bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 859: Characterizing SMARCA4/STK11/KEAP1 co-mutant lung adenocarcinoma
Authors: Costa Emily , Wohlheiter Corrin , Tischfield Samuel , Funnell Alister , Poirier JT , Villalonga Álvaro Quintanal , Sen Triparna , Rudin Charles
Published: June 2022

Lung adenocarcinoma (LUAD), constituting 50% of non-small cell lung cancer, is classically defined by the presence of driver mutations in oncogenes such as KRAS, EGFR, and BRAF. While the development of driver-targeted therapies has significantly improved survival in subsets of patients with LUAD, studies examining tumor sequencing data and clinical outcomes have shown that intra-driver genetic and phenotypic heterogeneity underlie differential patient responses to these therapies. In particular, co-occurring alterations in the genes SMARCA4 (BRG1), STK11 (LKB1), and KEAP1 are predictive of exceptionally poor prognosis and worse overall survival in metastatic LUAD patients. SMARCA4 loss is also a hallmark of undifferentiated, highly chemoresistant NSCLC tumors. While these co-mutations most often appear in KRAS-driven tumors, their association with poor outcomes is maintained regardless of driver status. Studies exploring the molecular features of SMARCA4-deficient and STK11/KEAP1 co-mutant LUAD have yielded translational insights specific to these subsets, establishing a rationale to characterize the unique biology of SMARCA4/STK11/KEAP1 (BLK) triple-mutant LUAD. To this end, we are profiling BLK LUAD through a combination of genomic, transcriptomic, epigenomic, and phenotypic assays in in vitro models. We have generated paired isogenic in vitro models of BLK LUAD via CRISPR-Cas9 knockout. Phenotypic analyses of these cell lines, whose engineered genotypes model varying combinations of SMARCA4, KEAP1, and STK11 loss, have revealed growth rate differences in vitro, and pairwise comparisons of RNA-sequencing in these lines show downregulation of lung differentiation markers and upregulation of metastasis-associated genes concomitant with SMARCA4 loss. In conclusion, we have generated isogenic preclinical models showing molecular and phenotypic features consistent with observations reported in BLK LUADs. Further molecular and functional characterization of these models may reveal mechanistic insights into the biology of this subset of aggressive tumors.

Citation Format: Emily Costa, Corrin Wohlheiter, Samuel Tischfield, Alister Funnell, JT Poirier, Álvaro Quintanal Villalonga, Triparna Sen, Charles Rudin. Characterizing SMARCA4/STK11/KEAP1 co-mutant 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 859.
Abstract 902: Identification, characterization, and targeting of desmoplastic small round cell tumor cancer stem cell-like cells
Authors: Magrath Justin W. , Hartono Alifiani B. , Lee Sean B.
Published: June 2022

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive form of pediatric cancer caused by a translocation between chromosomes 11 and 22, creating the novel EWSR1-WT1 fusion gene. Current therapy including surgery and the P6 chemotherapy regimen is insufficient, leading to a 5-year survival rate of only 15-25%. One potential explanation for this poor prognosis is the existence of a cancer stem cell (CSC) population with the ability to resist chemotherapy and cause both tumor recurrence and metastasis. While a CSC population has been identified in the closely related Ewing Sarcoma, most commonly caused by the EWSR1-FLI1 fusion gene, no CSC population has yet been identified in DSRCT. In this study, novel culture conditions were developed that enabled the formation of tumorspheres in vitro in the two commonly available DSRCT cell lines: JN-DSRCT-1 and BER-DSRCT. These novel culture conditions led to upregulation of stemness genes at the RNA and protein level including NANOG and SOX2, two genes that are associated with poor survival outcomes in TCGA sarcomas data. CCK-8 assay demonstrated an increased chemoresistance for tumorspheres versus normal adherent culture especially for doxorubicin, which was confirmed by reduction in PARP cleavage. This chemoresistance may be partially explained by a more quiescent CSC state as shown by reduced cell proliferation and a lower percentage of cells in the S and G2/M phases. However, cells grown in tumorspheres still robustly formed tumors in vivo, thus demonstrating the two hallmark CSC traits of chemoresistance and tumor formation. RNA-seq was performed to elucidate key differences between the adherent and sphere culture conditions and identify potential targets for the therapeutically important CSC-like population. Overrepresentation analysis and gene set enrichment analysis both showed upregulation of pathways related to chromatin assembly and disassembly, suggesting epigenetic changes as DSRCT cells move to a more stem-like state. RNA-seq further revealed kinases upregulated in tumorspheres including B-lymphocyte kinase (BLK) which previous studies have demonstrated is oncogenic, regulated by EWSR1-WT1, and not necessary for normal survival. BLK knockdown reduced CSC-like properties including abrogation of tumorsphere formation and reduction in the levels of SOX2 and NANOG. Together, this work for the first time identifies a CSC-like population in DSRCT and BLK as a potential DSRCT CSC target.

