bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3487: An atlas of substrate specificities for the human tyrosine kinome
Authors: Johnson Jared L. , Yaron Tomer M. , Cantley Lewis C. , Yaffe Michael B. , Turk Benjamin E. , Huntsman Emily M. , Hornbeck Peter V.
Published: April 2023

Numerous physiological processes are governed by tyrosine phosphorylation. The protein tyrosine kinases that carry this out are dysregulated in a variety of cancers and are the targets of efficacious drugs. For most tyrosine kinases, we have a very limited understanding of their downstream signaling pathways and phosphorylation targets. Conversely, thousands of tyrosine phosphorylation events have so far been experimentally identified, and the overwhelming majority of these do not have an associated kinase. In this work, we have utilized peptide arrays to determine the substrate sequence specificity for the human tyrosine kinome, comprising 46 receptor tyrosine kinases, 32 nonreceptor tyrosine kinases, and 14 noncanonical tyrosine kinases. Nearly every kinase we profiled favored tyrosines that were positioned next to phosphorylated amino acids, indicating that PTM priming and crosstalk are general properties of the tyrosine kinome. Comparisons with C. elegans orthologs suggested that tyrosine kinase substrate motifs have remained conserved throughout metazoan evolution. When we computationally utilized our dataset to identify kinases for tyrosine substrates, our predictions were in strong accordance with published reports. When applied to high-throughput tyrosine phosphoproteomics datasets, we could decipher the kinases involved in cellular responses to growth factors, oncogenic mutations, and targeted inhibitors. Together, this work uncovers fundamental rules of substrate selection for nearly the entire human tyrosine kinome and provides a comprehensive resource for connecting tyrosine phosphorylation events with their kinases and signaling pathways.

Citation Format: Jared L. Johnson, Tomer M. Yaron, Lewis C. Cantley, Michael B. Yaffe, Benjamin E. Turk, Emily M. Huntsman, Peter V. Hornbeck. An atlas of substrate specificities for the human tyrosine kinome [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 3487.
Abstract 3495: A kinome-wide CRISPR screen identifies CK1α as a novel target to overcome enzalutamide resistance of prostate cancer
Authors: Liu Jinghui , Zhao Yue , Allison Derek , He Daheng , Li Lang , Liu Xiaoqi
Published: April 2023

Background: Androgen deprivation therapy is the first line of therapy for localized prostate cancer (PCa), but most patients will eventually develop castration-resistant PCa (CRPC). Enzalutamide (ENZA), a second-generation androgen receptor (AR) antagonist, has significantly increased CRPC patient survival. However, resistance remains a prominent obstacle in treatment, illustrating the urgent need to develop new approaches to increase ENZA efficacy.

Methods: A Kinome-wide CRISPR-Cas9 screen was used to identify the targets related to ENZA response. In vitro cell viability and in vivo cell line or patient-derived xenografts were used to verify inhibiting CK1α overcoming ENZA resistance. RNA sequencing was used to address the functions of CK1α in ENZA resistance. In vitro kinase assay was used to study ATM phosphorylation by CK1α. Immunohistochemistry, immunofluorescence, immunoblot and immunoprecipitation assays were used to study the functions of CK1α in regulation of ATM and DNA damage response signaling. Bioinformatics analysis was used to confirm these findings with clinical patients’ database.

Results: Utilizing a kinome-wide CRISPR-Cas9 knockout screen, we identified casein kinase 1 alpha (CK1α) as a novel therapeutic target to overcome ENZA resistance. Depletion or pharmacologic inhibition of CK1α with a novel compound in clinical trial significantly enhanced EZNA efficacy in ENZA resistant cell lines and patient-derived xenografts. Mechanistically, CK1α phosphorylates the serine residue S1270 and modulates the protein abundance of ataxia-telangiectasia mutated (ATM), which is a primary initiator of DNA double-strand break (DSB)-response signaling, and is compromised in ENZA-resistant cells and patients. Inhibition of CK1α stabilizes ATM, resulting in the restoration of DSB-response signaling, and thus increases ENZA-induced cell death and growth arrest in an ATM-dependent manner.

Conclusions: Our study details an innovative therapeutic approach for ENZA-resistant PCa and characterizes a novel perspective for the function of CK1α in the regulation of DNA damage response signaling through phosphorylation of ATM S1270.

