bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1224: UMIBB: A novel nonparametric Bayesian method improves robustness and sensitivity of analysis in pooled CRISPR-Cas9 screens leveraging unique molecular identifiers
Authors: Choi Ashley , Meier Samuel , Fenoglio Silvia , Feng Tianshu , Engel Justin , Shen Binzhang , Liu Shangtao , Teng Teng , Khendu Tenzing , Huang Alan , Andersen Jannik , Pan Xuewen , Yu Yi
Published: June 2022

CRISPR-based functional genomics screens are a powerful tool for identifying synthetically lethal cancer drug targets. Current strategies for analyzing pooled CRISPR screens usually rely on signals from single guide RNAs (sgRNA) with differential relative abundance between two experimental conditions. However, conventional approaches are susceptible to false positives and false negatives driven by outlier cell clones, since the sgRNA abundance does not account for the heterogeneous phenotypes resulting from different editing outcomes of the same sgRNA. To overcome this, we added DNA barcodes to each sgRNA to create unique molecular identifiers (UMIs) for CRISPR libraries and developed a companion analytical platform that enables robust, industry-scale CRISPR screens. Here, we present UMIBB, a novel nonparametric Bayesian approach for analyzing UMI-CRISPR data. The number of UMIs with normalized count depletion or enrichment compared to the control experimental condition for each sgRNA is modeled by a beta-binomial distribution. The gene level statistics are derived by combining z-scores of the sgRNAs level posterior probabilities weighted by the number of UMIs in each sgRNA. This approach minimizes the impact of outlier cell clones on statistics and prioritizes genes with consistent count differentials across multiple UMIs in each gene. To assess the power of UMIBB, we benchmarked it on a low coverage (200X) genome-scale negative-selection screen, comparing with results from a high coverage (1000X) screen. These screens were conducted on KRAS mutant cancer celllines (A549) treated with trametinib or vehicle control. Despite the high noise level usually observed in lower coverage screens, our method was able to uncover most of the validated sensitizer genes for trametinib and achieved the highest sensitivity compared to conventional methods. Furthermore, we applied UMIBB on a genome-scale positive-selection screen and successfully identified novel genes (RAD18 and UBE2K) as key mediators of USP1 dependency in BRCA1 mutant cell lines. Our studies demonstrate that UMIBB is highly robust against false positives due to clonal heterogeneity and is more likely to identify true genetic interactions.

Citation Format: Ashley Choi, Samuel Meier, Silvia Fenoglio, Tianshu Feng, Justin Engel, Binzhang Shen, Shangtao Liu, Teng Teng, Tenzing Khendu, Alan Huang, Jannik Andersen, Xuewen Pan, Yi Yu. UMIBB: A novel nonparametric Bayesian method improves robustness and sensitivity of analysis in pooled CRISPR-Cas9 screens leveraging unique molecular identifiers [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 1224.
Abstract 1288: STING agonism enhances anti-tumor immune responses and therapeutic efficacy of PARP inhibition in BRCA-associated breast cancer
Authors: Jadhav Heta , Pantelidou Constantia , Kothari Aditi , Liu Renyan , Wulf Gerburg , Guerriero Jennifer L. , Shapiro Geoffrey I.
Published: June 2022

Poly (ADP-ribose) polymerase (PARP) inhibitors exert their efficacy by inducing synthetic lethal effects, as well as cGAS/STING-mediated immune responses in BRCA- and other homologous recombination repair-deficient cancer cells. Here, we investigated whether the immunologic and therapeutic effects of PARP inhibition in BRCA-deficient breast cancer models could be augmented by synthetic cyclic dinucleotide agonists of STING. In in vitro experiments, combined olaparib-mediated PARP inhibition and ADU-S100-mediated STING agonism induced a greater degree of STING pathway activation and proinflammatory cytokine production compared to monotherapies in BRCA1-deficient human MDA-MB-436 cells and mouse triple-negative breast cancer cell lines derived from a genetically engineered model of BRCA-deficient breast cancer. In vivo experiments were conducted in mice bearing completely established tumors derived from the genetically engineered mouse model expanded syngeneically. In this model, the combination also induced an improved immune response compared to either monotherapy alone, evidenced by a greater degree of cytotoxic T cell recruitment and activation, and enhanced dendritic cell activation and antigen presentation. Nanostring mRNA analysis indicated that combinatorial effects were the result of augmented interferon signaling and antigen processing, as well as heightened leukocyte and dendritic cell functions. The combination markedly improved anti-tumor efficacy in vivo compared to monotherapy treatment, with evidence of complete tumor clearance and prolongation of survival. Finally, in mice treated with the combination, tumors could not be re-established upon rechallenge, indicating the induction of immunologic memory. These results support the development of combined PARP inhibition and STING agonism in BRCA-associated breast cancer.

