bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2319: Niclosamide enhances the antitumor effect by inducing ubiquitination of β-catenin in pancreatic cancer
Authors: Kang HyeonWoong , Park JoonSeong , Lee YunSun , Lee Daeun , Kim HyoJung
Published: June 2022

Pancreatic Ductal Adenocarcinoma (PDAC) is known as one of the critical cancers that have a high morbidity and mortality worldwide. Due to lack of therapeutic agents, patients’ overall survival rate has been decreased. Gemcitabine is the only well-known drug to treat pancreatic cancer patients but less effective. Therefore, it is significantly required to discover and develop novel therapeutic agents for PDAC therapy. The present study represents the first study evaluating the anticancer efficacy of niclosamide in PDAC. Here, we found that niclosamide co-treatment with gemcitabine inhibited pancreatic cancer cells compared with gemcitabine alone by suppressing the Wnt/β-catenin signaling pathway. In addition, niclosamide co-treatment with gemcitabine inhibited cell proliferation as determined by MTS assay and induced cell apoptosis as determined by Annexin V and PI staining. Further, niclosamide induced G1 arrest of the cell cycle as evidenced by down-regulated CDK4/6 and Cyclin D1 expression. Our findings proved that combining niclosamide and Gemcitabine effectively down-regulates the tumor size and growth in pancreatic cancer. Altogether, our results indicate that niclosamide synergistically enhances the antitumor effect with treating gemcitabine by inducing the ubiquitination and degradation of β-catenin in pancreatic cancer and could be a novel potential candidate for PDAC therapy.Key words: PDAC, Niclosamide, Gemcitabine, β-catenin ubiquitination, Apoptosis, Cell cycle

Citation Format: HyeonWoong Kang, JoonSeong Park, YunSun Lee, Daeun Lee, HyoJung Kim. Niclosamide enhances the antitumor effect by inducing ubiquitination of β-catenin 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 2319.
Abstract 2320: Ivermectin suppresses pancreatic cancer via mitochondria dysfunction
Authors: lee daeun , park joon seong , lee yun sun , kang hyeon woong , kim hyo jung
Published: June 2022

Pancreatic cancer is an aggressive cancer characterized by high mortality and poor prognosis with survival of less than 5 years when advanced. Several studies showed that the ivermectin has antitumor effects on a variety of cancer cells. The present study represents the first study evaluating the anticancer efficacy of ivermectin in pancreatic cancer. We found that the anticancer effect of ivermectin in combination with gemcitabine on pancreatic cancer is more effective than gemcitabine alone. Ivermectin-gemcitabine combination inhibited cell proliferation via G1 arrest of the cell cycle, as evidenced by down-regulated cyclin D1 expression through mTOR/STAT3 signaling pathway. In addition, ivermectin-gemcitabine induced apoptosis by ROS generation and reduction of mitochondrial membrane potential (MMP), and blocked mitophagy. In vivo experiments also confirmed that ivermectin-gemcitabine groups significantly suppressed the tumor growth of pancreatic cancer compared with gemcitabine alone groups. These results indicate that ivermectin has a synergistic effect with gemcitabine in preventing cancer progression and could be a potential antitumor drug for the treatment of pancreatic cancer. Key words: Pancreatic cancer, Gemcitabine, Ivermectin, apoptosis, ROS, mitochondria dysfunction

Citation Format: daeun lee, joon seong park, yun sun lee, hyeon woong kang, hyo jung kim. Ivermectin suppresses pancreatic cancer via mitochondria dysfunction [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 2320.
Abstract 2338: Bioluminescent assays for measuringthemetabolic state of cancer and immune cells
Authors: Bach Maggie , Leippe Donna , Karassina Natasha , Valley Michael , Cali James , Vidugiriene Jolanta
Published: June 2022

In the tumor microenvironment, cancer and immune cells compete for limited nutrients and both must undergo complex metabolic changes to support their function. To better understand the underlying mechanisms of tumor growth, metastasis and adaptation, a comprehensive analysis of metabolic pathways is needed. Success of such studies requires rapid and reliable approaches for monitoring key metabolic pathways, including glycolysis, the pentose phosphate pathway, fatty acid metabolism, amino acid metabolism and TCA cycle. Due to its inherent robustness, sensitivity and broad dynamic range, bioluminescence detection provides an attractive opportunity for developing such assays. Here, we implement a panel of bioluminescent metabolite assays (glucose, lactate, glutamine/glutamate, triglyceride/glycerol, cholesterol esters/cholesterol) to investigate the metabolic state of immune and cancer cells. We demonstrate flexibility of the assays in various volumes (96-, 384- well plates), sample types (2D, 3D cultures), automation platforms, and workflows, including detecting multiple metabolites in the same sample and monitoring metabolite changes over time by assaying multiple samples. We validate the performance of the assays by using metabolic pathway-specific inhibitors and demonstrating the utility of the assays as early indicators of cytotoxic T-cell activation. Finally, since metabolism is a dynamic process guided by fuel availability, we show the importance of medium composition when designing studies and suggest guidance for future work. In conclusion, understanding the crosstalk between tumor cells and their microenvironment is required for next-generation drug development. Bioluminescent metabolite assays provide the sensitivity, throughput and robustness required for rapid evaluation of changes in major metabolic pathways and are well suited for novel inhibitor screening and drug development.

