bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5335: circAde: a circRNA-based system for prolonged and more effective treatment of cancer
Authors: O'leary Eoghan T. , Vikberg Sara , Foord Emilie , Wiklund Erik D. , Ottesen Lone , Levitsky Victor , Hansen Thomas B.
Published: April 2023

In recently performed phase I/II clinical studies, we have demonstrated successful delivery of vectorized transgene-encoding mRNA payloads to solids tumors with substantial clinical benefit. In patients, persistent and prolonged transgene expression correlated with positive clinical outcome, and thus temporal expansion of the transgene expression profile is expected to improve the therapeutic benefit even further. Recently, circular RNA (circRNA) has been discovered as a novel class of endogenously expressed RNA. CircRNAs, in contrast to mRNAs, are resistant to exo-nucleolytic decay which results in high intra-cellular stability and persistent expression within cells. Therefore, circRNA-based systems serve as an interesting new technology towards prolonged effects of treatment. Here, we show that our proprietary vector system, circAde, allows high expression of circRNAs through spliceosome-dependent biogenesis. By comparing the protein output from circAde and mRNA-based vectors, we show that the circAde-system out-performs conventional mRNA-based design, both in terms of transgene yield and temporal sustainability of expression. Moreover, using a panel of proof-of-concept-reporters, we show that certain design-features greatly influence the circRNA production and subsequent protein expression from the vector. Without the 5’cap, circRNAs are dependent on IRESs (internal ribosome entry sequences) for effective translation. By conducting two orthogonal IRES screens on 1000+ putative IRES elements, we have identified the most effective IRES sequence in melanoma and lung-cancer cells supporting effective circRNA production and high-yield protein expression, thereby improving our circAde vector system even further. Our results open a new platform towards development of novel, more effective strategies for the treatment of cancer and other settings in which durable transgene expression is desirable.

Citation Format: Eoghan T. O'leary, Sara Vikberg, Emilie Foord, Erik D. Wiklund, Lone Ottesen, Victor Levitsky, Thomas B. Hansen. circAde: a circRNA-based system for prolonged and more effective treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5335.
Abstract 5344: Novel melanoma leads demonstrate efficacy in A375 xenograft model in nude mice
Authors: Minond Dmitriy , Velayutham Sadeeshkumar
Published: April 2023

The purpose of this study was to evaluate efficacy of novel anti-melanoma leads for in vivo efficacy. Despite the recent advances in melanoma therapy, the need for new targets and novel approaches to therapy is urgent. We previously reported novel melanoma leads, 2155-14 and 2155-18, that work via binding and downregulating spliceosomal proteins hnRNPH1 and H2. To determine the therapeutic potential of these compounds we tested them in A375 cell-line derived xenograft in nu/nu mice. For determination of 2155-14 and 2155-18 potency in vivo, nude mice were inoculated subcutaneously with A375 cell line and tumors were allowed to reach 100mm3. Animals were then randomized into four groups to receive subcutaneous administration of vehicle, 10mg/kg vemurafenib, and 25mg/kg 2155-14 and 2155-18 three times per week for 15 days. The result revealed that both 2155-14 and 2155-18 significantly decreased the growth of A375 tumors, which was comparable to vemurafenib. These results are well correlated with the exposure of leads in plasma. In summary, these results suggest a therapeutic potential of targeting spliceosomal proteins hnRNPH1 and H2 and support potential of 2155-14 and 2155-18 as drug candidates for treatment of melanoma.

Keywords: Melanoma, drug discovery, A375, xenograft.

Citation Format: Dmitriy Minond, Sadeeshkumar Velayutham. Novel melanoma leads demonstrate efficacy in A375 xenograft model in nude mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5344.
Abstract 5345: The application of CDK16 inhibitors in the treatment of triple-negative breast cancer
Authors: Xiao Haihua , Jiang Huangyu , Feng Kunxian , Yang Mengli , Yang Jinping , Jian Chengfang , Tian Yuan , Vasilevich Natalya , Sun Lichun
Published: April 2023

Triple-negative breast cancer (TNBC) with estrogen receptor (ER), progesterone receptor (PR) and HER2 (c-erbB-2, neu) being negatively expressed in patient cancer tissues displayed its unique clinical pathological difference from other types of breast cancers. TNBC is more common in younger patients and more likely to have a family history of breast cancer. TNBCs more likely have poor prognosis and postsurgical recurrence, with strong invasiveness and metastasis. However, there is lack of effective and TNBC-targeting drugs. The non-typical CDK16 was found highly expressed in TNBCs and clearly associated with prognosis and survival of patients. CDK16 could promote the progression and metastasis of TNBC by phosphorylating PRC1. Knockdown of CDK16 could significantly suppress tumor growth and metastasis. Furthermore, in our studies, rebastinib acting as a CDK16 inhibitor displayed its strong suppression of TNBC tumor growth in a dose-dependent manner, indicating rebastinib or its analogs could be druggable and likely used to treat TNBC patients.

