bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2680: Combination of the Rac/Cdc42 inhibitor MBQ-167 with paclitaxel reduces metastasis in triple negative breast cancer
Authors: Cruz-Collazo Ailed M. , Grafals Nilmary , Borrero Luis D. , Dharmawardhane Suranganie
Published: June 2023

Metastasis represents a therapeutic challenge, especially in the case of triple negative breast cancer (TNBC), where patients often show recurrence and metastasis due to resistance to chemotherapy. The frontline therapy in TNBC is Paclitaxel, which targets actively dividing cells inducing subsequent apoptosis. Since dormant cells or metastatic cancer cells are not targeted by Paclitaxel, this strategy selects for non-dividing stem cells or more motile metastatic cells. Therefore, targeted treatment options are needed to overcome the recurrence and spreading of metastatic cells. We recently reported on MBQ-167, a Rac/Cdc42 inhibitor, which is highly effective in reducing tumor growth and metastasis in mouse models of TNBC (Cruz-Collazo et al., 2021, Molecular Cancer Therapeutics). Unpublished data show that in the MDA-MB-231 human TNBC model, MBQ-167 is as effective as Paclitaxel in reducing tumor growth and is more effective in metastasis prevention. Therefore, we hypothesize that MBQ-167 is a viable candidate for combined therapy with Paclitaxel and will reduce metastasis in TNBC. The purpose of this study is to test MBQ-167 in combination with Paclitaxel in the EGFR overexpressing MDA-MB-468 human TNBC cell line, which is highly malignant and also expresses the Rac1.B oncogenic splice variant. We found that MBQ-167 inhibits Rac, Rac1.B, and Cdc42 activation in this cell line. Next, we tested individual or combined MBQ-167 (50 mg/kg 5X a week) via oral administration and Paclitaxel (10 mg/kg 1X a week) via intraperitoneal administration in SCID mice bearing luciferase-tagged MDA-MB-468 tumors. Tumor growth and metastases were analyzed by chemiluminescence imaging followed by H&E staining of extracted lungs at necropsy. Similar to our studies with the syngeneic 4T-1/Balb/C model, in this MDA-MB-468 model, Paclitaxel increased metastasis while single agent MBQ-167 significantly reduced metastasis. MBQ-167 in combination with Paclitaxel reduced the enhanced lung metastasis in response to Paclitaxel. In conclusion, this study validates the clinical testing of MBQ-167 in combination with Paclitaxel as a potential combination therapy for TNBC.

Citation Format: Ailed M. Cruz-Collazo, Nilmary Grafals, Luis D. Borrero, Suranganie Dharmawardhane. Combination of the Rac/Cdc42 inhibitor MBQ-167 with paclitaxel reduces metastasis in 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 2680.
Abstract 2732: A self-replicating RNA precision medicine approach to therapeutic protein delivery of narrow therapeutic index biomolecules
Authors: Goldberg Zelanna , Maine Christian , Dailey Gabrielle P. , Domingo Christine , Picarda Gaelle , Little Hunter , Chou Annie , Sparks Jessica , Spasova Darina , Miyake-Stoner Shigeki , Rabiola Christopher A. , Crosby Erika J. , Hartman Zachary C. , Lyerly Herbert K. , Wang Nathaniel , Aliahmad Parinaz
Published: June 2023

Protein drug replacement using nucleic acid technologies has been a sought-after solution for in situ production of proteins with poor half-lives and challenging manufacturability. Linear mRNA approaches have failed clinically in this technological use case due to low levels of protein expression and poor durability. Self-replicating RNA (srRNA) is a new technology for expression of biotherapeutics combining the advantages of fully synthetic drug products with higher peak levels of protein expression with active expression persisting out to 49 days. RBI-2000 is a novel srRNA encapsulated in a lipid nanoparticle and encoding two distinct proteins on the same strand of RNA. The first protein is a pro-inflammatory multimeric cytokine to promote de novo immune cell generation and infiltration. The second molecule is a single chain high affinity protein antagonist of downstream mediators of the inflammasome to prevent sterile inflammation, aberrant angiogenesis, and tumor invasiveness. We selected a novel alphaviral vector which demonstrated an enhanced peak level of protein expression vs. the prototypical vector that has been used in other RNA products. These encoded proteins elicited a pharmacodynamic response that had no evidence of decay in the first 7 days following a single treatment down to doses of 0.01 mcg. Using an implanted MC38 tumor model, tumor control was obtained at the lowest, single dose tested at 0.1 mcg of RBI-2000. When tested in combination with a checkpoint inhibitor (CPI), a single dose of the CPI and a single dose of RBI-2000 at 10 mcg resulted in 80% tumor control in the mice (8/10 mice). Tumor re-challenge of the mice at a site distal to the original implantation showed 100% protection. Collectively, these data demonstrate the feasibility of novel srRNA vectors as a protein drug replacement approach including for the expression of complex, multimeric cocktails of proteins.

Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle P. Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shigeki Miyake-Stoner, Christopher A. Rabiola, Erika J. Crosby, Zachary C. Hartman, Herbert K. Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA precision medicine approach to therapeutic protein delivery of narrow therapeutic index biomolecules [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 2732.
Abstract 2733: TYMS as a novel target for AAV-based therapy
Authors: Vijayakurup Vinod , Meyer Benjamin , Nawab Akbar , Kaye Frederic J. , Guijarro Maria V. , Zajac-Kaye Maria
Published: June 2023

The purpose of this study is to develop a strategy for treating pancreatic neuroendocrine tumor (PanNET) through blocking Thymidylate Synthase (TYMS or TS), a DNA metabolic enzyme. Elevated TYMS levels are frequently observed in human PanNET and are associated with worse prognosis and resistance to cytotoxic agents such as 5FU. Elevated TYMS levels directly promote tumorigenesis in vitro and ectopic expression of human TS (hTS) in transgenic mice promotes adenoma development in the endocrine pancreas in vivo. Pancreatic islet tumor formation in hTS transgenic mice occurred with a long latency period, suggesting that additional somatic events are required for PanNET formation and progression. Men1 was shown to be a frequent target for somatic mutation in PanNETs (44% have MEN1 mutations). Therefore, we established a new mouse model designated hTS/Men1-/-, where hTS is overexpressed in pancreatic islet cells carrying conditional Men1 null alleles. We observed that ectopic hTS cooperates with Men1 inactivation to significantly accelerate PanNET progression and shorten survival. Since traditional TYMS inhibitors, such as 5-FU, consistently induce tumor resistance and cannot cure advanced disease, we have now tested the ability of TS-shRNA delivered using adeno-associated viral (AAV) vectors as a pharmacological safe and effective anti-tumor strategy. We packaged TS shRNA under the mouse insulin promoter (mIP) for islet-cell specific expression using capsid optimized Y to F double mutant AAV (AAV8-2M) to generate viral particles of scAAV8-2M-mIP-GFP-hTS-shRNA. We treated hTS/Men1-/- mice with intraperitoneal (IP) injection (1x1011 viral genomes/mice). Treated and control mice were analyzed for PanNET progression, TYMS expression and survival. In addition, we tested a human PanNET tumor cell line (BON) that was isogenic with or without hTS expression for tumor formation in xenograft model. We confirmed sustained reduction of TYMS levels 3 weeks after IP delivery of AA8-2M-mIP-GFP-hTS-shRNA into PanNET tissues of hTS/Men1-/- mice. In addition, AA8-2M-mIP-GFP-hTS-shRNA delivery blocked PanNET tumor progression and enhanced the survival of treated mice as compared to vector controls. We also confirmed that TYMS knock-down in BON cells by hTS-shRNA reduced xenograft tumor growth as compared to non-specific shRNA controls. In conclusion, the ability of AAV-mediated TS shRNA to block PanNET tumor progression directly demonstrates the importance of elevated TYMS in tumorigenesis and offers a novel anti-cancer strategy.

Citation Format: Vinod Vijayakurup, Benjamin Meyer, Akbar Nawab, Frederic J. Kaye, Maria V. Guijarro, Maria Zajac-Kaye. TYMS as a novel target for AAV-based therapy [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 2733.
Abstract 2863: Interferon-gamma induces NOS2 and COX2 in ER- breast cancer that drives increase metastatic potential
Authors: Cheng Yuk Sing Robert , Ridnour Lisa , Walke Abigail , Kedei Noemi , Wink Adelaide , Edmonston Elijah , Butcher Donna , Dorsey Tiffany , Heinz William , Bryant Richard , Kinders Robert , Lipkowitz Stanley , Wong Stephen , Pore Milind , Hewitt Stephen , McVicar Daniel , Anderson Stephen , Chang Jenny , Ambs Stefan , Lockett Stephen , Wink David
Published: June 2023

NOS2 and COX2 expression is significantly correlated with poor prognosis in ER- breast cancer. Multiple pro-oncogenic pathways involving metastasis, proliferation, angiogenesis, immunosuppression, and chemoresistance are stimulated in a synergistic manner by NOS2 and COX2, resulting in much more aggressive cancers. In this study, our single-cell RNAseq data demonstrated that certain combinations of cytokines, IFNg with TNFA or IL1B, drove NOS2 and COX2 expressions in vitro in the MDA-MB-231 cell line after 48 hours. Analysis of t-SNE plots indicates that the clustering patterns of NOS2 and COX2 are also related to cytokine expression. Moreover, according to our spatial proteomic analysis, CD8 that are associated with IFNg are more likely to have a positive response, indicating a dichotomy in the requirement for IFNg. To better understand this mystery, the spatial distribution of NOS2 and COX2 with CD8 cells in 21 ER- breast tumors was evaluated. In the presence of tumor-excluded CD8, high expression and clustering of NOS2-positive cells were seen near the tumor-stromal interface, showing that NOS2-positive cells were excluded from the tumor. Within the tumor nest, strong expression and population clustering of COX2 reached into the immune desert. In contrast, tumor-penetrant CD8 cells were few and poorly expressed. Metastasis and chemoresistance were among the leading causes of death in this cohort, and it was shown in vitro that IFNg/IL1B or TNFA increased tumor cell migration, suggesting that the interface of tumor with stromal restricted lymphoid aggregates provides the unique spatial environment to increase NOS2 expression and increase metastatic potential. The spatial study of the TME (tumor microenvironment) provides vital insight into how NOS2 and restricted CD8 lead to a higher probability of poor survival.

