bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1352: In vivo CRISPR screening identifies mediators of immune evasion in KRAS-mutant lung cancer
Authors: Boumelha Jesse , Romero-Clavijo Pablo , de Carné Sophie , Molina-Arcas Miriam , Downward Julian
Published: June 2022

Oncogenic KRAS mutations drive tumourigenesis in 30% of non-small cell lung cancer (NSCLC). Despite much effort, targeted therapies that aim to directly inhibit signalling pathways downstream of KRAS have limited clinical benefits for NSCLC patients, but the recent approval of PD-1/PD-L1 antibodies has led to striking durable responses. However, only a fraction of patients respond and therefore a deeper understanding of the mechanisms that drive immune evasion are required in order to broaden the clinical efficacy of immunotherapy. Increasing evidence suggests that oncogenic signalling pathways greatly influence the tumour immune landscape to impair anti-tumour immune responses. We therefore aim to understand the mechanisms by which KRAS signalling mediates immune evasion in lung cancer.

Current mouse models of KRAS-mutant lung cancer are poorly immunogenic, limiting investigations into tumour-immune interactions. To overcome this, we generated a novel transplantable KRAS-mutant lung cancer model, KPAR1.3, which triggers spontaneous anti-tumour immune responses and is sensitive to immune checkpoint blockade. To identify mechanisms of immune evasion we carried out an in vivo pooled CRISPR-Cas9 screen targeting 240 KRAS-regulated genes using this novel immunogenic model. This identified the prostaglandin synthase COX-2 as a mediator of immune evasion and its expression was driven by KRAS in multiple models of mouse and human lung cancer. Loss of COX-2 promoted a pro-inflammatory tumour microenvironment and sensitised tumours to both innate and adaptive anti-tumour immune responses. Furthermore, therapeutic COX inhibition extended the survival of tumour-bearing mice and synergised with PD-1 blockade. Together these data suggest that targeting KRAS-driven mechanisms of immune evasion could broaden the clinical efficacy of immunotherapy in KRAS-mutant NSCLC.

Since the approval of immunotherapy, a novel class of mutant-specific KRAS-G12C inhibitors have recently been approved for the treatment of KRAS-mutant NSCLC, however acquired drug resistance is common. We are therefore also currently investigating whether the inhibition of the COX-2/PGE2 axis enhances the therapeutic benefit of KRAS-G12C inhibitors.

Citation Format: Jesse Boumelha, Pablo Romero-Clavijo, Sophie de Carné, Miriam Molina-Arcas, Julian Downward. In vivo CRISPR screening identifies mediators of immune evasion in KRAS-mutant lung 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 1352.
Abstract 1354: Regulation and roles of polyamines in CD8+ T cell fate and function
Authors: Elmarsafawi Aya G. , Hesterberg Rebecca S. , Cleveland John L.
Published: June 2022

CD8+ T cells play central roles in tumor immune surveillance and are major effectors in adoptive cell therapy (ACT). Thus, strategies that enhance their functions are an urgent clinical need. Metabolic reprogramming determines T cell fate and function, where glycolysis principally drives an effector phenotype while oxidative phosphorylation (OxPhos) drives T cell memory [1]. However, the roles of amino acid catabolism in controlling effector and memory CD8+ T cell fate and function are less well understood [1, 2]. Glutamine is essential for nucleotide biosynthesis and as an anaplerotic fuel source for the tricarboxylic acid (TCA) cycle. Isotope tracing experiments with 13C glutamine or 13C arginine revealed that glutamine is the major source of polyamines in activated CD8+ T cells. Further, activation of CD8+ T cells provokes marked increases in the expression and activity of enzymes that direct polyamine biosynthesis, specifically ornithine decarboxylase (Odc), spermidine synthase (Srm) and spermine synthase (Sms), as well as to increases in the polyamines putrescine, spermidine and spermine. Notably, pharmacologic inhibition of Odc using difluoromethylornithine (DFMO), or genetic deletion of Odc using CD8+ T cells from CD4-Cre;Odcfl/fl mice, augments cytokine production in activated CD8+ T cells, including IFN-γ, TNF-α and granzyme B. Further, metabolic flux analysis indicates that DFMO treatment switches the metabolism of activated CD8+ T cells to rely on OxPhos, a phenotype found in CD8+ tissue resident memory (TRM) cells that play key roles in tissue and anti-tumor immunity anti-tumor reactivity. Indeed, inhibition or loss of Odc augments other TRM phenotypes, including increases in CD69high;CD44high;S1PR1low;CD62Llow cells that define the TRM phenotype. In addition, ACT of DFMO treated CD8+ T cells augments their survival in vivo and augments the generation of CD69+CD103+CD69+CXCR6+ and Ly6C+ TRMs in the bone marrow. Finally, DFMO treatment enhances IFN-γ and TNF-α production in human TILs, and co-treatment with TGF-β polarizes TILs into CD69+CD103+ and CD69+CD49a+ TRM-like cells. We conclude that polyamines are a metabolic check point in CD8+ TRM generation and are an actionable target to improve the anti-tumor immunity of T-cell based immunotherapies.

