR-T cell therapy through modulating the T cell metabolism. We are currently investigating the effect of MCJ-deficient CD8 CAR-T cells against solid malignancy.
Citation Format: Meng-Han Wu, Emily L. Giddings, Felipe Valenca-Pereira, Catherine Pham-Danis, Francesca Cendali, Michael C. Yarnell, Amanda J. Novak, Jorge Henao-Mejia, Richard A. Flavell, Angelo D'Alessandro, Mark Eric Kohler, Mercedes Rincon. Increasing mitochondrial respiration by silencing MCJ/DnaJC15 enhances CD8 CAR-T cell therapy efficacy [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 4013.
Abstract 4040: Hypoxia-induced HIF-1α/USP3 pathway promoting immune escape in lung squamous cell carcinoma
Authors: Zhang Xiuwen , Huang Hua , Ding Chen , Chen Chen , Li Yongwen , Liu Hongyu , Jun Chen 
Published: March 2024

Abstract
Background: Immune escape poses a significant mechanism for tumor progress as well as enormous challenge for the treatment of lung squamous cell carcinoma (LUSC). The close association between hypoxic environments and tumor immune escape, with PD-L1 serving as a key marker, has garnered considerable attention. This study aims to delve into the molecular mechanisms underlying the regulation of PD-L1 deubiquitination by the hypoxia-induced HIF-1α/USP3 pathway, shedding light on the immune escape mechanisms in LUSC.
Methods: NSCLC cells were cultured under hypoxia condition and the expression of USP3, HIF-1α and PD-L1 were measured by Western blot. Chromatin Immunoprecipitation (ChIP) and Co-immunoprecipitation (CO-IP) were employed to elucidate the direct regulatory mechanisms of the HIF-1α/USP3 pathway in PD-L1 deubiquitination. In vitro T cell-mediated killing assays was used to investigate the functional roles of USP3 in antitumor immunity in NSCLC.
Results: We demonstrated that hypoxia score was increased in LUSC and negatively correlated with CD8+ T cell infiltration. Hypoxia as a major factor triggering LUSC cell immunosuppression and against T cell surveillance via stabilization of PD-L1. Mechanistically, USP3, induced by HIF-1α, is required for hypoxia-mediated PD-L1 stabilization in LUSC cells. USP3 inhibits the ubiquitination and degradation of PD-L1. The combination of USP3 inhibitors with PD-1/PD-L1 monoclonal antibodies significantly enhances the anti-LUSC effect, possibly through promoting the infiltration of CD8+ T and NK cells.
Conclusion: This research unveils a novel mechanism by which hypoxia, through the HIF-1α/USP3 pathway, regulates PD-L1 deubiquitination, thereby promoting immune escape in LUSC. Furthermore, our study provides a new strategy involving USP3 inhibitors in combination with PD-1/PD-L1 monoclonal antibodies, offering novel insights for molecular targeted therapy and reversal of drug resistance in LUSC.
Citation Format: Xiuwen Zhang, Hua Huang, Chen Ding, Chen Chen, Yongwen Li, Hongyu Liu, Chen Jun. Hypoxia-induced HIF-1α/USP3 pathway promoting immune escape in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4040.