bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1723: Spatial analysis of tumor-infiltrated immune cells with highly specific RNAscope™ RNA-protein Co-detection assays
Authors: Dikshit Anushka , Basak Sayantani , Doolittle Emerald , Kovalenko Ilya , Bunting Michaeline
Published: June 2022

Interrogating complex tumor microenvironment requires a multi-omics approach that can provide high level of sensitivity and specificity. Identifying immune cell subsets within the tumor can be vital for predicting response and determining therapeutic efficacy. Detecting target immune cell markers using immunohistochemistry/Immunofluorescence (IHC/IF) and visualizing cytokine expression with in situ hybridization (ISH) can provide comprehensive information about the activation states of immune cells. Here, we demonstrate a newly developed integrated ISH and IHC/IF workflow compatible with manual and automated platforms that can substantially improve RNA-protein co-detection. We demonstrate the use of our RNA-Protein Co-detection assay in combination with the automated RNAscope Multiplex Fluorescent v2 assay, automated RNAscope Chromogenic Duplex assay and manual RNAscope Multiplex Fluorescent v2 assay to detect T cell markers, macrophage markers and checkpoint markers in the tumor microenvironment by using a microarray with different tumor samples. We identified CD4+ helper T cells and CD8+ cytotoxic T lymphocytes. Additionally, we determine the activation states of CD8+ T cells by visualizing IFNG, GZMB and IL-2 expression. We were also able to identify macrophages detected by CD68 protein expression and the M1 and M2 subsets were differentiated by using the M2-specific marker, CD163. We could also delineate tumor-stroma border in the samples by using the Pan-CK probe which distinctly marks the tumor cells and visualize the expression of immunoregulatory receptors PD-L1 and CTLA4 in the tumor cells. This assay is enabled for multiplexing by combining with our fluorescent assay platforms. Similarly, for researchers interested in detecting target expression while retaining morphological context, the codetection assay should be combined with our chromogenic assays. Overall, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies with minimal optimization.

Citation Format: Anushka Dikshit, Sayantani Basak, Emerald Doolittle, Ilya Kovalenko, Michaeline Bunting. Spatial analysis of tumor-infiltrated immune cells with highly specific RNAscope™ RNA-protein Co-detection assays [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 1723.
Abstract 1780: Functional inhibition of RNA-binding protein HuR reverses chemotherapeutic resistance in triple-negative breast cancer
Authors: Wei Lanjing , Zhang Qi , Zhong Cuncong , Aubé Jeffrey , Welch Danny R. , Wu Xiaoqing , Xu Liang
Published: June 2022

The 5-year relative survival rate of patients with triple-negative breast cancer (TNBC) is lower than the overall patients with breast cancer. The primary systemic treatment for TNBC remains to be chemotherapy. But chemoresistance frequently develops with the conventional usage of chemotherapy drugs, which results in poorer prognosis and higher recurrence of TNBC than other subtypes of breast cancer. Therefore, to improve the treatment for TNBC, overcoming chemoresistance is a critical challenge to conquer. The RNA-binding protein Hu antigen R (HuR) is a posttranscriptional regulator. It can stabilize target mRNAs through binding to U- or AU- rich elements mainly in 3’ untranslated region (UTR) of mRNA and upregulate their translation level in most cases. The encoded proteins of HuR target mRNAs are implicated in multiple cancer hallmarks, including chemotherapeutic sensitivity. The cytoplasmic accumulation of HuR is reported to contribute to chemoresistance in multiple cancer cells, and HuR inhibition sensitizes cancer cells to chemodrugs. We hypothesize that inhibition of HuR function by disrupting its interaction with mRNA can accelerate the decay of target mRNAs and thus reduce the translation level of proteins responsible for chemoresistance. Recently, our lab identified a small molecule HuR inhibitor, KH-3, which potently inhibits HuR function by disrupting HuR-mRNA interactions. In this study, KH-3 is used as a tool compound to investigate the roles HuR plays in chemoresistance development and evaluate whether HuR inhibition can enhance the efficacy of chemotherapy for TNBC cells. Two MDA-MB-231 cell sublines resistant to docetaxel (231-TR) or doxorubicin (231-DR) were generated in our lab. Compared to the parental cell line, two sub-lines exhibit similar sensitivity to KH-3, and KH-3 re-sensitizes chemoresistant cells to docetaxel or doxorubicin in the MTT-based cytotoxicity assay and the colony formation assay, indicating that HuR inhibition can overcome the acquired chemoresistance. The in vivo efficacy studies in orthotopic xenograft mouse models of human TNBC confirm that KH-3 synergizes docetaxel treatment. Regarding to mechanisms of action, several HuR direct target mRNAs implicated in chemoresistance were found upregulated in the resistant cells, which were reversed by KH-3 treatment. Detailed molecular mechanisms are now under investigation. This study suggests that HuR inhibition is a promising strategy to overcome the challenge of chemoresistance of TNBC.