Citation Format: Justin W. Magrath, Alifiani B. Hartono, Sean B. Lee. Identification, characterization, and targeting of desmoplastic small round cell tumor cancer stem cell-like 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 902.
Abstract 928: USP13 promotes development and metastasis of high-grade serous ovarian carcinoma in a novel mouse model
Authors: Kwon Juntae , Choi Hyungja , Ware Anna D. , Morillo Bernadette Cecilia , Wang Haiyang , Han Cecil
Published: June 2022

Epithelial ovarian cancer is the most lethal gynecologic malignancy and one of the most common causes of cancer mortality among women worldwide. Ubiquitin-Specific Peptidase 13 (USP13) gene copy is strongly amplified in human epithelial ovarian cancer, and high USP13 expression is correlated with poor survival outcomes, yet its pathological contribution to ovarian tumorigenesis remains unknown. Here, we crossed a conditional Usp13 overexpressing knock-in mouse with a conditional knockout of Trp53 and Pten mouse, and generated a novel ovarian cancer genetically engineered mouse model (GEMM), which closely recapitulates the genetic changes driving ovarian cancer in humans. Overexpression of USP13 with deletion of Trp53 and Pten in murine ovarian surface epithelium accelerated ovarian tumorigenesis and led to decreased survival in mice. Notably, USP13 greatly enhanced peritoneal metastasis of ovarian tumors with frequent development of hemorrhagic ascites. The primary and metastatic tumors exhibited morphology and clinical behavior similar to human high-grade serous ovarian cancer. Co-inhibition of USP13 and AKT significantly decreased the viability of the primary murine ovarian cancer cells isolated from the GEMM. USP13 also increased the tumorigenic and metastatic abilities of primary murine ovarian cancer cells in a syngeneic mouse study. These findings suggest a critical role of USP13 in ovarian cancer development and reveal USP13 as a potential therapeutic target for ovarian cancer.

Citation Format: Juntae Kwon, Hyungja Choi, Anna D. Ware, Bernadette Cecilia Morillo, Haiyang Wang, Cecil Han. USP13 promotes development and metastasis of high-grade serous ovarian carcinoma in a novel mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 928.
Abstract 950: Role of epithelial-mesenchymal plasticity in immune remodeling during metastasis
Authors: Visal Tanvi H.
Published: June 2022

Introduction: Breast cancer is the most prevalent type of cancer in women, accounting for 12% of all annual cancer cases worldwide with most deaths attributed to metastasis. It displays both inter- and intra-tumoral heterogeneity which is linked to epithelial-mesenchymal transition (EMT). This program involves the acquisition of a mesenchymal-like phenotype allowing cells to disseminate from the primary tumor and metastasize. However, recent studies show that tumor cells do not need to undergo complete EMT to gain invasive potential; instead, co-expression of both epithelial and mesenchymal markers is critical for metastasis. Such tumor cells are thought to display epithelial-mesenchymal plasticity (EMP) which bestows them with enhanced fitness and flexibility to fulfill diverse requirements of the metastatic cascade. Additionally, hybrid EM phenotype has been linked to immunesuppression but has been understudied due to lack to suitable models with intact immune system. Thus, this necessitates the mechanistic dissection of tumor immune microenvironment (TIME) in models with EM plasticity. To characterize the contribution of EMP to metastasis, we utilized syngeneic mouse mammary tumor cell lines, which are hybrid EM and investigated their proteomic and cytokine profile and identified several novel cytokines.

Methods: Reverse Phase Protein Array (RPPA) was performed to determine the proteomic profile of tumors with EM plasticity. In addition, Luminex assay was performed to assess the cytokine/chemokine profile in above tumor models.

Results: The RPPA involved a panel of 450 antibodies and showed that tumors with EM plasticity were enriched in specific immunesuppressive proteins. At the same time, Luminex analysis was suggestive of recruitment of tumor-associated macrophages (TAMs) in models with EM plasticity. Additionally, cells with EM plasticity showed the ability to induce tumor-promoting/M2-like macrophages (in vitro).

Conclusions: Our data indicates that cells/tumors with EM plasticity show differential protein expression than E and M counterparts and are enriched in specific immunesuppressive proteins. Moreover, such cells have immune networks (cytokines/chemokines) that promote recruitment of TAMs - the largest myeloid infiltrate in the microenvironment of solid tumors. Thus, our study invites more attention to investigate the mechanisms of immunesuppression and metastasis in tumors with EM plasticity.

Citation Format: Tanvi H. Visal. Role of epithelial-mesenchymal plasticity in immune remodeling during 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 950.
Abstract 964: Tumor cell plasticity promotes metastasis across heterogeneous tumors
Authors: Winkler Juliane , Tan Weilun , Pisco Angela O. , Goga Andrei , Darmanis Spyros , Werb Zena
Published: June 2022

Metastasis, the growth of secondary tumors in distant organs, is the major cause of cancer-related deaths due to insufficient therapeutic effects of conventional treatment. Individual tumor cells exhibit heterogeneous features and growth potentials within the same tumor that may influence metastasis formation. During the metastatic cascade, tumor cells adapt their phenotype to various microenvironments that are distinct from their original site resulting in heterogeneous metastatic cells that often are resistant to conventional treatment. The underlying mechanisms of why some tumor cells can give rise to metastases, whereas others cannot are remarkably poorly understood.