Citation Format: Jinghui Liu, Yue Zhao, Derek Allison, Daheng He, Lang Li, Xiaoqi Liu. A kinome-wide CRISPR screen identifies CK1α as a novel target to overcome enzalutamide resistance of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3495.
Abstract 35: Development of novel human hepatocellular carcinoma models using genetically modified human hepatocytes in FSRG humanized liver mice
Authors: Carbonaro Marisa , Bonetti Ciro , Huang Hui , Wang Kehui , Desclaux Mathieu , Gong Guochun , Lin John C. , Daly Christopher , Thurston Gavin , Li Zhe
Published: April 2023

The incidence of liver cancer is growing worldwide, and it is estimated that by 2025, over 1 million individuals will be affected by liver cancer every year. Hepatocellular carcinoma (HCC) accounts for about 90% of primary liver cancer cases and is one of the leading causes of death from cancer worldwide. Establishing animal models that faithfully replicate the human disease is critical to improve our understanding of HCC pathogenesis and to test new therapeutic strategies. Here, we describe a novel human HCC model using genetically modified human hepatocytes in a humanized liver mouse. Primary human hepatocytes (PHH) were modified ex vivo through lentivirus or Lipid Nanoparticle (LNP)-mediated delivery of CRISPR/Cas9 components, resulting in over-expression of oncogenes or inactivation of tumor suppressor genes. These genetically engineered PHH were engrafted into the livers of FSRG (FAH−/−, SIRPahu/hu, RAG2−/−, IL2Rg−/−) mice, developed by Regeneron VelociGene technology, in which mouse liver parenchyma can be replaced with human hepatocytes. Tumor formation was validated in livers engrafted with human hepatocytes after different combinations of genetic modification, including over-expression of cMYC plus HRASv12, and cMYC over-expression together with p53 KO. Tumor progression could be monitored by measuring human Alpha Fetoprotein (AFP) in serum of engrafted mice. Liver tumors were positive for fumarylacetoacetate hydrolase (FAH) and human Asialoglycoprotein Receptor 1 (ASGR1), confirming their human origin, as well as the proliferation marker, Ki67, and Glypican-3 (GPC3), which is frequently over-expressed in HCC and a potential therapeutic target. In these studies, mice were engrafted with a mix of lentivirus-transduced and non-transduced hepatocytes, resulting in humanized livers with both normal and transformed human hepatocytes, providing an ideal system to recapitulate neoplastic transformation and clonal expansion of human hepatocytes carrying oncogenic mutations as well as to test the specificity of novel therapies. By combining genetic manipulation of human hepatocytes with the FSRG humanized liver mouse platform, we have established a flexible system to model clinically relevant combinations of HCC mutations for both new target discovery and therapeutic testing.

Citation Format: Marisa Carbonaro, Ciro Bonetti, Hui Huang, Kehui Wang, Mathieu Desclaux, Guochun Gong, John C. Lin, Christopher Daly, Gavin Thurston, Zhe Li. Development of novel human hepatocellular carcinoma models using genetically modified human hepatocytes in FSRG humanized liver mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 35.
Abstract 3515: Histone demethylase KDM5D drives sex-specific differences in colorectal cancer
Authors: Li Jiexi , Lan Zhengdao , Liao Wenting , Liu Jielin , Jiang Shan , Shim Hong Seok , Slotnik Max , LaBella Kyle A. , Hsu Wen-Hao , Shang Xiaoying , Spring Denise , Wang Y Alan , DePinho Ronald A.
Published: April 2023

Gender exerts a profound impact on cancer incidence, spectrum and outcomes, yet the molecular genetic bases of such sex differences are ill-defined and presumptively ascribed to X-chromosome genes and sex hormones. Such sex differences are particularly prominent in colorectal cancer (CRC) where men experience higher metastases and mortality. A murine CRC model, engineered with an inducible transgene encoding oncogenic mutant KRASG12D and conditional null alleles of Apc and Trp53 tumor suppressors (designated iKAP), revealed higher metastases and worse outcomes specifically in males with oncogenic mutant KRAS (KRAS*) CRC. Integrated cross-species molecular and transcriptomic analyses identified Y-chromosome gene histone demethylase KDM5D as a transcriptionally up-regulated gene driven by KRAS*-mediated activation of the STAT4 transcription factor. KDM5D-dependent chromatin mark and transcriptome changes showed repression of regulators of epithelial cell tight junction and MHC class I complex components. Deletion of KDM5D in iKAP cancer cells increased tight junction integrity, decreased cell invasiveness, and enhanced cancer cell killing by CD8+ T cells. Conversely, iAP mice engineered with a Kdm5d transgene to provide constitutive KDM5D expression specifically in iAP cancer cells exhibited an increased propensity for more invasive tumors in vivo. Thus, KRAS*-STAT4-mediated upregulation of Y chromosome KDM5D contributes significantly to the sex differences in KRAS* CRC via its disruption of cancer cell adhesion properties and tumor immunity, thus providing an actionable therapeutic strategy for metastasis risk reduction for men afflicted with KRAS* CRC.

