bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6302: Home-based supportive care programs for quality-of-life outcomes among patients with advanced cancer: A systematic review
Authors: Hwang Inyoung , Wo Go-Un , Lee Sun-Young , Yoo Shin Hye , Kim Kyae Hyung , Kim Min-Sun , Shin Jeongmi , Jeong Hyejin , Jang Min Seol , Baek Sun Kyung , Jung Eun Hee , Lee Dong-Wook , Cho Belong , Park Jin Ho , Kwon Hyuktae
Published: March 2024

Objectives This study systematically reviews the literature on the effect of home-based supportive care (HbSC) programs on the quality of life (QoL) of patients with advanced cancer.

Methods: The research question ‘Do home-based supportive care programs for patients with advanced cancer improve their QoL?’ was addressed. After registering the plan with PROSPERO (CRD42021235007), literature published from 1 January 1990 to 11 July 2022 was searched on Ovid Medline, Embase, Cochrane library, Web of Science, and Google Scholar and reviewed for inclusion based on predefined criteria. This review only included trial studies published in English.

Results: Of 5288 articles identified, 17 studies were judged suitable for inclusion in this review. The components of HbSC programs included home visits, patient and caregiver education, home nursing, psychotherapy, exercise, and telephone consultation. Eight programs included conducting multidisciplinary team meetings. Improvements in QoL were reported in six studies. Specific areas of QoL improved when interventions included home nursing, psychotherapy, patient and caregiver education, and exercise.

Conclusion: HbSC programs appear to enable the improvement of the QoL of patients with advanced cancer. The area of QoL that shows improvement could vary depending on the HbSC components. More studies that address HbSC programs are needed to select patients at the proper time and provide suitable programs for patients to benefit most.

Citation Format: Inyoung Hwang, Go-Un Wo, Sun-Young Lee, Shin Hye Yoo, Kyae Hyung Kim, Min-Sun Kim, Jeongmi Shin, Hyejin Jeong, Min Seol Jang, Sun Kyung Baek, Eun Hee Jung, Dong-Wook Lee, Belong Cho, Jin Ho Park, Hyuktae Kwon. Home-based supportive care programs for quality-of-life outcomes among patients with advanced cancer: A systematic review [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 6302.
Abstract 631: Functional genetics of APR-246 rescuable TP53 mutants
Authors: Saunders Anais , Tong Caili , Karnezis Anthony N. , Leiserowitz Gary S. , Chein Jeremy R.
Published: March 2024

TP53, often referred to as the guardian of the genome, encodes for the p53 tumor suppressor protein, and is one of the most frequently mutated genes in human cancers. Some cancer sub-types, such as non-small cell lung carcinoma (NSCLC) and high grade serous carcinoma (HGSC), have high TP53 mutation rates reaching up to 56% and 96%, respectively. Given its multi-faceted role in human cancers, there have been several emerging therapeutic approaches targeting mutant p53. APR-246 is a small molecule drug currently in clinical trials for the functional rescue of p53 missense mutants, the most common type of p53 mutant protein, across various cancer types. APR-246 is proposed to bind mutant p53 and promote thermodynamic stabilization of the protein allowing it to regain wild-type-like conformation and consequentially regain WT-like functionality. However, there has yet to be a functional genomic screening of p53 mutants to systematically catalog which mutants are rescuable by APR-246. To address this critical gap, we have performed functional genetic screenings using a TP53 mutagenesis library. This pooled lentiviral library contains ~8,000 amino acid variants, spanning the entirety of the TP53 gene. Following stable transduction of this library into p53-null NSCLC and HGSOC cell lines, we performed functional screenings against sublethal doses of APR-246. Next-generation sequencing of the untreated and treated cell population allows for comparison of variant representation and determination of APR-246-sensitivity of p53 mutants. Preliminary statistical analysis of our screens has identified a pool of 117 “hit” mutants which are significantly depleted following treatment, indicative of APR-246-sensitivity. Two of these mutants, C242F and G266V, are non-functional, damaging mutations which occur in patients. Identifying p53 mutants that can be rescued by APR-246 will help identify which patients may benefit most from treatment with the drug.

Citation Format: Anais Saunders, Caili Tong, Anthony N. Karnezis, Gary S. Leiserowitz, Jeremy R. Chein. Functional genetics of APR-246 rescuable TP53 mutants [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 631.
Abstract 6321: Developing a lenalidomide-inducible safety switch for CAR T cell therapy
Authors: Sarkar Ditsa , Lin William , Kim Joanna Y. , Knudsen Nelson H. , Lane Isabel C. , Kienka Tamina , Kann Michael C. , Bouffard Amanda A. , Cheng Andy , Maus Marcela V. , Manguso Robert T. , Jan Max
Published: March 2024