Citation Format: Aya G. Elmarsafawi, Rebecca S. Hesterberg, John L. Cleveland. Regulation and roles of polyamines in CD8+ T cell fate and function [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 1354.
Abstract 1355: Lipid-rich foamy macrophages along with CD4 T cell-derived signaling drive aberrant lipid metabolism and are a cause of lung cancer development
Authors: Shi Gongping , Banerjee Sayantan , Li Xin , Jogdand Gajendra , Zhao Yongmei , Nagashima Kunio , Andresson Thorkell , Shetty Jyoti , Tran Bao , Hu Yinling
Published: June 2022

Previously we reported that bone marrow-derived macrophages are required for lung squamous cell carcinoma development in kinase-dead Ikkα knockin (KA/KA) mice. The KA/KA lungs display strikingly enlarged sizes, sustained inflammation with increased expression of multiple cytokines and chemokines and marked infiltrating macrophages and T cells, and tissue damage, which look like a “burning hill”. To determine a critical event for the pathogenesis of the lung disease and SCC development, in this study, we found that these F4/80+CD11b+CSF1R+ monocyte-derived macrophages isolated from KA/KA lungs showed enlarged sizes carrying many lipid droplets and highly expressed the genes that encode proteins involved in lipid metabolism compared to WT. To determine whether the lung lipid concentrations regulate foamy macrophage development, we incubated Raw macrophages with WT or KA/KA lung extract, respectively, and found that KA/KA lung extracts induced lipid droplets and elevated lipid levels and pathways for lipid metabolism compared to WT lung extracts. Furthermore, we found that cytokines including IL-4, IL-6, IL-33, and IFNb, promoted lipid metabolism in Raw macrophages incubated with WT lung extracts, and also stimulated lipid metabolism in human lung SCC SW900 cells. These results suggest that these cytokines, which were highly expressed in KA/KA lungs, contribute to aberrant lipid metabolism. Because IL-4, which is produced by Th2 T cells, promoted lipid metabolism in macrophages and lung SCC cells, we further showed that KA/KA lung CD4 T cells stimulated lipid gene expression in Raw macrophages compared to WT lung CD4 T cells. Depleting CD4 T cells indeed significantly decreased foamy macrophage formation in the lungs of KA/KA mice. Importantly, treatment with a lipid inhibitor reduced IL-4-mediated lipid metabolism in Raw macrophages and human lung SCC SW900 cells, as well as this treatment attenuated lung foamy macrophage numbers and dampened lung SCC development in irradiated KA/KA mice with KA/KA bone marrow transplants. These findings highlight that the interplay between CD4 T cells, macrophages, cytokines, and epithelial cells contribute to a series of biological events in a landscape scale, which promote lung disease and lung SCC development through aberrant lipid metabolism.

Citation Format: Gongping Shi, Sayantan Banerjee, Xin Li, Gajendra Jogdand, Yongmei Zhao, Kunio Nagashima, Thorkell Andresson, Jyoti Shetty, Bao Tran, Yinling Hu. Lipid-rich foamy macrophages along with CD4 T cell-derived signaling drive aberrant lipid metabolism and are a cause of lung cancer development [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 1355.
Abstract 1359: Selective HDAC6 inhibition by GB-1101 revokes tumor immune privilege and synergizes with immune checkpoint therapies to induce tumor regressions
Authors: Kotlarczyk Kari , Sepulveda Mario , Wang Tong , Murray Maggie , Gonzales Paul , Gately Stephen Thomas
Published: June 2022

HDAC6 is a cytoplasmic class IIB HDAC isoenzyme that is involved in the deacetylation of cytoplasmic proteins. HDAC6 also binds ubiquitinated proteins, facilitating the formation of the aggresome, to remove misfolded proteins. Previously, HDAC6-selective inhibitors were shown to decrease immunosuppression and enhance immune function of melanoma patient T-cells demonstrating an important role for HDAC6 inhibition for cancer immunotherapy. GB-1101 is a novel, orally bioavailable, nanomolar potent inhibitor of HDAC6. GB-1101 tested in vitro did not induce direct cytotoxicity to CD4+/CD8+ or NK cells at concentrations up to 300 nM. RNA-seq on GB-1101 exposed MHC-null human MSS colon cancer cell lines reveal potent induction of MHC Class I/II genes and expression numerous cancer neoantigens. To test the activity of GB-1101 in vivo, we established large (~450mm3) 4T1-luc syngeneic tumors and initiated treatment of GB-1101 given orally once daily alone and combined with anti-PD1, anti-CTLA4, and anti-PD1/anti-CTLA4 combination. The activity of anti-PD1 in this model was minimal (TGI: 3%); GB-1101 as a single agent showed more potent anti-tumor activity (TGI: 50%) compared to anti-PD1. We observed synergistic benefit when GB-1101 was combined with anti-PD1 (Combination TGI: 78%). GB-1101 showed comparable anti-tumor activity to single agent anti-CTLA4 (TGI: 43%) but revealed significant tumor regressions of large and well-established tumors when combined with anti-CTLA4 (Combination TGI: >100%). As a single agent, GB-1101 was superior to anti-PD1+anti-CTLA4 (TGI: 7%) and induced significant tumor regressions when combined with anti-PD1+anti-CTLA4 (Triple Combination TGI: >100%). In the TC-1, HPV-positive syngeneic model, GB-1101 reprogrammed the tumor immune microenvironment reducing the number of M-MDSC and TReg cells and increase the numbers of CD4+/CD8+ and NK cells. Taken together, these data support GB-1101 as an important new targeted epigenetic immunomodulator able to revoke immune privilege to enhance the clinical activity of immune checkpoint therapies.