Citation Format: Jiexi Li, Zhengdao Lan, Wenting Liao, Jielin Liu, Shan Jiang, Hong Seok Shim, Max Slotnik, Kyle A. LaBella, Wen-Hao Hsu, Xiaoying Shang, Denise Spring, Y Alan Wang, Ronald A. DePinho. Histone demethylase KDM5D drives sex-specific differences in colorectal 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 3515.
Abstract 3795: Deciphering the in vivo roles of a novel long non-coding RNA in chronic lymphocytic leukemia and Richter syndrome
Authors: Mohapatra Swati , Knutsen Erik , Gagea Mihai Iurascu , Fabris Linda , Calin George Adrian
Published: April 2023

The ultraconserved regions (UCRs) are the genomic segments manifesting perfect conservation between the orthologous genomes of humans, rats, and mice that get transcribed into mono-exonic long non-coding RNAs (lncRNAs) known as transcribed ultraconserved regions (T-UCRs). Increasing evidence demonstrates the importance of T-UCRs in human cancers, however, their involvement in the pathogenesis of Chronic Lymphocytic Leukemia (CLL) is poorly understood. Our lab has recently identified a lncRNA transcribed from a UCR and named it TRUC-16 (Translational Regulatory UltraConserved gene affecting p16), which is overexpressed in CLL and correlates with treatment-free survival. We have developed a transgenic C57BL6 mouse model to overexpress TRUC-16 in B cells. We monitored the number and development of B cell subsets over time in peripheral blood and lymphoid organs (lymph nodes, spleen, and bone marrow), and performed immunophenotyping to assess clonality and proportion of the cells considered to be the origin for CLL (i.e. B1 cells and IgM memory B cells that are CD5+). We followed mice survival (n=10 for each gender) and performed necropsy with histopathologic and immunophenotypic analysis. If blood sampling suggests developing a CLL-like state, we will monitor for evolution to Richter Syndrome by flow cytometry analysis and biochemistry markers. We have validated the TRUC-16 overexpression specific to the B-cell compartment in the mice cohorts and have observed lymphoma in several organs. Interestingly, TRUC-16 overexpressing mice showed significantly higher splenic weights compared to controls. The mice were found to develop adulthood lymphoma that we are now analyzing molecularly and phenotypically. We are planning to further characterize the subtype of lymphoma by utilizing specific diagnostic markers through immunohistochemistry and flow cytometry. This is the first-ever transgenic mice modeling overexpression of UCR in the context of cancer. Validation of the functional correlation between the TRUC-16 phenotype and CLL patients’ clinical data will provide a valuable resource to pursue a multitude of preclinical studies to find targets that might help better understand the disease pathophysiology.

Citation Format: Swati Mohapatra, Erik Knutsen, Mihai Iurascu Gagea, Linda Fabris, George Adrian Calin. Deciphering the in vivo roles of a novel long non-coding RNA in chronic lymphocytic leukemia and Richter syndrome. [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 3795.
Abstract 389: Focal adhension kinase (FAK) confers lenvatinib resistance in hepatocellular carcinoma via regulation of lysine deficient protein kinase 1(WNK1)
Authors: Hou Wei , Gad Shaimaa A. , Qiu Wei
Published: April 2023

Lenvatinib is a clinically effective multi-kinase inhibitor approved for first-line therapy of advanced hepatocellular carcinoma (HCC). Although resistance against lenvatinib often emerges and limits its anti-tumor activity, the underlying molecular mechanisms involved in endogenous and acquired resistance are still unidentified. Endogenous (SNU449 and SNU475) and established acquired (Hep3B-LR and Huh7-LR) lenvatinib resistant cells, as well as knockout mice were used in this study. As a result, we recognized focal adhension kinase (FAK) as a critical contributor to lenvatinib resistance in HCC. The elevated expression of FAK was observed in both established and endogenous lenvatinib-resistant HCC cells. Furthermore, inhibition of FAK reversed lenvatinib resistance in vitro and in vivo. Mechanistically, FAK promoted lenvatinib resistance through regulating lysine deficient protein kinase 1 (WNK1) signaling pathway. WNK1 inhibitor WNK463 re-sensitized either established LR HCC cells (Hep3B-LR and Huh7-LR) or endogenous resistant HCC cells (SNU449 and SNU475) to lenvatinib treatment. In addition, overexpression of WNK1 desensitized parental HCC cells (Hep3B-P and Huh7-P) to lenvatinib treatment. Conclusively, our results establish a crucial role and novel mechanism of FAK in lenvatinib resistance, and suggest that targeting the FAK/WNK1 axis is a promising therapeutic strategy in HCC patients showing lenvatinib resistance.