Citation Format: Haihua Xiao, Huangyu Jiang, Kunxian Feng, Mengli Yang, Jinping Yang, Chengfang Jian, Yuan Tian, Natalya Vasilevich, Lichun Sun. The application of CDK16 inhibitors in the treatment of triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5345.
Abstract 5347: Selective targeting deacetylase 3 (HDAC3) and HDAC8 by PROTACs
Authors: Xiao Yufeng , Hale Seth , Awasthee Nikee , Zhang Xuan , Liu Yi , Huo Zhiguang , Lyu Dongwen , Wang Lei , Zhang Weizhou , Mosteiro Megan , Liao Daiqing , Zheng Guangrong
Published: April 2023

Histone deacetylases (HDACs) are enzymes that play an essential role in multiple cellular processes such as DNA transcription, translation, replication, recombination, repair, and metabolism, and their dysregulation can be linked to many different diseases. Most commonly, the overexpression of HDACs is found in various cancer types including hematologic cancers, as well as solid malignancies. Of the many HDACs found in the body, class I HDACs, which consist of HDACs 1, 2, 3, and 8, play an essential role in activating oncogenes underlying tumorigenesis, disease progression, and treatment resistance. Several HDAC inhibitors (HDACis) have been approved for cancer treatment, however, they are pan-inhibitors. This lack of specificity poses many disadvantages for HDACis, including toxicity and other off-target effects. Isozyme-selective inhibitors may reduce these off-target effects and thus enhance their safety. Though favorable, selectivity is difficult to achieve from conventional inhibitors due to the highly homologous catalytic domain among HDAC isozymes. In addition, several HDAC isozymes have deacetylase-independent scaffolding functions that cannot be blocked by traditional inhibitors. Recently, there have been advances in Proteolysis Targeting Chimera (PROTAC), an emerging drug discovery technology designed to hijack cell’s existing protein degradation machinery, the ubiquitin-proteasome system (UPS) to selectively degrade target proteins. Selective degradation by PROTAC is a potential solution to many of the concerns associated with current HDACis. Previously, we reported an HDAC3-selective PROTAC, XZ9002, and following that we reported the discovery of PROTAC YX968, which can degrade both HDAC3 and HDAC8 isozymes with single-digit nanomolar DC50, this highly potent dual degrader exhibits distinct effects on modulating gene expression and is much more potent in inhibiting cancer cell proliferation compared to XZ9002. Based on this, we are further modifying the PROTACs to be selective for HDAC8. The HDAC3, HDAC8 selective degrader and HDAC3 and HDAC8 dual degrader we developed could be useful chemical probes to dissect the complex biological function of HDAC3 and HDAC8 and potential therapeutics for treating cancer.

Citation Format: Yufeng Xiao, Seth Hale, Nikee Awasthee, Xuan Zhang, Yi Liu, Zhiguang Huo, Dongwen Lyu, Lei Wang, Weizhou Zhang, Megan Mosteiro, Daiqing Liao, Guangrong Zheng. Selective targeting deacetylase 3 (HDAC3) and HDAC8 by PROTACs. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5347.
Abstract 5424: A deep learning approach (AI) which accurately identifies breast tumor cells, tumor infiltrating lymphocytes (TILS) and fibroblasts from H&E slides
Authors: da Silva Luiz Augusto Zillmann , Williams Alistair R. , Kubeyev Aidan , Giorni Andrea , Laurie Jordan , Sivasubramaniam Prabu , Foster Matthew , Griffiths Matthew , Asghar Uzma
Published: April 2023

Background: Histopathology assessments of cancers require highly skilled pathologists, are labor intensive and prone to errors without proper training or fatigue. Machine learning can assist pathologists by increasing efficiency and minimizing individual variability. This study adapted a deep learning model to reliably identify 3 cell types from triple negative breast cancers fixed on H&E slides and verified performance with an expert pathologist.

Methods: We apply the U-Net architecture to analyse pathology slides fixed with triple negative breast cancer (TNBC) tissue. The public dataset NuCLS was used for training. Semantic segmentation was used to identify single cells of 3 types: tumor cells, fibroblasts, and tumor infiltrating lymphocyte (TILs). For validation, the pathologist annotated 8 random H&E tiles. The 3 cell types accuracy for NuCLS and the model was evaluated by our pathologist.

Results: Overall, there was a 73% agreement with the pathologists (Pathologist vs. NuCLS). A set of 1,555 (90%) TNBC slides were used for training and 173 for validation (10%; unseen data). Table 1 outlines the accuracy metrics for each cell type and for each comparison. Compared to our pathologist, the model accurately identified TILs (62%), followed by fibroblasts (42%) and lastly tumor cells (26%). A significant source of discrepancy was variation in labeled single cell boundaries. The model was better at identifying TILs. The pathologist took 1.5 hours to annotate 8 tiles for the 3 cells and our model 644ms.

Conclusion: It is possible to develop a deep learning model to identify breast cancer cells, fibroblasts and TILs from H&E stained slides, with similar accuracy levels as a trained pathologist. The model performed better than a pathologist in identifying TILs, but both struggled with fibroblasts. Accuracy of 71% overall and 87% for TILs, motivates expansion to further datasets and other cancer types.