Citation Format: Heta Jadhav, Constantia Pantelidou, Aditi Kothari, Renyan Liu, Gerburg Wulf, Jennifer L. Guerriero, Geoffrey I. Shapiro. STING agonism enhances anti-tumor immune responses and therapeutic efficacy of PARP inhibition in BRCA-associated 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 1288.
Abstract 1302: Priming tumors using pH sensitizers potentiates the anti-tumor T cell response induced by immune checkpoint blockade therapy
Authors: Chin Renee , de la Cerda Jorge , Schuler William , Pagel Mark D.
Published: June 2022

While immune checkpoint blockade (ICB) treatments such as nivolumab and ipilimumab have spelled hope for many cancer patients, not all patients benefit from this form of therapy. One mechanism by which tumors suppress the anti-tumor T cell activity that results from ICB is through decreasing extracellular tumor pH (pHe), a function inherent to tumor cells due to the Warburg effect. We thus hypothesize that increasing pHe prior to ICB therapy potentiates the T cell-mediated anti-tumor effect of ICB treatment. To undergo this study, we first screened a panel of six inhibitors termed pH sensitizers, and found that esomeprazole significantly reduced the proton efflux rate (PER) of 4T1, but not B16-F10 cells. Furthermore, esomeprazole did not significantly reduce cell viability of either 4T1 or B16-F10 cells, and did not significantly inhibit T cell activity. Moving forward, our next objectives are to investigate the effects of esomeprazole and the combination of esomeprazole and ICB on tumor-bearing mice. Assessment of the tumor growth in response to esomeprazole and ICB and ex vivo interrogation of the immune cell infiltrate into the tumors will elucidate the efficacy of the treatment combination in terms of tumor control and anti-tumor immunity. Modification of pHe by esomeprazole will be examined by acidoCEST MRI, which will also provide insights as to the ideal pHe parameters for optimal ICB therapy in patients.

Citation Format: Renee Chin, Jorge de la Cerda, William Schuler, Mark D. Pagel. Priming tumors using pH sensitizers potentiates the anti-tumor T cell response induced by immune checkpoint blockade therapy [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 1302.
Abstract 1318: Interleukin-1 promotes enhanced functional expression of dual oxidase 2 in human pancreatic cancer cells
Authors: Mallick David J. , Wu Yongzhong , Konaté Mariam M. , Diebold Becky A. , Antony Smitha , Meitzler Jennifer L. , Jiang Guojian , Lu Jiamo , Roy Krishnendu , Doroshow James H.
Published: June 2022

Pancreatic inflammation contributes to increased risk of pancreatic adenocarcinoma (PDAC) development through the release of pro-inflammatory cytokines and generation of reactive oxygen species (ROS). Interleukin-1 (IL-1) is a major upstream pro-inflammatory cytokine secreted by malignant or microenvironmental cells that supports pancreatic tumorigenesis, invasiveness, and tumor heterogeneity. Previously, our laboratory has established that dual oxidase 2 (DUOX2), one of seven NADPH oxidase (NOX) family members, plays a critical role in H2O2-mediated host defense and chronic inflammation in the gastrointestinal tract. DUOX2 is upregulated and expressed along with its maturation factor, DUOXA2, in several human pancreatic cancer cell lines following exposure to various pro-inflammatory cytokines, including IFN-γ, IL-4, and IL-17A. Using a panel of PDAC cells lines, we report the induction of DUOX2/DUOXA2 mRNA and protein expression by both IL-1α and IL-1ß, two major agonists of IL-1. In BxPC-3, CFPAC-1 and Hs 766T (HTB-134) cells, prolonged treatment with IL-1α or IL-1ß results in transient activation of STAT1 and NF-kB signaling followed by prolonged induction of DUOX2/DUOXA2 mRNA and protein expression. Stimulation by IL-1α or IL-1ß results in a specific increase in the expression of DUOX2/DUOXA2; increased expression of other NOX family members was not observed. Activation of canonical IL-1 signaling in our PDAC cell lines was supported by the observation of IL-1-related upregulation of IL-8 (CXCL8) mRNA, a chemokine essential for neutrophil recruitment and a potent promoter of angiogenesis, in IL-1-treated PDAC cells. Moreover, when the anti-inflammatory compound anakinra (an IL-1R antagonist) was co-administered in vitro with IL-1, cytokine-induced DUOX2/DUOXA2 and CXCL8 induction was significantly suppressed. Finally, xenografts established with BxPC-3 cells demonstrated slower growth and significant downregulation of IL-1ß and DUOX2 when the immunosuppressive corticosteroid dexamethasone was administered. Given our previous findings that DUOX2 is highly expressed in the pancreas during episodes of chronic pancreatitis or pancreatic cancer, and contributes significantly to extracellular ROS formation, these studies suggest that IL-1 may play a role in promoting inflammation and tumorigenesis in pancreatic malignancies through upregulation of DUOX2.

Citation Format: David J. Mallick, Yongzhong Wu, Mariam M. Konaté, Becky A. Diebold, Smitha Antony, Jennifer L. Meitzler, Guojian Jiang, Jiamo Lu, Krishnendu Roy, James H. Doroshow. Interleukin-1 promotes enhanced functional expression of dual oxidase 2 in human pancreatic cancer cells [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 1318.
Abstract 1326: Loss of p53 sensitizes tumor cells to immune checkpoint blockade therapy via upregulation of IL-33
Authors: Gao David Shihong , Li Yang , Shoush Jason , Sun Runzi , Lu Binfeng
Published: June 2022