Citation Format: Maggie Bach, Donna Leippe, Natasha Karassina, Michael Valley, James Cali, Jolanta Vidugiriene. Bioluminescent assays for measuringthemetabolic state of cancer and immune 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 2338.
Abstract 236: Nuclear receptor 4A1 ligands target T-cell exhaustion in colorectal cancer
Authors: Mohankumar Kumaravel , Wright Gus , Kumaravel Subhashree , Shrestha Rupesh , Zhang Lei , Abdelrahim Maen , Chapkin Robert S. , Safe Stephen
Published: June 2022

Colorectal cancer (CRC) is a highly complex disease with multiple risk factors. The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in several cancers and is a negative prognostic factor for cancer patient survival. Previous reports indicate a potential role for overexpression of NR4A1 in T-cell exhaustion and in this study, we aim to investigate the antitumorigenic activity of two bis-indole derived ligands (DIMs) that act as receptor antagonists. Immune competent C57BL/6 mice and mouse MC-38 colon cancer cells were used and tumor Infiltrating Lymphocytes (TILs) were isolated from mice either untreated or treated with CDIM/NR4A1 antagonists. FACS analysis and Real -Time PCR were performed to determine expression of exhaustion markers in these tumor T-cell population. 1,1-Bis(3΄-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF3) and the 3,5-dichlorophenyl analog (DIM-3,5-Cl2) at doses of 2.5 and 7.5 mg/kg/d inhibited tumor growth and downregulated expression of PD-L1, an NR4A1-regulated gene in MC-38 - derived tumors and cells. The mouse MC-38 colon cancer cell line was used in a syngeneic mouse model of colon cancer and tumor infiltrating lymphocytes (TILs) from MC-38 cell-derived colon tumors exhibited multiple markers of T-cell exhaustion in both CD8+ and CD4+ T-cells. Lymphocytes from an enlarged spleen in these tumor-bearing animals also exhibited markers of CD8+ and CD4+ T-cell exhaustion. Analysis of CD8+ cells from TILs showed that treatment with the NR4A1 antagonists modulated expression of several genes associated with T-cell exhaustion namely a decrease in TOX, TOX2 and NFAT mRNAs, activation of T-Bet. The percentage of CD8+ and CD4+ T-cells from tumors and spleen expressing PD-1, 2B4 and TIGIT was decreased in the treated vs control mice and TIM3 expression was also decreased in CD8+ (tumors and spleen) and CD4+ (tumors) T-cells. These findings suggest that NR4A1 antagonists are highly effective as anticancer agents in this mouse syngeneic colon tumor model by inactivating the pro-oncogenic activities of NR4A1 in tumors and by remediating NR4A1-regulated T-cell exhaustion in tumor and splenic lymphocytes.

Citation Format: Kumaravel Mohankumar, Gus Wright, Subhashree Kumaravel, Rupesh Shrestha, Lei Zhang, Maen Abdelrahim, Robert S. Chapkin, Stephen Safe. Nuclear receptor 4A1 ligands target T-cell exhaustion 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 236.
Abstract 2393: Chemerin signaling presents a novel metabolic weakness in clear cell renal cell carcinoma (ccRCC)
Authors: Wang Dazhi
Published: June 2022

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer,accounting for 70-80% of the cases. While surgical resection of early-stage tumors remains the standard carefor patients, advanced ccRCC, metastatic or recurrent, portends a 5-year survival rate of only 12%, primarilydue to insensitivity toward conventional chemotherapy. ccRCC is identifiable histologically by its dramaticlipid storage phenotype, similar to adipocytes; yet the role of lipid metabolism in ccRCC is poorly understood.One of the well-established risk factors for ccRCC is obesity, and given that adipose tissue is known to producea variety of pro-adipogenic signaling molecules (called adipokines), we hypothesized that adipokine signalingmight be involved in the etiology of ccRCC. Our lab has recently identified a pro-tumorigenic adipokineprotein, chemerin (RARRES2), that is positively correlated with tumor aggressiveness. Suppression of chemerinvia either monoclonal antibody or siRNA led to reduction in lipid storage and significant tumor growthimpairment in both in vitro and in vivo models, suggesting the feasibility of targeting lipid metabolism as atreatment option in ccRCC patients. In this study, we investigate the signaling mechanisms of chemerin andhave found that chemerin drives downstream cellular activities and lipid deposition. We utilized CRISPR toknockout two candidate receptors of chemerin, GPR1 and CMLKR1, to gain insight into the signal transduction.CMLKR1 is a G-protein coupled receptor widely expressed on a variety of immune cells whereas GPR1expression is reported primarily in adipose tissues. While unknown on their detailed signaling cascadez, bothreceptors affect lipid metabolism related gene profiles in our ccRCC cell lines. Furthermore, both GPR1 andCMKLR1 knockout lead to growth suppression and decreased intracellular lipid deposition, highlighting theinvolvement of both receptors in regulating lipid metabolism and conferring growth advantage in ccRCC. Ourdata so far have shown the feasibility of identifying metabolic weaknesses in ccRCC, raising that possibility thattargeting lipid metabolism could be beneficial in ccRCC. Further investigation will be conducted to assess thetargetability of this ligand/receptor axis and various in vivo models including PDX and transgenic will be usedto assess the translational potential of our findings.

