bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3398: Exploring the biology of ctDNA release in colon cancer
Authors: Kisistok Judit , Henriksen Tenna Vesterman , Bramsen Jesper Bertram , Reinert Thomas , Mattesen Trine Block , Birkbak Nicolai Juul , Andersen Claus Lindbjerg
Published: June 2022

Background: Colorectal cancer is responsible for 9.4% of cancer-related deaths worldwide. Early detection of the disease has the potential to improve patient outcomes, therefore, developing an efficient and minimally invasive method for carrying out surveillance has been a clinically relevant effort in recent years. Circulating tumour DNA has garnered interest as a marker for early cancer detection, however, deeper understanding of the underlying biology of ctDNA release is needed in order to maximize its utility in clinical practice. In this project, we aimed to investigate whether a phenotype conductive to ctDNA shedding can be defined in a colorectal cancer cohort, including transcriptomic, genomic and clinical data collected from stage I to stage IV colon cancer patients at Aarhus University Hospital.

Methods: Transcriptomic data from 102 patients and whole exome sequencing data from 60 patients were analyzed. Differential gene expression and differential pathway enrichment analyses between ctDNA positive and ctDNA negative patients were conducted by performing Student’s t-test. The resulting p-values were adjusted according to the Benjamini-Hochberg method. The genes in the transcriptomic analyses were assigned to pathways with regard to the MSigDB Hallmark gene sets and pathway enrichment was assessed using Gene Set Variation Analysis (GSVA). Using an extended cohort of 537 patients, we performed Wilcoxon’s signed rank test to investigate the association between tumour size, histological subtypes and ctDNA shedding behavior. Additionally, we modelled the expected ctDNA fraction as a function of birth rate, death rate and tumour mass.

Results: In colon cancer, ctDNA release is primarily associated with tumour size, molecular subtypes and proliferative transcriptomic signatures. A moderate but significant correlation was observed between the tumour’s largest diameter and the detected ctDNA fraction. Furthermore, molecular subtypes associated with less aggressive disease tended to shed lower amounts of ctDNA. Notably, we have found that pathways associated with proliferation are enriched in the ctDNA positive subgroup.

Conclusions: Our results demonstrate that enhanced proliferative capacity in connection with tumour size and molecular subtypes may contribute to a higher ctDNA release rate. We anticipate our study to be a starting point for further investigation of ctDNA shedding dynamics in colorectal cancer.

Citation Format: Judit Kisistok, Tenna Vesterman Henriksen, Jesper Bertram Bramsen, Thomas Reinert, Trine Block Mattesen, Nicolai Juul Birkbak, Claus Lindbjerg Andersen. Exploring the biology of ctDNA release in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3398.
Abstract 343: New derivatives of 1,3-dioxoisoindoline: Potential breast cancer therapeutics that are S6K1 inhibitors
Authors: Kumar Satyendra , Komati Rajesh , Shaik Shahensha , Bratton Melyssa , Tran Linh , Sam Rion , Johnson-Henderson Elijah , Graham Breyanah , WIlliams Christopher , Sridhar Jayalakshmi
Published: June 2022

Currently, more than 3.8 million women in the United States are diagnosed with breast cancer which is the second leading cause of cancer-related death in women. The correlation of the ribosomal protein S6 kinase 1 (S6K1) activity and breast cancer is evident from the amplification of S6K1 localized chromosomal region 17q23 in 20% of primary breast cancers. S6K1 is a conserved serine/threonine protein kinase, is the principal kinase effector downstream of the PI3K/mTOR regulatory signaling pathway. Over-expression of S6K1 has been associated with cell transformation and elevated proliferation rates in tumors, poor prognosis and an increased risk of local recurrence. S6K1 has been found to play an important role in the progression of ER-positive (ER+) breast cancer, HER2 positive (HER2+) breast cancer and node-negative premenopausal breast cancer. Inhibition of this kinase can prove to be beneficial for the treatment of several types of breast cancer. Our group has identified new molecules that are derivatives of 6-amido-4-aminoisoindoline-1,3-dione core structure as S6K1 inhibitors with low micromolar inhibition potency (IC50 = 2-5 μM). These compounds also inhibited the growth of HER2+ (SKBR3 and HER2Δ16), ER+ and triple negative breast cancer cells (MDA-MB-231 and MDA-MB-468) with a range of EC50 values (2 - 2000 μM). The EC50 values of growth inhibition of these compounds varied by the type of breast cancer cells and were in direct correlation with the expression levels of S6K1 in those cell lines. These results clearly indicate that high efficacy S6K1 inhibitors can function as potential therapeutics for multiple types of breast cancer. Future work involves in-vivo studies to understand the efficacy and pharmacokinetics of the three compounds.