Recent studies suggest p53 contributes to poor survival in the context of immune checkpoint blockade (ICB). Mutations to p53 significantly improve the response rate to PD-1 ICB in lung cancer patients. Tumor-derived IL-33 is required for the anti-tumor immune response and efficacy of ICB. However, the relationship between p53 and IL-33 during ICB is unknown. In this study, we characterize the role of the p53/IL-33 axis in regulating the response of the tumor microenvironment to ICB. We use CRISPR-Cas9 to delete Trp53 from murine MC38 colon adenocarcinoma. Deletion of Trp53 and PD-1 ICB synergistically inhibits tumor growth. Il33 is upregulated in the Trp53-deficient and treated tumor cells, and its expression increases with time and response to treatment. Cd4+ and Cd8+ T cell infiltration increases adjacent to IL-33 expressing tumor cells, while Treg infiltration is decreased. Simultaneous deletion of Il33 in MC38 tumors reverses the efficacy PD-1 ICB. Trp53-deficient MC38 in ST2-/- (IL-33 receptor) mice also show no response to PD-1 ICB. Our findings depict a novel mechanism by which the loss of p53 in tumors treated with ICB unleashes the expression of tumoral IL-33 and induces downstream signaling. p53 mutations may be a double-edged sword for cancer, i.e. the loss of the tumor suppressor initially facilitates tumorigenesis, but eventual buildup of mutations and activation of damage pathways, such as NF-kB, leads to upregulation of danger signals in the tumor. These danger signals, such as IL-33, mediate the anti-tumor effect of ICB.

Citation Format: David Shihong Gao, Yang Li, Jason Shoush, Runzi Sun, Binfeng Lu. Loss of p53 sensitizes tumor cells to immune checkpoint blockade therapy via upregulation of IL-33 [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 1326.
Abstract 1342: Evaluating the role of antibody in pancreatic tumorigenesis
Authors: Foote Jeremy
Published: June 2022

Introduction: Infiltrating pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of 10%. The PDAC tumor microenvironment (TME) is characterized by a fibroinflammatory response mediated by a complex mix of cancer-associated fibroblast (CAF) subsets resulting significant deposition of extracellular matrix (ECM), poor vascularization, T cell exclusion, and recruitment of immune-suppressive myeloid cells. Collectively, these factors contribute to a resistance to aggressive chemotherapeutics and failure of checkpoint inhibitors. Preclinical studies as well as clinical efforts designed to deplete the dense tumor stroma or ECM have been successful, but unexpectedly accelerated tumor progression underscoring critical gaps in our understanding of the dynamics between tumor, stromal elements, and infiltrating immune cell subsets.

Rationale: In some cancer subtypes, B cells have been shown to suppress cancer progression through secretion of antibodies targeted to aberrantly expressed self-, stress-associated-, or tumor-specific antigens on neoplastic cells. A considerable body of literature also indicates that B cells may play a role in promotion of pancreatic tumor progression, however, the role of antibodies in PDAC progression is unclear. The major objective of our studies is to understand the significance of anti-tumor antibody responses in pancreatic tumorigenesis.

Methods: To address this question, we crossed the spontaneous mouse model of pancreatic cancer where Pdx1 drives Cre-mediated activation of a mutant KrasG12D allele on a heterozygous Tp53+/FL mutant background (KPC-WT mice) with animals that retain B cells that lack the ability to secrete antibody of any immunoglobulin (Ig) isotype (KPC-μS-AID mice).

Results: Kaplan-Meier survival studies demonstrate that loss of all circulating antibody accelerates progression to lethal PDAC in KPC-μS-AID versus KPC-WT mice (median survival of 3- versus 5-months, respectively), with increased incidence of lung and liver metastases. Further, loss of circulating antibodies increased the density of intratumoral neutrophils, reduced numbers of podoplanin+ CAFs and reduced ECM density. IgG subclass antibodies were localized at higher densities within stroma/ECM in pancreata from both mice and surgically resected tumor specimens from human PDAC patients. Loss of AID-dependent class switched antibodies similarly accelerated lethal PDAC in KPC-AID versus KPC-WT mice.

Conclusions: These findings implicate class-switched antibodies in delaying pancreatic tumor progression through potential regulation of intratumoral neutrophil density and ECM production within the PDAC TME.

Citation Format: Jeremy Foote. Evaluating the role of antibody in pancreatic tumorigenesis [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 1342.
Abstract 1356: In vivo epigenetic CRISPR screen identifies PRMT5 as an immune suppressor in pancreatic cancer
Authors: Hu Hai , Deng Jiehui , Li Fei , wang wei , Khodadadi-Jamayran Alireza
Published: June 2022

Pancreatic ductal adenocarcinoma (PDA) is a devastating disease in which the mortality rate approaches the incidence rate. Desmoplasia is a hallmark of PDA, which creates a mechanical barrier surrounding the tumor cells, prevents appropriate vascularization and leads to pool immune infiltration. PDA exhibits a “cold” tumor microenvironment characterized by a prominent myeloid cell infiltration, which is extremely immune suppressive. Therefore, PDA has been demonstrated to be resistant to immunotherapy. Indeed, single-agent immune checkpoint inhibitor therapy has failed in clinical trials for PDA patients. Targeting epigenetic modification is an attractive approach to enable the response to immunotherapy in multiple solid tumor models. To identify epigenetic modulators as potential novel immunotherapeutic targets in PDA, we performed an orthotopic in vivo epigenetic CRISPR screen. Prmt5 was screened out as one of the top candidates. Further validation studies showed that Prmt5 knockdown (KD) alone or in combination with anti PD-1 treatment significantly inhibited the tumor progression. PRMT5 shRNA in combination with PD1 blockade can dramatically altered the tumor immune microenvironment (TME) with influx of effector CD8 T cells and changes in the macrophage/myeloid populations. Collectively, our work describes PRMT5 as an attracting target which governs tumor immunity in PDA.