Table 1. - Accuracy metrics. Quality assessment8 H&E tiles Pathologist vs. NuCLS U-Net model vs. NuCLs U-Net model vs. Pathologist U-Net model vs. NuCLs (Full) Background 86% 71% 62% 73% Tumour 43% 35% 26% 71% Fibroblast 45% 39% 42% 31% TILs 34% 96% 62% 87% Overall 73% 74% 61% 71%

Citation Format: Luiz Augusto Zillmann da Silva, Alistair R. Williams, Aidan Kubeyev, Andrea Giorni, Jordan Laurie, Prabu Sivasubramaniam, Matthew Foster, Matthew Griffiths, Uzma Asghar. A deep learning approach (AI) which accurately identifies breast tumor cells, tumor infiltrating lymphocytes (TILS) and fibroblasts from H&E slides. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5424.
Abstract 5437: Vision transformers for breast cancer human epidermal growth factor receptor 2 (HER2) expression staging without immunohistochemical (IHC) staining
Authors: Ayana Gelan , Lee Eonjin , Choe Se-woon
Published: April 2023

Purpose: Staging of human epidermal growth factor receptor 2 (HER2) expression is crucial for evaluating the effectiveness of breast cancer treatment. However, it involves an expensive and challenging immunohistochemical (IHC) staining in addition to hematoxylin and eosin (H&E) staining. Here, we argue that customized vision transformers are effective in breast cancer HER2 expression staging using only H&E-stained images.

Methods: Our algorithm has three modules: a localization module to weakly localize the necessary features of an image via spatial transformers, an attention module to learn global features from the image using vision transformers, and a loss module that determines the closest one to a HER2 expression level from input images by calculating the ordinal loss. We utilized a publicly available breast cancer HER2 expression dataset, the breast cancer immunohistochemical image (BCI) dataset, which contains paired H&E and IHC stained images. Results were calculated with 95% confidence intervals and t-test was used to evaluate the significance of the proposed model against other models.

Results: Our approach achieved area under receiver operating characteristics curve (AUC), precision, sensitivity, and specificity of 0.9202±0.01, 0.922±0.01, 0.876±0.01, and 0.959±0.02, respectively, averaged over five-fold cross-validation, in staging HER2 expression. The proposed method showed better performance than the conventional vision transformer model and state-of-the-art convolutional neural network models with statistical significance of p<0.01 in all cases.

Conclusion: Our findings are important in assisting HER2 expression staging in breast cancer treatment, while avoiding the need for an expensive and time-consuming IHC staining procedure.

Comparison of the proposed model with state-of-the-art models. CNN-convolutional neural network Study Neural network Model AUC Couture et al. (2018) CNN VGG16 0.75 Shamai et al. (2019) CNN ResNet50 0.74 Yang et al. (2021) CNN ResNet50 0.76 Naik et al. (2020) CNN ResNet50 0.78 Khater et al. (2021) CNN ShuffleNet 0.75 Conde-Sousa et al. (2022) CNN EfficientNet 0.88 Farahmand et al. (2022) CNN InceptionV3 0.90 Proposed Vision transformer vitb_16 0.92

Citation Format: Gelan Ayana, Eonjin Lee, Se-woon Choe. Vision transformers for breast cancer human epidermal growth factor receptor 2 (HER2) expression staging without immunohistochemical (IHC) staining. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5437.
Abstract 5508: Artesunate increases enzalutamide efficacy in advanced prostate cancer
Authors: Wang Xinyi , Mao Fengyi , Liu Jinghui , Kong Yifan , he Dang , Wang Chi , Li Zhiguo , Liu Xiaoqi
Published: April 2023

Prostate cancer (PCa) is the most diagnosed worldwide. PCa development and progression require androgen receptor (AR) signaling, which stimulates its downstream gene expressions and cancer progression. While second-generation anti-androgen drugs plus androgen deprivation therapy (ADT) remains the first-line treatment for advanced prostate cancer patients, around one-third of patients will relapse in a short period. Advanced prostate cancer results in more mortalities than primary PCa patients. Evidence shows that the recurrence is caused by AR overexpression, AR variants, AR mutations, and signaling crosstalk. Thus, it is urgently needed to discover a novel therapeutic strategy for treating advanced prostate cancer. The heat shock protein family (HSP), including HSP90 and HSP70, play important roles in refolding aggregated protein for cancer cell proteomic equilibrium. HSP is induced primarily by heat shock factor (HSF1). As AR’s chaperone protein, HSP70 and HSP90 increase AR transcription activity. Inhibiting HSP70 and HSP90 promotes STUB1, an E3 ligase, binding to AR and AR - V7, and ubiquitination. Artesunate (ART) is a semi-synthetic ingredient from Artemisia annua and is the most common treatment for malaria throughout the world. It was approved for medical use by the FDA (Food and Drug Administration) (Food and Drug Administration). Recently ART has been unveiled for its anticancer properties. However, the efficacy of ART treatment in advanced prostate cancer and the direct target of ART have not been investigated yet. Herein, we have examined the efficacy of combining Enzalutamide (Enza) and ART in advanced prostate cancer cell lines. We also performed unbiased bioinformatics analysis using RNA seq results in enzalutamide-resistant cell line C4-2R cells and 22RV1 cells to investigate the cell response toward ART treatment. We identified ART could downregulate of AR signaling pathway. Moreover, we determined that ART treatment induces AR degradation in proteasome dependent manner. Interestingly, we found HSP70 and HSP90 are also decreased in RNA seq results. Taking these together suggests that ART may target HSF1 directly. Our results suggest ART induced AR degradation could be a promising clinical strategy for advanced prostate cancer.