Citation Format: Dazhi Wang. Chemerin signaling presents a novel metabolic weakness in clear cell renal cell carcinoma (ccRCC) [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 2393.
Abstract 2395: Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine
Authors: Barekatain Yasaman , Ackroyd Jeffrey , Yan Victoria , Khadka Sunada , Chen Ko-Chien , Kalluri Raghu , de Groot John , Huse Jason , muller Florian
Published: June 2022

Homozygous deletion of the CDK2NA locus frequently results in the co-deletion of methylthioadenosine phosphorylase (MTAP) in many fatal cancers such as glioblastoma multiform (GBM). In cell culture, cell lines with MTAP-deletions show elevations of its substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit PRMT5, which sensitizes MTAP-deleted cell lines to PRMT5 and MAT2A inhibition. While extensively corroborated in vitro, the clinical efficacy of these strategies ultimately relies on equally significant accumulations of MTA in human tumors. In this work, using comprehensive metabolomic profiling, we show that MTA is primarily secreted, resulting in exceedingly high levels of extracellular MTA in vitro. We further show that primary human glioblastoma tumors minimally accumulate MTA in vivo, which is likely explained by the metabolism of MTA by MTAP-competent stromal cells. Together, these findings highlight the metabolic discrepancies between in vitro models and primary human tumors and should thus be carefully considered in the development of the precision therapies targeting MTAPhomozygous deleted GBM.

Citation Format: Yasaman Barekatain, Jeffrey Ackroyd, Victoria Yan, Sunada Khadka, Ko-Chien Chen, Raghu Kalluri, John de Groot, Jason Huse, Florian muller. Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine [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 2395.
Abstract 2398: The tobacco cembranoid β-cembrenediol as promising prostate cancer recurrence suppressor lead
Authors: Mudhish Ethar A. , Siddique Abu Bakar , Abdelwahed Khaldoun S. , King Judy A. , Sayed Khalid A. El
Published: June 2022

Prostate cancer (PC) is the second leading cause of men death in the US, annually killing >34,000 men. Despite progress in early PC diagnosis and treatment, many patients have locally and distant disease recurrence. Therapeutic androgens deprivation have limited curative potential and associated with severe side effects and rapid resistance development justifying the dire need for discovery of novel recurrence inhibitors. (1S,2E,4R,6R,-7E,11E)-2,7,11-cembratriene-4,6-diol (β-CBT) and its 4-epi-analog (α-CBT) are the precursors to key flavor ingredients in leaves of most Nicotiana species. Nearly 40-60% of β- and α-CBT are purposely degraded during the commercial tobacco fermentation. Earlier, we reported the lack of β-CBT cytotoxicity below 50 µM while at 50 nM it reversed the calcitonin-stimulated decrease in transepithelial resistance and increased paracellular permeability in PC-3M PC cells. β-CBT also proven to stimulate tight junction formation between PC-3M cells, producing a tighter intercellular barrier. β-CBT 10-50 nM potently inhibited the calcitonin-stimulated PC-3M cells invasion in Matrigel assay. This study demonstrates the in vitro β-CBT anti-migratory (wound-healing assay) and anti-clonogenicity (colony-formation assay) activities against five PC cell lines, including the metastatic castration-recurrent human prostate cancer (mCRPC) CWR-R1ca. Meanwhile, β-CBT potently suppressed the in vivo locoregional and distant recurrences after the primary tumor surgical excision of the tumor formed by engrafting the human metastatic prostate PC-3M-Luc cells at the suprascupular region in male nude mice model. β-CBT treatments prevented organ and significantly suppressed bone metastasis and lacked any major toxicity over the 60-day study course. Therefore, β-CBT emerges as an effective promising PC recurrence and metastasis suppressive lead appropriate for near future use as a prospective nutraceutical.

Citation Format: Ethar A. Mudhish, Abu Bakar Siddique, Khaldoun S. Abdelwahed, Judy A. King, Khalid A. El Sayed. The tobacco cembranoid β-cembrenediol as promising prostate cancer recurrence suppressor lead [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 2398.
Abstract 2446: Low dose dimethyl sulfoxide (DMSO) downregulates the expression of androgen receptor (AR) and AR-variant 7 (AR-v7) in castration resistant prostate cancer (CRPC) cells
Authors: Khurana Namrata , Kim Hogyoung , Khan Talal , Kahhal Shohreh , Bukvic Amar , Abdel-Mageed Asim B. , Sikka Suresh C. , Mondal Debasis
Published: June 2022

The highly aggressive prostate cancer (PC) cells utilize androgen receptor (AR) signaling to facilitate their growth and metastasis. Despite androgen deprivation therapy (ADT), recurrence of castration-resistant prostate cancer (CRPC) often occur due to constitutive AR signaling via both full-length AR (AR-FL) and AR splice variants, primarily AR-V7. Therefore, safe strategies to suppress both AR-FL & AR-V7 expression in CRPC cells will be of significant benefit. Several plant-derived organosulfur compounds, e.g. sulforaphane and diallyl-trisulfide, can potently suppress AR expression in CRPC cells, but are yet to be clinically approved. We investigated whether dimethyl sulfoxide (DMSO) an approved organosulfur compound often used as a solvent for pharmaceutical agents, can similarly suppress AR levels in the CRPC cell lines, C4-2B and 22Rv1. Exposure to low dose DMSO (0.1-1%) was not cytotoxic to these CRPC cells, and less than 20% cytotoxicity was seen with 2.5% DMSO even at 96 h post-exposure. Interestingly however, DMSO dose-dependently suppressed AR-FL in C4-2B cells and both AR-FL and AR-V7 in 22Rv1 cells within 24 h post-exposure. Exposure to DMSO also downregulated the expression of hetero-nuclear ribonucleoprotein H1 (hnRNPH1), which is known to regulate AR expression and splicing. Although DMSO exposure showed a dose- and time-dependent effect on reactive oxygen species (ROS) production by PC cells, the AR-suppressive effect of DMSO was not altered by the antioxidant n-acetyl cysteine (NAC). Furthermore, although DMSO did not affect cell viability at these clinically-achievable concentrations, a significant (p<0.01) decrease in the migratory ability of both CRPC cell lines was clearly evident. Our novel findings indicate that safe doses of DMSO may be used to decrease AR expression and metastatic ability in CRPC cells.