While CAR T cell therapy is a medical breakthrough against various hematological malignancies, yet treatment failures often occur at least in part due to excessive engineered T cell activity leading to severe toxicities. To combat this, strategies pairing CAR T cells with safety systems, for example, suicide switches engineered to induce cell death are being actively pursued. However, existing designs have limited performance characteristics and generally use unapproved small molecule controllers or non-human sequences. To overcome these issues, we have developed a novel suicide switch leveraging the well-tolerated, FDA-approved anti-cancer drug lenalidomide, as a controller of the stability of fusion proteins tagged with a “degron” sequence. Specifically, we engineered a stoichiometric pair of transgenes using the apoptotic DNase pair, caspase activating DNase (CAD) and its degron tagged inhibitor (ICAD-degron). We hypothesized that the overexpressed ICAD-degron could restrain CAD, until lenalidomide treatment degrades ICAD to unleash CAD for apoptosis. We found that our system was stably overexpressed and rapidly induced cell death even at sub-nanomolar concentrations of lenalidomide. A comparison of our design with the frontline safety switch, inducible Caspase 9 in primary human T cells revealed that the lenalidomide-induced suicide switch enabled more complete depletion of engineered cells. We tested the effects of our suicide switch on CAR T cell function and found comparable proliferation and tumor cell cytolysis with fast depletion upon lenalidomide addition. In vivo, lenalidomide suicide switch CAR T cells exhibited similar anti-tumor function as control CAR T cells and were rapidly depleted after drug treatment. In summary, utilizing all human sequences and a clinically approved drug controller, we developed a suicide switch that is well tolerated and can rapidly induce cell death even at sub-therapeutic concentrations of lenalidomide. More broadly, chemogenetic regulation of stoichiometric protein pairs is a generalizable strategy for post-translational control of highly active transgenic elements.

Citation Format: Ditsa Sarkar, William Lin, Joanna Y. Kim, Nelson H. Knudsen, Isabel C. Lane, Tamina Kienka, Michael C. Kann, Amanda A. Bouffard, Andy Cheng, Marcela V. Maus, Robert T. Manguso, Max Jan. Developing a lenalidomide-inducible safety switch for CAR T cell 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 6321.
Abstract 6351: MUC1*-ADCs are effective against heterogeneous solid tumor cancers
Authors: Bamdad Cynthia Carol , Smagghe Benoit S. , Moe Scott T. , Carter Mark G. , Grant Trevor J. , Yi Kevin R. , Nash Michael J. , Marquez Jacy P. , Miller Natalie K. , Nash Jac-Leen S. , Miller Dan S. , Walkley Danica M. , Stewart Andrew K.
Published: March 2024

Purpose: To develop a MUC1*-ADC for the treatment of over 75% of solid tumor cancers.

Methods: MUC1* (muk 1 star) is a growth factor receptor created after cleavage and release of the N-terminal portion of MUC1. The targeting antibody, huMNC2, has already demonstrated safety and cancer selectivity in a 1st-in-human CAR-T clinical trial for treatment of metastatic breast cancers. MNC2 is an IgG1 antibody that only recognizes the conformational epitope created when MUC1 is cleaved by specific tumor-associated enzymes. Confocal microscopy and pHlourin2 were employed to measure antibody internalization as a function of time. We conjugated MNC2 to MMAE, MMAF, Dxd and exatecan via several different linkers. MUC1*-ADCs with DARs ranging from 4-8 were tested in vitro and in vivo for efficacy against a panel of solid tumor cancers. Heterogenous MUC1* tumors were made by mixing different ratios of mCherry wild-type cancer cells with GFP MUC1* overexpressed cancer cells. Resulting H scores ranged from 10-280 (max 300).

Results: Cancer cells internalized the MUC1* MNC2 antibody within 2 hours. In vitro, MUC1*-ADC IC50s ranged from 1.3nM to 20.0nM, depending on the linker-payload and cancer subtype, wherein the majority had an IC50 of about 10nM. In general, efficacy in animals was seen across multiple cancer sub-types treated with multiple MUC1*-ADCs. Efficacy in animals xenografted with heterogeneous MUC1* tumors showed a dependence on antigen density. Greatest efficacy was observed for breast cancer xenografts with an H Score >10 treated with MNC2-Deruxtecan. However, only pancreatic tumors with higher H Scores were completely eliminated by MNC2-MMAE, MNC2-Deruxtecan and MNC2-exatecan. MNC2-ADC and MNC2-CAR share the same antibody. No off-tumor toxicity was observed in animals treated with MNC2-ADC, which mimicked lack of off-target toxicities for MNC2-CAR T cells in humans.

Conclusions: These data, combined with MNC2-CAR T data from our 1st-in-human trial for metastatic breast cancer, supports a conclusion that the MUC1* antibody MNC2 is safe and could have high therapeutic value as a MUC1*-ADC for multiple solid tumors with both high and low antigen density.

Citation Format: Cynthia Carol Bamdad, Benoit S. Smagghe, Scott T. Moe, Mark G. Carter, Trevor J. Grant, Kevin R. Yi, Michael J. Nash, Jacy P. Marquez, Natalie K. Miller, Jac-Leen S. Nash, Dan S. Miller, Danica M. Walkley, Andrew K. Stewart. MUC1*-ADCs are effective against heterogeneous solid tumor 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 6351.
Abstract 6424: A patient centric full spectrum flow cytometry assay for comprehensive phenotyping of CAR T cells in clinical trials
Authors: Vaughan Keegan , Muthusamy Selvam , Zhao Coral , Carbajal Austin , Fu Charlie , Dakappagari Naveen , Alfonso Zeni
Published: March 2024

Purpose While there is a strong desire to fully understand next generation CAR-T product characteristics in clinical trials, limited amounts of blood specimens are available as the enrolled patients are very sick and lymphodepleted. To address this high unmet need, we developed a single tube 20-color full spectrum flow cytometry assay for an in-depth characterization of CAR-T cells that support fulfilment of five key clinical trial endpoints, namely, manufacturing success (fitness and memory state), pharmacokinetics, response or resistance (ICIs), efficacy (gross tumor burden) and safety (B cell regeneration) for use across a broad range of CAR-T therapies and clinical indications.