Citation Format: Wei Hou, Shaimaa A. Gad, Wei Qiu. Focal adhension kinase (FAK) confers lenvatinib resistance in hepatocellular carcinoma via regulation of lysine deficient protein kinase 1(WNK1) [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 389.
Abstract 3894: The role of ELF3 in acquired resistance to endocrine therapy in ER-positive breast cancer
Authors: Zhang Na , Zheng Rongbin , Brown Myles
Published: April 2023

Endocrine therapies targeting the estrogen receptor (ER) are the major treatments of ER+ breast cancer, however acquired resistance to endocrine therapy is almost unavoidable and is the main cause of death with breast cancer. The only FDA-approved selective estrogen receptor degrader (SERD) fulvestrant has been used in the second line treatment to overcome aromatase inhibitor- or tamoxifen-resistance. However, fulvestrant has poor pharmacokinetic properties, which has inspired the development of a new generation of oral SERDs including amcenestrant and giredestrant. To understand the mechanisms of acquired resistance to SERDs in ER+ breast cancer, we established ER+ breast cancer cell lines (MCF7 and T47D) resistant to fulvestrant, amcenestrant or giredestrant. We found that all of our MCF7 and T47D SERD-resistant cell lines showed cross-resistance to each of the three SERDs. We then performed multiple genome-wide profiling assays including CRISPR-KO screen, whole exome sequencing (WES), RNA-seq, ATAC-seq and H3K27ac ChIP-seq on the parental and SERD resistant cell lines. Our findings from the CRISPR-KO screens suggest that ETS transcription factor ELF3 is a potential driver of resistance to SERDs. ELF3 expression is negatively regulated by estrogen signaling. Cells that developed acquired resistance to different SERDs consistently show depletion of ER expression but also a dramatic increase of ELF3 expression, while knockdown of ELF3 in the resistant cells leads to cell growth inhibition, suggesting that ELF3 can drive cell survival in the SERD resistant cells. When comparing the open chromatin regions and active enhancer regions between SERD resistant and parental cells via ATAC-seq and H3K27ac ChIP-seq respectively, ELF3 DNA binding motif was found significantly enriched in SERD resistant cells versus parental cells further indicating that ELF3 may play an important role in the SERD resistant cells. ELF3 ChIP-seq was also performed to identify genes enriched with ELF3 binding sites in SERD resistant and parental cells. Pathway analysis of ELF3-targeted genes reveals that ELF3 may regulate cell metabolism specifically in SERD resistant cells but not in parental cells. Our study suggests that ELF3 may be a novel driver of acquired resistance to SERDs in ER+ breast cancer.

Citation Format: Na Zhang, Rongbin Zheng, Myles Brown. The role of ELF3 in acquired resistance to endocrine therapy in ER-positive 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 3894.
Abstract 3909: THY1 regulates therapy resistance in glioblastoma recurrence
Authors: Shankar Sunita , Ravikumar Visweswaran , Wang Hanxiao , Ji Sunjong , Hasan Zainab , Fehmi Ziad , Haydin Yacoub , Wahl Daniel R. , Rao Arvind , Rehemtulla Alnawaz , Al-Holou Wajd
Published: April 2023