Citation Format: Lanjing Wei, Qi Zhang, Cuncong Zhong, Jeffrey Aubé, Danny R. Welch, Xiaoqing Wu, Liang Xu. Functional inhibition of RNA-binding protein HuR reverses chemotherapeutic resistance 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 1780.
Abstract 1788: Engineered destabilized AU rich elements on the 3UTR of HER2 degrades HER2, inhibits proliferation, and induces apoptosis in HER2 positive trastuzumab resistant breast cancer cells
Authors: Awah Chidiebere U. , Dong Fu , Levine Fayola , Ash Leonard , Glemaud Yana , Ogunwobi Olorunseun
Published: June 2022

Resistance to molecular targeted therapy is a major challenge facing breast cancer patients and unfavorably impacts clinical outcomes, leading to hundreds of thousands of deaths yearly. Resistance to cancer therapies arises from many factors. One of the key factors driving resistance is oncogenes which initiate pro-growth, pro-survival and metastatic programs that enable cancers escape various therapies. In HER2+ breast cancer, the oncogene HER2 is the master driver of this tumor. Trastuzumab effectively targets HER2. While this is generally successful, some patients who have HER2+ breast cancer develop resistance to trastuzumab and there are limited treatment options for them. Moreover, for low HER2+ expressing breast cancer, there are limited treatment options for them. And in African American women who have HER2+ breast cancer, there is disparity in their treatment outcomes. We have discovered that 3’UTR of HER2 is enriched with poly U stabilizing AU rich elements (ARE). We have developed a novel technology wherein we engineered the stabilizing HER2 3’UTR ARE motifs to destabilizing motif in HER2+ and HER2+ trastuzumab resistant breast cancer cells to control HER2 transcript. We achieved complete degradation of HER2 within 4-8 days in HER2+ wildtype breast cancer cells and 9-11 days in HER2+ trastuzumab resistant breast cancer cells. We had loss of cancer cell viability by more than 90%. We also show that control of HER2 transcript downregulated HER2-dependent kinases, transcription factors, and HER2 interactome. Mechanistically, the control of HER2 transcript was achieved by destabilized ARE sequence specificity which triggered the proteins PARN, XRN1 and CNOT1 to degrade HER2 transcript and this led to induction of active caspase 3/7, reduction of cell size, and severe distortion and disruption of the cancer cell membrane leading to cell death. Taken together, we have a developed a novel approach to control HER2 transcript expression both on spatial and temporal scale. This novel approach offers applications for targeting HER2+ trastuzumab resistant breast cancer as well as other cancers resistant to HER2 targeted therapy and driven by pervasive oncogenic signals.

Citation Format: Chidiebere U. Awah, Fu Dong, Fayola Levine, Leonard Ash, Yana Glemaud, Olorunseun Ogunwobi. Engineered destabilized AU rich elements on the 3UTR of HER2 degrades HER2, inhibits proliferation, and induces apoptosis in HER2 positive trastuzumab resistant 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 1788.
Abstract 1792: FGFR3 mediated PARP1 tyrosine 158 phosphorylation promotes PARP inhibitor resistance
Authors: Chen MeiKuang , Gao Yuan , Xia Weiya , Wang Yu-Han , Litton Jennifer K. , Chu Yu-Yi , Meric-Bernstam Funda , Piwnica-Worms Helen , Arun Banu , Ahnert Jordi Rodon , Wei Yongkun , Chang Wei-Chao , Wang Hung-Ling , Tapia Coya , Albarracin Constance T. , Wang Shao-Chun , Wang Ying-Nai , Hortobagyi Gabriel N. , Lin Chunru , Yang Liuqing , Yu Dihua , Hung Mien-Chie
Published: June 2022

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), which induce DNA damage by inhibiting PARP1 enzymatic activity and trapping PARP on the damaged DNA, are used to eliminate BRCA1/2-mutated (BRCAm) cancer. However, clinical observations suggest that BRCAm tumors develop PARPi resistance. Current strategies to overcome PARPi resistance include impeding multiple DNA repair pathways to induce excessive DNA damage. Here, we propose a novel strategy targeting oncogenic receptor tyrosine kinases to enhance PARP trapping. By developing triple-negative breast cancer (TNBC) cells with acquired talazoparib resistance, we observed a high prevalence of activated fibroblast growth factor receptor 3 (FGFR3) kinase in these cells through kinase antibody array analysis. Mass spectrometry analysis and in vitro kinase assay suggested that FGFR3 phosphorylated PARP1 at tyrosine residues 158 and 176. Biochemistry studies suggested that only PARP1 tyrosine 158 phosphorylation contributes to PARPi resistance in the cells we developed. We then developed a monoclonal antibody against tyrosine 158 phosphorylated PARP1, and found that high-level PARP1 tyrosine 158 phosphorylation positively correlated with PARPi resistance in breast cancer patient-derived xenograft models. We further demonstrated that the combination of FGFR inhibitor and PARPi delayed DNA repair with prolonged PARP trapping. Moreover, synergy between PARPi and FGFR inhibition was observed in multiple TNBC cell lines with PARPi resistance in vitro. The combination of PARPi and FGFR inhibitor also showed synergism in vivo, and treatment with the combination of PARPi and FGFR inhibitor was tolerated in mouse models. These findings reveal that PARP1 tyrosine 158 phosphorylation facilitates resolving of the PARPi-induced PARP-trapping, and that the tyrosine 158 phosphorylated PARP1 may be an effective biomarker to indicate FGFR3 mediated PARPi resistance.