Citation Format: Satyendra Kumar, Rajesh Komati, Shahensha Shaik, Melyssa Bratton, Linh Tran, Rion Sam, Elijah Johnson-Henderson, Breyanah Graham, Christopher WIlliams, Jayalakshmi Sridhar. New derivatives of 1,3-dioxoisoindoline: Potential breast cancer therapeutics that are S6K1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 343.
Abstract 3432: A novel anti-CLDN6-CD137 bispecific antibody (NBL-028) for treating Claudin 6 positive solid tumors
Authors: Yu Raymond , Tong Yong , Lu Huarui , Danton Makenzie , Dillman Samantha , Dewe Joshua , Karchin Joshua , Patil Samruddhi , Lavery Danielle , Huang Haichun , Pei James , Liu Zhong , Li Han , Lei Ming
Published: June 2022

Claudin-6 is a tetraspan membrane protein associated with tight junction formation. Its expression in normal tissues is restricted to the fetal organs but is not detected in the adult tissues. Increased expression of CLDN6 has been shown in several human malignancies such as testicular, ovarian, uterine, liver and lung adenocarcinoma, and is associated with poor prognosis in these cancer patients. Therefore, CLDN6 is a promising tumor-associated antigen (TAA) for tumor-targeting therapeutics such as CART and T cell engaging bispecific antibodies. CD137 co-stimulation has been reported to lead to extended T-cell proliferation, reactivating anergic T cells, promoting memory T cell formation and maintenance. Activating CD137 with agonistic antibodies presents a great opportunity to improve the therapeutic efficacy of immune checkpoint inhibitors (ICIs) or overcome resistance to ICIs. Additionally, a bispecific antibody that activates CD137 signaling only in the presence of TAA would help reduce the dose-dependent hepatotoxicity that was observed in clinical trials with monoclonal anti-CD137 agonistic antibody due to the activation of CD137 signaling in liver resident Kupffer cells. NBL-028 is a novel tetravalent bispecific antibody uniquely designed to activate the CD137 co-stimulatory pathway in the tumor microenvironment through the CLDN6-mediated clustering of CD137. NBL-028 binds to Claudin 6 with high affinity and specificity, activates CD137 in a CLDN6-dependent manner with fast-on and fast-off CD137 binding properties. NBL-028 demonstrated strong CLDN6-dependent T-cell activation and T-cell mediated cytotoxicity in vitro. NBL-028 elicits potent anti-tumor effects and immunological memory in mouse models without detectable liver damages or systemic toxicity. The tumor infiltrated Lymphocyte (TIL) analysis also demonstrated that NBL-028 treatment induced CD45+ immune cells infiltration, decreased Treg/CD8 ratio and increased M1/M2 like macrophage ratio. Furthermore, NBL-028 is designed upon a proprietary molecular scaffold with superior developability features, demonstrated by stability, yield and ease to purify. NBL-028 is currently at the stage of IND-enabling activities with the aim to enter clinical studies for treating Claudin 6 positive solid tumors in 1Q 2023.

Citation Format: Raymond Yu, Yong Tong, Huarui Lu, Makenzie Danton, Samantha Dillman, Joshua Dewe, Joshua Karchin, Samruddhi Patil, Danielle Lavery, Haichun Huang, James Pei, Zhong Liu, Han Li, Ming Lei. A novel anti-CLDN6-CD137 bispecific antibody (NBL-028) for treating Claudin 6 positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3432.
Abstract 3437: Characterization of a potent and selective A2aR monoclonal antibody antagonist for the treatment of cancer
Authors: Hu Changyun , Zhang Jiandong , Liu Shuying , Zhao Xinyan
Published: June 2022

Introduction: Adenosine-mediated suppression of anti-tumor immunity has been shown to be one of the mechanisms for immunosuppression and resistance to immune checkpoint blockade (ICB) therapies such as anti-PD-1 or anti-PD-L1 monoclonal antibodies (mAbs). Therapeutic candidates (biologics and small molecule inhibitors(SMIs)) targeting ectonucleotidases CD39 and CD73, enzymes responsible for hydrolyzing ATP to adenosine, and SMIs targeting adenosine receptors are currently under active preclinical and early clinical development. Here we present the characterization of a novel, selective anti-human A2aR antagonist mAb that showed high potency in inhibiting adenosine mediated immunosuppressive functions to enhance anti-tumor immunity.

Methods: A panel of mAbs binding to human adenosine receptor A2aR (hA2aR) were generated via hybridoma technology. The specificity and cross-reactivity were measured by FACS using human A1R, A2aR, A2bR, A3R or mouse A2aR over-expressing cells, as well as human and cynomolgus monkey PBMC. The cellular potency of these mAbs was assessed by measuring decreased cAMP levels in engineered HEK293 cells stably over-expressing hA2aR, a Gs protein coupled receptor (GPCR), upon stimulation with agonist NECA. The ability of lead candidate AT-004 to reverse adenosine-mediated immune suppression of human CD8 T-cells was determined using standard CD3/CD28 activation conditions. The in vivo efficacy of humanized AT-004 is being evaluated using ICB treatment resistant solid tumor mouse models.

Results: AT-004 represents a panel of novel, selective mAbs. These mAbs specifically bind to human A2aR, but not to family members including A1R, A2bR and A3R. Cross-reactivity to cynomolgus monkey PBMCs was also observed. AT-004 inhibited NECA-mediated A2aR activation with a potency over 100-fold higher than SMIs e.g. AZD4635. AT-004 significantly reversed the ability of adenosine to suppress CD8 T cell activation as indicated by restoration of cell surface marker expression and increased levels of cytokines such as IFN-γ in the supernatants.

Conclusions: AT-004 is a potent, selective and highly potent antagonist of A2aR receptor. Considering its potential safety advantage as an mAb comparing to most known A2aR SMI antagonists in that it does not cross the blood brain barrier, AT-004 maybe a promising novel therapeutic candidate for immunotherapy, vital for enhancing anti-tumor responses in solid tumors that show an incomplete response or resistance to anti-PD-1 or anti-PD-L1 mAb treatment due to elevated adenosine level in tumor microenvironment (TME).