Citation Format: Kari Kotlarczyk, Mario Sepulveda, Tong Wang, Maggie Murray, Paul Gonzales, Stephen Thomas Gately. Selective HDAC6 inhibition by GB-1101 revokes tumor immune privilege and synergizes with immune checkpoint therapies to induce tumor regressions [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 1359.
Abstract 1386: MAPK inhibition remodels antigen presentation in pancreatic ductal adenocarcinoma
Authors: Creech Amanda L. , Le Thuc M. , Abt Evan R. , Capri Joseph R. , Donahue Timothy R. , Radu Caius G.
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a 5-year survival rate of less than 10%, highlighting the need for new therapeutic options. Hallmarks of this aggressive disease include constitutive activation of the MAPK signaling pathway via mutant KRAS (mKRAS) and an immunosuppressive tumor microenvironment (TME). Constituently-active KRAS has now been rendered targetable via clinical-stage mKRAS-specific small molecule inhibitors. While the impact of KRAS inhibition on tumor growth is well-studied, its ability to engender anti-tumor adaptive immune responses is not fully understood. Prior data generated by our group demonstrated mKRAS inhibition (mKRASi) increased expression of genes associated with antigen presentation in two models of PDAC in vitro. Additionally, proteomic analysis revealed increased expression of two tumor associated antigens (TAAs), MSLN and PSCA.

To better understand the alteration of antigen presentation in response to MAPK signaling perturbation, we measured surface expression of MHC-I and PD-L1 after mKRAS and/or SHP2 inhibition via flow cytometry in a PDAC-KRASG12C model in vitro. Results demonstrate an additive increase in MHC-I surface expression following inhibition of SHP2 and in combination with mKRASi that is more pronounced in the setting of type I interferon (IFN). Inhibition of MEK in a PDAC KRASG12D model recapitulates these findings. Addition of a JAK inhibitor did not suppress the additional MHC-I expression suggesting a potential JAK/STAT-independent mechanism for this observation.

To better understand the extent of alteration of presented epitopes in response to these perturbations we established an immunopeptidomics profiling workflow, using LC-MS/MS to identify peptide ligands eluted from MHC-I complexes after inhibition of mKRAS and/or SHP2. We additionally profiled epitope alterations after the addition of type I or II IFN. MAPK perturbation resulted in more unique epitopes identified, especially with dual inhibition of mKRAS and SHP2 and further potentiated by type I IFN. Furthermore, we were able to directly identify epitopes derived from MSLN and PSCA in our cell line models. This data demonstrates the potential for MAPK inhibition to render PDAC more sensitive to the adaptive immune system and synergize with immunotherapeutic treatment strategies. Additionally, direct identification of epitopes presented by cancer cells via mass spectrometry can aid in the rational design of targeted immunotherapies.

Citation Format: Amanda L. Creech, Thuc M. Le, Evan R. Abt, Joseph R. Capri, Timothy R. Donahue, Caius G. Radu. MAPK inhibition remodels antigen presentation in pancreatic ductal adenocarcinoma [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 1386.
Abstract 143: Novel HSP40 or J domain protein inhibitors that deplete misfolded mutant p53
Authors: Iwakuma Tomoo , Nishikawa Shigeto , Kaida Atsushi , Ranjan Atul , Parrales Alejandro , Alalem Mohamed A. , Johnson David K.
Published: June 2022

Background: Accumulation of oncogenic mutant p53 (mutp53) greatly contributes to cancer progression. Heat shock protein 40 (HSP40), also known as J-domain proteins (JDPs), has been implicated in stabilization of misfolded mutp53. Specifically, knockdown of DNAJA1, a member of HSP40/JDPs, results in degradation of mainly misfolded mutp53, leading to inhibition of tumor progression. Since no DNAJA1 inhibitors are currently available in clinics, these findings prompted us to identify potential DNAJA1 inhibitors which can induce mutp53 degradation.

Methods/Design: An in-silico docking study was performed for binding of the DNAJA1 J-domain to compounds in zinc data base, which identified compound Number7-3. Further analysis suggests three critical amino acids in DNAJA1 for the binding, including Y7, K44, and Q47.

Results: Eighteen commercially available analogs of Number7-3 were further examined. Of these, a compound, namely A11, had the strongest activity to deplete both DNAJA1 and misfolded mutp53. A11 reduced protein levels of misfolded mutp53 in concentration- and time-dependent manners without affecting mRNA levels of DNAJA1 and mutp53. Importantly, A11 showed minimal effects on the levels of wild-type p53 and DNA contact mutp53. Treatment of cells expressing misfolded mutp53 with A11 resulted in reduced filopodia formation and cell migration, which was not observed in cells lacking DNAJA1. Moreover, protein levels of a triple mutant DNAJA1 (Y7A/K44A/Q47A) were not altered by A11. Intriguingly, A11 reduced protein levels of several other HSP40/JDPs.

Conclusions: Our study, for the first time, has identified a small compound that depletes DNAJA1, leading to reduced levels of misfolded mutp53 and inhibited malignant properties of cancer cells.