Citation Format: MeiKuang Chen, Yuan Gao, Weiya Xia, Yu-Han Wang, Jennifer K. Litton, Yu-Yi Chu, Funda Meric-Bernstam, Helen Piwnica-Worms, Banu Arun, Jordi Rodon Ahnert, Yongkun Wei, Wei-Chao Chang, Hung-Ling Wang, Coya Tapia, Constance T. Albarracin, Shao-Chun Wang, Ying-Nai Wang, Gabriel N. Hortobagyi, Chunru Lin, Liuqing Yang, Dihua Yu, Mien-Chie Hung. FGFR3 mediated PARP1 tyrosine 158 phosphorylation promotes PARP inhibitor resistance [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 1792.
Abstract 1813: DIACC2010, a selective inhibitor of KIF20A
Authors: COLLETTE Yves , CASTELLANO Rémy , AURRAND-LIONS Michel , SICARD Hélène , VEY Norbert , BOUGERET Cécile
Published: June 2022

Mitotic kinesins are essential regulators of cancer cell replication and migration. The microtubule associated motor protein KIF20A (also called MKlp2), a member of the kinesin-6 family, plays an essential role during cytokinesis and is also involved in the fission of RAB6-positive vesicles from Golgi membrane, therefore contributing to intracellular vesicular trafficking (1). KIF20A plays a critical role in the development and progression of many cancers, and its high expression is associated with disease progression and poor survival outcome (2). More specifically, immature hematopoietic cells, exhibit high KIF20A expression, whereas mature peripheral blood cells do not (3). Herein we describe the preclinical anti-leukemic efficacy of DIACC2010, sole-in-class selective KIF20A inhibitor. DIACC2010 demonstrated potent and consistent cytotoxic activity in-vitro against a panel of 9 human AML cell lines, with median IC50 of 40 nM (range 10-77 nM). In the same experimental conditions, cytarabine (CYTA) had median IC50 of 207 nM (range 4-1580 nM). In parallel, no in-vitro toxicity of DIACC2010 was observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 >50 µM). DIACC2010 was subsequently evaluated in-vivo in xenograft models of CYTA-sensitive and CYTA-resistant AML cell lines: DIACC2010 significantly improved overall survival as compared to CYTA in all models, with dose-dependent efficacy. On a mechanistic standpoint, DIACC2010-exposed AML cells displayed characteristic Golgi scattering leading to cell death, as hallmarks of DIACC2010 mode of action and target engagement. Altogether, these results confirm the relevance of KIF20A-directed therapeutic approaches and support the development of DIACC2010 for the treatment of AML. (1) Coupling fission and exit of RAB6 vesicles at Golgi hotspots though kinesin-myosin interactions, S. Miserey-Lenkei et al, Nature com, 2017, 104:300 (2) Prognostic significance of KIF2A and KIF20A expression in human cancer. X. Li et al, Medicine, 2019, 98:46 (3) KIF20A, highly expressed in immature hematopoietic cells, supports the growth of HL60 cell line, H. Morita et al, Int J Immunol, 2018,108:607

Citation Format: Yves COLLETTE, Rémy CASTELLANO, Michel AURRAND-LIONS, Hélène SICARD, Norbert VEY, Cécile BOUGERET. DIACC2010, a selective inhibitor of KIF20A [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 1813.
Abstract 1837: Low dose actinomycin D preferentially activates p53-p21 pathway in aerodigestive tract cancers: Implication for cyclotherapy
Authors: Adeluola Adeoluwa A. , Amin A. R. M Ruhul
Published: June 2022

Actinomycin D (ActD) was the first anti-cancer antibiotic approved for the management of human cancers. However, the notorious toxicity profile limits its widespread application in cancers including cancers of the aerodigestive tract. Recent studies show that combining low-dose ActD with existing chemotherapies could potentially reduce the toxicity of chemotherapy drugs through p53 mediated cyclotherapy (protection of normal cells while eliminating cancer cells), a recently coined concept. An understanding of ActD’s effect on p53 signaling is critical for meaningful application of ActD in cyclotherapy-based combinations. This study evaluated the antitumor efficacy and mechanism of action of ActD in aerodigestive tract cancers. We found that ActD strongly inhibited the growth of a panel of aerodigestive tract cancer cell lines and induced efficient apoptosis although the sensitivity varies among cell lines. The IC50 values of ActD spanned between 0.021-2.96nM. Mechanistic studies revealed that ActD time- and dose-dependently increased the expression of total and phosphorylated p53 (ser15). Furthermore, ActD-induced apoptosis is dependent on p53 in cells expressing wild-type p53. Further studies demonstrated that ActD induced context-dependent differential expression of downstream targets p21 and PUMA without significant effects on p27. Interestingly, our study revealed that p53-p21 is the predominant pathway activated by low-dose ActD in cells with wild type p53. Therefore, our findings further rationalize the premise for future studies to explore the application of low-dose ActD as a chemoprotectant for cyclotherapy combinations in aerodigestive tract cancers. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

Citation Format: Adeoluwa A. Adeluola, A. R. M Ruhul Amin. Low dose actinomycin D preferentially activates p53-p21 pathway in aerodigestive tract cancers: Implication for cyclotherapy [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 1837.
Abstract 1868: Targeting adenosine deaminase enhances anticancer effects of 3'-deoxyadenosine in uveal melanoma
Authors: Lee Su-Chan , Alaali Lujain , Kwon HyukJean , Rigi Mohammed , Eberhart Charles G.
Published: June 2022

Uveal melanoma, which arises from melanocytes located in the uveal tract, is one of the most common primary intraocular cancers. GNAQ and GNA11 mutations, which are detected most of primary uveal melanoma, activate MAPK signaling pathway, increasing cancer cell proliferation, tumor progression and growth. BAP1 mutation is highly associated with liver metastasis and death. However, there are no good therapeutic compounds for uveal melanoma treatment in clinic. 3’-deoxyadenosine (cordycepin) is an adenosine analog which inhibits growth of several types of cancer. However, its anticancer effects have been showed in limited cancer cell lines. In addition, action mechanism of 3’deoxyadenosin is poorly understood. In this study, we found different adenosine deaminase (ADA) expression or activity affects different anticancer results in uveal melanoma. Inhibition of ADA using either siRNA or pharmacologic approaches sensitized tumors to 3’-deoxyadenosine in vitro and in vivo, resulting in increased apoptosis, reduced clonogenic capacity and slower migration of uveal melanoma cells in vitro. These potent ADA-dependent anti-tumor effects were also seen in various cancer cell lines. Therefore, we suggest the potential of adenosine deaminase (ADA) as both a biomarker predicting response to 3’-deoxyadenosine, and a target for combination therapy.