Citation Format: Changyun Hu, Jiandong Zhang, Shuying Liu, Xinyan Zhao. Characterization of a potent and selective A2aR monoclonal antibody antagonist for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3437.
Abstract 3440: Novel in vitro TME platform for rapid cancer therapeutic and target discovery
Authors: Matre Polina R. , Choi Byung-Kwon , Delgado Oliver , Westwick John K.
Published: June 2022

The tumor microenvironment (TME) plays an important role in malignances as tumors react with a multitude of changes and microenvironmental cues. TME components modulate tumor profiles and treatment responses, thereby affecting sensitivity to anti-cancer agents. The limited clinical success of therapeutics, including immunotherapies, emphasizes an unmet need to identify new targets and develop preclinical platforms to accelerate anti-cancer drug discovery. Although in vitro methods are essential in early drug development, standard approaches, which do not capture the complexity of TME, have been historically favored for both discovery and evaluation of potential therapeutics.

Resonant's IMPACT TME platform couples proprietary high-fidelity TME models with deep data-driven analytics for rapid and cost-effective discovery of novel targets and antibody-based therapeutics. Target and candidate selection is based upon a biology-forward approach driven by live animal immune systems and functional screening prior to target identification. Identified candidates are then prioritized via integration of publicly available information with internal functional and multi-omic data. Resultant targets display attributes, such as tumor selectivity and immune-modulatory capabilities, that are amenable to several therapeutic modalities, including antibody-drug conjugates (ADCs) and bispecifics. To date, Resonant has interrogated a variety of solid tumor types and thousands of candidates resulting in an expanding library of actionable antibodies and targets. Representative examples of in vitro and in vivo efficacy of selected candidates derived from the IMPACT TME platform are presented, highlighting their individual potential and validating the Resonant approach. As technological advances transform the landscape of therapeutics, the need for novel targets is burgeoning, and Resonant’s TME-driven platform provides a valuable tool to meet these demands.

Citation Format: Polina R. Matre, Byung-Kwon Choi, Oliver Delgado, John K. Westwick. Novel in vitro TME platform for rapid cancer therapeutic and target discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3440.
Abstract 347: Comparative activity of MBQ-167 metabolites in metastatic breast cancer
Authors: Medina Julia I. , Hernández Eliud , Vlaar Cornelis , Dharmawardhane Suranganie
Published: June 2022

Metastasis continues to be the most difficult phase for cancer treatment due to few targeted therapy options. Therefore, we focus on targeting the Rho GTPases Rac1 and Cdc42, key drivers of the primary steps of metastasis: cell migration and invasion. We developed MBQ-167 as a small molecule dual inhibitor for Rac1 and Cdc42 activation with IC50s of 103 and 78 nM, respectively. In metastatic human breast cancer cells, MBQ-167 inhibits the Rac/Cdc42 downstream effector p21-activated kinase (PAK)/LIM kinase/Cofilin signaling, lamellipodia extension, and thus, cell polarity, and migration. In addition, in metastatic breast cancer cells, MBQ-167 inhibits cell proliferation and cell cycle progression to ultimately induce apoptosis via anoikis. MBQ-167 inhibits tumor growth and metastasis in experimental and spontaneous HER-positive and Triple negative breast cancer (TNBC) mouse models. MBQ-167 has 35% bioavailability and is not toxic in rodent or canine models up to 1000 mg/kg. Therefore, we are further developing MBQ-167 as a lead anti metastatic cancer compound. The objective in this study is to elucidate potential activity of MBQ-167 metabolites. Prior work has identified MBQ-167 metabolites via LC MS/MS from a mammalian liver microsome (rats and human) assay. Four of the MBQ-167 metabolites with the highest MS peak area, were analyzed in metastatic breast cancer cell lines, for cell viability via MTT assays and apoptosis via caspase 3/7 assays. We report that the metabolites M6, M7, M8, and M9 did not inhibit cell viability or induce apoptosis at 250 or 500 nM in HER2-type MDA-MB-435, and TNBC MDA-MB-231 and MDA-MB-468 breast cancer cell lines, compared to MBQ-167. We next analyzed whether these metabolites could inhibit the activation of Rac and Cdc42 by analyzing the phosphorylation status of its downstream effector p-21 activated kinase isoforms 1/2/3 (PAK1/2/3). When MBQ-167 and metabolites at 250nM were incubated for 24h in HER2-type MDA-MB-435 metastatic cancer cells, and the lysates western blotted with antibodies to total PAK1/2/3 or phospho-Pak1/2/3, we found a decrease in the phosphorylation levels of PAK in response to MBQ-167 and metabolite M6. However, since the peak area of M6 in the microsome assay is only 6% compared to MBQ-167, we conclude that MBQ-167 is the major active compound with anti-metastatic cancer properties.