Citation Format: Hai Hu, Jiehui Deng, Fei Li, wei wang, Alireza Khodadadi-Jamayran. In vivo epigenetic CRISPR screen identifies PRMT5 as an immune suppressor in pancreatic 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 1356.
Abstract 1371: Rho-GEF hampers CD8+T cells-mediated immunosurveillance by reducing antigen presentation
Authors: Eyvani Haniyeh , Zhang Xinna
Published: June 2022

Immune response is a critical anti-tumor response to overcome tumorigenesis. In line with this, immune checkpoint blockade shows tremendous anti-tumor efficacy by enhancing cytotoxicity of CD8+ cytotoxic T lymphocytes (CTLs). However, cancer cells develop different mechanisms of immune evasion, among which suppression of immune recognition of somatic mutations via tumor antigen is prevalent. In fact, 65% to 90% of the cancers show reduced MHC-I antigen presentation. There is, nonetheless, little achievement toward the therapeutic approaches to restore antigen presentation on tumor cells. Colorectal cancer (CRC), the second deadliest cancer in U.S., shows increasing incidence and mortality rates over the past years. Standard conventional CRC treatments, surgery, chemotherapy and radiotherapy, often lead to many side effects due to their non-specificity and cytotoxicity. While immunotherapy is very promising, only a small population of CRC patients respond to the current immune checkpoint inhibitors. In this study, our objective was to harness a novel immunological approach to target CRC tumors. We developed a bioinformatics tool to create a pool of genes whose expression negatively correlates with cytotoxicity of CD8+ CTLs in the tumor microenvironment of CRC patients. Given the central role of antigen presentation in activating CD8+ CTLs, we further screened these genes based on the scope of their effects on antigen presentation. To this end, we developed stable clones of OVA expressing MC38 cells (MC38OVA) where OVA-derived peptide, SIINFEKL, will be bound to MHC-I complex for cell surface presentation. 4 genes with highest capacity to alter MHC-I cell surface levels were identified. Our consequent in vivo experiments and in vitro cytotoxicity assays identified that “Rho-GEF” gene was the top actionable target with striking suppressive effects on tumor growth. TCGA database analysis showed Rho-GEF is upregulated in 22% of CRC patients accompanied by a poor prognosis. Rho-GEF knockdown (KD) significantly inhibited tumor growth in immunocompetent mice whereas it only showed minor effects on immunocompromised mice, suggesting that the Rho-GEF may modulate tumor growth by regulating the interaction of tumor cells with non-tumor cells such as immune cells. Consistent with the major contribution of antigen presentation in promoting CD8+ CTLs activity, TNFα, IFNγ, and GZMB levels in CD8+ T cells isolated from Rho-GEF KD MC38-derived tumors showed a significant increase compared to the wild type tumors. Taken together, we introduced Rho-GEF as a potential clinical target involved in CRC immune evasion by modulating antigen presentation and CD8+ CTLs anti-tumor responses.

Citation Format: Haniyeh Eyvani, Xinna Zhang. Rho-GEF hampers CD8+T cells-mediated immunosurveillance by reducing antigen presentation [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 1371.
Abstract 1384: Immunopeptidome analysis of mouse MHC Class I
Authors: Nyayapathy Seeta , Jones Richard , Ford Michael , Pisano Michael , Rumble Julie M.
Published: June 2022

MHC class I is required for antigen presentation to CD8 T cells. These cell-surface proteins are heterodimers consisting of a variable alpha chain associated with β2 microglobulin and are expressed on nucleated cells at varying levels. The antigen specificity of the interaction between MHC, peptide and T cell receptor can be exploited in development of personalized cancer vaccines and other immunotherapies against cancer. MHC sequences are species-specific and there are many different haplotypes of MHC class I. For this reason, the antibodies used for immunoprecipitation of MHC complexes are a critical component of the workflow for identification of antigens presented by MHC. Subclasses of mouse MHC class I are divided into classical (H-2D, H-2K and H-2L) and non-classical (H-2Q, H-2M and H-2T), and haplotypes of each are known for homozygous inbred laboratory strains of mice. One common strain is C57BL/6, which expresses H2-Db and H2-Kb classical MHC molecules. Antibody clone M1/42, which reacts with H-2 expressed on nucleated cells from mice of a, b, d, j, k, s and u haplotypes, is commonly referred to as a pan-MHC class I antibody. Antibody clones Y-3 and HB-27 recognize H2-Kb and H2-Db haplotypes, respectively. In this study we aimed to compare different antibodies to mouse MHC class I complexes for their efficiency of recovery of associated peptides. Input was murine EL-4 cells which are T lymphoblast cells derived from a C57BL/6 background. We performed immunoprecipitation with different antibodies either singly or sequentially to enrich MHC class I, followed by gentle elution of associated peptides and finally peptide analysis by mass spectrometry. Each immunoprecipitation workflow yielded a different (but overlapping) cohort of peptides, suggesting optimal methodologies for analyses of immunopeptidomes from common laboratory mouse strains.