Citation Format: Namrata Khurana, Hogyoung Kim, Talal Khan, Shohreh Kahhal, Amar Bukvic, Asim B. Abdel-Mageed, Suresh C. Sikka, Debasis Mondal. Low dose dimethyl sulfoxide (DMSO) downregulates the expression of androgen receptor (AR) and AR-variant 7 (AR-v7) in castration resistant prostate cancer (CRPC) 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 2446.
Abstract 2448: Mmp-2 facilitates prostate cancer entry into and exit from dormancy
Authors: Nasr Mostafa
Published: June 2022

Prostate cancer (PC) patients often relapse with skeletal metastases years after remission. This is attributed to the dormancy program acquired by disseminated tumor cells (DTCs) in the bone microenvironment. Understanding the molecular mechanisms driving dormancy & reawakening (D&R) can reveal novel therapeutic opportunities. Matrix metalloproteinases (MMPs) are involved in each step of the metastatic cascade. Recent studies have shown upregulation of MMP-2 expression in D&R. To determine whether MMP-2 plays a causal role, we initially developed a model representing dormancy in PC cell lines using basal growth conditions and high seeding density. The generated dormant cells (D-Cs) formed clusters and had negative expression of Ki-67 (98% of cells, p = 0.007), Low ERK/P38 activity ratio (p = 0.002), and higher percentage of G0/G1 arrested cells (p = 0.0004). The clusters can remain dormant and viable even after long-term culture (>28 days). We observed that D-Cs can reawaken in-vitro by re-introducing serum-containing media. Next, we assessed MMP-2 expression in D-Cs and reawakened cells. We found MMP-2 expressed during entry into dormancy but highly upregulated during reawakening. Using a siRNA approach, we found that MMP-2 silencing prevented entry into dormancy by significantly inhibiting cluster formation and survival. To determine the role of MMP-2 in reawakening, we used a pharmacological approach due to the temporal nature of MMP-2 silencing by siRNA. Using a highly selective bisphosphonate-based MMP-2 inhibitor (ML115), we found that blocking MMP-2 activity prevented the reawakening of prostate cancer cells without impacting cell viability at 1µM as assessed by caspase-3 cleavage via immunoblotting. In conclusion, we developed an in-vitro PC-specific model for dormancy & reawakening. We found MMP-2 to be key for controlling entry and exit from dormancy in-vitro. We are currently elucidating the mechanisms through which MMP-2 mediates these effects. We also posit that our selective bone-targeting MMP-2 inhibitor may be effective in blocking the reawakening of dormant cells in-vivo.

Citation Format: Mostafa Nasr. Mmp-2 facilitates prostate cancer entry into and exit from dormancy [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 2448.
Abstract 2535: Omental preadipocytes support ovarian cancer tumorigenesis by mediating genes important for extracellular matrix reorganization
Authors: Waters Jennifer A. , Robinson Mikella , Gilbert Samuel F. , Urbano Ixchel , House Carrie D.
Published: June 2022

Ovarian cancer is the most lethal gynecological malignancy in the United States, with a known predilection for metastasizing to the omentum, a sheet of fatty tissue that encloses the abdomen. While it has been shown that ovarian cancer cells invade towards mature adipocytes in vitro, the contribution of stromal cells that reside in the omentum is not well understood. We hypothesize that preadipocytes, the precursor cells that give rise to omental adipocytes, are important mediators of cancer progression in the omental tumor microenvironment as they have a pro-inflammatory secretome that is distinct from that of mature adipocytes. To evaluate this hypothesis, we tested whether preadipocyte secreted factors altered tumorgenicity of ovarian cancer cells using colony formation and cell viability in vitro and a limiting dilution in vivo in a subcutaneous mouse model. The mouse studies showed that low dilutions of cancer cells require preadipocytes for engraftment and tumor formation. A transplantation assay showed that cancer cells require the presence of preadipocytes for sustained tumor formation capacity. In vitro co-culture assays revealed that preadipocytes secrete factors that increase clonogenicity and extend cell viability in serum-free conditions. To identify signaling pathways induced by preadipocytes we co-cultured cancer cells with either primary human omental preadipocytes or differentiated mature adipocytes derived from the same female donor and performed RNA sequencing and gene set enrichment analysis (GSEA). We identified differentially expressed genes in cancer cells that were unique to preadipocyte co-culture, including DCN, MMP2, COL6A2 and COL12A1, genes involved in extracellular matrix organization. The most significantly upregulated gene was insulin-like growth factor binding protein 5 (IGFBP5), which has functions in insulin-like growth factor signaling and ECM interactions. Ongoing studies using CRISPR edited cancer cells suggest IGFBP5 mediates the enhanced tumorigenesis of cancer cells in the presence of preadipocytes. Our findings highlight the role of preadipocytes in the ovarian tumor microenvironment and implicate IGFBP5 as a preadipocyte-mediated gene. Future studies will determine if there is a link between the upregulation of IGFBP5 and modulation of extracellular matrix proteins that promote cancer progression. Understanding these processes will enable the design of more effective therapies for treating ovarian cancer.