We established patient-derived xenograft (PDX) models of breast cancer with different metastatic potential and preserved tumor heterogeneity. We analyzed the intrinsic cellular programs of individual tumor cells that influence the metastatic potential of breast cancers using different single-cell RNA-sequencing protocols (MULTI-Seq and Smart-Seq2). Using gene expression profiling of matched primary tumor and metastatic cells of 13 human breast cancers, we demonstrated that while human tumors showed profound inter-patient heterogeneity, they also shared signatures of differentially expressed genes between primary tumor and metastatic cells. Tumors with similar metastatic capabilities shared similar signatures, suggesting potential targets to directly treat metastasis. Additionally, we found that the plasticity of tumor cells is a common feature across tumors that is beneficial for the formation of metastasis. Tumors with higher levels of plastic tumor cells have a greater ability to form metastases. Moreover, we identified a subset of tumor cells expressing both markers of epithelial and mesenchymal characteristics, demonstrating in-vivo evidence for tumor epithelial-mesenchymal plasticity (EMP) in the metastatic cascade. The EMP is a continuum of states with epithelial and mesenchymal cell states as the two extremes of this continuum. The identified transition cells express distinct markers suggesting that these cells may represent a novel and overlooked cell type within the EMP continuum. Given the importance of tumor plasticity in the metastatic cascade, inhibiting the subset of tumor cells that are currently undergoing EMP may represent a novel treatment strategy for metastasis.

Citation Format: Juliane Winkler, Weilun Tan, Angela O. Pisco, Andrei Goga, Spyros Darmanis, Zena Werb. Tumor cell plasticity promotes metastasis across heterogeneous 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 964.
Abstract 992: MUC13 enhances colorectal cancer metastasis through molecular interaction with YAP1 transcription factor
Authors: Doxtater Kyle , Tripathi Manish K. , Sekhri Radhika , Kotnala Sudhir , Hafeez Bilal , Khan Sheema , Zafar Nadeem , Yallapu Murali , Jaggi Meena , Chauhan Subhash
Published: June 2022

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. About 90% of all cancer-related deaths are due to the development of metastatic sites in the body. 40-50% of colorectal cancer patients develop metastasis at some point during their fight with the disease. Understanding the mechanism of metastasis in colorectal cancer is vital. Metastasis is a multistep process; anchorage-independent survival after intravasation of cells from the primary tumor site is a crucial step. In our lab, we have previously demonstrated that MUC13 plays an essential role in the CRC progression by modulating anti-apoptotic pathways and survival proteins expression. Under anchorage-independent growth conditions, we have identified that MUC13, in correspondence with YAP1, is the key upregulated protein and is responsible for increased survival. Isogenic CRC cell lines SW480 (non-metastatic) and SW620 (metastatic) on low adherence growth conditions are applied in these studies. In addition, lentiviral transduced stable overexpression and knockdown cell lines were generated for MUC13 and YAP1 mechanistic studies. The overexpression of MUC13 in non-metastatic SW480 cells (low MUC13 expressing) increased anchorage-independent survival and enhanced tumorigenesis compared to SW480+Vector cells, contrary results were found upon MUC13 knockdown in SW620 cells (high MUC13 expressing). In vitro results were recapitulated in the in vivo mouse model system and human CRC tissues. In Proximity Ligation Assays (PLA), we found an increased nuclear localization of the survival complex YAP1/β-catenin in MUC13 overexpressing cells. In contrast, MUC13 knockdown resulted to the lower aboudance of survival complex in the nucleus. Immunoprecipitation validates the protein-protein interaction between MUC13 and YAP1. YAP1 knockdown in MUC13 overexpressing cells showed a decrease in survival, indicating the necessary functional complex formation between MUC13 and YAP1. MUC13 and YAP1 expression in human CRC tissue were highest at Stage II. However, YAP1 expression increased when MUC13 was observed in the nucleus. This supports the notion that MUC13 is critical in enhancing CRC metastasis through molecular interaction with YAP1. For the first time, this study demonstrates complex formation between MUC13 and YAP1 and defines their role in CRC progression and metastasis.

Citation Format: Kyle Doxtater, Manish K. Tripathi, Radhika Sekhri, Sudhir Kotnala, Bilal Hafeez, Sheema Khan, Nadeem Zafar, Murali Yallapu, Meena Jaggi, Subhash Chauhan. MUC13 enhances colorectal cancer metastasis through molecular interaction with YAP1 transcription factor [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 992.
Abstract CT543: 18F-FDG PET/CT detects potentially malignant bone metastases in patients with suspected lesions: A prospective clinical trial
Authors: Zhao Hui , Chang Yujie , Jiang Zhiyuan , Wang Zhiyu , Ruan Shunyi
Published: June 2022