Citation Format: Seeta Nyayapathy, Richard Jones, Michael Ford, Michael Pisano, Julie M. Rumble. Immunopeptidome analysis of mouse MHC Class I [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 1384.
Abstract 1400: AS-1763: a highly potent, noncovalent and next generation BTK inhibitor for the treatment of patients with B-cell malignancies having C481S mutation in BTK
Authors: Kawahata Wataru , Asami Tokiko , Kiyoi Takao , Irie Takayuki , Kashimoto Shigeki , Furuichi Hatsuo , Sawa Masaaki
Published: June 2022

Introduction: Bruton's tyrosine kinase (BTK) has been recently recognized as a validated drug target for the treatment of B-cell malignances. The emergence of clinical resistance to the first-generation covalent BTK inhibitors is, however, becoming a serious concern. Patients are reported to develop resistance during the treatment due to substitution of cysteine residue at position 481 with serine (C481S mutation) in BTK, which prevents the covalent binding of the first-generation irreversible BTK inhibitors, resulting in the loss of inhibitory activities. Therefore, there is a high unmet medical need for new therapeutic approaches to overcome the BTK C481S-mediated resistance.

Material and methods: Compounds were tested in biochemical assays using recombinant human wildtype (WT) and C481S mutant BTKs. Cellular activity of AS-1763 was evaluated in human diffuse large B cell lymphoma (DLBCL) cell lines, OCI-Ly10 and its BTK[C481S] knock-in cells. The in vivo antitumor activity of AS-1763 was assessed in two tumor xenograft mouse models bearing WT or BTK [C481S] knock-in OCI-Ly10 cells.

Results: We have discovered AS-1763 as an orally available, highly potent, selective and reversible BTK inhibitor. AS-1763 showed potent inhibitory activities for WT BTK (an activated form, BTK[A]) and the C481S mutant with sub-nanomolar IC50s (IC50 = 0.85 and 0.99 nM for BTK[A] and BTK[C481S], respectively). In cellular assays, AS-1763 exhibited strong antiproliferative activities for WT and BTK [C481S] knock-in OCI-Ly10 cells. AS-1763 demonstrated significant tumor growth inhibition and tumor regression in WT as well as BTK[C481S] knock-in OCI-Ly10 xenograft model.

Conclusions: The preclinical data support the clinical development of AS-1763 in patients with B-cell malignancies both having WT and C481S mutation in BTK. The first-in-human Phase 1 clinical trial of AS-1763 in healthy volunteers (EudraCT 2020-005599-37) is currently ongoing, and the Phase 1b study in patients is expected to begin in 2022.

Citation Format: Wataru Kawahata, Tokiko Asami, Takao Kiyoi, Takayuki Irie, Shigeki Kashimoto, Hatsuo Furuichi, Masaaki Sawa. AS-1763: a highly potent, noncovalent and next generation BTK inhibitor for the treatment of patients with B-cell malignancies having C481S mutation in BTK [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 1400.
Abstract 1422: A novel agonist of CD137 shows immunoprevention efficacy against various tumors by invoking an innate immune surveillance mechanism
Authors: Shirwan Haval , Batra Lalit , Li Qingsheng , Malik Mohammad T. , Shrestha Pradeep , Egilmez Nejat , Yolcu Esma S.
Published: June 2022

Introduction: Immune checkpoints have been the focus of intense research due to their critical roles in regulating immune responses. The CD137 is an immune checkpoint stimulator has pleiotropic immune functions along with its canonical role in driving CD8+ T cell responses. Using a novel form of its natural ligand (SA-4-1BBL) as a single agent, we recently showed its robust cancer immunoprevention efficacy against various transplantable tumor types in mice. In this study, we report its efficacy in a spontaneous tumor model and elucidate the mechanistic basis of prevention.

Study design: Mice were treated subcutaneously with SA-4-1BBL protein twice, two weeks apart, and challenged with various tumor types 1-14 weeks later. Animals were monitored for tumor growth. The LSL-KrasG12D mice, as a model of spontaneous lung cancer, were intranasally infected with 2.5 × 107 PFUs of adenoviral-cre to induce cancer. Animals were treated with SA-4-1BBL twice, two weeks apart, pre or post-infection with adeno and euthanized at various times to collect the lung and lymphoid tissues for histopathology and flow cytometry.

Results: Pretreatment with SA-4-1BBL was effective in preventing the growth of various transplantable tumors, B16-F10 melanoma, 3LL lung cancer, A20 lymphoma, 4T1 breast cancer, in different mouse strains. Treatment with SA-4-1BBL induced a window of protection against tumors that lasted over 14 weeks. SA-4-1BBL treatment significantly reduced the lung tumor burden in LSL-KrasG12D mice that was associated with increased absolute numbers of CD137+CD4+CD44hi T cells and CD137+NKG2D+NK cells expressing IFN-γ and TNF-α. A blocking antibody to NKG2D reversed the immunopreventive efficacy of SA-4-1BBL in the 3LL and B16-F10 tumor models, providing direct evidence for the role of this receptor in cancer immunoprevention. Gamma/delta T cells did not play a role in the cancer prevention efficacy of SA-4-1BBL as mice lacking these cells showed protection.