Citation Format: Julia I. Medina, Eliud Hernández, Cornelis Vlaar, Suranganie Dharmawardhane. Comparative activity of MBQ-167 metabolites in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 347.
Abstract 3507: β-Hydroxy-β-methylbutyrate (HMB) sensitizes murine pancreatic ductal adenocarcinoma (PDAC) to immunotherapy
Authors: Etigunta Suhas K. , Coleman Michael F. , Liu Kristyn A. , Pfeil Alex J. , Cui Zhengrong , Hursting Stephen D.
Published: June 2022

Background: Pancreatic cancer is the fourth leading cause of cancer-related death in the United States. PDAC often presents at advanced tumor stages, resulting in poor survival rates. Immunotherapies such as inhibition of programmed death 1 (PD1) are effective in many cancers, but not in PDAC. HMB is a leucine metabolite that reduces PDAC progression and cachexia in association with enhanced antitumor immune responses. The purpose of this study was to determine if HMB treatment enhances immunotherapeutic response in PDAC.

Methods: C57BL/6J mice bearing subcutaneously transplanted PANC02 tumors were fed either a control (AIN93G) or HMB (1% w/w) supplemented diet. Once tumors were palpable, mice were randomized for treatment with vehicle or anti-PD1 immunotherapy (n=12-13/group). Tumors were harvested and global transcriptomic analysis was performed.

Results: Both HMB and anti-PD1 interventions alone were insufficient to reduce tumor growth. However, the combination of HMB and anti-PD1 resulted in significantly smaller tumors than the anti-PD1 alone group, indicating that HMB was able to stimulate PDAC response to ICI. Transcriptomic analysis revealed limited transcriptional effects attributable to immunosurveillance following treatment with either HMB or anti-PD1 alone; however, the combination produced a robust antitumor immune response. Digital cytometry analysis revealed altered immune cell composition in the tumor microenvironment.

Conclusion: The combination of HMB with anti-PD1 treatment elicited significant antitumor responses in a murine model of PDAC progression. Given that immunotherapy trials in PDAC have not yet proven effective, even when combined with cytotoxic chemotherapy, our preclinical findings support clinical evaluation of combined ICI and HMB regimens as a therapeutic strategy against PDAC.

Citation Format: Suhas K. Etigunta, Michael F. Coleman, Kristyn A. Liu, Alex J. Pfeil, Zhengrong Cui, Stephen D. Hursting. β-Hydroxy-β-methylbutyrate (HMB) sensitizes murine pancreatic ductal adenocarcinoma (PDAC) to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3507.
Abstract 3526: Host genetic susceptibility to gut microbiota-driven colorectal carcinogenesis
Authors: Vera-Urbina Ibis R. , Ayala Estefania , Rodriguez Ediel , Centeno-Girona Hilmaris , Casiano Leslie , Suarez Belisa , Cruz-Correa Marcia , Gonzalez-Pons Maria
Published: June 2022

Introduction: Colorectal cancer (CRC) is the 1st and 2nd leading cause of cancer death in men and women in Puerto Rico and the U.S., respectively. A dynamic balance between the host immune system and the gut bacterial communities is essential to protect colonic tissues against chronic inflammation, which may lead to CRC. The main goal of this study was to investigate if SNPs in the promoter regions of the IL-1β, IL-6 and IL-10, key cytokines that regulate gut inflammation, increase the risk for colorectal adenomas by enriching a subset of the toxin-producing gut microbiota (gene-environment interaction).

Methods: Using a case-control study design, associations between having pro-inflammatory SNPs and/or bacterial toxins in colonic mucosa or stool were assessed using odds ratios. TaqMan® SNP Genotyping Assays (ThermoFisher Scientific) for IL- 1β (rs1143627), IL-6 (rs1800795), and IL-10 (rs1800871) were performed according to the manufacturer’s recommendations. The presence of bacterial toxin genes (pks, TcPC, GelE, cnf-1, CDT, murB, and usp) in stool were determined using the QuantiTect SYBR Green PCR kit (QIAGEN).

Results: Our preliminary analyses showed that individuals with the IL-1β pro-inflammatory genotype were 1.5-times more likely to have colorectal adenomas (n=232; p=0.1). Detection of usp or pks in stool (n=147) was positively associated with having adenomas (OR=2.95; CI 95%= 0.92-9.45 and OR=2.91; CI 95%=0.58-14.50, respectively).

Conclusion: An evaluation of the association between having pro-inflammatory genotypes, the presence of bacterial toxin genes in stool, and colorectal adenomas using a larger sample size is warranted and currently underway. The integration of modifiable (e.g. gut bacterial toxins) and non-modifiable (e.g. host genetics) risk factors in a stratified model for CRC risk stratification and/or prevention may enhance current screening approaches.

Citation Format: Ibis R. Vera-Urbina, Estefania Ayala, Ediel Rodriguez, Hilmaris Centeno-Girona, Leslie Casiano, Belisa Suarez, Marcia Cruz-Correa, Maria Gonzalez-Pons. Host genetic susceptibility to gut microbiota-driven colorectal carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3526.
Abstract 3530: Microbial-Interleukin 17 receptor A (IL-17RA) signaling axis modulates tumor growth and microenvironment
Authors: Chandra Vidhi , Zhang Yu , Roux Olivereen Le , Petrosino Joseph , Kolls Jay , McAllister Florencia
Published: June 2022