Citation Format: Xinyi Wang, Fengyi Mao, Jinghui Liu, Yifan Kong, Dang he, Chi Wang, Zhiguo Li, Xiaoqi Liu. Artesunate increases enzalutamide efficacy in advanced prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5508.
Abstract 5635: Joint secondary transcriptomic analysis of triple negative breast cancer offers a window into new therapeutic possibilities and biomarkers
Authors: Rapier-Sharman Naomi , Poulsen Jenna Birchall , Dobbs Mauri E. , Pickett Brett E.
Published: April 2023

Breast cancer is a harmful growth of mutated breast tissue that is diagnosed in 2.3 million women yearly, and kills 685,000 patients (15%) each year around the world. Left unchecked, breast cancer can grow proliferatively and metastasize to new tissue sites, monopolizing the body’s energy supply and ultimately causing organ failure and death. Due to increased use of treatments targeting the breast tumor-specific markers Estrogen Receptor (ER), Progesterone Receptor (PR), and Human Epidermal growth factor Receptor 2 (HER2), patient survival of breast cancer has increased in recent years. However, patients with triple-negative breast cancer (TNBC), tumors lacking all three commonly-targeted biomarkers, suffer greater illness, experience more frequent relapse, and enjoy a shorter lifespan compared to other breast cancer patients due to the paucity of tumor-selective treatments for TNBC. Further research on TNBC is needed to understand targetable mechanisms and develop treatments capable of targeting the key mechanisms. To investigate the transcriptomic profiles of TNBC tumors, we performed a joint secondary analysis of publicly available RNA-sequencing data for TNBC, consisting of 214 relevant samples across 11 distinct studies in the NCBI Gene Expression Omnibus (GEO). We found ~12,500 significant differentially expressed genes (FDR p-value ⇐ 0.05) and 67 significantly modulated pathways (FDR p-value ⇐ 0.05). Additionally, our novel findings include candidates for drug repurposing, high-integrity predictive biomarkers, and potential new TNBC molecular mechanisms. We found several TNBC drug targets that have not yet been fully leveraged along with drugs that could likely be successfully repurposed for use in TNBC based on previous target history and our transcriptomic results. This joint secondary analysis opens the door to future therapeutic options that could increase patient survival once validated in the wet lab.

Citation Format: Naomi Rapier-Sharman, Jenna Birchall Poulsen, Mauri E. Dobbs, Brett E. Pickett. Joint secondary transcriptomic analysis of triple negative breast cancer offers a window into new therapeutic possibilities and biomarkers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5635.
Abstract 5646: Spatial transcriptomic profiling of the human and mouse retina prepared with CryoJane Tape Transfer System using GeoMx and CosMx spatial analysis
Authors: Glaser Charles , Ma Su , Yang Wei , Liang Yan , Beechem Joeseph , Bonilha Vera L. , Rao Sujata , Horrigan William , Anand-Apte Bela
Published: April 2023

The goal of this study is to identify key transcriptomic markers within layers of the retina by individually profiling layers using cellular and subcellular spatial transcriptomics, additionally, comparing the results between each level. Both human and mouse retina samples, prepared fresh frozen and fixed frozen, are analyzed using the GeoMx® Digital Spatial Profiler (DSP) and CosMx® Spatial Molecular Imager (SMI) using the whole mouse transcriptome atlas and 1000-plex mouse neuro panel, respectively. Samples are fixed using Cryo-Jane Taper Transfer system. Samples are mounted on to adhesive coated slides as well as adhesive tape to mount samples to glass slides. This method is used to secure fragile frozen tissue, such as the retina. Human and mouse samples were stained using immunofluorescent microscopy targeting neurofilament H (NF-H), glial fibrillary acidic protein (GFAP) and NeuN on DSP and 18s rRNA, amyloid-beta and GFAP on SMI. Staining allows for identification of structural layers in the retina. Simultaneously, regions of interest (ROI) for spatial profiling are selected based on immunofluorescent stains. On DSP, each sample had 3x ROIs in the photoreceptor layer, inner nuclear layer and ganglion cell layer, then, oligonucleotides were collected and sequenced. Finally, raw counts were Q3 normalized for analysis. For SMI data analysis, 6 field of views (FOVs) were put on each section to cover most regions with multiple layers. ~8000 genes were detected on human retina samples using DSP. Around 500 unique genes were detected between the photoreceptor and inner nuclear layer using DSP. Preliminary SMI results show we were able to identify cell types (amacrine, horizontal cell, biopolar cell, ganglion cell, etc) and cell specific markers for outer nuclear layer, inner nuclear layer and ganglion cell layer. Data between DSP and SMI showed high concordance with one another, identifying many genes in each layer.