Citation Format: Jennifer A. Waters, Mikella Robinson, Samuel F. Gilbert, Ixchel Urbano, Carrie D. House. Omental preadipocytes support ovarian cancer tumorigenesis by mediating genes important for extracellular matrix reorganization [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 2535.
Abstract 2555: Immunophenotyping early stage lung adenocarcinomas (LUAD) by histologic subtype
Authors: Xi Zhanhao , Pfefferkorn Roxana , Burks Eric , Beane Jennifer , Mazzilli Sarah
Published: June 2022

The immune microenvironment is thought to influence Lung Adenocarcinoma (LUAD) subtype formation, disease progression, and thus patient outcome. Despite recent advancements in screening, immunotherapy, and targeted therapy, the biology of aggressive LUAD is still poorly understood, and in-depth characterization associated with LUAD histologic subtypes and progression are needed. In this study, we sought to measure the immune environment associated with different LUAD subtypes using Imaging Mass Cytometry (IMC). IMC has emerged as a powerful new tool that enables the multiplex imaging of more than 50 markers simultaneously, quantify multiple protein expressions at a single cell level, and preserve tissue spatial information. We stained a tissue microarray of early stage LUAD patient samples, representing different LUAD subtypes, with a panel of 21 antibodies. These antibodies are designed to phenotype immune cell subsets, as well as using Ki67 to stain for highly proliferative epithelial cells. This will allow us to better understand how different immune cells are interacting with each other, as well as how they interact with highly proliferating epithelial cells. The resulting ablated samples generate images containing antibody intensity and coordinates, which we then perform cell segmentation to generate single cell data. The marker expression is then quantified, and single cell data analyzed using HistoCAT. Cell neighborhood interactions are analyzed based on spatial relationships, and similarly interacting cells are grouped into phenoclusters. Our results suggest that a Ki67 high phenocluster within solid LUAD subtypes have fewer macrophages (CD68, CD163) and T cell populations (CD8, CD4, FOXP3), however, are enriched for plasma B cells (CD20) and immune suppressive populations (PD1, PDL1). In contrast, a Ki67 high cluster within lepidic subtypes are enriched for of macrophage (CD68) and T cell populations (CD8, CD4, FOXP3). Using IMC, we have begun to identify immune subset shifts in LUAD. This early analysis of LUAD using IMC begins to shed light on the immune regulation that may indicate progression and/or guide intervention approaches.

Citation Format: Zhanhao Xi, Roxana Pfefferkorn, Eric Burks, Jennifer Beane, Sarah Mazzilli. Immunophenotyping early stage lung adenocarcinomas (LUAD) by histologic subtype [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 2555.
Abstract 2556: The protective role of mast cells against neuroendocrine prostate cancer depends on the release of cytokines mediated by intracellular osteopontin
Authors: Sulsenti Roberta , Frossi Barbara , Cancila Valeria , Enriquez Claudia , Ferri Renata , Sangaletti Sabina , Tripodo Claudio , Pucillo Carlo Emilio , Colombo Mario Paolo , Jachetti Elena
Published: June 2022

Fatal neuroendocrine prostate cancer (NEPC) often emerges in patients relapsing after hormone therapies. Besides, de novo NEPC can rarely occur in treatment-naïve patients. Treatment-related and de-novo NEPC have different genomic alterations but share a common transcriptional profile. Investigating the tumor microenvironment, we recently found that mast cells (MCs) accumulate within hormone-sensitive prostate cancer favoring its growth, whereas are excluded by de-novo NEPC both in patients and in the transgenic TRAMP spontaneous mouse model. TRAMP mice backcrossed with MCs-deficient KitWsh mice showed increased frequency of de-novo NEPC. The frequency of de-novo NEPC similarly raised also in TRAMP mice deficient for the matricellular protein osteopontin (OPN). Reconstituting KitWsh-TRAMP mice with wild type, but not with OPN-deficient, MCs lowered the frequency of NEPC to that of untreated TRAMP mice. We found that MCs stain positive for OPN in tumor sections and in vitro cultures, but release a tiny amount of OPN in supernatants if compared to NEPC cells. Notably, OPN has both secreted (sOPN) and intracellular (iOPN) forms; the latter can bind to MyD88 and regulate the signaling downstream toll-like receptors (TLRs). In vitro, wild type, but not OPN-/- or MyD88-/-, MCs inhibited the proliferation of NEPC cells. Also, in silico analyses showed that genes related to inflammatory response and TLRs signaling are down regulated in human and murine NEPC.Our data suggest that TLRs/MyD88/iOPN-mediated pathways induce MCs to release factor(s) able to restrain NEPC. Further studies are required to molecularly dissect this novel function of MCs, to identify actionable targets against NEPC.