Citation Format: Yuk Sing Robert Cheng, Lisa Ridnour, Abigail Walke, Noemi Kedei, Adelaide Wink, Elijah Edmonston, Donna Butcher, Tiffany Dorsey, William Heinz, Richard Bryant, Robert Kinders, Stanley Lipkowitz, Stephen Wong, Milind Pore, Stephen Hewitt, Daniel McVicar, Stephen Anderson, Jenny Chang, Stefan Ambs, Stephen Lockett, David Wink. Interferon-gamma induces NOS2 and COX2 in ER- breast cancer that drives increase metastatic potential [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 2863.
Abstract 2864: Selective suppression of melanoma lacking IFN-γ pathway by JAK inhibition depends on T cells and host TNF signaling
Authors: Shen Hongxing , Huang Fengyuan , Zhang Xiangmin , Ojo Oluwagbemiga A. , Li Yuebin , Trummell Hoa Quang , Anderson Joshua C. , Fiveash John , Bredel Markus , Yang Eddy , Willey Christopher , Chong Zechen , Bonner James , Shi Lewis Z.
Published: June 2023

Therapeutic resistance to immune checkpoint blockers (ICBs) is a pressing issue, limiting ICB efficacy to a small proportion of cancer patients. Concerted efforts from our group and others have identified that loss of IFN-γ signaling genes in melanoma is a major mechanism of resistance to ICBs. However, strategies of overcoming this resistance have been largely elusive. Moreover, given the indispensable role of T cells in governing ICBs, little is known about how tumor-intrinsic loss of IFN-γ signaling impacts tumor-infiltrating T cells (TILs). In this study, we report that melanomas with defective IFN-γ signaling (IFNγR1KO) have reduced infiltration and function of TILs. Intriguingly, IFNγR1KO melanoma cells harbor a network of constitutively active protein tyrosine kinases (PTKs) centered on JAK1/2, essential downstream components of the IFN-γ signaling. Additional mechanistic studies reveal that JAK1/2 activation is mediated by augmentation of the mTOR pathway. Importantly, targeting active JAK1/2 with FDA-approved Ruxolitinib (Ruxo) selectively suppresses growth of IFNγR1KO but not scrambled control melanoma, in a T cell and host TNF-dependent manner. Together, our results highlight an important role of tumor-intrinsic IFN-γ signaling in shaping TILs and manifest a therapeutic target that can be leveraged to bypass ICB resistance of melanomas defective of IFN-γ signaling.

Citation Format: Hongxing Shen, Fengyuan Huang, Xiangmin Zhang, Oluwagbemiga A. Ojo, Yuebin Li, Hoa Quang Trummell, Joshua C. Anderson, John Fiveash, Markus Bredel, Eddy Yang, Christopher Willey, Zechen Chong, James Bonner, Lewis Z. Shi. Selective suppression of melanoma lacking IFN-γ pathway by JAK inhibition depends on T cells and host TNF signaling [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 2864.
Abstract 29: Characterization of a murine intestinal cancer homograft model for therapeutic evaluation
Authors: Wang Jinxi , Chen Leilei , Zhang Likun , Zhu Jingqian , Bourre Ludovic , Wang Jingjing
Published: June 2023

Introduction: Intestinal cancers are one of the most lethal malignancies and are responsible for a mass of cancer-related deaths. The discovery of new drugs and therapeutic strategies is underway, and to complement this, the development of superior animal models is required for preclinical studies. Genetically engineered mouse models (GEMMs) mimic de novo human tumor growth within an intact tumor microenvironment and are potent immunocompetent models for studying pathogenesis and immuno-oncology of intestinal cancers. GEMMs with dysfunctional DNA mismatch repair (MMR) genes, like MHL1 and MSH2, manifest intestinal tumors related to microsatellite instability (MSI). However, the low incidence and distinct latency among individuals hamper the application of these GEMMs in preclinical research. In this study, a murine intestinal cancer homograft from GEMMs with MMR deficiency is established to enroll larger cohorts of mice for preclinical studies.

Methods: MMR-deficient GEMMs were generated by knockout of Mlh1 gene with a CRISPR-Cas9 system. Intestinal cancer development was investigated in 16 GEMMs. The developed tumor in the intestine of GEMM was isolated by surgery and inoculated into C57BL/6 mice subcutaneously to establish a murine homograft model. The tumor homograft was passaged and expanded in vivo, and stable tumor growth was established over consecutive passages. Histopathological studies were conducted for both the original and passaged tumors. Genomic mutations and gene expression were analyzed by whole exome sequencing and RNA-sequencing, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Therapeutic efficacy of chemotherapies and immune checkpoint inhibitors were evaluated in vivo for tumor growth inhibition.

Results: The intestinal tumors or lesions were observed in two of the sixteen Mlh1-KO mice with an incidence of 12.5%. A murine homograft model of small bowel adenocarcinoma was successfully established using the tumor located in jejunum of a Mlh1-KO mouse. This model was stably passaged in vivo with semblable pathological features as the original lesion in Mlh1-KO mice. The various degrees of TILs and response to treatments revealed the potential of this model for in vivo investigation of immune response.

Conclusion: Our murine intestinal cancer homograft model presented rapid and stable subcutaneous growth in immunocompetent mice, providing an efficient approach for intestinal cancer researchers and a superior preclinical model for new drug discovery.