Microbiota, both within the gut and the tumors, has emerged as a significant player influencing tumor growth and responses to therapies. Recent evidence links microbiota and pancreatic ductal adenocarcinoma (PDAC), an aggressive cancer surrounded by a highly immuno-suppressive tumor microenvironment which limits efficacy of most available therapies. The immunosuppressive tumor microenvironment of PDAC is partially facilitated by a proinflammatory cytokine Interleukin 17 (IL-17). IL-17 can be stimulated by intestinal commensal bacteria under normal physiological conditions. It is critical for microbial defense as well as immunopathology. Both IL-17 neutralization and antibiotics reduce murine PDAC growth. While the vital role of microbiota in affecting tumor immunity has been identified, there is still a gap of knowledge as to how microbes may regulate pro-tumorigenic IL-17 signaling. We wanted to investigate the systemic and local role of IL-17 in regulating the microbial-tumor immune axis. For this purpose, we genetically deleted the IL-17 receptor A (IL-17RA) in different compartments and evaluated tumor growth. We found that IL-17RA signaling was important for maintaining microbial homeostasis and its disruption resulted in differential tumor growth. Absence of IL-17RA signaling in the gut lead to local inflammation as well as systemic immune effects. Our data suggests that modulation of IL-17 signaling could serve as a therapeutic intervention to alter microbial mediated tumor effects. The significance of these findings may extend to other cancers as well.

Citation Format: Vidhi Chandra, Yu Zhang, Olivereen Le Roux, Joseph Petrosino, Jay Kolls, Florencia McAllister. Microbial-Interleukin 17 receptor A (IL-17RA) signaling axis modulates tumor growth and microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3530.
Abstract 3536: Modulating T cell tumor infiltration by modulating T cell migration
Authors: Johnston Adrian , Du Frisco , Wan Zeqi , Lee Cameron , Lim Yeongseo , Bessiake Michelle , Nair Praful , Wu Pei-Hsun , Wirtz Denis
Published: June 2022

Traditional assessments of T cells killing cancer cells are routinely done in two-dimensional cell culture wells and plates. The general methods comprise developing a cancer cell monolayer then overlaying it with T cells, or, worse, pre-mixing cancer cells with T cells before placing the mixture into culture wells/dishes. Such methods obviously force cancer cell-T cell interactions. This is not physiological, since T cells must migrate to tumors for cancer cell killing to even occur. Migration is a little-explored, yet central parameter ignored in 2D killing assays. There is no tumor cell killing by T cells if there is no T cell migration, biased or unbiased. Our work making use of novel 3D multi-compartments organoids systems identifies key molecules regulating pathways for T cell migration in 3D. We are currently testing molecules currently FDA-approved or in clinical trials that block or enhance T cell killing of cancer cells by blocking or enhancing T cell migration through a first-of-its-kind 3D killing assay that we developed.

Citation Format: Adrian Johnston, Frisco Du, Zeqi Wan, Cameron Lee, Yeongseo Lim, Michelle Bessiake, Praful Nair, Pei-Hsun Wu, Denis Wirtz. Modulating T cell tumor infiltration by modulating T cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3536.
Abstract 3537: Pharmacologic inhibition of the glycolytic pathway improves response to immune checkpoint blockade
Authors: Verma Svena , Serganova Inna , Budhu Sadna , Dong Lauren , Ko Myat , Mangarin Levi , Merghoub Taha , Wolchok Jedd , Zappasodi Roberta
Published: June 2022

Preferential engagement in glycolysis is a hallmark of cancer cells and contributes to the progression and metastasis of many tumor types, including melanoma and triple-negative breast cancer (TNBC). Tumor reliance on glycolysis is emerging as a mechanism of resistance to immunotherapy, due in part to lactate-mediated immunosuppression and competition for glucose between effector T cells and tumor cells within the tumor microenvironment. Elevated serum lactate dehydrogenase (LDH) levels are associated with poor outcomes in cancer patients, and we observed that serum lactate and LDH levels correlate with primary tumor burden in mice. We recently demonstrated that genetic dampening of LDH subunit A in 4T1 TNBC results in improved and long-lasting anti-tumor responses to CTLA-4 blockade in mice. Therefore, we hypothesize that combining LDH inhibitors (LDHi) with CTLA-4 blockade will be an effective strategy to combat resistance to anti-CTLA-4 therapy. Since activated T cells rely on glycolysis, we first determined that glycolytic cancers overexpress LDH (compared with immune cells) by analyzing single-cell transcripts from patient melanoma biopsies. LDHA gene expression is significantly higher in malignant cells than infiltrating CD8+ T cells, and we replicated these findings at the protein level in whole cell lysate from B16-F10 melanoma and 4T1 TNBC tumor cells and tumor-antigen specific T cells in vitro. We show that LDHi reduces tumor lactate production and glucose consumption without inhibiting anti-tumor T-cell killing in vitro. The anti-tumor effect of LDH inhibition requires adaptive immunity, as daily treatment with LDHi results in reduced tumor burden in immunocompetent but not immune-deficient mice. Finally, we show that targeting LDH in combination with CTLA-4 blockade is more effective in slowing B16-F10 growth compared with CTLA-4 blockade alone, and that this combination promotes effector T cell activation while destabilizing regulatory T cell function.