Citation Format: Roberta Sulsenti, Barbara Frossi, Valeria Cancila, Claudia Enriquez, Renata Ferri, Sabina Sangaletti, Claudio Tripodo, Carlo Emilio Pucillo, Mario Paolo Colombo, Elena Jachetti. The protective role of mast cells against neuroendocrine prostate cancer depends on the release of cytokines mediated by intracellular osteopontin [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 2556.
Abstract 2581: IL-15 expressing MSCs for NK cell-mediatedcancer immunotherapy
Authors: Sehgal Drishti , Shanas No'ad , Prabha Swayam
Published: June 2022

Mesenchymal stem cells (MSCs) offer the possibility of achieving highly selective, tumor-targeted drug delivery because of their ability to home to malignant tissues. IL-15 is currently in clinical trial either alone or as an adjuvant for certain types of metastatic solid tumors. Systemic administration of IL-15 is associated with severe toxicities including hemodynamic imbalance, lymphopenia, and other cytokine-related complications. We hypothesized that tumor targeted delivery of IL-15 using MSCs can  improve efficacy and reduce toxicity  associated with IL-15.  MSCs were engineered to express IL-15 using a lentiviral system (pLenti-C-Myc-DDK-P2A-Puro vector; Invitrogen) tagged with C-terminal MYC/DDK tag for easy detection and purification with anti-DDK antibody. We achieved stable transfection of IL-15 in MSCs (5.7 ng/106 MSCs in 24 hrs; ELISA). Bioactivity of secreted IL-15 was confirmed by splenocyte proliferation assay. We then tested the anticancer activity of IL-15 secreting MSCs in a syngeneic LL/2 lung cancer model. Animals treated with IL-15 secreting MSCs demonstrated significant inhibition of tumor growth and prolonged survival compared to mice treated with IL-15 alone. It was further demonstrated that IL-15 expressing MSCs enhance tumor infiltration of various immune cells. These studies demonstrate the potential of developing novel MSC-based immunotherapy constructs with reduced toxicity.

Citation Format: Drishti Sehgal, No'ad Shanas, Swayam Prabha. IL-15 expressing MSCs for NK cell-mediatedcancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2581.
Abstract 2601: Concomitant treatment of ovarian cell lines with Tumor Treating Fields (TTFields) and PARP inhibitors
Authors: Martinez-Conde Antonia , Dor-On Eyal , Haber Adi , Giladi Moshe , Weinberg Uri , Palti Yoram
Published: June 2022

Background: PARP inhibitors (PARPi) are an effective treatment option for ovarian cancer patients harboring deleterious BRCA mutations. While such mutations prevail in one third of ovarian cancer patients, most of the other patients gain little survival benefit by the use of PARPi. Tumor Treating Fields (TTFields) are alternating electric fields with antimitotic effects on cancerous cells, also shown to induce a state of BRCAness in several types of cancer cell lines. The current study aimed to explore the potential of combining TTFields with PARP inhibition for the treatment of ovarian cancer in vitro.

Methods: Ovarian carcinoma cells A2780 (BRCA1/2 wild type) and OVCAR3 (BRCA1 wild type, BRCA2 deep deletion) were treated with TTFields using the inovitro system. Treatment was applied for 72 h at an intensity of 1.2 V/cm RMS and frequency of 200 kHz. The efficacy of concomitant application of TTFields with the PARP inhibitors niraparib and olaparib was examined via measurements of cell survival, colony forming ability, overall effect (multiplicity product of cytotoxicity and clonogenicity), and apoptosis induction.

Results: A2780 or OVCAR3 cells treated with TTFields displayed about 50% reduction in cell counts. Co-application of TTFields with PARPi enhanced the cytotoxic effect compared to that demonstrated by TTFields or PARPi alone. Concomitant application also resulted in reduced clonogenicity, enhanced overall effect, and increased apoptosis.

Conclusions: The results suggest potential benefits for TTFields concomitant with PARP inhibiting agents for treatment of ovarian cancer.

Citation Format: Antonia Martinez-Conde, Eyal Dor-On, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Concomitant treatment of ovarian cell lines with Tumor Treating Fields (TTFields) and PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2601.
Abstract 2634: Vasoactive intestinal polypeptide (VIP) regulates ZEB1 expression in cancer
Authors: Rao Ishani H. , Dhamsania Rohan , Waller Edmund K. , Chandrasekaran Sanjay
Published: June 2022

Background: In cancer immunology, VIP is a neuropeptide with pleiotropic effects that include inhibiting T cell activation, increasing Treg frequency, and augmenting the myeloid-derived suppressor cells function. VIP expression by cancer may represent a paracrine immune-checkpoint pathway while simultaneously having autocrine effects on cancer growth. We used an in silico model to identify genes whose over-expression in cancer was correlated with VIP expression and confirmed the role of VIP receptor (VIPR) signaling in vitro using VIPR inhibitors.

Methods: The TCGA PanCan dataset (UCSC Xena) was used to compare mRNA expression data of VIP versus 760 genes involved in 48 cancer related pathways. Associations by cancer histology and germ layer were calculated. A Pearson’s Coefficient cutoff of R > [0.3] defined a significant gene association. VIP and target gene expression were confirmed and quantified by western blot analysis in cancer cell lines and VIPR signaling was inhibited using ANT008, a novel VIPR inhibitor.

Results: Significant associations were observed between VIP expression and 10 genes (Table 1). Strong associations (R > [0.4]) were observed in cancers derived from ectodermal and mesodermal germ layers. The strongest gene association was for VIP and ZEB1 in stomach adenocarcinoma (STAD, R = 0.7643). In vitro experiments using Jurkat T cells confirmed expression of VIP and ZEB1 proteins. Treatment with the VIPR inhibitor ANT008 (10μM) reduced VIP and ZEB1 protein expression by 50%. A similar fold increase in ZEB1 expression was observed with exogenous VIP. PCR studies are currently underway.