Glioblastoma (GBM), is the most common primary brain tumor. It is a lethal disease with a median survival of 15 months characterized by treatment resistance, aggressive brain invasion, and inevitable recurrence. Less than 10 percent of patients survive beyond 5 years. Here, we developed an intracranial longitudinal in vivo recurrence model using HF2303, a patient derived explant, to produce paired specimens (pre- and post-recurrence) following temozolomide(TMZ) and radiation(IR) treatment. Mouse brain tissues from the two groups were compared to identify genes and pathways driving treatment resistance. This analysis showed an elevated THY1 expression in recurrent HF2303 tumors compared to naïve tumors. Increased Thy1 expression was also associated with a more mesenchymal phenotype. THY1 expressing cells from naïve HF2303 were also resistant to TMZ/IR treatment in an intracranial model. In U87 neurospheres, overexpression of THY1 increased resistance to TMZ/IR. Interestingly, stable knockdown of Thy1 in GBM cells and human derived GBM organoids (GBOs) expressing high THY1 levels led to reduced cell proliferation. Yet, an inducible knockout of THY1 using CRISPR/Cas9 in human GBOs showed increased sensitivity to TMZ/IR. This suggests a clear link between THY1 expression and TMZ/IR resistance.Single cell RNA sequencing of over 25 primary GBM and 4 recurrent tumors (spanning over 120,000 cells) shows that cells expressing high levels of THY1 are associated with two distinct populations; neuronal precursor-like (NPC-like) and mesenchymal-like (MES-like). Furthermore spatial transcriptomics from 3 primary GBMs suggests that high THY1 expressing cells are colocalized with macrophages within the perivascular niche contributing to the mesenchymal phenotype. We will present these studies and our more recent work analyzing spatial recurrent GBM samples. Particularly, we will address how THY1 expression in these spatially distinct NPC-like and MES-like differ and how each of these states may contribute towards TMZ/IR resistance.

Citation Format: Sunita Shankar, Visweswaran Ravikumar, Hanxiao Wang, Sunjong Ji, Zainab Hasan, Ziad Fehmi, Yacoub Haydin, Daniel R. Wahl, Arvind Rao, Alnawaz Rehemtulla, Wajd Al-Holou. THY1 regulates therapy resistance in glioblastoma recurrence. [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 3909.
Abstract 391: Adenylosuccinate lyase enzyme is essential for growth of endocrine therapy resistant ER+ breast cancer cells
Authors: Yadav Anil K. , Clarke Robert , Sengupta Surojeet
Published: April 2023

The endocrine therapy (ET) that targets estrogen synthesis (aromatase inhibitors) or estrogen receptor mediated signaling (tamoxifen, or Fulvestrant) is the mainstay therapy for estrogen-receptor positive (ER+) breast cancer. However, emergence of resistance to ET is a major clinical challenge as it leads to relapse and disease progression that cannot be further treated with any targeted therapy. We studied the role of a key enzyme involved in de-novo purine biosynthesis, adenylosuccinate lyase (ADSL), in the growth and proliferation of ER+, ET-resistant breast cancer cells. ADSL enzyme is responsible for the catalysis of two non-sequential steps in de-novo purine synthesis pathway that produces aminoimidazole carboxamide ribonucleotide (AICAR) and adenosine monophosphate (AMP) as well as generates fumarate as a byproduct. Here we show that siRNA mediated depletion of ADSL in two different ER+, ET-resistant cell lines, namely LCC9 and T47D-4HT cells, block the proliferation of the cells in 2-D cell culture as well as in spheroid formation. Our investigation revealed that reduced growth of cells after ADSL-depletion was due to cell-cycle perturbation in both LCC9 and T47D-4HT cells and not cell death. Intriguingly, the cell cycle was blocked at different phases in LCC9 and T47D-4HT cells. After ADSL knock-down the LCC9 cells were arrested at G1/S boundary with concurrent elevated levels of cyclin D1 protein whereas the T47D-4HT cells were blocked at S/G2-M boundary with reduced levels of cyclin D1. In addition, we observed that ADSL deficient cells had low levels of ATP in conjunction with attenuated mitochondrial membrane potential. Notably, addition of low concentration of AICAR (but not AMP, adenosine, or fumarate) to ADSL deficient LCC9 cells partially rescue reduced proliferation of cells as well as restored the mitochondrial membrane potential. Our data establishes that ADSL enzyme function is critical for proliferation of ET-resistant ER+ breast cancer cells and its deficiency impair the mitochondrial function and energy production. We propose that ADSL is a potentially novel therapeutic target for ER+, endocrine therapy resistant breast cancer.

Citation Format: Anil K. Yadav, Robert Clarke, Surojeet Sengupta. Adenylosuccinate lyase enzyme is essential for growth of endocrine therapy resistant ER+ breast 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 391.
Abstract 3916: role of her3 v104l mutation on tumor growth and her3 stabilization
Authors: Mishra Rosalin , Kilroy Mary Kate , Feroz Wasim , Patel Hima , Alanazi Samar , Garrett Joan T.
Published: April 2023