Citation Format: Svena Verma, Inna Serganova, Sadna Budhu, Lauren Dong, Myat Ko, Levi Mangarin, Taha Merghoub, Jedd Wolchok, Roberta Zappasodi. Pharmacologic inhibition of the glycolytic pathway improves response to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3537.
Abstract 3545: New hybrid molecular modalities comprised of DNA-origami and interfering peptides as inhibitors of protein-protein interactions
Authors: Roman Jessica , Carter Rachel , Luchini Alessandra , Liotta Lance , Veneziano Remi , Haymond Amanda
Published: June 2022

Immunotherapy has shown tremendous promise in the treatment of blood cancers; however, treatment typically fails when attempted with solid tumors, such as breast cancer. However, the cases where immunotherapies have been successful in breast cancer, the results have been remarkable: full remission of stage 4 disease has been observed. This highlights the need to elucidate how the tumor microenvironment (TME) fosters a context of molecular crosstalk that is hostile to inflammatory, anti-tumor immunological responses that present a significant roadblock to consistently successful immunotherapy. Aberrant IL-33 present in the TME is believed to be one of these major factors that drives tumor progression via its ligand, ST2, and the subsequent recruitment of IL1RAcP. This activates MyD88 cascades in dysfunctional myeloid immune cells and myeloid derived suppressor cells (MDSCs), eliciting immunosuppression that promotes tumor tolerance and subsequent disease progression. To this end, preventing IL-33/ST2 signaling activation is an attractive target to possibly manipulate immune cells to recognize and mount a response against tumor cells. Our previous work has identified 4 IL1RAcP hotspot residues of interaction between it and the ST2/IL-33 complex: R157, K238, K343, and K346. Here, we designed peptides, which mimic these hotspots and the adjacent residues that interact with the ST2/IL-33 complex. Wild type peptides inhibit trimer formation in vitro as determined by immunoprecipitation and chemical crosslinking experiments. Modifications introduced to increase rigidity and stability increased affinity in vitro and showed inhibition of downstream signaling in reporter gene cell models.

Citation Format: Jessica Roman, Rachel Carter, Alessandra Luchini, Lance Liotta, Remi Veneziano, Amanda Haymond. New hybrid molecular modalities comprised of DNA-origami and interfering peptides as inhibitors of protein-protein interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3545.
Abstract 3572: Oncolytic virotherapy with the HSV-1 (VC2) vaccine strain induces anti-tumor T-cell responses in the murine 4T1 breast cancer model
Authors: Nabi Rafiq , Musarrat Farhana , Chouljenko Vladimir , Kousoulas Konstantin
Published: June 2022

The tumor microenvironment (TME) plays important roles in suppression of anti-tumor immune responses. Immune suppression is achieved by multiple mechanisms including interfering with antigen presentation and inhibition of effector T-cell tissue infiltration. Altering specific TME properties can reduce immune-suppression and improve anti-tumor immunity with abscopal effects. Oncolytic Virotherapy (OV) is a promising approach for cancer treatment. We evaluated the efficacy of the HSV-1 (VC2) vaccine strain for the treatment of breast cancer using the murine stage-4 (4T1) syngeneic and immunocompetent breast cancer mouse model. Following Intratumoral (IT) injection of VC2 in mice bearing implanted 4T1 tumors, treatment efficacy was evaluated by monitoring tumor weight, immune infiltration, and metastasis in the lung. Animals treated with VC2 via the IT route exhibited significantly reduced metastasis and improved intra-tumoral T-cell infiltration. Improved T-cell infiltration was not due to viral replication since improved parameters were observed during late times post VC2 treatment when no viral replication was detectable. The total area of metastasis in the lung was significantly reduced compared to control groups. VC2 treated animals via the IT route had significantly higher cytotoxic splenocytes, evident by the in-vitro killing of 4T1 cells, compared to the control group. This improved cytotoxic activity, intratumoral T-cell infiltration, and reduced metastasis in the lung suggest that IT administration of VC2 altered TME in a way that induced better antigen recognition, processing, and presentation resulting in improved anti-tumor immune responses with an abscopal effect in the lung. Further understanding of the mechanisms involved in the observed improved anti-tumor immune responses will help improve treatment efficacy and provide new approaches for the treatment of solid tumors.

Citation Format: Rafiq Nabi, Farhana Musarrat, Vladimir Chouljenko, Konstantin Kousoulas. Oncolytic virotherapy with the HSV-1 (VC2) vaccine strain induces anti-tumor T-cell responses in the murine 4T1 breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3572.
Abstract 358: Restoring necroptosis in colorectal cancer cells to overcome chemotherapeutic resistance
Authors: Ermine Kaylee , Chen Dongshi , Wang Peng , Yu Jian , Zhang Lin
Published: June 2022

Colorectal cancer (CRC) is a leading cause of cancer related deaths in the United States. CRC patients are often treated with chemotherapeutic drugs, such as 5-fluorouracil (5-FU), and resistance to chemotherapy is a major clinical problem. It is well-established that 5-FU induces apoptosis in cancer cells, but apoptosis inhibition does not block the anticancer effect of 5-FU, suggesting a role of non-apoptotic cell death. Necroptosis is a regulated form of necrosis controlled by Receptor-Interacting Protein kinase 1 (RIP1), RIP3, and Mixed Lineage Kinase domain-Like (MLKL). TCGA RNA-Seq data reveal that RIP1 and RIP3 are frequently downregulated in CRC, with RIP3 downregulation correlating with worse clinical outcomes. We found that loss of RIP3 expression in CRC cells is associated with decreased 5-FU sensitivity. RIP3 silencing in CRC was shown to be caused by promoter hypermethylation. However, the exact functional role of RIP3 in response to anticancer therapies remains unclear. Our studies reveal that RIP3-expressing CRC cells were able to undergo both apoptosis and necroptosis in response to 5-FU treatment, which was abrogated upon knock-out or inhibition of RIP3. Restoration of RIP3 in RIP3-silenced CRC cells by transfection enhanced 5-FU sensitivity, along with restored necroptosis and enhanced apoptosis. Furthermore, we have shown that RIP3 expression can be restored in RIP3-silenced CRC cells by the DNA methyltransferase 1 inhibitor 5-aza-2’-deoxycytidine (5-aza-dC), which also enhances 5-FU sensitivity. This effect of 5-aza-dC is partially dependent on RIP3 and suppressed by a RIP3 inhibitor. Together, our results suggest RIP3 plays a critical role in response to 5-FU and other chemotherapeutic agents, and that restoring RIP3 expression can be used to potentiate chemotherapy. These results provide novel insight into the functional role of RIP3 in CRC chemotherapy, which could be explored for developing improved and personalized CRC treatments based on RIP3 status.