Citation Format: Su-Chan Lee, Lujain Alaali, HyukJean Kwon, Mohammed Rigi, Charles G. Eberhart. Targeting adenosine deaminase enhances anticancer effects of 3'-deoxyadenosine in uveal melanoma [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 1868.
Abstract 1883: Large scale viability screening with PRISM underscores non-inhibitory properties of small molecules
Authors: de Waal Lucian , Larpenteur Kevin , Wang Li , Zhu Cong , Bryan Jordan , Kocak Mustafa , Aghia Corrie , Bittker Joshua , Jones Victor , Vrcic Anita , McBride Edward , Bender Samantha , Gore Kalea , Li Frank , Chen Zhenghao , Tsherniak Aviad , Subramanian Aravind , Koller Daphne , Settleman Jeffery , Mader Chris C. , Stokoe David , Firestone Ari , Roth Jennifer A. , Golub Todd R.
Published: June 2022

Oncology drug discovery has been dependent on phenotypic screening that lead to discovery of many drugs that remain essential cancer treatments to this day. Historically, logistical and technological limitations have hampered the scale of phenotypic small molecule screening across genomic diverse models. Until recently, large scale phenotypic screening across many cell lines, reflecting the somatic diversity in cancer, has not been possible. The PRISM technology does allow for a high-throughput approach to phenotypic viability screening by pooling cell lines and deconvoluting their response to small molecules using unique integrated DNA-barcodes. Here, we report on the results of a large phenotypic screening effort to identify novel small molecules and targets that can expand our knowledge on cancer vulnerabilities and can spur development of phenotypic drug discovery. A library of 18.000 compounds was assembled based on structural diversity and ability to induce gene-expression changes. Of these 18.000 compounds, around 3.500 were found to reduce cell viability of at least 5 cell lines in the PRISM assay. Of the cytotoxic compounds, we did not identify many small molecules that correlated strongly with CRISPR KO dependency data from project Achilles, and instead found that gene-expression correlations hold much more predictive value. By using additional approaches, we were able to identify the target of select novel cytotoxic small molecules and their respective mechanism-of-action. To our surprise, we have identified few compounds that act as canonical inhibitors of cancer drivers, and instead found compounds that act as molecular glues, prodrugs or inhibitors of more general cellular processes. These findings make a strong case for large scale phenotypic viability screening across genomically diverse models as a complementary approach to target identification next to target based drug discovery.

Citation Format: Lucian de Waal, Kevin Larpenteur, Li Wang, Cong Zhu, Jordan Bryan, Mustafa Kocak, Corrie Aghia, Joshua Bittker, Victor Jones, Anita Vrcic, Edward McBride, Samantha Bender, Kalea Gore, Frank Li, Zhenghao Chen, Aviad Tsherniak, Aravind Subramanian, Daphne Koller, Jeffery Settleman, Chris C. Mader, David Stokoe, Ari Firestone, Jennifer A. Roth, Todd R. Golub. Large scale viability screening with PRISM underscores non-inhibitory properties of small molecules [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 1883.
Abstract 1949: Circulating tumor cells presence shorten the cancer specific survival in colorectal cancer: 10 years follow-up study
Authors: Srovnal Josef , Skalicky Pavel , Rehulkova Alona , Vyslouzil Kamil , Vidlarova Monika , Vrbkova Jana , Hajduch Marian
Published: June 2022

Introduction: Colorectal cancer (CRC) is one of the most common cancers worldwide and metastases-related death is the main cause of its high mortality. Circulating/disseminated tumor cells (CTCs/DTCc) are considered as precursors of distant metastatic spread and can act as an independent prognostic biomarker. The impact of CTCs/DTCs presence in perioperative period on long-term cancer specific survival (CSS) was assessed.

Patients and methods: The presence of CTCs/DTCs in peripheral blood, tumor-draining blood, and bone marrow of 188 colorectal cancer patients was analyzed before, immediately after, and one month after surgery using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) to detect the carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) mRNA-positive CTCs. The trial was registered at ClinicalTrials.gov (NCT 03337347), patients were followed-up and the cause of death was determined.

Results: Over a median follow-up period of 116.5 months, 81 of 148 (54.7%) stage I-III CRC patients died. In total, 40.7% of them (33/81) died of cancer. The presence of CTCs/DTCs did not correlate with any conventional clinical or pathological features (tumor node metastasis (TNM) stage, grade, KRAS mutations or microsatellite instability). Patients with CTCs/DTCs in the peripheral blood and bone marrow one month after surgery had significantly shorter CSS (p=0.013, HR=2.6; resp. p=0.019, HR=2.3) than those without CTCs/DTCs. Shorter overall survival was also observed in patients with tumor-draining blood CEA mRNA-positive CTCs before surgery (p=0.014).