Citation Format: Jinxi Wang, Leilei Chen, Likun Zhang, Jingqian Zhu, Ludovic Bourre, Jingjing Wang. Characterization of a murine intestinal cancer homograft model for therapeutic evaluation [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 29.
Abstract 2909: Genetically engineered Natural Killer cells as an immunotherapeutic approach for the treatment of neuroblastoma
Authors: Croom-Perez Tayler J. , Oyer Jeremiah L. , Hasan Md Faqrul , Robles-Carrillo Liza D. , Tullius Brian P. , Copik Alicja J.
Published: June 2023

In this study we test if knockout of DNAM-1 competing inhibitory receptors on Natural Killer (NK) cells improves cytotoxic response against neuroblastoma cell lines. Neuroblastoma and other peripheral nervous cell tumors account for 6% of all pediatric cancer. While the 5-year survival rate has greatly improved, the prognosis of children with high-risk neuroblastoma at diagnosis is less than 50%. Immune checkpoint therapy is a promising new application for pediatric oncology; however, neuroblastoma is often immunologically “cold” and current immune checkpoint targets are not abundant in pediatric solid tumors. To improve response to checkpoint blockade and directly target tumor cells for elimination, this study evaluated the use of NK cells. NK cells prime the immune system for successful response to cancer immunotherapy. NK cells activated with particle technology (PM21-NK cells) are highly cytotoxic, produce IFNγ and induce PD-L1 on cancer cells. Although highly cytotoxic, PM21-NK cells express inhibitory receptors that compete for ligands that trigger NK cell lysis, such as PVR, a ligand highly expressed on neuroblastoma cells. We knocked out expression of multiple PVR inhibitory receptors in PM21-NK cells to further enhance cytotoxicity and prevent exhaustion. Three inhibitory receptors on NK cells that bind PVR - TIGIT, CD96, and PVRIG were knocked out alone or in combination and effector functions of these edited NK cells tested. Cytotoxicity against 3D spheroids of the neuroblastoma cell line SK-NA-S cells was assessed and compared to wild type PM21-NK cells. Knockout (KO) of TIGIT enhanced NK cell killing compared to wildtype NK cells (72%±10 vs 55%±3% killing) as well as PVIRG KO (84±3% vs 46±6% killing) and CD96 KO (62%±9% vs 46±6% killing) at a 1:3 E:T ratio at 72 h. Double or triple KO of these receptors resulted in further increased killing of cancer cells. The knockouts of each of the receptors could affect cytokine production and cytokine responsiveness and this will be examined. Overall, PM21-NK cells show promise for therapeutic use against neuroblastoma and preventing the PVR-mediated inhibitory axis signaling enhances their function.

Citation Format: Tayler J. Croom-Perez, Jeremiah L. Oyer, Md Faqrul Hasan, Liza D. Robles-Carrillo, Brian P. Tullius, Alicja J. Copik. Genetically engineered Natural Killer cells as an immunotherapeutic approach for the treatment of neuroblastoma [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 2909.
Abstract 2916: CAR-NK cells generated with CAR-RNA-LNP express high cytotoxic activity against tumor cells
Authors: Golubovskaya Vita M. , Sun Jinying , Sienkiewicz John , Huang Yanwei , Zhou Hua , Deng Gongping , Harto Hizkia , Xu Shirley , Berahovich Robert , Wu Lijun
Published: June 2023

NK (natural killer) cells have several advantages compared to T cells such as no cytokine release syndrome (CRS), neurotoxicity and GVHD in allogenic setting to kill tumors. We developed CAR-NK platform using RNA-LNP (lipid-nanoparticles) technology. High expansion of NK cells (up to 2000-fold) was achieved using K562 feeder cells with stable expression of 4-1BBL and IL-21. We showed efficient encapsulation of BCMA and CD19-CAR RNA into LNP that resulted in up to 99% expression of CAR+ CD56+ NK cells after transfection. The BCMA-CAR-NK cells more effectively than NK cells killed MM1S, H929 and RPMI8226 multiple myeloma cells and secreted IFN-gamma in a dose-dependent manner. In addition, frozen BCMA-CAR-NK cells were viable and expressed similar cytotoxic activity to not frozen expanded CAR-NK cells. In addition, frozen-thawed BCMA-CAR-NK cells showed 93% CAR expression at 24 hours post-thaw which remained >70% at 48 hours post-thaw. In a similar way, CD19-CAR-NK cells generated with CD19-CAR-LNP killed CD19-positive Raji and Daudi target cells and secreted high level of IFN-gamma. This, frozen/thawed CAR-NK cells demonstrated high in vitro cytotoxic activity against target cells. Manufacturing of NK cells using G-Rex system resulted in 1862-fold expansion at day 13 and 2450-fold expansion of 97% CD56+ NK cells at day 14. Transfection of BCMA-CAR RNA-LNP into 13 day expanded NK cells resulted in 95% BCMA-CAR-NK positive cells. Thus, CAR-NK cells generated with RNA-LNP technology can be used in future pre-clinical studies.

Citation Format: Vita M. Golubovskaya, Jinying Sun, John Sienkiewicz, Yanwei Huang, Hua Zhou, Gongping Deng, Hizkia Harto, Shirley Xu, Robert Berahovich, Lijun Wu. CAR-NK cells generated with CAR-RNA-LNP express high cytotoxic activity against tumor cells [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 2916.
Abstract 3107: Development of novel JAK2 inhibitors and PROTACs as chemical probes for the investigation of JAK2 inhibition and degradation
Authors: Humphrey Briley A. , li Bao , Ferreira Renan B. , Ranatunga Sujeewa R. , Rowsell Tegan M. , Sun Luxin , Lawrence Harshani R. , Schonbrunn Ernst , Reuther Gary , Lawrence Nicholas J.
Published: June 2023