Study Design: The single tube 20-parameter full spectrum flow cytometry (FC) assay was designed to include CD45, HLA-DR, CD3, CD4, CD8, CD56 as backbone markers, CD45RA, CCR7, CD95, CD25 and CD127 antibodies allowed for the identification of all major subsets of CD4 and CD8 T cells. CXCR3, CD38, PD1, LAG3, and TIM3 were used for identification of activation/exhaustion phenotypes, while CD20 and HLA-DR were useful for identification of B cells and monocytes respectively. Additionally, we left two parameters (PE and AF647) open for customized CAR-T detection reagents or other customized markers. This assay was developed using Cytek® Northern Lights™ and frozen PBMCs.

Results This robust 20-color full spectrum FC assay allowed a thorough analysis of the major T cell populations cells. FMO (Fluorescence-Minus-One) control studies confirmed the specificity of the antibodies and of the panel in identifying Naïve, EMRA, CM, EM, Th17, T stem cell memory and regulatory T cells with minimum background on all parameters. This method showed to be highly reproducible and can be adapted for use with many CAR-T detection reagents.

Conclusion In summary, we developed a robust 20-parameter multiparameter full spectrum flow cytometry assay focused on different populations of T cells in a single tube. The inclusion of drop ins for prospective CAR-T cell detection reagents allows for easy integration of future advances in CAR-T cell research. This universal panel shows great potential for usage across CAR-T clinical trials, as it serves as a valuable tool understand product fitness, PK, biomarker monitoring and disease burden simultaneously.

Citation Format: Keegan Vaughan, Selvam Muthusamy, Coral Zhao, Austin Carbajal, Charlie Fu, Naveen Dakappagari, Zeni Alfonso. A patient centric full spectrum flow cytometry assay for comprehensive phenotyping of CAR T cells in clinical trials [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 6424.
Abstract 6564: SOS1 inhibitor treatment improves response to Venetoclax in acute myeloid leukemia
Authors: Kostyrko Kaja , Hinkel Melanie , Rees Matthew G. , Ronan Melissa M. , Roth Jennifer A. , Gerlach Daniel , Waizenegger Irene
Published: March 2024

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. First-line treatment of AML patients consists primarily of chemotherapy, sometimes combined with a targeted therapy. However, elderly patients unfit for chemotherapy receive venetoclax, a BCL2 inhibitor, combined with azacitadine or decitabine. While responses in these patients are high (50-70%, with median OS of 10-17 months), around 30-50% of them do not respond and most relapse within 1-10 years. Increased MAPK pathway activity or low frequency RAS pathway mutations have been identified as key mediators of resistance to standard-of-care targeted therapies for AML, including venetoclax. Here, we found that in DepMap whole-genome CRISPR screens, leukemia cell lines, including AML, show a particular dependency on SOS1. A subsequent PRISM screen with a selective inhibitor of SOS1 confirmed these results. To validate these results in vitro, we treated 7 AML cell lines showing strongest dependency on SOS1 based on DepMap and PRISM screens, with SOS1i or a SHP2 inhibitor. We found that in 4 of 7 cell lines treatment with SOS1i or SHP2i strongly impaired proliferation. In 3 of 4 responding cell lines, the anti-proliferative effect with either SOS1i or SHP2i was stronger than azacytidine single agent treatment. We next treated 29 primary ex vivo cultures of AML with SOS1i and found that the treatment induced strong or intermediate anti-proliferative effect in 21 of 29 models (72%). In two AML PDX models, treatment with SOS1i as a single agent resulted in approximately 25% reduction of human CD45+ AML blast cells in both models. Venetoclax treatment led to approximately 75% decrease in AML blasts, while the combined venetoclax and SOS1i treatment resulted in near complete eradication of the disease. Annexin V staining of AML monoblasts demonstrated that this decrease was due to an increase in apoptosis in the combination groups. In summary, we demonstrate that SOS1 inhibitor treatment may be used to improve responses to venetoclax in AML patients.