Conclusion: The presence of CK20 mRNA-positive CTCs/DTCs in blood and bone marrow after surgery was confirmed as an independent negative prognostic factor for the long-term cancer-specific survival in stage I-III colorectal cancer patients.The study was supported by Ministry of Health of the Czech Republic (NV18-03-00470), Ministry of Education, Youth and Sport of the Czech Republic (LM2018132; LM2018133), Palacky University Olomouc (LF 2021_019) and European Regional Development Fund (ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868, ACGT CZ.02.1.01/0.0/0.0/16_026/0008448).

Citation Format: Josef Srovnal, Pavel Skalicky, Alona Rehulkova, Kamil Vyslouzil, Monika Vidlarova, Jana Vrbkova, Marian Hajduch. Circulating tumor cells presence shorten the cancer specific survival in colorectal cancer: 10 years follow-up study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1949.
Abstract 2052: LAMP1 targeting of the large T antigen of Merkel cell polyomavirus elicits potent CD4+ T cell responses, tumor inhibition, and provides rationale for first-in-human trial
Authors: Rosean Claire Buchta , Karkada Mohan , Koelle David M. , Nghiem Paul , Heiland Teri
Published: June 2022

The majority of Merkel cell carcinomas (MCC), a rare and highly aggressive type of neuroendocrine skin cancer, are associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV integrates into the host genome, resulting in expression of a truncated form of the viral large T antigen (LT) in infected cells and thus making LT an attractive target for therapeutic cancer vaccines. We designed a cancer vaccine that promotes potent, antigen-specific CD4+ T cell responses to MCPγV-LT. To activate antigen-specific CD4+ T cells in vivo, we utilized our nucleic acid platform, UNITE࣪ (UNiversal Intracellular Targeted Expression), which fuses a tumor-associated antigen with lysosomal-associated membrane protein 1 (LAMP1). This lysosomal targeting technology results in enhanced antigen presentation and a balanced T cell response. LTS220A, encoding a mutated form of MCPγV-LT that diminishes its pro-oncogenic properties, was introduced into the UNITE࣪ platform. In pre-clinical studies, vaccination with LTS220A-UNITE࣪ (ITI-3000) induced antigen-specific CD4+ T cells that were sufficient to delay tumor growth, and this effect was dependent on their ability to produce IFNγ. Moreover, ITI-3000 induced a favorable tumor microenvironment (TME), including significantly enhanced numbers of CD4+ and CD8+ T cells as well as NK and NKT cells. These findings strongly suggest that in pre-clinical studies, DNA vaccination using the UNITE࣪ platform enhances CD4+ T cell responses to MCPγV-LT that result in significant anti-tumor immune responses. We are planning a first-in-human (FIH) Phase 1 open-label study to evaluate the safety, tolerability, and immunogenicity of ITI-3000 in patients with polyomavirus-positive MCC. Patients will receive up to four intramuscular vaccinations of 4mg of ITI-3000 using the PharmaJet Stratis® needle-free injection system. The primary objectives will be safety and tolerability, observing dose-limiting toxicities, serious adverse events, standard clinical assessments, and safety laboratory parameters. Immunogenicity of the vaccine will be measured by peripheral blood assessments of T cell activation using ELISpot and flow cytometry assays.

Citation Format: Claire Buchta Rosean, Mohan Karkada, David M. Koelle, Paul Nghiem, Teri Heiland. LAMP1 targeting of the large T antigen of Merkel cell polyomavirus elicits potent CD4+ T cell responses, tumor inhibition, and provides rationale for first-in-human 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 2052.
Abstract 2089: Evaluating in vivo efficacy of anti-TSLP antibodies in humanized B-hTSLP/hTSLPR mice
Authors: Zhang Shujin , Nie Yanhui , Chang Jiahui , Chromikova Veronika , Yu Luke (Zhaoxue) , Kuraguchi Mari
Published: June 2022

Expression of the epithelial cytokine, thymic stromal lymphopoietin (TSLP), has been observed across several barrier surfaces and plays a critical role in both physiological and pathological processes. Via T helper (Th) 2 cytokine production, TSLP has been implicated in asthma, atopic dermatitis, inflammatory bowel disease, multiple sclerosis, and chronic obstructive pulmonary disease. This range in which TSLP exerts its effects on inflammatory diseases makes the TSLP/TSLPR axis an attractive therapeutic target. At Biocytogen, we generated a novel TSLP/TSLPR double humanized mouse model that enables in vivo assessment of human anti-TSLP and anti-TSLPR antibodies. In our B-hTSLP/hTSLPR mice, the murine Tslp and Tslpr genes are replaced with the human TSLP and TSLPR genes by homologous recombination. ELISA analysis showed that human TSLP (after induction with calcipotriol) was exclusively detectable in homozygous B-hTSLP/hTSLPR mice compared to wild type mice. Similarly, flow cytometry analysis indicated TSLPR expression was detectable in monocytes, neutrophils, macrophages, total DCs, cDC1, cDC2, Mo-DC and Pre-DC. Percentages of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD8+ T cells, CD4+ T cells and Tregs in homozygous B-hTSLP/hTSLPR mice were comparable to those in the wild type mice, demonstrating that introduction of human TSLP and TSLPR in place of their mouse counterparts did not impair the overall development, differentiation, or distribution of these cell types. Furthermore, using an asthma mouse model via induction with ovalbumin (OVA), we showed that an anti-TSLP antibody (Tezepelumab, synthesized in house) was efficacious in controlling asthma progression in B-hTSLP/hTSLPR mice. Altogether, B-hTSLP/hTSLPR mice are a promising model for preclinical in vivo pharmacodynamic assessment of TSLP antibodies.