Tumor metastasis to the skeleton affects over 70% cancer patients, more than any other site of metastasis Skeletal related events (SREs) included spinal cord compression, fracture, bone radiation or surgery, and tumor-related hypercalcemia are severe complications of bone metastases, which impair patients life quality and require particular treatments The gold standard for the definitive diagnosis is still pathological examination, which needs pre-biopsy imaging review to improve the diagnosis efficacy Classic imaging review method for being CT (Computed Tomography) and 18F-FDG PET/CT Research has shown that 18F-FDG PET/CT is superior to CT in detecting bone metastasis, however, it is not validated by a prospective study Besides, as SUVmax is a metabolic parameter in oncology imaging, we can serve as a reliable semiquantitative indicator to differentiate bone lesions We conducted a prospective clinical trial at Shanghai Sixth People’s Hospital Inclusion criteria were as follows: referred for diagnostic biopsy on a suspicion of metastatic disease Exclusion criteria included contraindications for biopsy 205 patients participated in the cohort study, then were randomized into PET/CT and CT group They underwent 18F-FDG PET/CT and CT separately to confirm accurate location, then both received CT-guided bone biopsy to confirm The sensitivity, specificity and accuracy were calculated In PET/CT group, malignant bone metastases were found in 83/137 patients and biopsies proved 80 were actual metastases In CT group, 48 malignant bone metastases were spotted and 47 were truly decisive The sensitivity, specificity and accuracy of 18F-FDG PET/CT and CT are 99% vs 87%, 95% vs 93%, and 97% vs 88% A chi-square test was utilized to analyze The sensitivity of 18F-FDG PET/CT is better than CT (P=0 0065), but the specificity and accuracy had no differences, indicating 18F-FDG PET/CT could detect more bone metastatic lesions potentially than CT We also found metastasis malignant lesions had higher bone SUVmax than benign (median 9 3 vs 4 25, p <0 001) ROC curves were asked to evaluate the differential efficacy of SUVmax In all 137 patients, the SUVmax 6 3 showed an AUC of 0 874 to predict malignant metastases Our research shows that [18F]FDG PET/CT has greater sensitivity in detecting malignant bone metastases than CT, and SUVmax can be different criteria for determining the malignancy of bone lesions

Citation Format: Hui Zhao, Yujie Chang, Zhiyuan Jiang, Zhiyu Wang, Shunyi Ruan. 18F-FDG PET/CT detects potentially malignant bone metastases in patients with suspected lesions: A prospective clinical trial [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 CT543.
Abstract LB004: Upregulation of STAT3 signaling in tumor cells fosters a TME-dependent secondary resistance of BRCA1-deficient HGSOC to PARP inhibition
Authors: Ding Liya , Wang Qiwei , Kearns Michael , Jiang Tao , Cheng Xin , Qian Changli , Zhao Jean
Published: June 2022

PARP inhibitors (PARPi) have demonstrated potent therapeutic efficacy in patients with ovarian cancer. However, acquired resistance to PARPi is a major challenge in the clinic. Therefore, understanding the resistance mechanism and developing new treatment approaches are important to overcome therapeutic resistance to PARP inhibition. By using our GEM of Brca1-deficient ovarian tumor model, we uncovered a new mechanism underlying a secondary resistance to PARP inhibition mediated by tumor associated macrophages (TAMs) in the tumor microenvironment (TME). Mechanistically, PARP inhibition induced the STAT3 signaling pathway in tumor cells, which in turn promoted pro-tumor polarization of TAMs in the TME of ovarian cancer. Ablation of STAT3 in tumor cells mitigated polarization of M2-like pro-tumor macrophages in the TME and increased tumor infiltration T cells in response to PARP inhibition. Furthermore, we demonstrated that STING agonists reprogramed myeloid cells in the TME of ovarian tumor into antitumor M1-like macrophages and activated dendritic cells (DCs) in a myeloid cell STING-dependent manner. These findings were recapitulated in patient-derived PARPi-resistant ovarian tumor models. Finally, we show that STING agonism was able to overcome the secondary resistance to PARPi in ovarian cancer rendered by immunosuppressive TME. Our study elucidates a new mechanism of PARPi resistance and provides a new treatment strategy to overcome the TME-induced therapeutic resistance to PARP inhibition in ovarian cancers.

Citation Format: Liya Ding, Qiwei Wang, Michael Kearns, Tao Jiang, Xin Cheng, Changli Qian, Jean Zhao. Upregulation of STAT3 signaling in tumor cells fosters a TME-dependent secondary resistance of BRCA1-deficient HGSOC to PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB004.
Abstract LB021: Mechanosensitive hormone signaling promotes mammary progenitor expansion and breast cancer progression
Authors: Northey Jason J. , Yui Yoshihiro , Hayward Mary-Kate , Stashko Connor , Kai FuiBoon , Mouw Janna , Thakar Dhruv , Lakins Jonathon , Ironside Alastair , Samson Susan , Mukhtar Rita , Hwang E. Shelley , Weaver Valerie
Published: June 2022

Tissue stem-progenitor cell frequency has been implicated in tumor risk and progression. Tissue-specific factors linking stem-progenitor cell frequency to cancer risk and progression remain ill defined. Using a genetically engineered mouse model that promotes integrin mechanosignaling with syngeneic manipulations, spheroid models, and patient-derived xenografts we determined that a stiff extracellular matrix and high integrin mechanosignaling increase stem-progenitor cell frequency to enhance breast tumor risk and progression. Studies revealed that high integrin-mechanosignaling expands breast epithelial stem-progenitor cell number by potentiating progesterone receptor-dependent RANK signaling. Consistently, we observed that the stiff breast tissue from women with high mammographic density, who exhibit an increased lifetime risk for breast cancer, also have elevated RANK signaling and a high frequency of stem-progenitor epithelial cells. The findings link tissue fibrosis and integrin mechanosignaling to stem-progenitor cell frequency and causally implicate hormone signaling in this phenotype. Accordingly, inhibiting RANK signaling could temper the tumor promoting impact of fibrosis on breast cancer and reduce the elevated breast cancer risk exhibited by women with high mammographic density.