Citation Format: Kaja Kostyrko, Melanie Hinkel, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Daniel Gerlach, Irene Waizenegger. SOS1 inhibitor treatment improves response to Venetoclax in acute myeloid leukemia [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 6564.
Abstract 6569: KLIPP: Precision targeting of cancer with CRISPR
Authors: Ljungman Mats , Yang Huibin , Hulbatte Radhika Suhas , Gratsch Natalie , Kelher Alan , Raupp John , Wang Yin , Dorsey Kristen Hong , Halseth Troy , Schwendeman Anna , Palmbos Philip
Published: March 2024

Current cancer therapeutic treatments are typically associated with significant collateral damage to normal tissues and the emergence of treatment resistance. Thus, there is an unmet need to develop therapies that are uniquely cancer specific and unhindered by the emergence of resistance. We have developed an RNA therapy approach based on CRISPR technology, known as “KLIPP”, that targets cancer-specific structural variant junctions (SVJs), a so far untapped cancer “Achilles heel”, that are highly abundant across cancer types. KLIPP uses a “split enzyme” system consisting of dCas9 conjugated to an endonuclease, Fok1, that requires dimerization for its activity. By designing sgRNAs that bind to sequences flanking the cancer-specific SVJs, Fok1-dCas9 dimers are formed, leading to the induction of DNA double strand breaks (DSBs) specifically at SVJs in cancer cells. We verify induction of DSBs by γH2AX foci staining in the nucleus of KLIPP-treated cells and resulting loss of clonogenic survival and induction of apoptosis, while cells lacking the targeted SVJs are unaffected. Importantly, we show elimination of orthotopic bladder cancer tumors in vivo after turning on expression of the KLIPP reagents by doxycycline in the drinking water. Finally, we show successful lipid nanoparticle delivery of KLIPP reagents to orthotopic bladder tumors in vivo using catheter-mediated delivery, leading to specific targeting of bladder cancer cells. Independently of the cancer-driving mutations, the omnipresence of cancer-specific SVJs is exploited by KLIPP to force cancer cells into apoptosis while leaving normal cells unperturbed. We believe that the successful implementation of precision KLIPP therapy could be transformative for the treatment of cancer patients.

Citation Format: Mats Ljungman, Huibin Yang, Radhika Suhas Hulbatte, Natalie Gratsch, Alan Kelher, John Raupp, Yin Wang, Kristen Hong Dorsey, Troy Halseth, Anna Schwendeman, Philip Palmbos. KLIPP: Precision targeting of cancer with CRISPR [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 6569.
Abstract 657: Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects
Authors: Kim Miyeon , Kim Dongsu , Jeon Yeejin , Yoon Kyeong Jin , Jang Anna , Lee Mijung , Jeon Dahye , Jang Soyeon , Kim Jinhwan , Kim Eunji , Park Jihoon , Hong Victor , Jung Hayoun , Choi Sungpil
Published: March 2024

KRAS is the most frequently dysregulated oncogene with high prevalence in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC). Currently, FDA-approved sotorasib and adagrasib provide ground breaking through therapies for cancer patients harboring KRAS G12C mutation. However, there is still high unmet medical needs for inhibitors targeting broader KRAS-driven tumor, especially a substantial unmet medical need for agents that can penetrate BBB to control the brain metastases. SHP2, a key activator of KRAS located in the upstream of RAS pathway, facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state serving as a key mechanism of KRAS activation. Therefore, SHP2 could be a promising therapeutic target to shut down the oncogenic RAS pathway as a broad combination partner with RAS/RAF/MEK/ERK pathway inhibitors. Here we report a highly potent and selective brain penetrant SHP2 inhibitor that is designed to target solid tumors metastazied to brain. KT-01766 showed excellent potency against SHP2 protein in various biochemical and cellular assays and showed the robust synergistic effect with RAS pathway inhibitors. KT-01766 as a mono therapy and in combination with KRAS G12C inhibitors demonstrated great anti-tumor efficacy with no apparent toxicity in mouse xenograft models harboring KRAS mutations. Furthermore, robust anti-tumor acitivty was confirmed in intracranial brain tumor model, supporting its potential to control brain-metastasized tumors. The present data suggest that KT-01766 is a potential candidate for combination therapy with agents targeting RAS pathway, as it is able to effectively suppress tumor growth both systemically and within the brain in KRAS mutated cancers.

Citation Format: Miyeon Kim, Dongsu Kim, Yeejin Jeon, Kyeong Jin Yoon, Anna Jang, Mijung Lee, Dahye Jeon, Soyeon Jang, Jinhwan Kim, Eunji Kim, Jihoon Park, Victor Hong, Hayoun Jung, Sungpil Choi. Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects [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 657.
Abstract 6600: Investigating molecular regulators of a dual identity state in lung adenocarcinoma
Authors: Fort Gabriela , Camolotto Soledad , Tariq Rushmeen , Fang Pengshu , Gillis Katherine L. , Stubben Chris , Snyder Eric L.
Published: March 2024