Citation Format: Tomoo Iwakuma, Shigeto Nishikawa, Atsushi Kaida, Atul Ranjan, Alejandro Parrales, Mohamed A. Alalem, David K. Johnson. Novel HSP40 or J domain protein inhibitors that deplete misfolded mutant p53 [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 143.
Abstract 1434: Molecular characteristics of breast cancer patients subjected to screening for germline predisposition in a population-based observational study
Authors: Nacer de Oliveira Deborah F. , Vallon-Christersson Johan , Ehrencrona Hans , Kvist Anders , Borg Åke , Staaf Johan
Published: June 2022

An estimated 5-10% of breast cancer cases carry germline mutations in genes associated with a moderate to strong breast cancer risk, like BRCA1 and BRCA2. These mutations can influence disease onset, progression, biology of the tumor, potential for therapy, risk of recurrence and cancer risk of close relatives. This warrants clinical germline screening for such gene variants according to national guidelines on patient selection. In this study we aimed to molecularly and clinicopathologically contrast clinically screened patients and their tumors against unscreened patients. For this we used a population-based breast cancer cohort from southern Sweden with 6660 patients (14% of which had been screened) with accompanying RNA-sequencing and clinicopathological registry data. Screening subsets were analyzed within five clinically relevant breast cancer subgroups based on hormone receptor and lymph node status. Among other features, we investigated clinicopathological characteristics, risk of tumor recurrence, survival outcomes, tumor proliferation markers, immune cell content, genes differentially expressed and the pathways in which they were involved. Most differences were found in the triple negative breast cancer (TNBC) subgroup: when compared to patients that had not been screened, the screened subset contained younger individuals showing better overall survival, more proliferative tumors with upregulated genes that belong to DNA replication and double-strand break repair processes, lower proportion of M2 macrophages and higher proportion of T-helper 2 cells. As so, Swedish screening guidelines divide TNBC cases into two separate subsets possibly by selecting distinct underlying biological and mutational mechanisms associated with DNA repair deficiency (e.g., through BRCA1/2-deficiency), a disparity not seen in other breast cancer clinical subgroups. Understanding the molecular landscape of defined screening populations may improve future guideline updates, which in turn would also improve patient and resource handling.

Citation Format: Deborah F. Nacer de Oliveira, Johan Vallon-Christersson, Hans Ehrencrona, Anders Kvist, Åke Borg, Johan Staaf. Molecular characteristics of breast cancer patients subjected to screening for germline predisposition in a population-based observational study [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 1434.
Abstract 1466: MYC localizes to the nuclear pore to promote DNA repair during replication stress
Authors: Cohn Gabriel , Daniel Colin J. , Liefwalker Daniel F. , Sears Rosalie C.
Published: June 2022

Genomic instability is a hallmark of cancer which promotes oncogenic mutations in pancreatic ductal adenocarcinoma (PDAC), leading to more aggressive tumors with greater potential for drug resistance. Deregulation of the master transcription factor, MYC, is found in virtually all PDAC tumors and promotes genomic alterations by increasing replication stress, augmenting DNA repair pathways, and promoting cell survival. To resolve replication stress, stalled forks are trafficked to the nuclear pores where the necessary machinery accumulates. Activated MYC is spatially reorganized to the nuclear pores as well, a mechanism that is exacerbated in cancer. Whether MYC activity at the nuclear pores contributes to resolving replication stress in PDAC remains to be known. To investigate this mechanism, we are creating genomic and proteomic tools to determine MYC’s function at the nuclear pores during replication stress induced by olaparib treatment in PDAC cells. We found that olaparib significantly increases MYC accumulation at the nuclear pore in low-passage cell lines we established from PDAC patient tumors. Furthermore, we showed that MYC interacts with the nuclear pore resident SUMO protease, SENP1, which plays a key role in resolving stalled forks. Our preliminary data suggest a role for MYC resolving replication stress at nuclear pores, and could indicate MYC activity as a therapeutic target in olaparib-resistant PDAC.

Citation Format: Gabriel Cohn, Colin J. Daniel, Daniel F. Liefwalker, Rosalie C. Sears. MYC localizes to the nuclear pore to promote DNA repair during replication stress [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 1466.
Abstract 1477: Circulating miR-125a-3p and miR-451a may be liquid biopsy biomarkers for the diagnosis of breast cancer
Authors: Yousefi Hassan , Eastlack Steven C. , Fong Jordyn T , Okpechi Samuel C. , Zabaleta Jovanny , Buckner Lyndsey R. , Trapani Donnalee N , Sims Erin , McDaniel Alison McDaniel , Scuderi Heather , Lakey Meredith A. , Cole John T. , Alahari Suresh K.
Published: June 2022

Background: Breast cancer is the most prevalent female cancer. Emerging evidence suggests that the unique serum miRNAs can assist in the early detection and diagnosis of breast cancer. Cell-free circulating miRNAs (about 22 nucleotides) are potential diagnostic tools and minimally invasive biomarkers for breast cancer detection. This study aimed to identify specific microRNAs in the serum of patients with breast cancer, which may serve as promising diagnostic and prognostic biomarkers and therapeutic targets.

Material and methods: Next-generation sequencing and quantitative real-time PCR (qRT-PCR) array analyses of microRNAs in sera from different subtypes of breast cancer patients and normal female individuals were performed. The differentially expressed microRNAs were verified in serum samples from 100 breast cancer patients. The predictive values of the selected microRNAs were statistically analyzed, determined by the correlation between microRNA expression and tumor parameters. Datasets were graphed using GraphPad Prism Software 8.0. and analysis was done using one-way ANOVA with unpaired two-tailed Student’s t-test.