Citation Format: Shujin Zhang, Yanhui Nie, Jiahui Chang, Veronika Chromikova, Luke (Zhaoxue) Yu, Mari Kuraguchi. Evaluating in vivo efficacy of anti-TSLP antibodies in humanized B-hTSLP/hTSLPR mice [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 2089.
Abstract 2093: PEGylated IL-2 with enhanced anti-tumor efficacy and safety profile
Authors: Song Yuntao , Li Hui , Zhang Wei , Fu Dayan , Xu Meng
Published: June 2022

Introduction: Interleukin-2 (IL-2) can activate and expand tumor killing lymphocytes. High doses of IL-2 are needed to activate CD8+ T cells. Severe toxic side effects associated with high doses, as well as the poor PK profile of IL-2, have limited its clinical usage. Various strategies have been employed to prolong half-lives of IL-2 and enable selectivity or bias towards receptor binding to reduce toxicity and maintain good efficacy. Among these, certain PEGylation strategies have generated promising clinical results, but also showed some major limitations. We report a unique PEGylation strategy to produce PEGylated IL-2 with a therapeutic window more than two times larger than the reference PEGylated IL-2.

Methods: CT26 murine syngeneic model was used for evaluation of in vivo efficacy and safety. Each 6-8 weeks old female BALB/c mouse was inoculated subcutaneously in the right front flank region with CT26 tumor cells (5 x 105). Tumors were allowed to grow to mean size ~110 mm3 before randomization and treatment. Body weights and tumor volumes were measured three times per week. The tumor-free mice at the end of the study were re-challenged with CT26 colon tumor cells on the left side.

Results: Utilizing our proprietary PEGylation technology, rhIL-2 was covalently connected with multiple PEG polymers through releasable linkers (RL). The releasable linkers are designed to modulate the release rate of the active IL-2 species. In CT26 syngeneic mouse model, CS-PEG-IL-2 showed significantly enhanced efficacy and safety over IL-2 and reference PEGylated IL-2. rhIL-2 (2 mg/kg, bid x 5, two cycles, i.p.) resulted in complete response (CR) in one out of eight (1/8) mice, while 3 mg/kg dose resulted in death in 8/8 mice. With the reference PEGylated IL-2, 3/8 CR were obtained at 3 mg/kg dose (q1w x 4, i.v.), and 6 mg/kg dose resulted in death in 8/8 mice. Whereas, with three CS PEGylated IL-2 analogs, 5/8 and 6/8 CR were achieved at 3 mg/kg and 6 mg/kg dose respectively, and no obvious toxicity was observed even at the highest dose. After re-challenged with another inoculation of CT26 cancer cells, the CR mice remained tumor-free until the end of the study at day 158.

Conclusions: CS unique PEGylation strategy generated PEG-IL-2 with superior anti-tumor efficacy and safety profiles. Preclinical results support clinical evaluation of this novel class of PEGylated IL-2 analogs.

Citation Format: Yuntao Song, Hui Li, Wei Zhang, Dayan Fu, Meng Xu. PEGylated IL-2 with enhanced anti-tumor efficacy and safety profile [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 2093.
Abstract 2151: Identification and proteogenomic characterization of novel lung adenocarcinoma subtypes with therapeutic relevance
Authors: Roh Whijae , Geffen Yifat , Miller Mendy , Anand Shankara , Kim Jaegil , Heiman David , Meyerson Matthew , Laird Peter , Cherniack Andrew ,   NCI CCG Tumor Molecular Pathology (TMP) Analysis Working Group, Getz Gad
Published: June 2022

Lung adenocarcinoma (LUAD) is one of the most common cancer types with various available treatment modalities. However, better biomarkers of response are still needed for further improving precision medicine. Therefore, a robust LUAD subtyping can substantially aid in determining the most effective therapies that target subtype-specific vulnerabilities. In this study, we integrated multiple datasets: (i) the full 509 LUAD patient cohort from The Cancer Genome Atlas (TCGA) project, (ii) cancer vulnerability data in LUAD cell lines from the Broad Institute’s DependencyMap, and (iii) proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) LUAD patients. Using these datasets, we identified 5 expression subtypes (S1-S5) with unique proteogenomic and dependency profiles that increased the resolution of previously defined subtypes (Proximal Inflammatory [PI]; Proximal Proliferative [PP]; and Terminal Respiratory Unit [TRU]). S4-associated cell lines exhibited specific vulnerability to CDK6 and CDK6-cyclin D3 complex gene, CCND3. S3 was characterized by dependency on CDK4, immune-related expression patterns, and altered MET signaling. Experimental validation showed that S3-associated cell lines responded to MET inhibitors, which also led to increased PD-L1 expression. Finally, we identified a small set of biomarkers for S3 and S4 that could be used in the clinic to classify patients into our therapeutically relevant subtypes. Overall, our lung adenocarcinoma expression subtypes, especially S3 that represents 20% of LUAD patients and S4 that represents 25% of LUAD patients, and their biomarkers could help identify patients likely to respond to CDK4/6, MET, or PD-L1 inhibitors, potentially improving patient outcome.