Citation Format: Jason J. Northey, Yoshihiro Yui, Mary-Kate Hayward, Connor Stashko, FuiBoon Kai, Janna Mouw, Dhruv Thakar, Jonathon Lakins, Alastair Ironside, Susan Samson, Rita Mukhtar, E. Shelley Hwang, Valerie Weaver. Mechanosensitive hormone signaling promotes mammary progenitor expansion and breast cancer progression [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 LB021.
Abstract LB036: Molecular insight into ERG transcriptional activity to unravel novel therapeutic options for ERG-positive castration resistant prostate cancer
Authors: Lorenzin Francesca , Fracassi Giulia , Ciani Yari , Gasperini Paola , Rickman David , Demichelis Francesca
Published: June 2022

Gene fusions of the AR-regulated gene TMPRSS2 with the ETS family transcription factor ERG characterize about 40% of castration resistant prostate cancer (CRPC) - an advanced and lethal stage of prostate cancer. AR-mediated overexpression of ERG hijacks AR inhibiting lineage-specific differentiation and increases cell invasion and migration. In vivo work revealed a causal role of ERG in initiating prostate cancer. Notably, in vitro and in vivo models of CRPC endogenously bearing T2ERG depend on ERG activity for proliferation and survival. Thus, pursuing the development of ERG-based therapeutic strategies represents a promising route for CRPC treatment, although major challenges in targeting transcription factors such as ERG have been reported. Yet, synthetic lethality approaches including genome-wide loss-of-function screens can be employed to avoid direct ERG targeting. Here, we used metastatic PCa cell lines (LNCaP, 22Rv1 and DU145) engineered with inducible systems to overexpress ERG to show that high levels of ERG expression lead to an apparent reduction in the fitness of AR-positive and negative cells. Prolonged overexpression of a mutant form of ERG, which is not able to bind DNA, does not affect cell viability indicating that ERG DNA binding and transcriptional activity are necessary for the induction of this phenotype. The ERG-mediated reduction in cellular fitness is influenced by cell adhesion and is characterized by cell cycle arrest mediated by several cell cycle inhibitors induced by ERG. As revealed by gene expression analyses and functional assays, ERG induces a less proliferative, more dedifferentiated and invasive phenotype, which lead to an apparent reduction of cellular fitness in vitro, but is characteristic of more aggressive tumor cells in vivo. Altogether, these results underscore the reliability of the developed in vitro models for ERG activity, which will be used for screening to identify therapeutic vulnerabilities unlocked by ERG in CRPC. Importantly, we also uncovered confounding factors (i.e. loss of ERG-positive cells not due to screen selection) that could impinge on the results arising from in vitro screens.

Citation Format: Francesca Lorenzin, Giulia Fracassi, Yari Ciani, Paola Gasperini, David Rickman, Francesca Demichelis. Molecular insight into ERG transcriptional activity to unravel novel therapeutic options for ERG-positive castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB036.
Abstract LB045: β-catenin antagonist peptide, ST316, attenuates Wnt-dependent oncogenic activity
Authors: Ghamsari Lila , Gallagher Erin , Darvishi Emad , Leong Siok , Koester Mark , Ramirez Rick , Gonzales Jerel , Merutka Gene , Kappel Barry , Rotolo Jim A.
Published: June 2022

Constitutive activation of the Wnt/β-catenin signaling pathway underlines many breast and colorectal cancers, promoting cancer cell survival, proliferation and migration. B-cell CLL/lymphoma 9 protein (BCL9), a co-activator of β-catenin transcriptional activity, is overexpressed in many tumors and facilitates β-catenin nuclear localization. Disruption of the β-catenin and BCL9 interaction has been shown genetically to suppress oncogenic Wnt/β-catenin transcription, without impacting β-catenin homeostatic functions, and represents a powerful approach to inhibit this previously ‘undruggable’ target. Here, we investigate the impact of ST316, a peptide antagonist of β-catenin that inhibits its interaction with BCL9, on cells displaying hyperactivation of the Wnt/β-catenin pathway. Administration of ST316 to HCT116 human colorectal carcinoma cells, which carry a degradation-escape, gain-of-function mutation in β-catenin, dramatically reduced nuclear β-catenin localization. Furthermore, ST316-mediated loss of nuclear β-catenin was associated with a significant decrease in the expression of Wnt/β-catenin target genes in HCT116 cells stimulated with Wnt3a, including Axin2, hTCF7, Fox01 and CDK4. ST316 reduced β-catenin target gene expression by up to ∼70%, whereas no impact was observed following administration of a negative control peptide. A significant anti-proliferative and dose-dependent cytotoxic effect was observed on HCT116 cells 48 hours post ST316 exposure, with an EC50 of 4.4 µM, and on patient-derived, APC-mutant colorectal adenocarcinoma and breast carcinoma cells 72 hours post ST316 exposure, with EC50 values ranging from 1.5 to 2.6 µM. To extend these findings, we investigated the impact of ST316 on HCT116 tumors in vivo. ST316 (5 mg/kg) administered three times weekly by subcutaneous injection resulted in 99.6% tumor growth inhibition vs. vehicle controls (p<0.05), while a negative control peptide had no significant impact on tumor growth. These data demonstrate the potential of ST316 as a potent therapeutic candidate for Wnt/β-catenin-dependent cancers.