Result: High serum levels of miR-125a-3p and miR-451a were found to be associated with breast cancer tumor subtype, size, and lymph node metastasis. Further functional in vitro experiments and enrichment analysis showed that overexpression and knockdown of miR-451 might significantly affect tumorigenic signaling pathways in TNBC cells.

Conclusion: Altogether, our data suggested that serum levels of microRNAs miR-125a-3p and miR-451a can potentially serve as liquid biopsy biomarkers in breast cancer.

Citation Format: Hassan Yousefi, Steven C. Eastlack, Jordyn T Fong, Samuel C. Okpechi, Jovanny Zabaleta, Lyndsey R. Buckner, Donnalee N Trapani, Erin Sims, Alison McDaniel McDaniel, Heather Scuderi, Meredith A. Lakey, John T. Cole, Suresh K. Alahari. Circulating miR-125a-3p and miR-451a may be liquid biopsy biomarkers for the diagnosis of 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 1477.
Abstract 1487: Role of micro-RNA199a-3p in lymph node stromal extracellular vesicles on tumorigenesis in colorectal cancer
Authors: Baer Hallie , Simon Jessica , McKean-Baste Lara , Ray Alicia N. , Green Heather , Maresh Grace , Zhang Xin , Marjolin David , Paruch Jennifer , Li Li
Published: June 2022

Introduction: MicroRNAs (miRNAs) are short non-coding RNAs implicated in post-transcriptional regulation of gene expression. Abnormal expression of miRNAs in cancer is associated with enhanced tumorigenesis and metastatic potential. Our previous data has shown that extracellular vesicles (EV) secreted by human lymph node stromal cells (LNSC) enhance tumorigenesis in colorectal cancer (CRC). By systemic searching and modifying miRNAs carried by EV through overexpression and inhibition on LNSC, we aim to demonstrate their effect on CRC progression in vitro and orthotopic mouse model.

Methods: Total microRNA sequencing was analyzed using in-silico computer prediction models. Four overexpressed miRNAs in the LNSC-EV were selected (miR-155-5p, miR-199a-3p, mi-143-3p, and mi-214-3p). Tumor cell proliferation assay was performed using a CRC SW620 cell line transfected with selected miRNA mimics or inhibitors or controls. In our mouse model, miRNA transfected SW620 cells tagged with luciferase/RFP, were injected into the rectal submucosa of NOD/SCID mice. Tumor growth and metastases were observed by weekly bioluminescent imaging (BLI). At week 7, tumors were weighed and BLI of the tumors and organs were recorded. Immunohistochemistry staining was performed on the paraffin sections of tumors, livers and lung collected from these mice.

Results: In vitro proliferation assays, cells transfected with the mimic of miRNA 199a-3p significantly increased CRC SW620 cell proliferation (p<0.01) while cells treated with the inhibitor showed decreased proliferation (p<0.05). In our orthotopic mouse model, mice with SW620 cells transfected with the mimic had increased frequencies of tumorigenesis (94% vs. 14%) and liver and lung metastasis comparing to control group (56% vs 9.5%, 50% vs 0%). Mice injected with SW620 cells transfected with the miRNA inhibitor had decreased tumor growth compared to the negative control (p<0.05).

Conclusion: Our study shows that miRNAs carried in LNSC-EV are crucial for CRC progression. Specifically, inhibition of miRNA-199a-3p may serve as a therapeutic target in the treatment of CRC. Understanding the role and mechanisms of this miRNA in CRC could lead to the improved management of patients with CRC.

Citation Format: Hallie Baer, Jessica Simon, Lara McKean-Baste, Alicia N. Ray, Heather Green, Grace Maresh, Xin Zhang, David Marjolin, Jennifer Paruch, Li Li. Role of micro-RNA199a-3p in lymph node stromal extracellular vesicles on tumorigenesis in colorectal 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 1487.
Abstract 1489: MicroRNA-1 targets CXCR4/FOXM1/RRM2 axis regulating small cell lung cancer growth and metastasis
Authors: Khan Parvez , Siddiqui Jawed A. , Maurya Shailendra Kumar , Mirzapoiazova Tamara , Kanchan Ranjana , Venkata Ramakanth Chirravuri , Atri Pranita , Tang Wemin , Perumal NaveenKumar , Kshirsagar Prakash , Fatima Mahek , Khan Md Arafat , Chaudhary Sanjib , Rehman Asad Ur , Lakshmanan Imayavaramban , Mahapatra Sidharth , Kulkarni Prakash , Ganti Apar Kishor , Jain Maneesh , Oupicky David , Salgia Ravi , Batra Surinder Kumar , Nasser Mohd Wasim
Published: June 2022