Lung adenocarcinoma (LUAD) is the largest subtype of lung cancer and is the most common cause of cancer related death. LUAD progression is driven by both genetic alterations and epigenetic changes that confer increased lineage plasticity and heterogeneity, but underlying mechanisms dictating cell identity in LUAD remain incompletely understood. The lineage defining transcription factors, NKX2-1 and FoxA1/2, coordinately maintain a pulmonary identity in LUAD. Recent work suggests that NKX2-1-positive (NKX2-1+) LUAD evolves from an alveolar-like state and adopts various disparate cell fates that drive progression, including “dual-identity” states, characterized by acquisition of gastrointestinal (GI) and pulmonary-like states in the same cell. Using sophisticated genetically engineered mouse models and 3D organoid cultures, we demonstrated that the gastrointestinal transcription factor HNF4α, which robustly marks the dual-identity state, is critical for growth in NKX2-1+ LUAD. We performed bulk RNA-seq and single cell RNA-seq in vitro and in vivo, and we found that loss of HNF4α alters the differentiation state of NKX2-1+ LUAD, causing cells to shed elements of their gastric identity in favor of alternate differentiation states. Using paired chromatin immunoprecipitation sequencing and RNA-seq in dual-identity organoids, we found that HNF4α can access and activate many of its canonical gastrointestinal target genes despite being aberrantly expressed in the lung, where it is typically epigenetically silenced early in development. Surprisingly, HNF4α also colocalizes with NKX2-1 and FoxA1/2 and binds at novel sites, suggesting that HNF4α acquires expanded activity in this state beyond its canonical roles. Together, our work unveils key insights into the mechanisms by which lineage defining transcription factors regulate cellular identity and growth in LUAD.

Citation Format: Gabriela Fort, Soledad Camolotto, Rushmeen Tariq, Pengshu Fang, Katherine L. Gillis, Chris Stubben, Eric L. Snyder. Investigating molecular regulators of a dual identity state in lung adenocarcinoma [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 6600.
Abstract 6621: Development of a melanocyte lineage-specific knockout system in zebrafish
Authors: Perlee Sarah , Ma Yilun , Swanson Jacob , Ming Zhitao , Xia Julia , Lionnet Timothée , McGrail Maura , White Richard
Published: March 2024

Zebrafish have emerged as an ideal model organism for investigating melanocyte and melanoma biology. While CRISPR-Cas9 technology has significantly advanced our understanding of gene functions in zebrafish, two challenges remain: 1) how do we knock out embryonic lethal genes in adult melanocytes? and 2) how do we knock out tumor promoting genes (rather than tumor suppressors)? To address this, we have developed a new transgenic line in which Cas9 is knocked into the melanocyte-specific endogenous mitfa locus. To test this system in normal melanocytes, Tg(mitfa:Cas9) embryos were injected with a plasmid containing albino sgRNA. Cells with integration of this plasmid exhibited almost complete loss of pigmentation, demonstrating the high efficiency of our approach. This system can now be rapidly used to knock out embryonic essential genes specifically in melanocytes and evaluate the phenotype of adult fish. In melanoma, the commonly available zebrafish lines overexpress the oncogene BRAFV600E in melanocytes with germline knockout of tp53. This is not comparable to the situation in humans, where global p53 loss is rare. By utilizing our mitfa:Cas9 system, we have generated tumors with melanoma-specific loss of tp53 or ptena/b, thereby eliminating non-specific tumors that originate from knockout of tumor suppressors in other cell types. Using this system, we are now testing whether knockout of tumor promoting genes delays melanoma onset and progression, something that has previously been difficult to efficiently achieve in zebrafish. In addition to specificity, an additional advantage of our system is that we can create nearly complete knockout of genes in the melanocytes by the F1, rather than the F2 generation. This saves several months of time and reduces the number of uninformative animals without the relevant genotype. Collectively, our system provides a highly efficient mechanism to study essential genes in melanocytes and melanoma in the zebrafish system.

Citation Format: Sarah Perlee, Yilun Ma, Jacob Swanson, Zhitao Ming, Julia Xia, Timothée Lionnet, Maura McGrail, Richard White. Development of a melanocyte lineage-specific knockout system in zebrafish [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 6621.
Abstract 6629: Cancer-associated nerves regulate anti-tumor immunity in pancreatic cancer
Authors: Xu Ruiyuan , Song Jianlu , Chen Yuan , Yin Xinpeng , Wang Chengcheng , Zhao Yupei
Published: March 2024

Nerves serve an emerging component of the tumor microenvironment (TME), contributing to tumorigenesis and cancer progression. Neural invasion (NI) is a common feature of pancreatic ductal adenocarcinoma (PDAC), accounting for distant metastasis and local recurrence. However, the mechanism by which tumor associated nerves (TANs) regulate anti-tumor immunity remains largely elusive. Here, we investigated the association between TANs and immunosuppressive TME to develop more effective therapeutic strategies for PDAC. Using tumor samples of PDAC patients, we identified that the nerves invaded by cancer cells showed increased adrenergic activation accompanied with no parasympathetic or sensory innervation changes. Patients with severe NI were associated with increased catecholamine levels. Additionally, single-cell RNA-sequencing and spatial transcriptome analysis of tumors from patients with PDAC revealed that peri-neural niches of severe NI patients were characterized by decreased CD8+ T cells and increased activity of M2 polarization of tumor-associated macrophages (TAMs), and regulatory T (Treg) cells, which was validated by multiplexed immunofluorescence staining. We further identified that CD8+ T cells expressing β-adrenaline receptors (β-ARs) reduced proliferation and cytokine production when exposure to catecholamines in a dose-dependent manner or co-cultured with dorsal root ganglia pretreated with cancer cells. KrasLSL-G12D; p53LoxP; Pdx1-CreER (KPC) mice treated with catecholamines accelerated tumor progression by inhibiting T cell-mediated antitumor responses, which was abrogated by surgical sympathectomy of KPC mice. Especially, in an orthotopic PDAC model, nonselective β-blocker propranolol in combination with immune checkpoint blockade (ICB) significantly enhanced active CD8+ T cells with concurrent reduced tumor growth. We suggest a novel regulatory mechanism of inhibiting anti-tumor immunity that can promote cancer progression mediated by TANs invasion. These data establish a close connection between the nerves, cancer cells, and immune cells and provide the potential novel targets of therapeutic approaches in PDAC.