Citation Format: Charles Glaser, Su Ma, Wei Yang, Yan Liang, Joeseph Beechem, Vera L. Bonilha, Sujata Rao, William Horrigan, Bela Anand-Apte. Spatial transcriptomic profiling of the human and mouse retina prepared with CryoJane Tape Transfer System using GeoMx and CosMx spatial analysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5646.
Abstract 5653: LCB97, an antibody drug conjugate (ADC) targeting a novel tumor antigen for the treatment of multiple solid tumors
Authors: Jeong Yeojin , Spohn Gunther , Mehli Flavio , Chung Chul-Woong , Park Changsik
Published: April 2023

LCB97 is an antibody-drug conjugate (ADC) directed against a novel tumor antigen, composed of monomethyl auristatin E (MMAE) as payload (DAR 4) and the humanized IgG1 antibody AFF4 generated by Elthera. AFF4 targets a novel antigen preferentially expressed in various solid tumors including pancreatic cancer, ovarian cancer, colon cancer, breast cancer and melanoma. LCB97 generated by using LCB’s ConjuAllTM technology with AFF4 showed potent cytotoxicity in various cancer cell lines including breast cancer (BC), ovarian cancer (OC), pancreatic cancer (PaC), small cell lung cancer (SCLC), colorectal cancer (CRC) and melanoma. LCB97 also showed excellent anticancer efficacy in several cell line-derived xenograft (CDX) models, including BC, CRC, OC and melanoma. Preliminary toxicity assessment in cynomolgus monkeys (an AFF4 cross reactive species) demonstrated that LCB97 was well tolerated within the estimated therapeutic index of ~20 for repeat dosing. In conclusion, LCB97, generated by combining theAFF4 monoclonal antibody of Elthera and the highly innovative ConjuAllTM platform technology of LegoChem Biosciences is a promising next-generation ADC for the treatment of various solid cancers, including BC, CRC, melanoma, OC and PaC.

Citation Format: Yeojin Jeong, Gunther Spohn, Flavio Mehli, Chul-Woong Chung, Changsik Park. LCB97, an antibody drug conjugate (ADC) targeting a novel tumor antigen for the treatment of multiple solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5653.
Abstract 5875: Rab37 mediates intracellular trafficking and membrane presentation of PD-1 in T cells to foster an immunosuppressive microenvironment in lung cancer
Authors: Kuo Wan-Ting , Kuo I-Ying , Wu Shih-Ting , Su Wu-Chou , Wang Yi-Ching
Published: April 2023

Programmed cell death protein 1 (PD-1) expressed on the surface of CD8+ T cells is known as an immune checkpoint protein. High expression of PD-1 leads to T-cell dysfunction in the tumor microenvironment (TME). Our published studies show that Rab37 small GTPase-mediated vesicular trafficking of cell surface proteins and cytokines is critical in immunosuppressive TME. Here, we identify novel mechanisms of intracellular trafficking and plasma membrane presentation of PD-1 mediated by Rab37. Confocal immunofluorescence (IF) and vesicles isolation data demonstrated that PD-1 colocalized with Rab37-specific vesicles in T cells in a GTP-dependent manner. Total internal reflection fluorescence imaging, membrane fractionation, and flow cytometric analyses confirmed the dynamic trafficking and membrane presentation of PD-1 by Rab37. In addition, sucrose density gradient centrifugation and IF revealed that the level of glycosylated wild-type PD-1 in membrane compartment was more than glycosylation mutant PD-1, suggesting that glycosylation mutant PD-1 delayed in recruitment to the Rab37 vesicles and thus stalled in membrane presentation. Furthermore, T cell proliferation and activity were downregulated in Rab37 wild-type splenocytes co-cultured with cancer cells compared to those from Rab37 knockout mice using various T cell functional assays. Clinically, the multiplex IF-immunohistochemical assay indicated that the tumor infiltrated Rab37+/PD1+/TIM-3+/CD8+ exhaustion T cells were more in late staged lung cancer patients compared to those in early staged patients. Importantly, patients with Rab37+/PD-1+/TIM-3+/CD8+ tumor infiltrated T cells expression profile correlated with poor overall survival. Our results provide first trafficking mode of PD-1 mediated by Rab37 small GTPase and novel evidence of the tumor promoting function of Rab37/PD-1 axis in T cells of the tumor microenvironment.

Citation Format: Wan-Ting Kuo, I-Ying Kuo, Shih-Ting Wu, Wu-Chou Su, Yi-Ching Wang. Rab37 mediates intracellular trafficking and membrane presentation of PD-1 in T cells to foster an immunosuppressive microenvironment in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5875.
Abstract 5880: The role of SOAT1 on CD8+T cells-mediated immune response in ovarian cancer
Authors: He Jiangnan , Siu Michelle K.Y. , Long Runying , Zhang Ruiqian , Yung Mingo M.H. , Ngan Hextan Y. S. , Chan Karen K.L.
Published: April 2023

Introduction: Sterol-O acyltransferase 1 (SOAT1) functions by converting cholesterol and acyl-CoA into cholesterol ester and coA-SH. High SOAT1 expression with its mediated tumorigenesis has been shown in multiple cancers. Studies showed that SOAT1 inhibition by its inhibitors augmented the outcome of immunotherapies, in terms of the immune checkpoint blockades, cancer vaccines, and CAR-T cells, by boosting the function of T cells. However, in order to have a better understanding of the role played by SOAT1 in the tumor microenvironment of ovarian cancer (OC) and optimize strategies of combination immunotherapy, it’s necessary to detect the role of SOAT1 in OC cells on T cell-mediated immune response.

Methods: SOAT1 knockdown was manipulated by siRNAs transfection. SOAT1 was inhibited by avasimibe and OC cells were treated in different doses of avasimibe. qPCR was performed to detect gene expressions in ovarian cancer cells. T cells, isolated and activated from healthy donors, were cocultured with SOAT1-silenced and avasimibe pre-treated OC cells and their tumor-conditioned medium (TCM). Flow cytometry analysis was used to measure cytotoxic secretory molecules of human T cells, like IFN-r, TNF-a, and Granzyme B.