Small cell lung cancer (SCLC) is a high-grade neuroendocrine metastatic lung cancer subtype having a universal relapse and poor prognosis. Limited or extensive-stage SCLC patients have limited therapeutic options due to a lack of potential drug targets. In the quest for novel therapeutic molecules, we performed micro-RNA sequencing from the serum samples and also analyzed bulk RNA-sequencing data from tumors of SCLC patients. We found a consistent downregulation of miR-1 in SCLC. These results were recapitulated in SCLC cell lines and tumor tissues compared to their matched normal. To assess the therapeutic potential of miR-1, we overexpressed miR-1 in SCLC cell lines that translated into decreased cell growth and oncogenic signaling. Mechanistic studies revealed that CXCR4 is a direct target for miR-1 in SCLC. Intracardiac injection of SCLC cell lines in the mouse models showed that overexpression of miR-1 decreases the distant organ metastasis, whereas miR-1 sponging potentiates aggressiveness and metastasis. Furthermore, we identified FOXM1-RRM2 as a unique downstream target of the miR-1/CXCR4 axis involved in the growth and metastasis of SCLC. Our results showed that FOXM1 directly binds to the promoter site of RRM2 and regulates its activity. Overexpression of miR-1 decreases the expression and activity of FOXM1-RRM2 through CXCR4 that reduces cell growth and metastasis. Taken together, our results suggest that miR-1 decreases SCLC metastasis by targeting the CXCR4/FOXM1-RRM2 axis and has a high potential for the development of novel SCLC therapies.

Citation Format: Parvez Khan, Jawed A. Siddiqui, Shailendra Kumar Maurya, Tamara Mirzapoiazova, Ranjana Kanchan, Ramakanth Chirravuri Venkata, Pranita Atri, Wemin Tang, NaveenKumar Perumal, Prakash Kshirsagar, Mahek Fatima, Md Arafat Khan, Sanjib Chaudhary, Asad Ur Rehman, Imayavaramban Lakshmanan, Sidharth Mahapatra, Prakash Kulkarni, Apar Kishor Ganti, Maneesh Jain, David Oupicky, Ravi Salgia, Surinder Kumar Batra, Mohd Wasim Nasser. MicroRNA-1 targets CXCR4/FOXM1/RRM2 axis regulating small cell lung cancer growth and metastasis [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 1489.
Abstract 1554: Functional analysis of human circular RNA_0001587 and its target miRNAs in oral cancer
Authors: Khan Muhammad Jawad , Rabia Ghulam , Dandare Abdullahi , Awan Hassaan Mehboob , Raja Afraz Ahmad
Published: June 2022

Oral cancer is the sixth most aggressive malignancy around the world and accounts for 90% of head and neck lesions. It is the most prevalent type of cancer in South Asian countries and contributes to around 25% of all new cancer cases in the region. The involvement of non-coding circular RNAs (circRNAs) in various disease pathology has been reported. However, their role as diagnostic or therapeutic biomarkers is still unclear and needs to be further explored. This study was aimed to identify the role of hsa_circ_0001587 and competing endogenous RNA (ceRNA) mechanism in early diagnosis or as a therapeutic target for oral cancer. For this purpose, lists of circRNAs, miRNAs, and differentially expressed genes (DEGs) involved in oral squamous cell carcinoma (OSCC) were identified from the reported literature. Circinteractome was used to obtain target miRNAs that potentially bind to hsa_circ_0001587. Five miRNAs were selected for further in silico analysis on the basis of maximum target sites. Data were subjected to functional enrichment analysis, pathway analysis, and gene homology studies.Our results showed active participation of both hsa_circ_0001587 and its target miRNAs (miR-548c-3p, miR-520h, miR-1827, miR-607, and miR-579) in oral carcinogenesis via the regulation of genes including AKT, GADD45, PKA, PKC, and CASP8. Dysregulation of these genes disturbed the immune system, signaling pathways, cellular compartment activities, and molecular functions, indicating their role in tumor initiation and progression. Furthermore, it was predicted that hsa_circ_0001587 enhances the expression of genes for the RAS signaling pathway via its sponging effect on miR-548c-3p and miR-607. Noncoding RNAs including hsa_circ_0001587 and target miR-548c-3p and miR-607 play a critical role in the pathogenesis of OSCC. It is therefore suggested that hsa_circ_0001587 and its target miRNAs could be used as potential biomarkers for oral cancer.

Citation Format: Muhammad Jawad Khan, Ghulam Rabia, Abdullahi Dandare, Hassaan Mehboob Awan, Afraz Ahmad Raja. Functional analysis of human circular RNA_0001587 and its target miRNAs in oral 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 1554.
Abstract 1563: Spatial transcriptomics reveals heterogeneity and pathway dependencies of cancer associated fibroblasts in pancreatic ductal adenocarcinoma
Authors: Caligiuri Giuseppina , Thalappillil Jennifer , Hinds Juliene , Courtois Elise T. , Flynn William F. , Robson Paul , Dobin Alexander , Park Youngkyu , Tuveson David A.
Published: June 2022