Citation Format: Ruiyuan Xu, Jianlu Song, Yuan Chen, Xinpeng Yin, Chengcheng Wang, Yupei Zhao. Cancer-associated nerves regulate anti-tumor immunity in pancreatic 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 6629.
Abstract 663: Irreversible covalent azetidine-based small molecule inhibitors of Stat3 activity block growth of human pancreatic tumors
Authors: Chen Yue , Zhai Ning , Yue Peibin , Brotherton-Pleiss Christine , Fu Wenzhen , Nakamura Kayo , Chen Weiliang , Tius Marcus , Lopez-Tapia Francisco , Turkson James
Published: March 2024

The incidence and death rates for pancreatic adenocarcinoma in the US have been increasing by about 1% per year and the death rate has increased by approximately 0.2% yearly. The five-year survival rate has remained below 10% for many years. With surgery, radiation, and chemotherapy being the main treatment options to extend survival or for symptom relief, there is an urgent unmet need for effective new treatment options. Signal Transducer and Activator of Transcription (Stat) 3, one of the Stat family members, is aberrantly activated in human pancreatic and many other cancers. Aberrantly-active Stat3 promotes abnormal tumor-cell intrinsic and extrinsic mechanisms, including dysregulation of gene expression and mitochondria energy metabolism in the tumor cells, and suppression of immune cell functions in the tumor microenvironment. Stat3 is therefore an important target for therapeutic development. Herein we present two small molecules, H182 and H333, which are covalent inhibitors of Stat3 and that potently block Stat3 DNA-binding activity in vitro, with IC50 0.66-0.98 µM. H182 and its structural analog, H333 bind Stat3 via irreversible covalent interactions with key cysteine residues in the Stat3 DNA-binding domain to induce a time-dependent inhibition of Stat3 activity. Both compounds inhibit intracellular constitutive Stat3 tyrosine phosphorylation and induce loss of viable cells and apoptosis in vitro of human pancreatic Panc-1 and Mia-Paca2 cells. H333 is 13-fold more specific and H182 is 4-fold more specific against tumor cells over normal cells. Furthermore, MiaPaca-2 cells treated with H182 and analyzed by Seahorse showed dramatically decreased mitochondria respiration. Notably, xenograft models of MiaPaca2 treated with H182 or H333 via i.p. every other day for 27 days showed dramatic growth inhibition. Collectively, our results identify H182 and H333 as therapeutically-viable small molecules with unique irreversible mechanism of Stat3 inhibition which accounts for their strong antitumor effects against human pancreatic tumor xenografts.

Citation Format: Yue Chen, Ning Zhai, Peibin Yue, Christine Brotherton-Pleiss, Wenzhen Fu, Kayo Nakamura, Weiliang Chen, Marcus Tius, Francisco Lopez-Tapia, James Turkson. Irreversible covalent azetidine-based small molecule inhibitors of Stat3 activity block growth of human pancreatic tumors [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 663.
Abstract 6631: Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of immunosuppression
Authors: Kartalia Emma , Leatherman James M. , Lee Jae W. , Jaffee Elizabeth M. , Weeraratna Ashani T. , Zabransky Daniel J.
Published: March 2024

Advancing age is the strongest independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC) and a negative prognostic factor. PDACs are typically immunologically “cold” tumors and do not often respond to currently approved immunotherapy treatment approaches. How aging may alter non-malignant cells, such as fibroblasts and immune cell populations, in the PDAC tumor microenvironment (TME) to decrease anti-tumor immune responses is not understood.

We identified that aged microenvironments significantly alter the growth and progression of PDAC tumors. Orthotopic KPC and Panc02 mouse PDAC cell line injections into the pancreata of aged (>64-week-old) compared to young (6-8-week-old) C57BL/6 mice result in more rapid tumor growth and increased frequency of liver metastases in vivo. Tumors from aged mice have high expression of the TGFβ family member GDF-15 and hyperactivation of AKT signaling driven by the release of secreted factors from aged pancreatic fibroblasts.

We found that in aged microenvironments there is a marked reduction in the number of CD8+ T cells and an increase in FoxP3+ regulatory T cells in primary orthotopic pancreatic tumors using the KPC and Panc02 models. In addition, we performed portal vein injections to establish liver metastases to study the differences in immune cell infiltration in metastases from young vs. aged microenvironments. Aged mice form more and larger liver metastases compared to young mice, and metastatic lesions in aged mice have decreased CD8+ T cell infiltration when compared to young mice.

Since tumors in aged microenvironments have increased AKT activation compared to young microenvironments, we next tested if AKT signaling was sufficient to alter immune cell infiltration in PDAC tumors. In young mice, treatment with recombinant GDF-15, which activates AKT signaling, reduced CD8+ T cell infiltration into tumors. Conversely, in KPC tumors grown in aged mice treated with the AKT inhibitor MK-2206, there was a trend toward increased CD8+ T cell infiltration and a significant reduction in immunosuppressive FoxP3+ Tregs.