Inhibition and, more recently, degradation of protein kinases is an important therapeutic strategy to modulate disregulated signaling pathways in the cell cycle. Janus kinase 2 (JAK2) is a protein kinase involved in the JAK/STAT pathway which is one of the 12 core cancer pathways; thus, the regulation of JAK2 is important in the maintenance of normal cell function. A fragment-based drug discovery approach was taken to synthesize novel JAK2 inhibitors based using building blocks suitable for PROTAC development. Small molecule inhibitors were analyzed through computational analysis to probe the binding interactions in the hinge region of the ATP active site of JAK2. PROTACs were designed to investigate the degradation of JAK2 based on small molecule ligands with high binding affinity to JAK2. Pyrrolopyrimidine and pyrimidine scaffolds were used to probe the hydrogen bonding interactions in the hinge region to investigate binding affinity. Modification of A-ring and B-ring fragments of the inhibitors and PROTACs were used to develop SAR through data obtained by functional assays, including differential scanning fluorimetry and 33P HotSpot™ kinase assay, in addition to in-vivo cell viability assays as models for myeloproliferative neoplasms. Several key pyrrolopyrimidine and pyrimidine core containing inhibitors and PROTACs bind strongly to JAK2 and modulate JAK2 activity in appropriate cancer cell lines.

Citation Format: Briley A. Humphrey, Bao li, Renan B. Ferreira, Sujeewa R. Ranatunga, Tegan M. Rowsell, Luxin Sun, Harshani R. Lawrence, Ernst Schonbrunn, Gary Reuther, Nicholas J. Lawrence. Development of novel JAK2 inhibitors and PROTACs as chemical probes for the investigation of JAK2 inhibition and degradation [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 3107.
Abstract 3117: Molecular and clinical correlates of genetic ancestry in a diverse, admixed cancer dataset
Authors: Postel Mackenzie D. , Teer Jamie K. , Eschrich Steven A. , Dutil Julie , Siegel Erin M. , Cress William D. , Carpten John D. , Craig David W.
Published: June 2023

The objective of this study is to determine whether genetic ancestry analysis can provide additional insight into the genetic determinants of cancer disparities distinct from socially constructed variables like self-reported race and ethnicity. The Oncology Research Information Exchange Network (ORIEN) “Avatar” project is a diverse dataset of extensive, longitudinal clinical and molecular data from 18 cancer centers across the U.S.; the ORIEN Intermember Ancestry Project cohort described in this study is a subset of the larger dataset. Using paired tumor/germline whole exome sequencing data from ORIEN, the Genome Aggregation Database (gnomAD) dataset of ancestry reference individuals, and the software tools ADMIXTURE and RFMix, we have estimated genetic ancestry for cancer cases from both ORIEN and The Cancer Genome Atlas (TCGA). The ORIEN dataset has 2.6X greater representation of self-reported Hispanic/Latinos than does TCGA, with Hispanic/Latinos making up 9% of the ORIEN dataset vs. 4% in TCGA. As expected, individuals of Hispanic/Latino ethnicity are heterogeneous in their admixture proportions of Indigenous American, African, and European ancestries. We performed statistical analyses in R to identify molecular and clinical correlations with ancestry. This study highlights the clinical utility of genetic ancestry analysis as an approach to understand the genetic contributions to cancer disparities in diverse, admixed populations.

Citation Format: Mackenzie D. Postel, Jamie K. Teer, Steven A. Eschrich, Julie Dutil, Erin M. Siegel, William D. Cress, John D. Carpten, David W. Craig. Molecular and clinical correlates of genetic ancestry in a diverse, admixed cancer dataset [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 3117.
Abstract 3166: CanSig: De novo discovery of shared transcriptional states in cancer single cells
Authors: Yates Josephine , Barkmann Florian , Czyz Pawel Piotr , Kraft Agnieszka , Beerenwinkel Niko , Boeva Valentina
Published: June 2023

Tumor heterogeneity is regarded as a significant obstacle to successful personalized cancer medicine. Specifically, multiple cancer types have been shown to exhibit heterogeneity in the transcriptional states of malignant cells even within the same tumor. Some of these specific transcriptional states of malignant cells have been linked to cancer relapse and resistance to treatment. However, today there is no universal and easy-to-use computational method to extract information about shared transcriptional states from single-cell RNA sequencing measurements (scRNA-seq).

To reliably identify shared transcriptional states of cancer cells, we propose a novel computational tool, CanSig. CanSig automatically preprocesses, integrates, and analyzes cancer scRNA-seq data from multiple patients to provide novel signatures of shared transcriptional states; it also associates these states to known and potentially targetable biological pathways. CanSig jointly analyzes cells from multiple cancer patients while correcting for batch effects and differences in gene expressions caused by genetic heterogeneity. For this, CanSig automatically infers copy number variations in malignant cells and trains a deep learning architecture based on conditional variational autoencoders integrating scRNA-seq measurements.

In our benchmarks, CanSig shows state-of-the-art performance in data integration and reliably re-discovers known transcriptional signatures on four previously published cancer scRNA-seq datasets. For instance, CanSig re-discovers four main cellular states of glioblastoma cells previously reported by Neftel et al., Cell, 2019.

We further illustrate CanSig’s investigative potential by uncovering signatures of novel transcriptional states in several cancer types; some of the novel signatures are linked to such cancer hallmarks as cell migration and proliferation and are enriched in more advanced tumors. We also uncover signatures associated with specific genomic aberrations such as gene copy number gains.