Citation Format: Whijae Roh, Yifat Geffen, Mendy Miller, Shankara Anand, Jaegil Kim, David Heiman, Matthew Meyerson, Peter Laird, Andrew Cherniack, NCI CCG Tumor Molecular Pathology (TMP) Analysis Working Group, Gad Getz. Identification and proteogenomic characterization of novel lung adenocarcinoma subtypes with therapeutic relevance [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 2151.
Abstract 2159: PRMT5 inhibitor PRT543 displays potent antitumor activity in U2AF1S34F and RBM10LOF spliceosome-mutant non-small cell lung cancer in vitro and in vivo
Authors: Carter Jack , Ito Koichi , Thodima Venkat , Sivakumar Monisha , Hulse Michael , Rager Joseph , Vykuntam Komali , Bhagwat Neha , Vaddi Kris , Ruggeri Bruce , Scherle Peggy
Published: June 2022

PRMT5 (protein arginine methyltransferase 5) is a major Type II PRMT, which catalyzes the symmetric dimethylation of protein arginine residues (sDMA). As an epigenetic regulator, PRMT5 plays essential roles in promoting cancer growth and survival, including through mechanisms that control alternative splicing and RNA processing, and the expression of DNA damage repair genes. Spliceosome mutations have been suggested to represent a potential biomarker for PRMT5 inhibitors, and our previous work highlighted this sensitivity in SF3B1R625C/G expressing uveal melanoma cells. Here, we highlight in vitro and in vivo activity of PRT543, a potent, selective, and orally available PRMT5 inhibitor, in cancer cells harboring mutations in other spliceosome factors such as U2 small nuclear RNA auxiliary factor 1 (U2AF1) and RNA binding motif protein 10 (RBM10). Mutations in U2AF1 (including S34F hotspot mutations) and RBM10 (primarily loss of function (LOF) mutations) occur in 5-10% of all non-small cell lung cancers (NSCLC). Cell proliferation (10-day assay) was assessed in a panel of NSCLC cell lines treated with PRT543, either wild-type or harboring U2AF1S34F or RBM10LOF mutations. Strikingly, both U2AF1S34F and RBM10LOF cell lines were significantly more sensitive to PRT543 compared to wild-type cell lines. Furthermore, PRT543 induced significant dose-related tumor growth inhibition at well-tolerated doses in cell-line derived xenograft (CDX) models harboring the U2AF1S34F or RBM10LOF mutation. Consistent with our previous findings in other tumor types, PRT543 decreased expression of DNA damage repair-associated genes (e.g. BRCA1, RAD51AP1, FANCA, and FANCL) in U2AF1S34F or RBM10LOF mutant NSCLC cells. Combination with PRT543 increased the effectiveness of specific chemotherapeutic agents in both in vitro and in vivo (CDX) models of U2AF1S34F and RBM10LOF NSCLC. Efficacy studies in patient-derived xenograft (PDX) models, as well as genomic profiling of spliceosome-mutant cellular models in response to PRT543 are ongoing. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831).

Citation Format: Jack Carter, Koichi Ito, Venkat Thodima, Monisha Sivakumar, Michael Hulse, Joseph Rager, Komali Vykuntam, Neha Bhagwat, Kris Vaddi, Bruce Ruggeri, Peggy Scherle. PRMT5 inhibitor PRT543 displays potent antitumor activity in U2AF1S34F and RBM10LOF spliceosome-mutant non-small cell lung cancer in vitro and in vivo [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 2159.
Abstract 2177: Myeloid-derived arginase depletes microenvironmental arginine in PDAC tumors and leads to activation of arginine de novo biosynthesis in cancer cells
Authors: Apiz-Saab Juan , Muir Alex
Published: June 2022

Arginine is an amino acid critical for various cellular processes, not only protein synthesis but also metabolism of other essential metabolites, like polyamines, as well as a signaling factor for pathways such as the growth regulator mTOR. Previously, our group measured arginine levels in the interstitial fluid of tumors (TIF) of pancreatic ductal adenocarcinoma (PDAC) murine models and found extremely low arginine levels (2-5 uM) in the tumor microenvironment (TME). Despite near complete absence of this critical nutrient in the TME, pancreatic tumors exhibit aggressive growth. We have sought to understand both how the PDAC TME becomes arginine limited and how PDAC cells adapt to proliferate in the absence of arginine. Using genetically engineered mice, we find that arginase activity in the myeloid compartment of PDAC tumors is responsible for arginine depletion in the TME. Staining of Arg1+ myeloid populations in human PDAC samples suggest a similar mechanism reduces arginine availability in human PDAC tumors as well. We then leveraged our newfound knowledge of PDAC TIF composition to develop a novel ex vivo cell culture media formulation with physiologically relevant nutrient levels and monitored arginine acquisition pathways using isotope tracing and metabolomics assays to determine how PDAC cells cope with arginine deprivation. Under TME nutrient conditions, PDAC cells consume available citrulline and use it to produce arginine by de novo synthesis. Starving cells of citrulline or genetically perturbing arginosuccinate synthase (ASS1), key enzyme in arginine biosynthesis, significantly reduces PDAC cellular arginine and proliferative capacity. Immunohistochemical analysis of both human and mouse PDAC tumors indicates that the de novo arginine synthesis pathway is highly expressed in PDAC but not in untransformed pancreas, suggesting a key role for this pathway in PDAC progression. Altogether, we find that myeloid-derived arginase challenges PDAC cells by limiting arginine availability and suggest that de novo arginine synthesis may be a critical metabolic pathway that enables PDAC tumors to cope with this metabolic challenge.