Conclusions: The results demonstrate autocrine signaling between VIP expression and cancer associated gene pathways. ZEB1 is a known EMT (epithelial mesenchymal transition) regulator, and our in vitro studies novelly link VIP to EMT and demonstrate that this pathway is therapeutically targetable using a VIPR inhibitor. Further studies in the pertinent tumor histologies are warranted.

Table 1. Genes All Samples Ectoderm (All) Mesoderm (All) Endoderm (All) MAPK3 0.3120 0.3537 0.2252 0.3380 ZEB1 0.3621 0.4341 0.4901 0.3348 NOS2 0.3611 0.3186 0.4849 0.3428 TEK 0.3113 0.3336 0.5131 0.2600 PTCH1 0.3031 0.4040 -0.0061 0.2879 EI4G1 -0.3216 -0.4268 -0.3779 -0.2064 GMPS -0.3074 -0.4698 -0.3005 -0.0928 CDK2 -0.3186 -0.4687 -0.2345 -0.0563 RUVBL1 -0.3135 -0.3270 -0.4716 -0.1886 TIMELESS -0.3036 -0.4284 -0.3717 -0.1505

Citation Format: Ishani H. Rao, Rohan Dhamsania, Edmund K. Waller, Sanjay Chandrasekaran. Vasoactive intestinal polypeptide (VIP) regulates ZEB1 expression in 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 2634.
Abstract 2645: Targeting mRNA processing pathways in ovarian cancer with a small molecule inhibitor
Authors: Tailor Dhanir , Tsai Chia-Feng , Honkala Alexander , Li Wenqi , Liu Tao , Pejovic Tanja , Malhotra Sanjay V.
Published: June 2022

mRNA processing related pathways, including spliceosome and ribosome are over-expressed and unregulated in cancer cells. To investigate the mRNA processing pathways in patient samples, we performed a comprehensive, tandem mass tag (TMT) labeling-based proteome and phosphoproteome profiling of the normal and ovarian cancerous tissues from patients treated with different chemotherapy revealed that malignant cells have overexpression of mRNA processing pathways compared to normal cells. For this analysis, equal amount of tissues were homogenized, and tissue lysates were collected and processed for the quantitative proteomics and phosphoproteomics analysis. KEGG pathway enrichment analysis revealed that spliceosome, basal transcription factors, mRNA surveillance, ribosome and ribosome biogenesis in eukaryotes pathways are upregulated in cancer cells compared to control. On other hand, pathways associated with metabolism and fatty acid biosynthesis were downregulated in malignant cells. Moreover, kinase Enrichment Analysis shows that CDK2, ATM, ATR, and MAPK associated kinases are upregulated in cancer cells compared to normal. Based on proteome analysis of patient samples, we investigated the pharmacological inhibition of Y-box binding protein 1 (YB-1). This is an RNA binding protein and key regulator of pre-mRNA alternative splicing and processing. We recently showed that a natural product-derived small molecule (SU056) inhibits YB-1 activity. We found that SU056 binds to the YB-1 and blocks the RNA binding pocket. Proteomics results suggest that treatment with this drug strongly inhibits the spliceosome pathway. SU056 treatment arrests the ovarian cancer cells in G1 phase and inhibits the CDK2 and Cyclin E. SU056 inhibits the tumor progression and metastasis in the ID8 mice model and sensitizes the OVCAR8 NSG mice model for paclitaxel treatment. Our study provides a compelling case for YB-1 inhibition using SU056 as a potential therapy for ovarian cancer.

Citation Format: Dhanir Tailor, Chia-Feng Tsai, Alexander Honkala, Wenqi Li, Tao Liu, Tanja Pejovic, Sanjay V. Malhotra. Targeting mRNA processing pathways in ovarian cancer with a small molecule inhibitor [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 2645.
Abstract 2651: The tankyrase inhibitor OM-153 demonstrates anti-tumor effect with a therapeutic index above 10
Authors: Brinch Shoshy Alam , Amundsen-Isaksen Enya , Espada Sandra , Aizenshtadt Aleksandra , Holmen Lone , Hammarström Clara , Høyem Merete , Scholz Hanne , Grødeland Gunnveig , Sowa Sven T. , Galera-Prat Albert , Lehtiö Lari , Meerts Ilonka A.T.M. , Leenders Ruben G. G. , Wegert Anita , Krauss Stefan , Waaler Jo
Published: June 2022

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) poly-ADP-ribosylate target proteins, including AXIN proteins, to earmark them for degradation by the ubiquitin-proteasomal system. Hence, inhibition of TNKS1/2 can stabilize AXIN proteins, and consequently β-catenin degradosomes, resulting in inhibition of WNT/β-catenin signaling. Although several potent small-molecules have been developed to inhibit TNKS1/2 and oncogenic WNT/β-catenin signaling, also showing convincing anti-tumor effects in numerous mouse cancer models, there are currently no TNKS1/2 inhibitors available in the clinic. The development has mainly been disadvantaged by concerns over previous reports unfolding intestinal toxicity, apparently caused by on-target and WNT/β-catenin signaling pathway-specific side effects, and a deficient therapeutic index. Here we show that the novel, highly potent and selective1,2,4-triazole-based TNKS1/2 inhibitor OM-153, displaying improved ADME-properties and mouse pharmacokinetics, reduced WNT/β-catenin signaling and tumor progression in COLO 320DM colon cancer xenografts upon peroral (PO) administration of 0.33-10 mg/kg twice daily (BID). In addition, combined OM-153 and anti-PD-1 treatment conferred a synergistic anti-tumor effect in a B16-F10 mouse melanoma model. In a 28-day repeated dose mouse toxicity study, body weight loss, intestinal damage and tubular damage in the kidney was documented after PO BID administration of 100 mg/kg. In contrast, in mice treated PO BID using 10 mg/kg, the intestinal architecture was intact and no atypical histopathological changes were observed in other organs, while clinical biochemistry and haematological analyses did not identify any changes indicating considerable toxicity. The results provide a scaffold for using OM-153 as a potential anti-cancer treatment and additional preclinical and clinical evaluations.