In conclusion, CanSig detects shared transcriptional states in tumors using as input scRNA-seq data for cells with different genetic backgrounds and possibly exhibiting strong batch effects. It can significantly facilitate the analysis of scRNA-seq cancer data and efficiently identify transcriptional signatures linked to known biological pathways. The CanSig method implemented in Python is available at https://github.com/BoevaLab/CanSig

Citation Format: Josephine Yates, Florian Barkmann, Pawel Piotr Czyz, Agnieszka Kraft, Niko Beerenwinkel, Valentina Boeva. CanSig: De novo discovery of shared transcriptional states in cancer single cells [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 3166.
Abstract 3263: Implications for cancer viroimmunotherapy: a new adenovirus capsid sensor
Authors: Gillard Andrew Gregg , Shin Dong Ho , Laspidea Virginia , Jiang Hong , Xuejun Fan , Yi Yanhua , Alonso Marta M. , Fueyo Juan , Gomez-Manzano Candelaria
Published: June 2023

The median survival for glioblastoma (GBM) patients is 12-15 months, with a five-year survival rate of less than 10% despite aggressive treatment with surgery, radiotherapy, and chemotherapy. Oncolytic virotherapy is emerging as a promising approach to reprogramming the immunosuppressed microenvironment of GBMs and restoring anti-tumor immunity. Delta-24-RGD is an oncolytic adenovirus that has shown favorable results in phase I clinical trials (NCT00805376; NCT03178032). However, immune responses functionally cleared the virus within weeks of administration, limiting anti-cancer efficacy to a subset (~20%) of patients. A better understanding of the innate immune response against adenoviruses may provide the rationale for developing improved anticancer approaches. Using bulk RNA sequencing, we have examined the transcriptome of normal lung fibroblasts infected with wild-type adenovirus. Ingenuity pathway analysis (IPA) showed upregulation of several immune response-related pathways, including interferon response factor 3 (IRF3). Interestingly, these analyses also showed an unexpected upregulation of the Non-POU Domain Containing Octamer Binding (NONO) pathway during adenoviral infection. In fact, upstream regulators analyses showed a 7-fold increase in the NONO pathway in two different cell lines. Increases in NONO expression changes in response to adenovirus treatment were validated by western blot. Furthermore, co-immunoprecipitation assays showed that NONO binds to adenoviral capsid proteins during infection and to the foreign DNA sensor cyclic GMP AMP synthase (cGAS), which is critical for the activation of the innate immunity, via IRF3. In our proposed model, NONO protein binds to the adenoviral capsid protein and associates with the sensor cGAS in the nucleus to enable sensing of viral nucleic acid. In conclusion, we uncovered a new mechanism for detecting adenovirus infection that involves the interaction of adenoviral capsid proteins with members of the NONO pathway in the nucleus of the infected cell. Further studies are in progress to examine to which extent adenoviruses unable to bind NONO may persist longer in the infected tumors than unmodified adenoviruses and whether the persistence of the virus in the tumor correlates with enhanced anti-glioma effect.

Citation Format: Andrew Gregg Gillard, Dong Ho Shin, Virginia Laspidea, Hong Jiang, Fan Xuejun, Yanhua Yi, Marta M. Alonso, Juan Fueyo, Candelaria Gomez-Manzano. Implications for cancer viroimmunotherapy: a new adenovirus capsid sensor [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 3263.
Abstract 3265: Temozolomide combined with ipilimumab + nivolumab enhances T cell killing of MGMT-expressing, MSS colorectal cancer cells
Authors: Gonzalez Zenovia , Carlsen Lindsey J. , El-Deiry Wafik S.
Published: June 2023

Colorectal cancer (CRC) is the third most frequently diagnosed cancer and third-deadliest cancer globally. Approximately 95% of patients with metastatic CRC have tumors that are microsatellite stable (MSS) and do not respond to immune checkpoint inhibitors (ICI). Results from the 2022 MAYA clinical trial suggest that the DNA-damaging agent temozolomide (TMZ), which is usually used to treat glioblastoma (GBM), sensitizes patients with MSS, MGMT-silenced CRC to ipilimumab + nivolumab ICI. The benefit of adding ipilimumab + nivolumab to TMZ and the impact of MGMT silencing remain unclear. Here, we aimed to determine in a controlled in vitro system if adding ICI to TMZ enhances T cell killing of MSS CRC cells. We also aimed to determine the role of MGMT in this response. MGMT protein expression was measured by western blot in CRC (n=4) and GBM (n=4) cell lines. Cell viability assays were conducted to measure sensitivity to TMZ. CRC and GBM cells were co-cultured with TALL-104 cells -/+ TMZ and -/+ ICI to measure TALL-104-mediated cell death. Western blot analysis indicated that CRC cells had significantly elevated MGMT expression as compared to GBM cells likely due to MGMT promoter methylation in GBM cells. In line with this, CRC cells were slightly more resistant to TMZ (IC50=100-150 µM) compared to GBM cells (IC50=10-90 µM) after five days of treatment. TMZ + ICI sensitized MGMT-expressing, MSS CRC cells to T cell killing. TMZ alone did not enhance T cell killing of MSS or MSI CRC cells but did enhance T cell killing of T98G GBM cells. Our results indicate that TMZ sensitizes MSS, MGMT-expressing CRC cells to ipilimumab + nivolumab ICI. Importantly, this suggests that TMZ-mediated sensitization to ipilimumab + nivolumab is independent of MGMT status and the patient cohort that may benefit TMZ + ipilimumab + nivolumab may be expanded to CRC patients with MGMT-expressing, MSS tumors.