Citation Format: Juan Apiz-Saab, Alex Muir. Myeloid-derived arginase depletes microenvironmental arginine in PDAC tumors and leads to activation of arginine de novo biosynthesis in 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 2177.
Abstract 2178: Diet-modulated SCAP-SREBP signaling is essential for acinar cell differentiation, pancreatic morphogenesis, and pancreatic adiposity
Authors: Lilly Anna C. , Surumbayeva Aizhan , Nguyen Theodore , Astsaturov Igor , Golemis Erica A.
Published: June 2022

High levels of dietary fats are associated with pancreatitis, diabetes, and pancreatic cancer. Sterol regulatory element binding protein Cleavage-Activating Protein (SCAP) is an essential intermediate in lipid-regulated activation of Sterol Regulatory Element Binding Protein (SREBP) transcription factors, which induce expression of genes regulating lipid homeostasis. We previously demonstrated that the elevated SREBP1 arising from inhibition of the cholesterol biosynthetic pathway in mice genetically predisposed to pancreatic cancer additionally transcribed genes that induced epithelial-mesenchymal transition, promoting basal rather than glandular cancer. To further evaluate SCAP and SREBP function in the pancreas, we developed a new mouse model with selective pancreatic knockout of the Scapf/f gene under the control of the Pdx1-Cre promoter (ScapΔpanc mice).

Although size of the pancreas in ScapΔpanc neonates was unaffected, and number of islets was normal, there was a reduced number of acinar cells, and some evidence of disorganized tissue structure. Preliminary histopatholgical assessment of pancreatic tissue of ScapΔpanc neonates indicated no acinar cell apoptosis, and unimpaired acinar proliferation, suggesting loss of Scap may cause a differentiation defect. Further supporting this idea, siRNA depletion of Scap in the rat AR42J acinar cell line resulted in loss of markers of mature acinar cell identity. ScapΔpanc pancreata underwent progressive and selective loss of acinar cells as mice aged, with three-month old mice having large areas of acinar cell loss, and mislocalization of ductal cells. Coupled with this loss was a large increase in pancreatic adipocytes and mesenchymal cells, resulting in a highly disorganized tissue morphology accompanied by signs of inflammation. These phenotypes were exacerbated in mice maintained on high fat or high carbohydrate diet. Together, these results imply that SCAP and SREBP signaling in the pancreas plays an important role in mediating dietary modulation of pancreatic development, acinar cell homeostasis, and restraint of pancreatic adiposity. Intrapancreatic fat, associated with obesity and chronic pancreatitis, is a known risk factor for pancreatic cancer, and SCAP polymorphisms have been to interact with diet in regulating obesity and blood pressure in humans; we propose SCAP-SREBP signaling in the pancreas may mediate dietary promotion of pancreatic cancer risk.

Citation Format: Anna C. Lilly, Aizhan Surumbayeva, Theodore Nguyen, Igor Astsaturov, Erica A. Golemis. Diet-modulated SCAP-SREBP signaling is essential for acinar cell differentiation, pancreatic morphogenesis, and pancreatic adiposity [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 2178.
Abstract 2179: Increased BCAT1 expression creates a pseudo-hypoxic environment that promotes cell proliferation in a subtype of glioblastoma
Authors: Fala Maria , Ros Susana , Sawle Ashley , Brindle Kevin M.
Published: June 2022

Background: BCAT1 (Branched Chain Amino Transferase 1) has been shown to be upregulated in human IDH (isocitrate dehydrogenase) wild-type glioblastoma (GBM) and BCAT1 knockdown leads to reduced cell proliferation in vitro and in vivo1. Here we investigated the role of BCAT1 in human patient-derived glioblastoma cells and the corresponding orthotopically implanted xenografts to understand how upregulation of the enzyme leads to a more aggressive phenotype.

Methods: The expression of BCAT1 was investigated in a panel of patient derived IDH wild-type glioblastoma cells and BCAT1 expression modulation was achieved using lentiviral expression of a doxycycline-inducible shRNA, to knockdown BCAT1 and expression of a BCAT1 cDNA to overexpress BCAT1. Effects of BCAT1 knockdown and overexpression were investigated in vitro in cell culture and in vivo, following intracranial implantation of mice with the relevant cells.

Results: Variable levels of BCAT1 expression were identified in different patient-derived cell lines. RNA sequencing analysis of BCAT1 knockdown in BCAT1 expressing glioblastoma cells revealed that knockdown led to significant downregulation of HIF 1α (Hypoxia Inducible Factor 1α) and its targets, which was confirmed by Western Blot analysis of Carbonic Anhydrase IX (CAIX) and Hexokinase II (HKII) in cell and tumor lysates. The opposite was observed following BCAT1 overexpression. BCAT1 knockdown also led to higher DNA demethylation levels. Treatment with membrane permeant dimethyl-alpha ketoglutarate mimicked the effect of BCAT1 knockdown. These data suggest that decreased cell proliferation following BCAT1 knockdown in BCAT1 expressing cells is mediated by accumulation of alpha-ketoglutarate, a substrate of BCAT1. Cells that had no BCAT1 expression showed a similar growth profile and overexpression had no effect on proliferation, suggesting that these cells are not dependent on BCAT1 expression.

Conclusion: We conclude that in a subtype of GBM characterized by upregulated BCAT1, BCAT1 expression results in lowered levels of alpha-ketoglutarate, leading to reduced prolyl hydroxylase activity and stabilization of HIF 1α and therefore a pseudo-hypoxic state, resulting in a transcriptome that favors a high proliferation rate. This makes BCAT1 a potential therapeutic target, for tumors that show BCAT1 upregulation.1. Tönjes M, Barbus S, Park YJ, et al. BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1. Nat Med. 2013;19(7):901-908. doi:10.1038/nm.3217

Citation Format: Maria Fala, Susana Ros, Ashley Sawle, Kevin M. Brindle. Increased BCAT1 expression creates a pseudo-hypoxic environment that promotes cell proliferation in a subtype of glioblastoma [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 2179.