Pancreatic ductal adenocarcinoma (PDA) is a deadly disease characterized by an immunosuppressive microenvironment and a dense stroma that encapsulates the tumor, making therapeutic targeting particularly challenging. One of the main cellular components of PDA tumor microenvironment (TME) is cancer associated fibroblasts (CAFs), whose function is to sustain tumor growth and provide structural support to the TME. Previously, we established the existence of three transcriptional subtypes of CAFs, myCAF, iCAF and apCAF, each with distinct functions and location in respect to cancer cells. Two of these subtypes, myCAF and iCAF, display a highly plastic potential in vitro and can transdifferentiate into one another upon activation of specific signaling pathways. The plasticity observed supports the existence of transitional CAF sub-states that have previously eluded observation due to technical limitations to their isolation. Through a combination of spatial transcriptomics and fluorescent in situ hybridization (FISH) on tumor samples obtained from KPC (KrasLSL-G12D; p53LSL-R172H; PDX-CRE) and FPC (KrasFrt-LSL-G12V-Frt; p53LSL-R172H; PDX-CRE; Rosa26FlpOERT2) mice, two PDA mouse models displaying a Kras G12D and G12V mutation respectively, we were able to confirm the presence of the previously identified CAF subtypes and two additional CAF sub-states. These sub-states are associated with the expression of specific markers and display different pathway enrichment. Notably, Kras G12D and G12V mutations generate similar organization of the stroma and heterogeneity of the CAF population. Subsequently, to understand the contribution of activating Kras mutation to these CAF phenotypes, we utilized the unique feature of the FPC mouse model which allows the excision of Kras G12V once the tumor has fully developed. Through FISH analysis, we observed deep reorganization of the stroma and a shift in marker expressions within the CAF populations upon Kras excision. Overall, the data here presented offers insight into the diversity of PDA CAFs and the role of mutated Kras in regulating CAF subtypes and stromal organization.

Citation Format: Giuseppina Caligiuri, Jennifer Thalappillil, Juliene Hinds, Elise T. Courtois, William F. Flynn, Paul Robson, Alexander Dobin, Youngkyu Park, David A. Tuveson. Spatial transcriptomics reveals heterogeneity and pathway dependencies of cancer associated fibroblasts in pancreatic ductal adenocarcinoma [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 1563.
Abstract 1581: Uncovering mechanisms of biogenesis and secretion of small extracellular vesicles in non small cell lung cancer (NSCLC)
Authors: Vargas Zulaida M. Soto , Sohal Ikjot Singh , Hasan Humna , Arteaga David , Kasinski Andrea L.
Published: June 2022

Non-small cell lung cancer (NSCLC), the most common type of lung cancer, is the leading cause of cancer related deaths in United States. Only 24% of NSCLC patients survive 5-years post diagnosis, which could be attributed to the lack of efficient treatment strategies at the metastatic stage. Thus, understanding the biological mechanisms that promote NSCLC metastasis is critical for developing effective cancer-specific therapeutic agents. The development of cancer metastasis is greatly driven and influenced by intercellular communication. Key mediators of cell-to-cell communication are small extracellular vesicles (sEVs) of approximately 100-150 nm in diameter, often referred as exosomes. Previous report revealed that increased levels of exosomes in NSCLC patients correlated with lower overall survival. Indeed, secretion of sEVs by cancer cells can induce malignant transformation of non-cancerous cells nearby or at distant locations through influencing pre-metastatic niche formation. Therefore, biogenesis and release of cancer derived sEVs are determinant steps for the development of tumor metastasis. However, the mechanisms through which sEVs are produced and secreted by cancer cells are yet to be determined. Here, we isolated and characterized sEVs from a panel of NSCLC cell lines (H358, Calu6, H460, SKMES-1) and non-tumorigenic cells (BEAS-2B and HBEC) to identify mechanisms involved in the elevated secretion of EVs by cancer cells. Contrary to what has been previously reported in literature, our data demonstrated that not all cancer cell lines release high levels of sEVs. Our data suggests that elevated secretion of sEVs could be driven by specific mutations and oncogenic signaling in cancer cells. Among all the cell lines tested, H358 cells which harbor the infamous G12C mutation in KRAS, had the highest sEV release. Therefore, we hypothesize that the KRASG12C can drive high sEV production and secretion in H358 cells. Our preliminary data suggests that siRNA mediated knockdown of oncogenic KRAS signaling in H358 cells significantly decreases secretion of sEVs. Ongoing studies aim to pinpoint the detailed mechanisms by which cancer cells utilize KRAS oncogenic signaling to promote EV biogenesis pathways and tumorigenesis. The current study will provide the foundation to developed new therapeutic strategies against tumor sEVs-driven metastasis.

Citation Format: Zulaida M. Soto Vargas, Ikjot Singh Sohal, Humna Hasan, David Arteaga, Andrea L. Kasinski. Uncovering mechanisms of biogenesis and secretion of small extracellular vesicles in non small cell lung cancer (NSCLC) [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 1581.
Abstract 161: Regulation of Abl at mitotic centrosomes and survival by ILK in glioblastoma cells
Authors: Dresselhuis Jonathan , Brown Maddisen , Robinson Nicole , Tan Jacqueline , Cox Michael E. , Mills Julia
Published: June 2022