Conclusions: These results demonstrate that SA-4-1BBL invokes an innate immune surveillance mechanism orchestrated by cross-communication between memory-like CD4+ T cells and NK cells with long-term protection against various tumor types. Funded in parts by NIH R41CA199956, T32HL134644, NCI R25CA134283, and Kentucky KSTC-184-512-16-237 awards.

Citation Format: Haval Shirwan, Lalit Batra, Qingsheng Li, Mohammad T. Malik, Pradeep Shrestha, Nejat Egilmez, Esma S. Yolcu. A novel agonist of CD137 shows immunoprevention efficacy against various tumors by invoking an innate immune surveillance mechanism [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 1422.
Abstract 1423: NKX3.1 expression predicts response to metformin intervention in prostate cancer
Authors: Papachristodoulou Alexandros , Dutta Aditya , Abate-Shen Cory
Published: June 2022

The goal of precision prevention is to tailor interventions based on individualized patient cancer risk and likelihood of progression. This approach is particularly relevant for prostate cancer since it is characteristically indolent, and thus delaying disease progression can be highly advantageous for patients. Therefore, identifying molecular features predictive of cancer progression and response to specific interventions is vital to improve precision prevention in prostate cancer. In this regard, expression levels and localization of the tissue-specific homeobox protein, NKX3.1, helps distinguish men with high risk for developing more aggressive prostate cancer. Our recent study revealed a novel function for NKX3.1 in mitochondria, where it regulates aberrant mitochondrial oxidative stress to suppress prostate cancer initiation. Lower NKX3.1 expression levels at gene and protein levels are associated with poor outcome and higher risk for aggressive prostate cancer. We are now investigating the role of NKX3.1 as a prognostic marker and potential target for prostate cancer precision prevention interventions. Our new preliminary data indicate a role for metformin as a preventative intervention in contexts of reduced NKX3.1. Specifically, we have shown that metformin reduces oxidative stress and suppresses cancer initiation in the prostates of Nkx3.1 mutant mice. Moreover, studies in human prostate cancer cells as well as human prostate cancer organotypic assays revealed that metformin can indeed, restore mitochondrial function and inhibit tumorigenicity in the absence of NKX3.1. Although men on active surveillance for prostate cancer may benefit from metformin intervention, it has been unclear whether metformin is effective in delaying disease progression and, if so, whether specific patients are more likely to benefit. Our new data implicate NKX3.1 status as a predictor of response to metformin, suggesting that it can stratify high-risk prostate cancer patients most likely to benefit from such intervention during active surveillance.

Citation Format: Alexandros Papachristodoulou, Aditya Dutta, Cory Abate-Shen. NKX3.1 expression predicts response to metformin intervention in prostate 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 1423.
Abstract 1431: ADO contributes to tumour initiating phenotypes
Authors: Lee Sandy Che-Eun Serena , Pyo Andrea Hye An , Koritzinsky Marianne
Published: June 2022

2-aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a role in both metabolism and protein stability. ADO directly metabolizes cysteamine to produce hypotaurine and taurine in mammals. ADO has also been recently identified to promote oxygen dependent stability of a subset of substrates involved in the N-degron pathway in mammals (IL32, RGS4 and RGS5). The ability of ADO to target protein stability of signaling molecules suggests that it may have the potential to transduce rapid responses to hypoxia and affect tumour initiation and progression phenotypes. Here, we have successfully knocked down and knocked out ADO using two independent siRNAs and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system, respectively. We have assessed proliferation and migration through the Incucyte® ZOOM system by imaging cell confluency over time. Survival was assessed through a clonogenic assay. siRNA mediated knockdown of ADO in cervical (HeLa and SiHa), pancreatic (Panc1 and Capan2) and liver (SNU499 and Huh6) cancer cell lines drastically reduced proliferation, survival, and migration in normoxia. These results were also replicated in hypoxia (0.2% O2) across all 6 cell lines. Out of the 6 cell lines, the liver cancer cell lines were most drastically affected by the knockdown of ADO. This phenotype was replicated in the ADO KO cell lines. Taken together, these data suggest that expression of ADO may contribute to phenotypes that induce aggressive tumour phenotypes by targeting the stability of specific proteins and altering cellular metabolism in mammals.

Citation Format: Sandy Che-Eun Serena Lee, Andrea Hye An Pyo, Marianne Koritzinsky. ADO contributes to tumour initiating phenotypes [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 1431.
Abstract 1464: Zaniya Mark
Authors: Mark Zaniya A. , Li Guoliang , Matusik Robert , Chen Zhenbang
Published: June 2022