Together, these data highlight age-dependent differences in the immune infiltration in PDAC tumors. They suggest that activation of AKT signaling can drive features of immunosuppression in PDAC, providing the rationale for targeting AKT signaling to improve antitumor immune responses in PDAC.

Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Elizabeth M. Jaffee, Ashani T. Weeraratna, Daniel J. Zabransky. Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of 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 6631.
Abstract 669: Interrogating circadian clock compounds as an adjuvant to chemoradiation against GBM
Authors: Chan Priscilla , Wu Qiulian , Hovsepyan Anahit , Mkhitaryan Seda , Karapetyan Gevorg , Shah Khalid , Wakimoto Hiroaki , Kamenecka Theodore , Solt Laura A. , Cope Jamie , Hirota Tsuyoshi , Moats Rex A. , Rich Jeremy N. , Kay Steve A.
Published: March 2024

Despite the extensive efforts of researchers and physicians over the past two decades, glioblastoma (GBM) remains the most prevalent brain tumor type with a grim average survival time of 8 months following diagnosis irrespective of treatment. Additionally, the current standard-of-care has not changed from maximal surgical resection followed by concurrent chemoradiation from its establishment in 2005. One of the major challenges that limits progress in treating GBM is the presence of GBM stem cells (GSCs) that are highly invasive, reaching far past the tumor margins, are resistant to both chemotherapy and radiation, and can recapitulate the primary tumor following resection. Recent studies have found that GSCs, however, are highly sensitive to targeting of core circadian clock components, particularly positive regulators of the clock Brain and Muscle ARNTL-Like 1 (BMAL1) and Circadian Locomoter Output Cycles Protein Kaput (CLOCK). Here we employ the use of small molecule compounds that upregulate the activity of the negative regulators of the clock in order to suppress BMAL1::CLOCK transcriptional activity or repress BMAL1 transcription. We find that these compounds not only have a much more potent effect against GSCs compared to temozolomide (TMZ) chemotherapy but also, they display combination effect against both GSCs and differentiated GSCs (DGCs) when combined with TMZ. These results suggest that the blood brain barrier (BBB) penetrant circadian clock compounds can be utilized as an adjuvant to current GBM therapies as they can effectively target both the primary tumor cells and GSCs.

Citation Format: Priscilla Chan, Qiulian Wu, Anahit Hovsepyan, Seda Mkhitaryan, Gevorg Karapetyan, Khalid Shah, Hiroaki Wakimoto, Theodore Kamenecka, Laura A. Solt, Jamie Cope, Tsuyoshi Hirota, Rex A. Moats, Jeremy N. Rich, Steve A. Kay. Interrogating circadian clock compounds as an adjuvant to chemoradiation against GBM [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 669.
Abstract 6736: Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration
Authors: Li Keyu , Wang Junke , Espinoza Birginia , Xiong Yirui , Niu Nan , Wang Jianxin , Jurcak Noelle , Rozich Noah , Osipov Arsen , Henderson MacKenzie , Funes Vanessa , Lyman Melissa , Blair Alex B. , Herbst Brian , He Mengni , Yuan Jialong , Trafton Diego , Yuan Chunhui , Wichroski Michael , Liu Xubao , Wei Yuquan , Zheng Lei
Published: March 2024

Objective: Due to the challenge for intratumoral administration, innate agonists have not made it beyond preclinical studies for efficacy testing in most of tumor types. Pancreatic ductal adenocarcinoma (PDAC) has a T-cell excluded or deserted tumor microenvironment. Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD-1 antibody (a-PD-1) treatment.

Design: Using a transplant murine model with spontaneously formed liver metastasis and also the genetically engineered KPC mouse model that spontaneously develops PDAC, we compared the antitumor efficacy between intrahepatic/intratumoral and intramuscular systemic administration of BMS-986301, a next-generation STING agonist. Flow cytometry, Nanostring, and cytokine assays were used to evaluate local and systemic immune responses.

Results: The study demonstrated that administration of STING agonist systemically via intramuscular injection is equivalent or potentially superior to its intratumoral injection in inducing both effector T cell response and antitumor efficacy. Compared to intratumoral administration, T cell exhaustion and immunosuppressive signals induced by systemic administration were attenuated. Nonetheless, either local or systemic treatment of STING agonist was associated with increased expression of CTLA-4 in the tumors. However, the combination of a-PD-1 and anti-CTLA-4 antibody with systemic STING agonist demonstrated the antitumor efficacy in the KPC mouse spontaneous PDAC model. Our study also demonstrated the feasibility and antitumor efficacy of systemic administration of BMS-986299, a new NLRP3 agonist.

Conclusion: For the first time, our study supports the clinical development of innate agonists via systemic administration, instead of local administration, for treating PDAC.