Results: The downregulation of intracellular cytotoxic cytokines was shown in the T cells cocultured with SOAT1-silenced and avasimibe-pretreated OC cells or treated by their TCM, thereby impairing the cytotoxic capacity of T cells, compared to the corresponding control group. qPCR results showed that several immunosuppressive cytokines, like IL-6, IL-8, TGF- were upregulated in the SOAT1-silenced or avasimibe-treated OC cells.

Conclusion: SOAT1 inhibition in ovarian tumor cells impairs CD8+ T cell cytotoxic function, to some extent, via the upregulation of immunosuppressive-related cytokines.

Citation Format: Jiangnan He, Michelle K.Y. Siu, Runying Long, Ruiqian Zhang, Mingo M.H. Yung, Hextan Y. S. Ngan, Karen K.L. Chan. The role of SOAT1 on CD8+T cells-mediated immune response in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5880.
Abstract 5901: Microbiome and genomic profiling in rectal adenocarcinoma
Authors: Hein David , Coughlin Laura , Poulides Nicole , Koh Andrew , Sanford Nina
Published: April 2023

Introduction: Recent studies have identified bacteria associated with colorectal cancer (CRC), with some data supporting causality in preclinical CRC models. We examined the gut microbiome composition and tumor genomic profiles in a diverse cohort of rectal cancer patients.

Methods: Twenty-two patients with newly-diagnosed, Stage II-IV rectal cancer without pathogenic germline mutations (mismatch repair genes, MUTYH) were included in the study. Gut microbiome profiling was performed on genomic DNA extracted from collected fecal samples (16S rRNA sequencing, V4 region). Tumor specimens were tested for somatic and germline mutations using the Tempus xT 648-gene panel and whole transcriptome RNA sequencing, providing information on tumor mutation burden (TMB), pathogenic KRAS mutation status and consensus molecular subtype (CMS). We then assessed differential abundance of microbial taxa (using ANCOM-BC) between the following groups: high vs low TMB (dichotomized at median, 3.7 mut/MB), presence versus absence of pathogenic KRAS mutation, and CMS subtype.

Results: The median age of study subjects was 49 years (range 37-78). Racial/ethnic breakdown was as follows: 2 non-Hispanic Asian, 4 non-Hispanic Black, 5 non-Hispanic White, and 11 White Hispanic. Twelve tumors carried a pathogenic KRAS variant and the median TMB was 3.7 m/MB (range 0.5-8.9 m/MB). Among 20 tumors that underwent whole transcriptome RNA sequencing, 1 was categorized as CMS1, 8 CMS2, 3 CMS3, 7 CMS4, and 2 CMS undetermined. There were no statistically significant differences in gut bacteria taxa abundance by KRAS mutation status or among CMS subtypes. The group with greater than median TMB, however, had statistically significant enrichment of Pepostreptococcus stomatis (18.4 fold increase, p=0.039 after Benjamini-Hotchberg adjustment with FDR = 0.05).

Conclusions: In this pilot study, we found that CRC patients with higher TMB had an enrichment of Peptostreptococcus stomatis, a bacteria associated with colonic dysplasia in mouse models and enriched in CRC patients as compared to healthy controls. (PMID 28823860, 35292756) Higher TMB is a predictive biomarker for response to immunotherapy across cancer types including CRC. Since the gut microbiome is potentially modifiable, further research is needed to better elucidate the connection between microbiome and CRC carcinogenesis including specific mutation profiles, which could pave the way for microbiome-related risk reduction or therapeutic strategies in CRC.

Citation Format: David Hein, Laura Coughlin, Nicole Poulides, Andrew Koh, Nina Sanford. Microbiome and genomic profiling in rectal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5901.
Abstract 5985: Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression
Authors: Chen Ching-Tso , Shao Yu-Yun , Hsu Hung-Wei , Wo Rita R. , Wang Han-Yu , Cheng Ann-Lii , Hsu Chih-Hung
Published: April 2023

Background: PD-1 blockade has been approved as treatment for advanced HCC. We previously demonstrated the efficacy of CDK9 inhibition in reducing the expression of inducible proteins and as a treatment for HCC (Shao et al. Oncology 2022). We thus examined the influence of CDK9 inhibition on the expression of PD-L1 in HCC and the potential of improving the efficacy of PD-1 blockade with the combination of CDK9 inhibitors.

Methods: We first examined the influence of specific CDK9 inhibitors, AZD4573 and atuveciclib, on interferon-γ (IFN-γ) induced PD-L1 expression of human HCC cell lines HuH7 and Hep3B. Overexpression of CDK9 and siRNA to CDK9 were used to confirm the findings. Similar experiments were repeated in the mouse HCC cell line BNL. To test the in vivo efficacy, we orthotopically implanted BNL cells in the subcapsular area of BALB/c mice, which were then treated with vehicle, CDK9 inhibitors, anti-PD-L1 antibodies, or their combination. To compare with tumor sizes, all mice were sacrificed after 14 days of treatment. To compare survival, the mice were only sacrificed if they met the criteria of animal euthanasia.