Citation Format: Kaylee Ermine, Dongshi Chen, Peng Wang, Jian Yu, Lin Zhang. Restoring necroptosis in colorectal cancer cells to overcome chemotherapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 358.
Abstract 3606: Lenalidomide switch control of CAR T-cell phenotype and function via degradable membrane-bound IL-7
Authors: Kann Michael C. , Tepper Alexander , Silva Harrison J. , Lane Isabel C. , Bouffard Amanda A. , Larson Rebecca C. , Ebert Benjamin L. , Maus Marcela V. , Jan Max
Published: June 2022

CAR T cells have had marked success in the treatment of hematologic malignancies such as B-cell acute lymphoblastic leukemia and multiple myeloma. However, some patients fail to have sustained responses, in large part to due limited persistence of their CAR T cells. Previous attempts to prolong CAR T cell survival have attempted to combine CAR T cell therapy and cytokine therapy (IL-2, IL-15) to increase persistence, but this combination of T cell activation and high levels of systemic cytokines can lead to toxicities. To enhance CAR T cell persistence and while mitigating potential cytokine toxicities, we present a bicistronic CAR T engineered to express a membrane-bound drug-degradable IL-7 (mbdIL7) on its surface in addition to a 2nd generation anti-CD19 CAR.

The mbdIL7 is composed of an IL-7 molecule anchored to the cell by a CD80 hinge/transmembrane domain and followed by a cytoplasmic super-degron tag and drives JAK/STAT5 signal transduction. Both mbdIL7 abundance and STAT5 phosphorylation can be efficiently suppressed with lenalidomide, an FDA approved drug, at sub-therapeutic doses. The mbdIL7 can supplant the requirement of exogenous cytokines during manufacturing and leads to a more central memory-biased T cell phenotype. mbdIL7 CAR T cell proliferation remains antigen-dependent. These novel mbdIL7 CAR T cells have similar in vitro effects to traditional CAR T against models of leukemia and lymphoma, but have distinct transcriptional profiles associated with lower levels of exhaustion. In an orthotopic xenograft lymphoma model, mbdIL7 CAR T cells demonstrate markedly enhanced anti-tumor activity compared to traditional anti-CD19 CAR T cells. In addition, mbdIL7 CAR T cells have increased expansion/persistence in the blood, bone marrow, and spleen. The inclusion of a membrane-bound degradable IL-7 into the backbone of a traditional anti-CD19 CAR allows for spatial and temporal control of a pro-persistence cytokine leading to a more effective CAR T treatment. This presents a potential controllable CAR T for patients that are currently not responding conventional FDA-approved products.

Citation Format: Michael C. Kann, Alexander Tepper, Harrison J. Silva, Isabel C. Lane, Amanda A. Bouffard, Rebecca C. Larson, Benjamin L. Ebert, Marcela V. Maus, Max Jan. Lenalidomide switch control of CAR T-cell phenotype and function via degradable membrane-bound IL-7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3606.
Abstract 362: MYCN-amplified neuroblastoma is addicted to iron and vulnerable to ferroptosis
Authors: Floros Konstantinos V. , Johnson-Berro Mia O. , Kurupi Richard , Fairchild Carter K. , Dalton Krista , Hu Bin , Puchalapalli Madhavi , Dozmorov Mikhail G. , Koblinski Jennifer E. , Olzmann James A. , Cowart Lauren A. , Faber Anthony C.
Published: June 2022

MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN-amplified neuroblastoma contributes to a large percent of pediatric cancer-related deaths. Therapy improvements for this subtype of cancer are a high priority. Ferroptosis is an iron-dependent, oxidative form of cell death that is counteracted mainly by the production of Glutathione Peroxidase 4 (GPX4), a phospholipid hydroperoxidase that is produced through the glutathione pathway. The identification of cancers that may benefit from ferroptosis inducers are just emerging. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1 (TFR1). Accumulating iron causes reactive oxygen species (ROS) production, and MYCN-amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. By performing metabolomics, we demonstrate that the transsulfuration pathway is also activated by MYCN. The increased activation of both pathways leads to cysteine accumulation that results in inhibition of lipid peroxidation. Utilizing drugs that target the main components of the glutathione and transsulfuration pathway we sensitize the MYCN neuroblastomas to ferroptotic cell death. These data provide novel insights into how MYCN alters the transcriptome in neuroblastoma to confer growth and survival advantages and simultaneously sheds light on the mechanism of action of ferroptosis inducers with potential application in other types of cancer.