Citation Format: Keyu Li, Junke Wang, Birginia Espinoza, Yirui Xiong, Nan Niu, Jianxin Wang, Noelle Jurcak, Noah Rozich, Arsen Osipov, MacKenzie Henderson, Vanessa Funes, Melissa Lyman, Alex B. Blair, Brian Herbst, Mengni He, Jialong Yuan, Diego Trafton, Chunhui Yuan, Michael Wichroski, Xubao Liu, Yuquan Wei, Lei Zheng. Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration [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 6736.
Abstract 678: Experimental therapy of triple negative breast cancer with next-generation of MDM2 inhibitors
Authors: Wang Wei , Chen Hao , Xie Yang , Datta Sayantap , Li Wei , Zhang Ruiwen
Published: March 2024

Patients with triple-negative breast cancer (TNBC) typically experience minimal benefits from the existing hormonal or trastuzumab-based therapies, necessitating reliance on chemotherapy as the predominant treatment approach, which, unfortunately, is limited by insufficient efficacy, significant side effects, and the development of drug resistance. These issues highlight an urgent demand for the discovery and development of more effective and safer therapeutics for TNBC. Overexpression and amplification of the MDM2 oncogene frequently occur in TNBC, which is associated with invasive and high-grade/late-stage tumors, metastasis, and recurrence, and is an independent negative prognostic factor for TNBC patients. Extensive studies have indicated that MDM2 represents a novel molecular target for TNBC. Thus far, multiple strategies aiming at MDM2 inhibition have not succeeded in Phase II-III clinical trials, largely due to factors like inadequate efficacy, significant drug toxicity, and emergence of resistance. In addition, TNBC tumors commonly harbor mutant p53, and the existing MDM2 inhibitors that block p53-MDM2 binding and depend on wild-type p53 expression in the tumor to exert anticancer effects would have little or no clinical responses or even induce resistant clones by increasing MDM2 expression and its oncogenic activity. Therefore, there is a need for developing new-generation MDM2 inhibitors that are p53-independent. We recently discovered a novel class of MDM2 degraders, representing a paradigm shift in the strategy of targeting MDM2. One of these analogs, WW68, was identified as a potent and selective MDM2 inhibitor. Its in vitro and in vivo anti-breast cancer activities and underlying mechanisms of action were evaluated in breast cancer cell lines with various p53 backgrounds. Our results demonstrate that WW68 effectively induces MDM2 protein degradation. It selectively inhibits breast cancer cell growth, decreases cell proliferation, and induces G2/M phase arrest and apoptosis in breast cancer cells, regardless of p53 status. Notably, in clinically relevant TNBC PDX models, WW68 inhibited MDM2 and suppressed tumor growth without causing host toxicity. In conclusion, WW68 represents a new class of MDM2 inhibitor that exerts its anticancer activity through directly down-regulating MDM2, and may be developed as a novel anti-TNBC therapeutic agent. (Supported by NIH R01CA214019 and R01CA240447; and DoD W81XWH2010011)

Citation Format: Wei Wang, Hao Chen, Yang Xie, Sayantap Datta, Wei Li, Ruiwen Zhang. Experimental therapy of triple negative breast cancer with next-generation of MDM2 inhibitors [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 678.
Abstract 6781: Maintaining the tumor microenvironment: Serum-free cell culture for an advanced 3D in vitro tissue model
Authors: Siebenmorgen Alina , Wöhle Christian , Lamfried Katharina , Carrera Lilian Martinez , Heller Bianca , Lupar Katharina , Bergamini Giovanna , Poeckel Daniel , Hardt Olaf , Theek Benjamin
Published: March 2024

Cancer research has made significant progress in recent years, especially in the field of immunotherapy that uses immune modulators. A significant portion of these novel therapeutic strategies focuses on manipulating the tumor microenvironment (TME). The TME is a complex structure that comprises various cellular elements, including cancer cells, immune cells, and structural components, such as secreted factors and extracellular matrix proteins. However, replicating the human TME in experimental murine models has proved challenging. Therefore, researchers have started to develop ex vivo cultivation protocols of tumor tissues, particularly precision-cut slices (PCS), that offer a three-dimensional representation of the TME. It is essential to select the best culture conditions to ensure the preservation of the TME that closely resembles the in vivo conditions. To further this research, a serum-free cell culture medium has been developed to propagate primary epithelial tumor cells originating from pancreatic, renal, and ovarian tissues. The optimized media formulation was successfully employed in the derivation and expansion of tumor cell lines, sourced from both primary and xenotransplanted tumors. An additional serum-free cell culture medium has been designed to cultivate primary lung cancer cells. Suitable culturing conditions have been introduced that maintain TME composition ex vivo cultured PCS prepared from fresh non-small cell lung carcinoma (NSCLC) tissue. Lung TumorMACS Medium and a serum-containing medium were compared, and it was found that the cellular composition of PCS cultivated in Lung TumorMACS was comparable to the original composition. At the same time, PCS cultivated in Lung TumorMACS showed a lower expression of stress genes, compared to the serum-containing alternative.

Citation Format: Alina Siebenmorgen, Christian Wöhle, Katharina Lamfried, Lilian Martinez Carrera, Bianca Heller, Katharina Lupar, Giovanna Bergamini, Daniel Poeckel, Olaf Hardt, Benjamin Theek. Maintaining the tumor microenvironment: Serum-free cell culture for an advanced 3D in vitro tissue 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 6781.