Citation Format: Shoshy Alam Brinch, Enya Amundsen-Isaksen, Sandra Espada, Aleksandra Aizenshtadt, Lone Holmen, Clara Hammarström, Merete Høyem, Hanne Scholz, Gunnveig Grødeland, Sven T. Sowa, Albert Galera-Prat, Lari Lehtiö, Ilonka A.T.M. Meerts, Ruben G. G. Leenders, Anita Wegert, Stefan Krauss, Jo Waaler. The tankyrase inhibitor OM-153 demonstrates anti-tumor effect with a therapeutic index above 10 [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 2651.
Abstract 2664: Development of covalent KRASG12C inhibitor APG-1842 for the treatment of solid tumors
Authors: Gu Shaoulai , Li Qiang , Li Chao , Wang Qixin , Fang Douglas D. , Wang Shaomeng , Yang Dajun , Zhai Yifan
Published: June 2022

The KRAS gene, which encodes a guanosine triphosphate (GTPase) protein, is frequently mutated in solid cancers. The KRASG12C mutation aberrantly attenuates GTPase activity, leading to accumulation of the GTP-bound activated form of the KRAS protein and activation of downstream signaling pathways. This genotype occurs in approximately 13% of non-small-cell lung cancers (NSCLCs) and 1% to 3% of colorectal cancers and other solid tumors. In this study, investigational agent APG-1842 was characterized as a potent, selective, and covalent KRASG12C inhibitor that irreversibly and specifically blocks KRASG12C in an inactive guanosine diphosphate (GDP)-bound state and downregulates KRAS-dependent signaling.

A RAS-GTP pull-down assay demonstrated that APG-1842 (at a concentration as low as 12 nM) completely blocked GDP-GTP nucleotide exchange, which stabilized an inactive GDP-bound form of KRASG12C and thereby inhibited RAS and RAF binding in KRASG12C-mutant human NSCLC NCI-H358 cells. In these cells, APG-1842 dose dependently inhibited KRAS-dependent signaling target proteins, including phosphorylated extracellular signal-regulated kinase (ERK), S6, and serine/threonine protein kinase Akt (protein kinase B), with a half-maximal inhibitory concentration (IC50) value of 4 nM for ERK1/2 phosphorylation. Using a panel of 18 cancer cell lines representing multiple cancer types in cell viability assays, we found that APG-1842 selectively inhibited cell proliferation in KRASG12C-mutated, but not non-KRASG12C or KRAS wild-type, cell lines.

Oral APG-1842 in mice carrying cell-derived NCI-H358 NSCLC subcutaneous xenografts exerted significant antitumor efficacy at doses ranging from 3 to 100 mg/kg, resulting in tumor regression at 30 mg/kg and above. Consistent with the cellular results, APG-1842 dose dependently inhibited ERK1/2 and S6 phosphorylation in xenografts after 6 and 24 hours of treatment, correlating with systemic and tumor drug exposures. Significant antitumor activity of APG-1842 was also confirmed in a panel of 8 KRASG12C-mutated patient-derived xenograft models derived from patient samples of lung, colorectal, and gastric cancers.

Collectively, these preclinical results suggest that APG-1842 is a potent, bioavailable, and highly selective KRASG12C inhibitor, laying a foundation for clinical development of this agent for patients with KRASG12C-mutant solid tumors.

Citation Format: Shaoulai Gu, Qiang Li, Chao Li, Qixin Wang, Douglas D. Fang, Shaomeng Wang, Dajun Yang, Yifan Zhai. Development of covalent KRASG12C inhibitor APG-1842 for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2664.
Abstract 2720: Unbiased identification of RNA splicing-defined patient populations in lung adenocarcinoma
Authors: Crowther Audrey
Published: June 2022

Aberrant alternative splicing is known to directly contribute to tumor progression in lung adenocarcinoma (LUAD). While the action of individual splicing variants as cancer drivers have been characterized in LUAD, the global splicing landscape and how that corresponds to patient prognosis is poorly understood. Using whole transcriptome RNA-Seq data provided from The Cancer Genome Atlas (TCGA), we applied a new computational pipeline, OncoSplice, to identify potentially novel prognostic disease splicing subtypes and elucidate the biological mechanisms underlying these subtypes. OncoSplice identified over 30 novel splicing-defined subtypes of LUAD, several of which correlate with worse patient survival. Additionally, we identified patient clusters that were significantly enriched in mutations for RNA binding proteins, such as Cap Methyltransferase 2 (CMTR2) and RNA Binding Motif 10 (RBM10). More specifically, mutations in the splicing factor RBM10 were found to be enriched but not requisite for the dominant poor prognosis splicing subtype in LUAD. In non-mutant patients within this subset, RBM10 gene expression downregulation was induced, mimicking the mutation phenotype. Hence, we find alternative non-genetic pathways for impacting splicing in LUAD that leads to poor prognosis through disruption of canonical splicing.

Citation Format: Audrey Crowther. Unbiased identification of RNA splicing-defined patient populations in lung 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 2720.