We aimed to determine if naturally occurring HER3 mutations could drive oncogenic activity of HER2+ HCC1569 cells in which endogenous HER3 was knocked out using CRISPR-Cas9 technology. A series of HER3 mutations found in breast cancer patients (F94L, V104L, G284R, D297Y, T355I, and E928G) were introduced via lentiviral transduction and stable cell lines were generated in HER3 knock out (KO) HCC1569 cells and were maintained in 0.5 mg/mL puromycin. Our data indicated that cells stably transduced with HER3V104L mutation had significantly higher cell growth with higher p-HER3 expression versus wild-type (wt) and empty-vector (EV) HER3. We found that HCC1569HER3KO cells with WT and V104L were sensitive to increasing concentration of neratinib (0.1-0.5 µM) as indicated by crystal violet assay. Next we examined if V104L mutation rendered resistance to another FDA- approved irreversible HER2 tyrosine kinase inhibitor, tucatinib. Our results showed that V104L cells were sensitive to higher concentration of tucatinib compared to neratinib. In other experiments, we used COS7 cells to examine the signaling pathways of HER3V104L mutation. Our western blot data demonstrated that transient transfection of COS7 cells with HER3V104L mutant significantly upregulated p-HER3 and p-HER2 expression versus WT HER3 in a NRG- dependent manner. In addition, we found that the V104L mutation stabilizes HER3 protein expression independent of ligand stimulation. Our western blot data showed that V104L induced HER3 stabilization is independent on HER3 binding partners HER1, HER2 and HER4. Our cycloheximide chase data indicated that V104L mutation stabilizes HER3 expression in COS7 cells as well as low HER3 expressing colon cancer cell line, SNUC5. Our western blot data indicates that V104L mutation activates ERK signaling in NRG dependent manner over AKT pathway in COS7 cells. In addition, we are using various PDXs with different HER3 mutations (V104M and E928D) to determine the role of these driver HER3 mutations in cancer and how this can be targeted in the clinic using HER targeted therapy.

Citation Format: Rosalin Mishra, Mary Kate Kilroy, Wasim Feroz, Hima Patel, Samar Alanazi, Joan T. Garrett. role of her3 v104l mutation on tumor growth and her3 stabilization. [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 3916.
Abstract 3955: Functional evaluation of pancreatic cancer prognostic marker, LXN, utilizing multiomics analysis based on patient-derived cancer model
Authors: Park Chan Hee , Lee Hee Seung , Kim Jin Su , Tae Yoo Keung , Lee Jin Young , Bang Seungmin
Published: April 2023

Background: Pancreatic cancer is the most dreadful malignant tumor and expected becoming the second cause of death in 2030. Despites of vigorous studies, there were no definitive prognostic biomoleculars marker for pancreatic cancer. Herein, we already established primary pancreatic cancer cell lines from the real patients of pancreatic cancer, using conditionally reprogrammed cell culture (CRC). We tried to find novel prognostic biomarkers with multi-omics analysis of our primary pancreatic cancer cell lines.

Methods: In this study, we selected pancreatic cancer prognostic markers using established CRCs and FFPE samples and validated the molecular mechanism of LXN. As the first step, we performed both transcriptome and proteome analysis using RNA sequencing and liquid chromatography-tandem mass spectrometry (LC/MS-MS) with 6 primary pancreatic cancer cell lines established by CRC. After analysis, we mined several candidate markers of prognosis including latexin (LXN) and sialic acid acetylesterease (SIAE). And we validated these markers clinicopathologically using immunohistochemical (IHC) staining of 136 tissues from a different set of pancreatic cancer patients. And we confirmed the knock-down effect of LXN using siLXN in pancreatic cancer cells.

Results: We selected 2 highly prognostic markers with LXN and SIAE after transcriptome and proteome analysis. In these genes, high LXN expression group showed longer median overall survival (OS) than low expression group, respectively (14.8 vs. 10.4 months, P-value=0.28), respectively. For the evaluation of IHC, we established 3D organoid model using CRCs and confirmed the correlation between mRNA expression and protein expression with western blotting and IHC. The expression of Akt/mTOR was decreased after siLXN treatment compared to control groups. In order to validation as a prognostic marker, we stained formalin-fixed, paraffin-embedded (FFPE) slide from 136 pancreatic cancer patients. After IHC analysis, LXN high group (H-score > 12) showed significantly longer OS than low LXN group (52 vs. 28 months, P-value = 0.043).

Conclusion: In this study, we found LXN as a prognostic marker with inhibition of proliferation utilizing patient-derived cancer model and FFPE samples.