Citation Format: Lila Ghamsari, Erin Gallagher, Emad Darvishi, Siok Leong, Mark Koester, Rick Ramirez, Jerel Gonzales, Gene Merutka, Barry Kappel, Jim A. Rotolo. β-catenin antagonist peptide, ST316, attenuates Wnt-dependent oncogenic activity [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 LB045.
Abstract LB077: Reengineering ponatinib to minimize cardiovascular toxicity
Authors: Hnatiuk Anna Pavlovna , Bruyneel Arne A. , Taylor Dhanir , Pandrala Mallesh , Dheeraj Arpit , Li Wendy , Fernandez Ricardo Serrano , Feyen Dries A. , Wu Michelle M. , Amatya Prashila , Morgado Isabel , Wiebking Volker , Porteus Matthew H. , Malhotra Sanjay , Mercola Mark
Published: June 2022

Introduction and Purpose of the Study: The advent of small molecule Tyrosine Kinase Inhibitors (TKIs) has revolutionized cancer treatment and greatly improved patient survival. However, the life-threatening cardiovascular toxicity of many TKIs present major concerns. Ponatinib is the most effective treatment for patients harboring the common T315I mutation of BCR-ABL that drives Chronic Myeloid Leukemia (CML) yet is one of the most cardiotoxic of TKIs. Ponatinib-induced events include myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. The aim of this study is to reengineer the chemical structure of ponatinib to minimize its adverse cardiovascular effects by using in vitro and in vivo models of cardiovascular toxicity.

Experimental Procedures: We developed in vitro cardiovascular toxicity assays using human iPSC-cardiomyocytes (hiPSC-CMs) and human microvascular endothelial cells (HMVECs), and assessed anti-tumor efficacy in T315I mutated K562 CML cells. The cardiotoxicity was determined by impairment of cardiomyocyte contractility function, the vascular toxicity was defined by inability to form capillary-like networks in vasculogenesis assays. In vitro screening of ponatinib analogues probed chemical moieties responsible for its cardiovascular toxicity that were confirmed using mice xenograft mice models with human T315I mutant K562 cells.

Summary of Unpublished Data: Compared to ponatinib, refined analogues showed markedly decreased adverse effects on HMVECs vasculogenesis and hiPSC-CMs contractility in vitro. The therapeutic window was increased in vivo, leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increasing serum levels of cardiac troponin. The kinase inhibition profiles of the “safe” analogues compared to ponatinib identified candidate mediators of ponatinib-induced toxicity. Validation by selective knockdown revealed likely mediators of cardiotoxicity, including FGFR.

Conclusions: We succeeded in engineering a cardiovascular safe TKI that retained effective therapeutic properties against BCR-ABL T351 mutated form of CML, but with substantially decreased cardiovascular toxicity.

Citation Format: Anna Pavlovna Hnatiuk, Arne A. Bruyneel, Dhanir Taylor, Mallesh Pandrala, Arpit Dheeraj, Wendy Li, Ricardo Serrano Fernandez, Dries A. Feyen, Michelle M. Wu, Prashila Amatya, Isabel Morgado, Volker Wiebking, Matthew H. Porteus, Sanjay Malhotra, Mark Mercola. Reengineering ponatinib to minimize cardiovascular toxicity [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 LB077.
Abstract LB084: AXL as a therapeutic target in adenoid cystic carcinoma: preclinical evaluation of AXL targeting antibody-drug conjugate (ADCT-601)
Authors: Humtsoe Joseph O. , Jones Leilani , Naara Shorook , Zammarchi Francesca , Burr Nicole Spardy , van Berkel Patrick H. , Ha Patrick K. , Kang Hyunseok
Published: June 2022

Background: Adenoid cystic carcinoma (ACC) is a rare cancer of salivary gland cancer with a significant unmet clinical need. Conventional systemic therapy has limited efficacy and there are currently no FDA approved drugs for these patients with metastatic disease. Advances in molecular profiling led identification of potential therapeutic targets, and previous studies with proteomic analysis identified AXL as a potential therapeutic target in ACC. AXL, a member of the TAM tyrosine kinase receptor family has been generally associated with poor prognosis, and its overexpression and activation is implicated in conferring resistance to conventional systemic therapies in solid tumors. ADCT-601 is an antibody drug conjugate targeting human AXL carrying novel pyrrolobenzodiazepine dimer cytotoxin as the payload.

Methods: The goal of this study is to determine the in vitro and in vivo anti-tumor activity of ADCT-601 in ACC cell lines and patient-derived xenograft (ACCX) models with varying AXL expression determined by Western blot. ADCT-601 treated cell lines in vitro were analyzed by MTT-based cytotoxic assay and immunoblotting, and anti-tumorigenicity was assessed in two ACCX models.

Results: ADCT-601 induced a potent dose-dependent cytotoxic effect in ACC cell lines, but its cytotoxic activity was attenuated after AXL knockdown. Biochemical data indicated that ADCT-601 treatment led to DNA damage and induction of apoptotic factors accompanied by phosphorylation of gamma-H2AX. In high AXL expressing ACCX6 model, a single dose of either 0.5 or 1.0 mg/kg ADCT-601 significantly suppressed tumor growth resulting 3/5 partial responders (PRs) at 1mg/kg and 2/5 PRs at 0.5 mg/kg, compared to the vehicle or isotype-ADC control treated mice (0/5 PR and 1/5 PR, respectively). ADCT-601 appears to eliminate tumor, as no evidence of recovery in tumor growth was observed during a 60-day period. In low AXL expressing ACCX9 model, ADCT-601 treatment (at either the 0.5 or 1.0 mg/kg dose) delayed tumor growth compared to isotype-ADC control, but did not lead to durable partial responses.