Prostate cancer (PCa) is one of the most common types of cancers diagnosed in American men. Moreover, PCa malignancy disproportionally strikes more on African American (AA) men than any other ethnic groups including Caucasian American (CA) men. Forkhead Box A1 (FOXA1) is frequently mutated in hormone receptor-driven cancers including PCa. FOXA1 mutation rates in AA PCa specimens are 3-fold higher than that in CA PCa. By annotating the FOXA1 mutation landscape two hotspots were defined in AA PCa patients located in the forkhead domain: D226N and R219C. FOXA1 expression and functions are regulated by lysine [K]-specific demethylase 5B (KDM5B). KDM5B contributes to the activation of signaling pathways that promote cancer progression. Here in this project, we will focus on the mechanistic role of FOXA1 dysregulation and its interaction with KDM5B and AR on PCa progression in AA vs CA PCa cells. Therefore, we hypothesize that the dysregulation of FOXA1 plays an essential role in the progression of PCa by altering chromatin remodeling and AR interaction, and FOXA1 mutation promotes more aggressive malignancies in AA PCa cells. In order to investigate the biological function and regulation of FOXA1 in PCa cells we first need to knockout (KO) the endogenous expression using CRISPR technology and then knockin (KI) the unique mutants R219C and D226N. These in vitro studies of FOXA1 mutations found in AA PCa will be critical to determine the biological functions of these mutants for PCa progression. The findings from this study will provide valuable insights into the contributions of the FOXA1 regulation and will add to our knowledge base for the potential development of a novel therapeutic strategy to reduce PCa disparities between AA and CA populations. This project was supported, in part, by MVTCP Cancer Partnership Grant U54 CA163069.

Citation Format: Zaniya A. Mark, Guoliang Li, Robert Matusik, Zhenbang Chen. Zaniya Mark [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 1464.
Abstract 1470: Characterization of ER receptor transcription complex using super resolution STORM microscopy
Authors: Akhshi Tara , Brown Myles
Published: June 2022

Steroid/thyroid hormone receptors are ligand-dependent transcription factors (TFs) that play fundamental roles in cell development and homeostasis. They bind to their DNA response elements and modulate the transcription of target genes via interaction with co-regulators termed co-activators or co-repressors. Estrogen Receptor α (ERα) is an important member of this family which is expressed in in hormone receptor positive (ER+) breast cancer, one of the leading causes of death among women. Current therapeutic options involve endocrine therapies, however despite its effectiveness, a significant number of patients eventually experience resistance to these treatments, leading to disease recurrence and relapse. One of the major hurdles to combat resistant cells, is lack of comprehensive information about ERα transcriptional complex activity. While biochemical assays have provided significant information on interaction with its coregulators, it is not clear how the dynamic between these transpire. This is while ERα is a highly dynamic protein with a fast rate of interaction with coregulators and cycling between chromatin, nucleus, and cytosol. Consequently, it is critical to imply techniques that allow direct visualization of ERα activity and gain spatiotemporal information on its regulation. One of the major limitations to conduct such studies was due to the resolution limits inherited with light microscopes. However, the emerge of super resolution microscopes, offers a promising tool to overcome this challenge. Here, we imply super resolution microscopy to examine the mechanism of ERα transcriptional activity in the presence of estrogen as well as compounds that are currently used for endocrine therapy, Tamoxifen and Fulvestrant. We determine how each of these compounds influence ERα interaction with its co-activators (P300, SRC-3), and its co-inhibitor (NCoRI), and how these interactions modulate chromatin activity. Obtaining such information of ERα dynamics is critical to map the mechanism of its activity under different treatments, and thus is key to overcome some of the hurdles faced in endocrine therapy.

Citation Format: Tara Akhshi, Myles Brown. Characterization of ER receptor transcription complex using super resolution STORM microscopy [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 1470.
Abstract 1494: 5-methyltetrahydrofolate (5-MTHF) as a superior ligand for delivery of small RNAs for cancer therapy
Authors: Kasireddy Sudarsan R. , Abdelaal Ahmed M. , Low Philip S. , Kasinski Andrea L.
Published: June 2022

MicroRNAs (miRNAs) are small noncoding RNAs that regulate a wide array of genes. Because miRNA downregulate translation proteins associated with cancer progression, miRNA-based therapeutics have great potential as anticancer drugs. Nonetheless, the therapeutic potential of miRNA replacement is limited due to lack of safe and efficient delivery vehicles, and inability to target miRNAs to the intended cells/tissues. Thus, identifying molecules that are able to deliver therapeutic miRNA is of critical need. This could be achieved by targeting receptors that are overexpressed on target tumor cells. For example, the folate receptor (FR) is overexpressed on various tumor types including breast, lung, ovarian and brain tumors. Our laboratory has previously developed a unique strategy that directly links a miRNA mimic to the high affinity FR ligand, folate (FolamiRs). In the present work, to further improve the potency of FolamiRs, we introduce the reduced folate metabolite, 5-methyltetrahydrofolate (5-MTHF) that maintains nanomolar affinity for the FR at pH 7, but disengages from the FR at a significantly higher pH than that of folate, which is important when including an endosomal escape agent that typically disrupts endosomal acidification. We synthesize and conjugate 5-MTHF ligand to the potent tumor suppressor miRNA-34a (5-MTHF-miR-34a). We show that 5-MTHF-miR-34a uptake is specific to the cells overexpressing FR and represses target gene expression. Our data supports the use of 5-MTHF in combination with the endosomal escape agent, nigericin, to achieve robust cytosolic localization of therapeutically relevant miRNAs such as miR-34a. This work provides the foundation for future studies using 5-MTHF- conjugated therapeutic agents for targeting additional FR expressing cells, including myeloid derived suppressor cells (MDSCs) and macrophages. Collectively these studies will pave the way for developing 5-MTHF-based small RNA conjugates for treating FR-expressing cancers. The details of these intriguing results will be discussed in the poster.