Citation Format: Chan Hee Park, Hee Seung Lee, Jin Su Kim, Yoo Keung Tae, Jin Young Lee, Seungmin Bang. Functional evaluation of pancreatic cancer prognostic marker, LXN, utilizing multiomics analysis based on patient-derived cancer model. [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 3955.
Abstract 3956: Identifying PIKfyve as potential target in clear cell renal cell carcinoma with a loss of the von Hippel-Lindau tumor suppressor gene
Authors: Bouhamdani Nadia , Turcotte Sandra
Published: April 2023

Background. Clear cell renal cell carcinoma (ccRCC) is the most frequent type of cancer in the kidney. These tumors are highly vascular and correlate with poor prognosis. Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is a truncal event of ccRCC carcinogenesis, which yields new insights for targeted therapy. Our studies identified a small molecule, STF-62247, that is toxic to cells lacking VHL compared to RCC with a functional gene. We reported that STF-62247 blocks late stages of autophagy by targeting lysosome dynamics. Moreover, we identified lysosomal vulnerability in VHL-mutated ccRCCs, which could lead cells to death.

Goal. This study aims to recognize and characterize STF-62247 potential targets.

Results. Using biochemical approaches, our results indicated that STF-62247 cytotoxicity is driven by PIKfyve inhibition. Binding affinity between PIKfyve and STF-62247 is around 5 nM. Intracytoplasmic vacuoles and enlargement of endolysosomes were observed, which was prevented by adding exogenous PI(3,5)P2. Moreover, PIKfyve inhibitors such as apilimod, YM201636, Vacuolin-1, APY0201 sensitize ccRCC with a loss of VHL. Finally, genetic knockdown of PIKfyve was achieved by CRISPR/Cas9 leading VHL-inactivated cells to death.

Conclusion. Altogether, our studies identified, for the first time, PIKfyve as potential target for kidney cancer cells with a loss of VHL.

Citation Format: Nadia Bouhamdani, Sandra Turcotte. Identifying PIKfyve as potential target in clear cell renal cell carcinoma with a loss of the von Hippel-Lindau tumor suppressor gene. [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 3956.
Abstract 4015: EGFR activation via c-Jun axis promotes adaptive resistance to third generation ALK-TKI lorlatinib in ALK-rearranged non-small cell lung cancer cells
Authors: Katayama Yuki , Yamada Tadaaki , Morimoto Kenji , Ozasa Hiroaki , Tokuda Shinsaku , Takayama Koichi
Published: April 2023

Anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs), including lorlatinib have shown dramatic efficacy in patients with ALK-rearranged lung cancer; however, complete response in these patients is rare, as a small population of tumor cells survives due to adaptive resistance. In this preclinical study, we investigated the adaptive resistant mechanisms to lorlatinib and its therapeutic strategies to overcome, using in vitro and in vivo experiments. We have identified EGFR as an adaptive resistance mechanism of lorlatinib using phospho-RTK array and siRNA library. Cell line-based assay showed that exposure of lorlatinib accelerated EGFR signal activation as an adaptive response via activation of JNK cascade in ALK-rearranged lung cancer cells. Knockdown for EGFR suppressed the survival signal of ALK lung cancer cells when treated with lorlatinib. The combination of lorlatinib and erlotinib, a first-generation EGFR-TKI, was shown to enhance cell growth inhibition by inducing cell apoptosis via suppressing the anti-apoptotic factor Bcl-xl compared with lorlatinib monotherapy. Moreover, cell line-derived xenograft model indicated that the combination of lorlatinib plus erlotinib enhanced anti-tumor effects compared with lorlatinib monotherapy. Taken together, our results displayed that EGFR activation plays an important role in the initial adaptive resistance to lorlatinib for ALK-rearranged lung cancer. The combination of lorlatinib and erlotinib is expected as the promising strategy for those tumors.

Citation Format: Yuki Katayama, Tadaaki Yamada, Kenji Morimoto, Hiroaki Ozasa, Shinsaku Tokuda, Koichi Takayama. EGFR activation via c-Jun axis promotes adaptive resistance to third generation ALK-TKI lorlatinib in ALK-rearranged non-small cell lung 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 4015.
Abstract 4083: In vivo efficacy of LGR5-targeting CAR-T cell therapies developed for the treatment of colorectal cancer
Authors: McPeake Dylan , Tyllis Timona , Abbott Caitlin , Foeng Jade , Bandara Veronika , Gundsambuu Batjargal , Rohani-Rad Elaheh , Napoli Silvana , Sadlon Timothy , Barry Simon , McColl Shaun
Published: April 2023