Conclusion: This study demonstrated that ADCT-601 induced a potent and specific in vitro and in-vivo anti-tumor activities in AXL expressing ACC models and suggests further development of ADCT-601 in biomarker driven clinical trials.

Citation Format: Joseph O. Humtsoe, Leilani Jones, Shorook Naara, Francesca Zammarchi, Nicole Spardy Burr, Patrick H. van Berkel, Patrick K. Ha, Hyunseok Kang. AXL as a therapeutic target in adenoid cystic carcinoma: preclinical evaluation of AXL targeting antibody-drug conjugate (ADCT-601) [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 LB084.
Abstract LB127: Reduced expression of annexin-a6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells
Authors: Williams Stephen D. , Widatalla Sarrah E. , Sakwe Amos M.
Published: June 2022

The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying rapid tumor progression and/or therapeutic resistance in solid tumors, including the hard-to-treat triple-negative breast cancer (TNBC) subtype. Here, we assessed the contribution of the tumor suppressor, Annexin A6 (AnxA6) in the metabolic adaptation of rapidly growing versus invasive TNBC cells as well as in Lapatinib resistant (Lap-R) TNBC cells. AnxA6 expression in cultured TNBC cell lines, Lap-R cells and in crude isolates of mitochondria was determined by Western blotting and/or immunofluorescence assays. The oxygen consumption rates (OCR), extracellular acidification rates (ECAR) and mitochondria activity were assessed using the Seahorse XF Analyzer. Intracellular metabolites were detected by 1H-NMR analysis. Using model proliferative basal-like and invasive mesenchymal-like TNBC cell lines, we show that TNBC cells also exhibit metabolic heterogeneity. Down regulation of AnxA6 in TNBC cells attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity, resulting in a quiescent cellular energy phenotype. We further show that AnxA6-depletion was associated with decreased lipid droplet accumulation and the lipolytic phenotype by enhancing the uptake and mitochondrial fatty acid oxidation for ATP production. Chronic Lapatinib-induced up regulation of AnxA6 in AnxA6-low TNBC cells reversed their lipolytic to a more lipogenic/glycolytic metabolic phenotype with gluconeogenic precursors as additional metabolites. Collectively, these data suggest that the expression status of AnxA6 in TNBC cells underlies significant metabolic adaptation during stress and/or chronic Lapatinib treatment and provide additional insights into the potential of AnxA6 as a biomarker for not only therapeutic intervention but also the metabolic subtyping of TNBC subsets.

Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. Reduced expression of annexin-a6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB127.
Abstract LB138: DAXX interacts with sterol regulatory element-binding proteins (SREBPs) to promote oncogenic lipogenesis and tumorigenesis in triple-negative breast cancer
Authors: Mahmud Iqbal , Tian Guimei , Hutchison Tarun , Kim Brandon , Liao Daiqing
Published: June 2022

Elevated lipid metabolism including lipogenesis is a major metabolic feature in cancer cells. In breast and other cancer types, genes involved in lipogenesis are highly upregulated, but the mechanisms that control their expression remain poorly understood. DAXX modulates gene expression through binding to numerous transcription factors although the functional impact of these diverse interactions remains to be defined. Our recent analysis indicates that DAXX is overexpressed in diverse cancer types and metastases. However, mechanisms underlying DAXX’s oncogenic function remains elusive. Using global integrated transcriptomic and lipidomic analyses, we show that DAXX plays a key role in lipid metabolism in triple-negative breast cancer (TNBC) cells. DAXX depletion attenuates, while its overexpression enhances, lipogenic gene expression, lipid synthesis and tumor growth. Mechanistically, DAXX interacts with SREBP1 and SREBP2 and activates SREBP-mediated transcription. DAXX associates with lipogenic gene promoters through SREBPs. Underscoring the critical roles for the DAXX-SREBP interaction for lipogenesis, SREBP2 knockdown attenuates tumor growth in cells with DAXX overexpression, and a DAXX mutant unable to bind SREBPs are incapable of promoting lipogenesis and tumor growth. In TNBC patients, DAXX expression levels are increased in breast cancer brain metastasis and correlate with poor patient survival. Our results identify the DAXX-SREBP axis as an important pathway for tumorigenesis in TNBC. (This work is supported by Florida Department of Health Grants.)