Many cancer cells possess supernumerary centrosomes, having more than one centrosome at each pole during mitosis which can lead to aberrant mitotic division and cell death. To prevent this fate, cancer cells cluster their centrosomes at two poles during division to divide in a pseudobipolar manner. As the presence of supernumerary centrosomes is rare in normal cells, targeting the declustering of supernumerary centrosomes in cancer cells is a promising strategy for cancer treatment. Integrin-linked kinase (ILK) is an oncogenic protein and chemotherapeutic target shown to be found at the centrosome where it has a role in facilitating centrosome clustering. Inhibition of ILK, via an anti-ILK chemotherapeutic drug QLT-0267, has been shown to increase centrosome declustering causing mitotic arrest and cell death. However, not all cancers cells are susceptible to anti-ILK treatment alone. Therefore, we investigate a combination treatment strategy targeting ILK and another oncogenic kinase, Abelson kinase (Abl). Both ILK and Abl are involved in cancer cell survival, regulated by the same signalling pathways and share common binding partners that occur at the centrosome and are critical for mitosis. The ILK inhibitor QLT-0267 induced a significant sub-G1 peak following propidium iodide FACS analysis and an increase in MTT labelling, indicating that QLT-0267 significantly decreased survival in glioblastoma cell lines. These effects were augmented in the presence of imatinib, an Abl inhibitor. Moreover, QLT-0267 and imatinib, in combination, significantly increased mitotically arrested cells over the ILK inhibitor alone. Increased levels of cytosolic Abl have been associated with its transformative abilities. The ILK inhibitor effects on survival correlated with its ability to decrease cytosolic Abl levels and inhibited Abl’s localization to mitotic centrosomes in dividing glioblastoma cells. These effects were reversed by the proteasomal inhibitor MG132 (a drug that inhibits the degradation of Abl). Together these results indicate that combination treatment with QLT-0267 and imatinib are more effective then QLT-0267 alone at decreasing successful mitoses and survival in glioblastoma cells. Future studies will unravel the relationship between these oncogenic kinases at centrosomes and its effect on cancer cell cycle progression and survival.

Citation Format: Jonathan Dresselhuis, Maddisen Brown, Nicole Robinson, Jacqueline Tan, Michael E. Cox, Julia Mills. Regulation of Abl at mitotic centrosomes and survival by ILK in glioblastoma 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 161.
Abstract 1736: Local treatment of lung cancer by inhaled monoclonal antibodies: A pre-feasibility screening approach for spray drying
Authors: Landry Madeleine , Pluntze Amanda , Shepard Kimberly
Published: June 2022

Local treatment of asthma and COPD by inhaled therapies has been standard of care for decades. A similar approach for lung cancer treatment could bring many benefits to the patient, including ease of administration, non-invasiveness, and potential for reduced side effects. Spray drying is a versatile, scalable manufacturing technique used to engineer therapies for delivery to the lung by dry powder inhaler. It is applicable to nearly all classes of molecules, including RNA, small molecules and proteins. Spray dried inhalation powders eliminate the need for cold chain storage, and can improve product shelf life compared with liquid formulations, improving access for developing nations and rural populations. Recently, inhaled delivery for a VEGF inhibitor, bevacizumab, has shown in preclinical studies to reduce tumor burden in rats at 1/10th the dose of injected bevacizumab, when combined with chemotherapy. To enable the next generation of inhaled monoclonal antibody (mAb) therapies by spray drying, mAb structural integrity is key. Spray dried materials undergo thermal and shear stresses, which can damage delicate mAbs. Herein we identified a set of analytical tests to evaluate the upper limits of heat and shear mAbs can withstand during spray drying. We exposed five cancer-relevant, FDA-approved mAbs to varying heat and shear stress then characterized changes to primary (1°), secondary (2°), and tertiary (3°) structure and binding activity indicative of degradation. Using the following analytical techniques we detected graduations of degradation: capillary electrophoresis-isoelectric focusing (1°), Fourier transform infrared spectroscopy (2°), Raman spectroscopy (2°), size exclusion chromatography (3°), absorbance and fluorescence spectroscopy (3°), dynamic light scattering (3°), multi-cell differential scanning calorimetry (3°), SDS-PAGE (3°), and binding activity ELISAs. Using these techniques, we developed a materials-sparing protocol to first assess whether an antibody is a candidate for spray drying, and second, to identify ideal process conditions for mAb spraying drying success. This approach will help to accelerate the development of inhaled protein therapies for lung cancer and other lung diseases.

Citation Format: Madeleine Landry, Amanda Pluntze, Kimberly Shepard. Local treatment of lung cancer by inhaled monoclonal antibodies: A pre-feasibility screening approach for spray drying [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 1736.
Abstract 1743: Identification of mesothelin binding peptide for targeted therapy against pancreatic cancer
Authors: Park Min-Sung , Lee Byungheon
Published: June 2022

Pancreatic cancer is the main cause of cancer-related deaths worldwide and is difficult to diagnose before the extensive local invasion and distant organ metastasis. Mesothelin is abnormally overexpressed in tumors such as ovary cancer and pancreatic cancer. Studies show a significant link between mesothelin overexpression and short survival in patients with pancreatic cancer. In this study, we screened a phage displayed-peptide library for peptides that selectively bind to mesothelin using mesothelin-overexpressing cells. After five rounds of screening, we selected a 9-mer peptide (named MSLN-Pep) that preferentially bound to mesothelin-high pancreatic cancer cells such as ASPC-1 and Panc-1 cells over mesothelin-low cells such as HEK 293 cells. MSLN-Pep was efficiently internalized into ASPC-1 cells and inhibited the cell migration and invasion while little affected the phosphorylation of Akt. Moreover, a MSLN-Pep-guided proapoptotic peptide (MSLN-Pep-KLA) exerted selective cytotoxicity against pancreatic cancer cells over mesothelin-low cells. MSLN-Pep-KLA when combined with gemcitabine, a chemotherapeutic agent against pancreatic cancer, sensitized ASPC-1 cells to the gemcitabine treatment. These results suggest that MSLN-Pep is a promising tool for a targeted therapy against pancreatic cancer expressing mesothelin at high levels.

Citation Format: Min-Sung Park, Byungheon Lee. Identification of mesothelin binding peptide for targeted therapy against 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 1743.