Results: Both HuH7 and Hep3B cells had minimal PD-L1 expression, which however could be induced using IFN-γ. AZD4573 and atuveciclib decreased the IFN-γ induced PD-L1 expression of HuH7 and Hep3B cells in a dose dependent manner. The flow cytometry results confirmed that membrane PD-L1 expression was also decreased by CDK9 inhibition. Overexpression of CDK9 decreased the influence of these CDK9 inhibitors on PD-L1 expression. CDK9 knockdown using siRNA reduced the IFN-γ induced PD-L1 expression. AZD4573 and atuveciclib also reduce the PD-L1 expression in BNL cell lines. In the orthotopic animal model, the tumors of the sacrificed mice were significantly smaller in mice treated with atuveciclib and an anti-PD-L1 antibody than mice treatment with either alone. The survival of mice treated with the combination was also longer than mice treated with each therapy alone.

Conclusion: We demonstrated that CDK9 inhibition could reduce the IFN-γ induced PD-L1 expression of HCC cells. Combination of CDK9 inhibitors and anti-PD-L1 antibodies was more effective than either therapy alone.

Citation Format: Ching-Tso Chen, Yu-Yun Shao, Hung-Wei Hsu, Rita R. Wo, Han-Yu Wang, Ann-Lii Cheng, Chih-Hung Hsu. Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5985.
Abstract 5988: A non-canonical CDK9 complex mediates endocycling in polyaneuploid cancer cell (PACC) state
Authors: Lee Cheng-fan , Loycano Michael , LOFTUS Luke , Kostecka Laurie , Li Melvin , Amend Sarah , Pienta Kenneth
Published: April 2023

Once prostate cancer has spread from its primary site, treatment is limited to systemically administered therapy. While tumors initially respond to these therapies, eventually, all therapies fail, and the tumor recurs. Ours and others' recent studies have shown the presence of a universal mechanism of resistance: accession of the polyaneuploid cancer cell (PACC) state. The PolyAneuploid Transition (PAT) is initiated when a cancer cell is subjected to external stress, including therapy (we have tested three classes of chemotherapy and radiation), and accesses an alternative non-mitotic cell cycle known as endocycling, resulting in a greater-than-G2 polyploidization of the aneuploid genome (polyaneuploidy). While in this pro-survival PACC state, we have shown that cells are resistant to all forms of cytotoxic therapy. Initial RNA sequencing of cells that have accessed the PACC state in response to multiple classes of chemotherapy, including docetaxel and cisplatin, demonstrate a remodeled transcriptional profile that is distinct from control cultures. We hypothesize that accession of the resistance-mediating PACC state requires transcriptional remodeling. Our data show that cells in the PACC state had increased overall transcriptional activity. The multi protein complex pTEFB (positive transcription elongation factor) is a critical driver of RNA Polymerase II function and is composed of multiple subunits, including CDK9, a cyclin regulatory subunit, and a transcription factor. CDK9 phosphorylation was significantly elevated during accession and persistence in the PACC state. Genetic inhibitionof CDK9 by siRNA led to decreased expression of cyclin A which plays a role in S phase, both in the mitoticcycle and in the PACC endocycle. Interestingly, despite increased CDK9 levels, known CDK9 pTEFb complex members cyclin T1 and BRD4 were both decreased in cells in the PACC state. We treated cancer cells induced to enter the PACC state upon exposure to cisplatin with pharmaceutical inhibitors of CDK9, Flavopiridol and Fadraciclib. Combination of CDK9 inhibition with chemotherapy showed a suppressive effect on the PACC population. Our findings therefore suggest that a non-canonical CDK9 complex could be critical role for the endocycling to access and maintain the PACC state and represents a therapeutic candidate to eliminate the resistance-mediating PACC state.

Citation Format: Cheng-fan Lee, Michael Loycano, Luke LOFTUS, Laurie Kostecka, Melvin Li, Sarah Amend, Kenneth Pienta. A non-canonical CDK9 complex mediates endocycling in polyaneuploid cancer cell (PACC) state. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5988.
Abstract 6011: Methylation profiling reveals the role of PRC2 in regulating DNA methylome changes in malignant peripheral nerve sheath tumors
Authors: Stehn Christopher M. , Bhunia Minu , Williams Kyle B. , Larsson Alex T. , Largaespada David A.
Published: April 2023

Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant cancer predisposition syndrome defined by germline deletion of one NF1 allele. Somatic loss of heterozygosity in the remaining NF1 allele in Schwann lineage cells gives rise to benign tumors known as plexiform neurofibromas. These tumors have a high risk of developing into cancerous lesions known as malignant peripheral nerve sheath tumors (MPNST) through the sequential deletion of CDKN2A/B followed by recurrent mutations in SUZ12 or EED, constituting the inactivation of polycomb repressor complex 2 (PRC2). PRC2 is a transcriptional repressor complex involved in the silencing of several genes throughout cell development and differentiation. Loss of this complex leads to a depletion in trimethylation marks on Histone H3 Lysine 27 (H3K27me3) and the subsequent gain of acetylation marks on the same lysine residue (H3K27Ac). However, full range of epigenetic consequences of these events are currently not well characterized, especially with respect to MPNST formation and development. The Largaespada lab has developed Schwann cell lines engineered to harbor deletions in NF1 and either SUZ12 or EED and assayed the methylation status of 850,000 CpG sites using the EPIC array. The resulting methylation profiles reveal a large degree of change in promoter methylation between cell lines with and without PRC2 inactivation. These changes, however, appear to be inconsistent. To further understand this phenomenon, I have analyzed differential methylation patterns between lines with SUZ12/EED deletions on top of NF1 deletion compared to those with NF1 deletion alone. Pathway analysis of genes whose promoters are differentially methylated upon PRC2 reveals a bias towards developmental modules such as morphogenesis and neuronal differentiation, suggesting that DNA methylation is a mechanism by which PRC2 loss promotes stemness in Schwann lineage cells prior to MPNST formation. Additionally, differentially methylated regions were overlapped with functional non-coding elements in the genome to understand potential ripple effects in the epigenome. These findings underline the importance of DNA methylation in contributing to tumor formation and define unique functions by which PRC2 specifically leverages diverse methylation states to drive malignant transformation in Schwann lineage cells.