Citation Format: Zenovia Gonzalez, Lindsey J. Carlsen, Wafik S. El-Deiry. Temozolomide combined with ipilimumab + nivolumab enhances T cell killing of MGMT-expressing, MSS colorectal cancer cells [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 3265.
Abstract 3347: Characterization of extracellular POTE-2 protein in liver cancer
Authors: Lopez Samantha , Doxtater Kyle D. , Anilkumar Adithya , Leslie Sophia , Sanchez Amayrani , Ezell Kristopher , Chauhan Subhash , Tripathi Manish K.
Published: June 2023

Liver cancer is projected to be the 5th and 7th leading cause of mortality in males and females in 2022. The top two forms of liver cancer diagnosed are hepatocellular carcinoma and cholangiocarcinoma, with a percentage of 75% and 11%. In recent years, Texas has had the highest incidence, with a rate of 11.6 per 100,000, with the Hispanic population having one of the highest rates. In South Texas, the Rio Grande Valley contains a high Hispanic population and faces multiple health disparities leading to a higher prevalence of liver cancer. These factors and the insufficient diagnostic methods for liver cancer lead to a need to identify faster and early biomarkers. Recently, we have identified an extracellular cancer-testis antigen, POTE-2, which belongs to the POTE gene family. Our preliminary data show that POTE-2 has an increased expression in liver cancer and can be associated with cancer progression. In this study, we will discuss the characterization of the extracellular face of POTE-2 in liver cancer. Lentiviral plasmids created an overexpressing and a knockdown POTE-2 cell line. The exosomes and conditioned media were collected from the cell lines to analyze POTE-2 protein expression through immunoblotting and ELISA. Antibodies targeting varying protein regions were produced and characterized for use in extracellular characterization. Exosome isolation and collection of conditioned media showed the expression of extracellular POTE-2 protein. Using varying antibodies targeting different regions determined the protein domain cleaved through secretion. These studies will aid in discovering novel characteristics of POTE-2 protein function and further explore its potential in liver cancer progression.

Citation Format: Samantha Lopez, Kyle D. Doxtater, Adithya Anilkumar, Sophia Leslie, Amayrani Sanchez, Kristopher Ezell, Subhash Chauhan, Manish K. Tripathi. Characterization of extracellular POTE-2 protein in liver 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 3347.
Abstract 3607: RNA-binding proteins are essential for EGF-induced fibronectin expression and metastasis of head and neck squamous cell carcinoma
Authors: Chang Ting-Wei , Chang Wen-Chang , Chen Ben-Kuen
Published: June 2023

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the precise mechanism involved in modulating EGF-associated metastasis in HNSCC remains unclear. In this study, we reveal that the concurrent expression of EGFR and fibronectin (FN1) was highly associated with the stages of HNSCC progression. EGF-induced fibronectin/integrin axis enhanced tumor-endothelial cell interaction and increased endothelial permeability, conferred HNSCC cell metastasis. Mechanistically, we found that ERK and NF-κB pathway activation was essential for EGF-induced fibronectin expression. In addition, EGF-enhanced mRNA stability of fibronectin occurred through the 5’ untranslated region (5’-UTR) of the gene and was mediated by nucleolin (NCL) and hnRNPA2/B1, which were also involved in the regulation of epithelial-mesenchymal transition gene expression. Intriguingly, the in vivo study showed that depletion of NCL, hnRNPA2/B1, and inhibition of the fibronectin/integrin axis significantly inhibited EGF-promoted HNSCC metastasis in the lung. Our data suggests that the nucleolin/hnRNPA2/B1-regulated fibronectin is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.

Citation Format: Ting-Wei Chang, Wen-Chang Chang, Ben-Kuen Chen. RNA-binding proteins are essential for EGF-induced fibronectin expression and metastasis of head and neck squamous cell carcinoma. [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 3607.
Abstract 3642: SPHK1 mediated regulation of antitumor immunity in ovarian cancer
Authors: Gupta Prachi , Kumar Sudhir , Kadamberi Ishaque Pulikkal , Mittal Sonam , George Jasmine , Geethadevi Anjali , Chaluvally-Raghavan Pradeep , Pradeep Sunila
Published: June 2023

Introduction: Sphingosine kinase 1 (SPHK1) is an enzyme that helps to synthesize biolipid sphingosine-1-phosphate (S1P). We showed extracellular vesicles facilitate an increased accumulation of S1P in the ovarian tumor microenvironment (TME). S1P enhances the exhaustion of CD8 T cells, thus downregulating anti-tumor immunity. Furthermore, we also found less ovarian tumor burden in SPHK1−/− mice compared to WT mice. Based on our preliminary data, we hypothesized that the SPHK1 depleted tumor microenvironment increases the infiltration of the immune cells and its activity downregulates the tumor progression.

Methods: SPHK1 global knockout mice and WT C57BL/6 mice were used in the study. Tumor cells (ID8 P53−/− BRCA−/−) were injected, and tumor progression was studied. Immunophenotyping was performed by flow cytometry on ascites and tumor samples. Activation of CD8 T cells, B cells, and dentritic cells were studied by flow cytometry. Bulk RNA sequencing was performed on SPHK1−/− and WT tumors.