Citation Format: Konstantinos V. Floros, Mia O. Johnson-Berro, Richard Kurupi, Carter K. Fairchild, Krista Dalton, Bin Hu, Madhavi Puchalapalli, Mikhail G. Dozmorov, Jennifer E. Koblinski, James A. Olzmann, Lauren A. Cowart, Anthony C. Faber. MYCN-amplified neuroblastoma is addicted to iron and vulnerable to ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 362.
Abstract 3622: DNA damage drives DNA methylation and 3D chromatin organization alterations in glioblastoma
Authors: Modrek Aram S. , Do Catherine , Zhang Zeyan , Deng Yingwen , Karp Jerome , Ezhilarasan Ravesanker , Cova Giulia , Snuderl Matija , Tsirigos Aristotelis , Skok Jane , Sulman Erik P.
Published: June 2022

In glioblastoma, treatment with radiation and chemotherapy leads to DNA-damage and most DNA breaks are faithfully repaired, but the impact on the epigenome has not been thoroughly investigated. Since epigenetic alterations in DNA-methylation can impact the binding of factors involved in chromatin folding, such as CTCF as well as transcription factors that bind regulatory elements, the downstream impact on gene regulation could lead to the emergence of treatment resistance. Here, using radiation, we show there are significant and wide-spread alterations in DNA-methylation after treating multiple glioblastoma cultures. However, it is difficult to study these alterations because breaks are introduced at different sites in each cell, resulting in irreproducible DNA methylation alterations between replicates and cell lines. To circumvent this issue, we have developed a “multi-cut” CRISPR-Cas9 DNA break system that targets 142 or 483 pre-defined loci. We find that induction of mapped genome-wide cuts reproduces a similar level of toxicity as standard doses of radiation and we characterize the kinetics of overall cellular resolution of DNA damage using immunofluorescence markers and COMET assays. In order to assess repair efficiency (i.e., deletions, translocations, complete repair), we have designed a custom panel of DNA probes that flank the 142 or 483 sites to allow for high coverage sequencing of each cut site. To understand how DNA damage may lead to local epigenetic alterations and 3D chromatin organization changes, we performed HiC (genome-wide chromosome conformation capture), before and after cut induction, and subject cultures to further downstream analysis. Our findings show significant mega-base scale alterations in chromatin contacts centered around cut sites. To understand how these alterations may contribute to treatment resistance after DNA damage, we show these alternating chromatin contacts result in new promoter and enhancer interactions, which may drive gene-expression alterations after DNA damage at nearby loci. These results provide a mechanistic view of the interplay between DNA damage, DNA methylation and genome re-organization in glioblastoma. Importantly, these studies shed light on how these alterations evolve and their effect on local gene expression and ensuing treatment resistance.

Citation Format: Aram S. Modrek, Catherine Do, Zeyan Zhang, Yingwen Deng, Jerome Karp, Ravesanker Ezhilarasan, Giulia Cova, Matija Snuderl, Aristotelis Tsirigos, Jane Skok, Erik P. Sulman. DNA damage drives DNA methylation and 3D chromatin organization alterations in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3622.
Abstract 3658: Ototoxicity of chemotherapy and radiation agents used in pediatric cancer treatment
Authors: Moore Brittney T. , Tsien Fern , Sheets Gabrielle , Umrigar Ayesha , Doss Jordan , Norman Michael , Stark Matthew , Prasad Pinki , Musso Amanda , Hicks Chindo , Otohinoyi David , Zabaleta Jovanny , Li Li
Published: June 2022

Many highly effective therapies used to treat cancer have been associated with irreversible development of detrimental neurological sequelae following the survival of childhood and adolescent cancer. While cancer treatment-induced late effects vary due to patient-related risk factors, cancer survivors who received chemotherapy or radiation may develop conductive or sensorineural hearing loss (SNHL). The purpose of this project is to determine which neuroinflammation-associated genes and biological pathways are affected by pediatric cancer treatments, and which treatment protocols are associated with increased ototoxicity. We hypothesize that cancer treatment causes abnormal gene expression of neuroinflammation and deafness-associated genes. The hypothesis will be tested with two specific aims. Aim I is to perform a retrospective review of diagnostic audiograms from pediatric cancer survivors enrolled in the Treatment After Cancer and Late Effects Center at Children’s Hospital New Orleans, Louisiana (CHNOLA). Aim II is to perform Nanostring neuroinflammation gene expression analysis followed by Ingenuity Pathway Analysis (IPA) of brain autopsy specimens from deceased pediatric patients who have previously undergone cancer treatment (Pathology Department, CHNOLA). Preliminary results revealed abnormal upregulation or downregulation of key genes, some of which are related to hearing loss and cochlear neuron degeneration when compared to age-matched controls (e.g., GJA1 and CASP3). The present study may provide information regarding which cancer treatment agents are ototoxic and reveal the candidate risk genes and pathways that contribute to auditory late effects. The long term goal of this project is to identify the needs of cancer survivors who are affected by treatment-induced hearing loss and provide them and their families with access to educational materials, medical resources, and social support to increase their health-related quality of life.

Citation Format: Brittney T. Moore, Fern Tsien, Gabrielle Sheets, Ayesha Umrigar, Jordan Doss, Michael Norman, Matthew Stark, Pinki Prasad, Amanda Musso, Chindo Hicks, David Otohinoyi, Jovanny Zabaleta, Li Li. Ototoxicity of chemotherapy and radiation agents used in pediatric cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3658.