Citation Format: Iqbal Mahmud, Guimei Tian, Tarun Hutchison, Brandon Kim, Daiqing Liao. DAXX interacts with sterol regulatory element-binding proteins (SREBPs) to promote oncogenic lipogenesis and tumorigenesis in triple-negative 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 LB138.
Abstract LB200: Pdgf/Pi3k/akt axis activation by hey1-ncoa2 fusion and potential target in the treatment of mesenchymal chondrosarcoma
Authors: Qi Wenqing , Wang Lu
Published: June 2022

Mesenchymal chondrosarcoma is a rare and often aggressive cancer mostly affecting children and young adults. Genetically, this tumor is mainly characterized by the recurrent HEY1-NCOA2 fusion. Localized tumor is managed surgically; traditional chemotherapy and radiation therapy, however, did not appear to substantially improve the event-free survival rate of mesenchymal chondrosarcoma patients. It is clear that better therapeutic options are needed for the treatment of this tumor. In our previous study, an in vitro model for studying mesenchymal chondrosarcoma tumorigenesis was developed using stably transduced iPSC-derived mesenchymal stem cells (iPSC-MSCs) with inducible-expression of HEY1-NCOA2. With the in vitro model, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq to identify HEY1-NCOA2-dependent transcriptional regulation and demonstrated the dramatic activation of PDGF-PDGFR and PI3K-AKT signaling pathways (manuscript in review). Our data also suggested that PDGFB is one of the direct targets of HEY1-NCOA2 fusion protein. In the present study, with the in vitro iPSC-MSC model, we verified that HEY1-NCOA2 fusion protein, but not wild-type HEY1 or NCOA2, dramatically increased phospho-AKT (Ser473, a readout of PI3K) expression by western blotting. Further on, we tested the effect of two kinase inhibitors CP-673451 and PF-04691502, a potent and selective PDGFR inhibitor and the mTOR/PI3K inhibitor, respectively. Stably transfused iPSC-MSC were treated with the kinase inhibitors, respectively, at different dosages (0.1 µM, 0.5 µM and 1.0 µM) for one week. At the concentration of 1.0 µM, CP-673451 efficiently suppressed AKT phosphorylation by ~23% as well as suppressed cell growth by ~67% which is significantly higher than that observed in the controls (e.g. ~20% suppression in HEY1- and NCOA2- expression iPSC-MSCs). Similarly, at the concentration of 0.5 µM, PF-04691502 suppressed the HEY1-NCOA2-expression iPSC-MSCs growth by ~24% more than that in the control (p=0.02). In summary, both CP-673451 and PF-04691502 were active against the in vitro mesenchymal chondrosarcoma model tested. Based on these data, the potential benefit of targeting PDGF/PI3K/AKT signaling pathways in the treatment of mesenchymal chondrosarcoma was suggested.

Citation Format: Wenqing Qi, Lu Wang. Pdgf/Pi3k/akt axis activation by hey1-ncoa2 fusion and potential target in the treatment of mesenchymal chondrosarcoma [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 LB200.
Abstract LB214: Discovery of EPIM-001, a tumor targeted engineered IL2 with biased IL2Rβ agonist activities
Authors: Zhao Kehao , Hassan Sam , Chen Yan , Nguyen Jenna , Jiang Ning , Zhang Gening , Li Irene , Feng Ryan
Published: June 2022

Background: The cytokine Interleukin 2 (IL2) has been approved for cancer treatment. However, its application has been limited due to severe toxicities associated with its high concentration administration. These side effects are attributed to the strong binding of IL2 to its receptor IL2Rα which is enriched on regulatory T Cells and Lung epithelial cells. We have engineered IL2 to significantly reduce IL2Rα binding and enhance IL2Rβ binding. We further developed EPIM-001, a bi-functional IL2Rβ agonist and Anti-PDL1 biologic, aiming to treat a broad set of solid tumors with improved therapeutic index.

Methods: We used mSCAFold࣪, a unique protein engineering platform to redefine and improve pharmacologic properties of the cytokine through structure-based rationally designed libraries and mRNA display selection. The optimal IL2 candidate with enhanced IL2Rβ and reduced IL2Rα binding was engineered with an internally discovered anti-PD-L1 antibody to generate EPIM-001, a bispecific IL2/PD-L1 biologic drug candidate. EPIM-001 was characterized in vitro for receptor binding activity, immune cell activation, proliferation, and tumor cell killing assays. In vivo efficacy was evaluated in xenograft humanized mouse models, and PK/PD and toxicity has been studied in Non-Human Primates (NHPs).

Results: We have shown that EPIM-001 is a strong agonist of IL2Rβ, with significantly reduced binding to IL2Rα. It demonstrated much stronger CD8+ T cells and NK cell activation and proliferation compared WT-IL2, whilst only weaker activation and minimal proliferation with Tregs. In vitro cell killing assays showed that EPIM-001 promoted better immune cell killing of PD-L1 expressing target cells than WT IL2. In xenograft in vivo studies, EPIM-001 has shown significant tumor growth inhibition against the human lung cancer cell line H1975, which whilst expressing high levels of PD-L1, does not respond to PD-L1/PD-1 blockade. Furthermore, in the human breast cancer cell line MDA-MB-231, EPIM-001 showed stronger tumor growth inhibition compared to Keytruda. PK/PD and toxicity studies in NHPs showed that EPIM-001 is well tolerated. EPIM-001 greatly expanded CD8+ T cells, whilst having minimal effect on TRegs and Eosinophils associated with toxicity.

Conclusions: EPIM-001 is a promising immune modulating drug candidate that selectively activates CD8+ T cells and NK Cells with strong anti-tumor activity and safe profile for both immune checkpoint sensitive and insensitive PD-L1 positive tumors.

Citation Format: Kehao Zhao, Sam Hassan, Yan Chen, Jenna Nguyen, Ning Jiang, Gening Zhang, Irene Li, Ryan Feng. Discovery of EPIM-001, a tumor targeted engineered IL2 with biased IL2Rβ agonist activities [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 LB214.