Results: SPHK1−/− mice showed regression in tumor growth in multiple tumor models. Tumor tissues from SPHK1−/− mice enhanced infiltration of CD4 and CD8 T cells, B cells, and dentritic cells compared to WT mice. Furthermore, infiltrated immune cells were active in SPHK1−/− mice. Bulk RNA sequencing also showed enhanced immune response in SPHK1−/− tumors compared to WT tumors.

Conclusion: SPHK1−/- environment decreased the tumor growth by enhancing the infiltration and activation of immune cells. We are currently focusing on identifying the immune cells which are critical players in this process. We will further implement these findings in human immune cells for clinical benefits for ovarian cancer patients.

Citation Format: Prachi Gupta, Sudhir Kumar, Ishaque Pulikkal Kadamberi, Sonam Mittal, Jasmine George, Anjali Geethadevi, Pradeep Chaluvally-Raghavan, Sunila Pradeep. SPHK1 mediated regulation of antitumor immunity 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 3642.
Abstract 3799: Regulation of p16-E2F1-CDK4/6 signaling by TRUC-16 ultraconserved long non-coding RNA
Authors: Fabris Linda , Knutsen Erik , Bertilaccio Maria Teresa S. , Calin Steliana , Bayraktar Recep , Eisenhoffer George T. , Girnita Leonard , Calin George A.
Published: June 2023

High phylogenetic conservation of genomic loci is a potential marker of essential biological functionality. Ultraconserved elements (UCEs) are DNA sequences entirely conserved through millions of years of evolution, but their functions are not well understood. Several long non-coding RNAs (lncRNAs) are transcribed from UCEs and play active roles in cancer development. Using a combination of in vitro and in vivo experiments with two animal models and 4 patient cohorts, we discovered several remarkable aspects of ultraconservation. Using a multi-step genetically defined carcinogenesis model, we identified one UCE, uc.206, and its associated overexpressed lncRNA TRUC-16 as a regulator of cell proliferation in chronic lymphocytic leukemia (CLL). High levels of TRUC-16 inhibit p16INK4A translation, causing increased proliferation through an E2F1-mediated mechanism in CLL patients, as well as in zebrafish and murine models. uc.206/TRUC-16 function is extremely conserved throughout evolution. We found cell cycle-related effects of TRUC-16 in both mice and zebrafish, in which we observed increased proliferation of blood cells after overexpression of the human TRUC-16. High levels of CDK4/6 consequent to TRUC-16 overexpression and p16 decrease can be specifically targeted in both CLL and Richter’s Transformation (RT), a rare but deadly consequence of CLL. Inhibition of CDK6 is a therapeutic option never tested in CLL, but in use for another type of B cell malignancy, such as the mantle cell lymphoma. Our results highlight the multifaceted contribution of a UCE to human tumorigenesis and open new possibilities for the use of long-noncoding RNAs in cancer therapies.

Citation Format: Linda Fabris, Erik Knutsen, Maria Teresa S. Bertilaccio, Steliana Calin, Recep Bayraktar, George T. Eisenhoffer, Leonard Girnita, George A. Calin. Regulation of p16-E2F1-CDK4/6 signaling by TRUC-16 ultraconserved long non-coding RNA. [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 3799.
Abstract 38: PBMC donor selection affects engraftment rates and onset of graft versus host disease in humanized NCG mice for use in cancer studies
Authors: Bronson Steven , Rowe Jenny , Eberle Christoph , Festin Stephen
Published: June 2023

Preclinical studies examining tumor biology and immunity are limited by differences between the mouse and human immune systems. Humanized mouse models offer a unique tool to assess the anti-tumor response by recapitulating aspects of human tumor biology. Humanized mice are created by injecting either human CD34+ hematopoietic stem cells or peripheral blood mononuclear cells (PBMCs) into an immunodeficient mouse strain. Here we utilized a PBMC humanized NCG mouse model to investigate human T-cell mediated anti-tumor response (>95% CD3+ T-cells by day 21). The challenges encountered with this model include the onset of graft versus host disease (GvHD) and donor variability leading to donor specific engraftment rates. Consistency is required with humanized PBMC models to ensure their use as an appropriate research model. Therefore, we examined the relationship between donor selection and engraftment rates as well as the onset of GvHD. Inter-donor variability was assessed by collecting PBMCs from multiple healthy donors. Intra-donor consistency was assessed by collecting PBMCs from the same donors at subsequent collections and comparing to initial human immune cell profiling by flow cytometry. NCG mice were injected with 1 × 107 PBMCs from individual donors. Animals were weighed three times a week to monitor for changes in body weight and general health status. Peripheral blood was collected at day 9, 21 and 42 for flow cytometry screening of human immune cell engraftment (hCD45+, hCD3+, hCD4+ and hCD8+) at the initial collection. Human PBMCs isolated from additional collections from the same donors were also injected into NCG mice and flow cytometry was performed on the new cell lots at day 21. Donors were grouped into categories based on engraftment rates, which were donor dependent. Donor variability is an important aspect to consider when humanizing mice with human PBMCs. PBMC humanized models are important for the study of pre-clinical therapeutics, but donor engraftment rates must be considered when engrafting the mice to create a consistently humanized mouse.

Citation Format: Steven Bronson, Jenny Rowe, Christoph Eberle, Stephen Festin. PBMC donor selection affects engraftment rates and onset of graft versus host disease in humanized NCG mice for use in cancer studies [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 38.