Citation Format: Christopher M. Stehn, Minu Bhunia, Kyle B. Williams, Alex T. Larsson, David A. Largaespada. Methylation profiling reveals the role of PRC2 in regulating DNA methylome changes in malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6011.
Abstract 6012: Comparison of pre-analytical methods for DNA methylation analysis of cfDNA
Authors: Tanic Miljana , Emiloju Oluwadunni , Dhami Pawan , Beck Stephan
Published: April 2023

Liquid biopsies in the form of cell-free circulating tumor DNA (ctDNA) isolated from blood plasma or serum have shown great promise to be used as minimally-invasive markers in cancer patients providing an insight into the composition of both primary tumor and metastases, and an opportunity to assess clonal dynamics throughout the course of disease. CtDNA carries not only tumor- specific changes in its sequence but also distinctive epigenetic marks, namely DNA methylation patterns similar to the patterns in the primary tumor. Deregulation of tissue-specific DNA methylation patterns is an early event in carcinogenesis holding great promise to be exploited for early detection of cancer. However, in contrast to mutation detection, targeted DNA methylation sequencing of cfDNA remains a challenge due to low concentration and heavy fragmentation. The limiting step in detection of any analyte is the recovery of the DNA molecules. The pre-analytical steps in the DNA methylation detection pipeline include extraction of cfDNA molecules and their conversion using sodium bisulfite. Bisulfite conversion is a harsh chemical reaction that induces further fragmentation and degradation of already scarce cfDNA. In this study we compared commercial methods for cfDNA extraction and for bisulfite conversion to evaluate the yield, degradation and procedural loss. We compared five commercial kits for cfDNA extraction from blood plasma, three column-based (Qiagen’s QIAamp Circulating nucleic acids kit, Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification and Macherey-Nagel’s NucleoSpin® Plasma XS) and two based on magnetic beads cleanup (Bioo Scientific’s Next Prep magnetic Cell-Free Circulating DNA isolation kitand Applied Biosystems’ Magmax Cell-Free Circulating DNA isolation kit. The kits used for bisulfite conversion were Zymo EZ DNA Methylation Lightning with column based purification and with magnetic beads purification, Zymo EZ DNA Methylation Direct with column based purification and with magnetic bead purification, Qiagen Epitect Fast DNA Bisulfite and Diagenode Premium Bisulfite kits. Same samples were processed by each of the kits, Sample degradation was determined using Agilent Bioanalyzer dsDNA kit. Yield was evaluated by Picogreen, absolute qPCR for single-copy gene and for contamination of gDNA by a B-cell specific assay, while procedural loss was determined based on the recovery of a synthetic spike-in.

Citation Format: Miljana Tanic, Oluwadunni Emiloju, Pawan Dhami, Stephan Beck. Comparison of pre-analytical methods for DNA methylation analysis of cfDNA. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6012.
Abstract 6022: The importance of DNA methylation to breast cancer cell phenotype and its role in the anticancer activity of withaferin-A
Authors: Brane Andrew , Tollefsbol Trygve O.
Published: April 2023

Breast Cancer (BC) is a widespread malignancy that affects the lives of millions of women each year. Prevention of BC through administration of epigenetically active dietary phytochemicals has received increased interest in recent years due to its relatively low cost, few side effects and potential to reduce the financial and healthcare burdens associated with BC. Many phytochemicals’ anticancer functions are thought to arise from their effects on the epigenome, although the relative importance of specific genes’ epigenetic states remain unclear. To elucidate this, we utilized human BC cell lines in combination with CRISPR-dCas technology tied to methylation modifiers to explore the importance of gene specific DNA methylation to cancer cell phenotype in both naïve BC cells and BC cells treated with the phytochemical Withaferin A (WA). We found that demethylation of promoters of the tumor suppressors p21 and p53 results in increased gene expression, while methylation of the promoter of the oncogene CCND1 results in decreased in gene expression. Additionally, changes to biological processes were associated with tumor suppressor promoter methylation state, including increases in cell cycle arrest for a demethylated p21 promoter and decreases in cell viability for a demethylated p53 promoter. We also found that downregulation of p21 through targeting DNMT3A to its promoter was able to ablate the anticancer function of WA in both MDA-MB-231 and MCF-7 cells. Taken together, these results highlight the potential importance of DNA methylation changes for cancer cell phenotype and a possible mechanism of action for the phytochemical WA in BC prevention.

Citation Format: Andrew Brane, Trygve O. Tollefsbol. The importance of DNA methylation to breast cancer cell phenotype and its role in the anticancer activity of withaferin-A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6022.