Citation Format: Sudarsan R. Kasireddy, Ahmed M. Abdelaal, Philip S. Low, Andrea L. Kasinski. 5-methyltetrahydrofolate (5-MTHF) as a superior ligand for delivery of small RNAs for cancer therapy [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 1494.
Abstract 1509: Genome-wide CRISPR/Cas9 library screening identified THRAP3 as a critical driver for resistance to ATR inhibitor in sarcomas revealing synthetic lethality with ATM inhibition
Authors: Spalato Mariella , Laroche-Clary Audrey , Chaire Vanessa , Italiano Antoine
Published: June 2022

Background: ATR plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. We have previously demo started that ATR inhibition is associated with anti-tumor activity in pre-clinical models of soft-tissue sarcomas (STS) (Laroche et al., 2020). We decided to investigate the mechanisms of resistance to ATR inhibition in sarcomas.

Methods: We used a CRISPRa was to identify genes that mediate resistance to AZD6738 in STS cells. The anti-tumor activity of AZD6738 (ATR inhibitor) and AZD0156 (ATM inhibitor) was investigated in a panel for 10 STS cell lines and two patient-derived xenografts (PDX) established at Institut Bergonié (Bordeaux, France).

Results: We used a CRISPR approach in a STS cell line resistant to AZD6738 and we made a negative-selection screen, to identify genes whose silencing conferred sensitivity to low doses of ATR inhibition. Among these genes, we identified THRAP3 which encodes a protein phosphorylated in response to DNA damage and primarily by ATR. THRAP3 is involved in the maturation and further export of transcripts encoding the ATM and its depletion results in deficient processing of transcripts encoding the ATM kinase. We validate the result of our CRISPR analysis by demonstrating that selective THRAP3 inhibition reduce significantly ATRi IC50. Given the role of role of THRAP3 on the regulation of ATM activity, we decided to investigate the potential synergy between ATR and ATM inhibition in vivo and in vitro. AZD6738 and AZD0156 displayed a synergistic or additive effect in above all the STS cell lines tested. We noted that ATRi treatment reduced phosphorylation of the ATR kinase target CHK1 but increased ATM phosphorylation, suggesting that ATM pathway takes over for DNA Damage Response and cells survival. In contrast, both ATR and ATM pathway were inhibited when AZD6738 and AZD0156 were used in combination, as shown by reduction of CHK1 and ATM phosphorylation. We validated our in vitro results in an in vivo model of liposarcoma xenograft in which AZD6738 and AZD0156 had synergistic anti-tumor activity.

Conclusion: Our study demonstrates synthetically lethal interactions between ATM and ATR in vitro and in vivo. This combination deserves further investigaitons in sarcomas.

Citation Format: Mariella Spalato, Audrey Laroche-Clary, Vanessa Chaire, Antoine Italiano. Genome-wide CRISPR/Cas9 library screening identified THRAP3 as a critical driver for resistance to ATR inhibitor in sarcomas revealing synthetic lethality with ATM inhibition [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 1509.
Abstract 1527: Developing a multifaceted approach to target androgen receptor (AR) in castration resistant prostate cancer
Authors: Saran Uttara , Chandrasekaran Balaji , Tyagi Ashish , Tapadar Subhasish , Wu Bocheng , Adegboyega Oyelere , Damodaran Chendil
Published: June 2022

Androgen receptor (AR) signaling is also regulated epigenetically by histone-modifying enzymes (histone deacetylases-HDACs) that result in the development and progression of prostate cancer (CaP). It has been established that the inhibition of HDACs suppresses CaP proliferation by inhibiting AR signaling via multiple mechanisms, including AR degradation. Hence, the goal of this study was to develop small molecules that employ multiple mechanisms to inhibit AR expression and promote its degradation, which would effectively control the emergence and treatment of castration-resistant prostate cancer (CRPC). Several rationally designed antiandrogen equipped HDAC inhibitors (HDACis) were synthesized, and their efficacy on CRPC growth was examined both in vitro and in vivo models. Results demonstrated that these compounds' target (HDAC and AR) binding properties translated into potent anticancer activity against AR+ CRPC cell lines and to a lesser extent in AR- CRPC cell lines. In addition, among these compounds, we found that SB-146, a nontoxic agent, not only inhibited the growth of AR+ CRPC cells at nanomolar IC50s without sparing toxicity to healthy prostate epithelial cells. Molecular analysis confirmed SB-146 downregulated the expressions of both AR and AR-spice variants (AR-SV) in CRPC cells. Further analyses revealed downregulation of AR downstream (PSA, HOXB13 and FOXA1) targets as well as AR-SV’s (NUP210 and SLC3A2) in CRPC cells. SB-146 was also able to overcome DHT-induced AR and AR-SV signaling in C4-2B and 22Rv1 cells. Assessment of SB-146’s therapeutic efficacy on in vivo models revealed that oral administration of this compound abrogated AR+ and AR-SV (C4-2B and 22Rv1) tumor growth in xenotransplanted nude mice models. Finally, immunohistochemical analysis confirmed SB-146’s target of both AR and AR-SVs on xenografted tumor tissues. Together these results confirm that SB-146 is a potent agent that inhibits AR signaling in CRPC cells.

Citation Format: Uttara Saran, Balaji Chandrasekaran, Ashish Tyagi, Subhasish Tapadar, Bocheng Wu, Oyelere Adegboyega, Chendil Damodaran. Developing a multifaceted approach to target androgen receptor (AR) in castration resistant prostate 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 1527.