While CAR-T cells have demonstrated significant efficacy in the treatment of haematological malignancies, there are many challenges to overcome in developing CAR-T cell therapies against solid tumours. In particular, the scarcity of recognised effective target antigens in solid tumours remains a significant hurdle. Leucine-rich repeat-containing G-protein coupled receptor (LGR5) is a cancer stem cell marker, which mediates important roles in tumor initiation and metastasis. Upregulated LGR5 expression in malignant cell contexts highlights this surface receptor as a promising target for CAR-T cell therapy. In this study, we evaluated the efficacy of LGR5-targeting CAR-T cells against a range of human cancers. We demonstrate that LGR5 is expressed, both in vitro and in vivo, by numerous human cancers, including colorectal, ovarian, hepatic, brain, and pancreatic cancer. LGR5-targeting CAR-T cells effectively kill LGR5-expressing cancer cells in in vitro cytotoxicity assays and display significant anti-tumor activity in vivo when administered at 1, 2 or 3 weeks post-tumor inoculation in a subcutaneous human colorectal cancer xenograft mouse model. Furthermore, LGR5-targeting CAR-T cells that induce primary tumor rejection confer complete protection against a secondary tumor challenge 5 weeks post-T cell transfer. In vivo spatiotemporal distribution and repeated dosing studies demonstrate that LGR5-targeting CAR-T cells accumulate at the tumor site until tumor elimination, do not persist in off-target organs by d28 post-tumor inoculation and are safe and well-tolerated under the conditions tested. Together, these results provide the foundation for furthering the development of LGR5-targeting CAR-T cells for use in the clinic against colorectal cancers and other solid tumours.

Citation Format: Dylan McPeake, Timona Tyllis, Caitlin Abbott, Jade Foeng, Veronika Bandara, Batjargal Gundsambuu, Elaheh Rohani-Rad, Silvana Napoli, Timothy Sadlon, Simon Barry, Shaun McColl. In vivo efficacy of LGR5-targeting CAR-T cell therapies developed for the treatment of colorectal 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 4083.
Abstract 4148: Immunophenotyping of responses to immunotherapy in syngeneic tumor models using multiplex immunofluorescence compared to multiparametric flow cytometry
Authors: Metzger Philipp , Castro Carla , Obodozie Cynthia , Weber Holger
Published: April 2023

In the last decades, several major breakthroughs in cancer treatment were achieved through a better understanding and therapeutic targeting of the immune system. Multiparametric flow cytometry is a robust and powerful tool to evaluate the frequency and activation status of a large variety of immune cells. However, for flow cytometric analysis tumor, tissue must be digested into a single cell suspension, and thereby the information on the tumor architecture as well as the spatial distribution of immune cells is lost. The distribution of immune cells in the tumor microenvironment is often heterogeneous and a favorable immune cell distribution is crucial for an efficacious anti-tumor immunotherapy. The recent advances in multiplex immunofluorescence (mIF) allows the analysis of multiple parameters simultaneously so that the frequency as well as the spatial distribution of multiple immune cells can be analyzed.

In this study, immunotherapy-responsive tumor cell lines (colon carcinoma cell lines MC38-CEA and CT26wt as well as melanoma cell line Clone M3) were implanted in the mammary fat pad (subQperiorTM), which leads to reduce ulceration rate and homogenous tumor growth compared to subcutaneous implantation. Anti-PD-1 immunotherapy reduced the tumor growth in all three tumor models significantly. Formalin-fixed paraffin-embedded tumor samples were collected from control and anti-PD-1 treated animals. Murine specific InSituPlex® (ISP) technology (Ultivue) was used to perform multiplex immune profiling of the markers CD3, CD4, CD8 and FOXP3 on mouse whole slide FFPE tumor serial sections. Slides were then imaged for high quality images of the four targets and downstream analysis performed. The evaluated T cell frequency and spatial distribution was compared to flow cytometric data of anti-PD-1 treatment in these models.

In summary, flow cytometry and multiplex immunofluorescence (mIF) are valuable tools in the evaluation of immune responses in pre-clinical tumor models. Both technologies enable an improved understanding of treatment responses and facilitate the development of novel treatment modalities as well as the identification of potential predictive biomarkers.

Citation Format: Philipp Metzger, Carla Castro, Cynthia Obodozie, Holger Weber. Immunophenotyping of responses to immunotherapy in syngeneic tumor models using multiplex immunofluorescence compared to multiparametric flow cytometry. [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 4148.
Abstract 4150: Novel preclinical model for the evaluation of cytokine release syndrome in response to CD19xCD3 bispecific antibody in humanized mice
Authors: Shah Deep S. , Simion Catalina , hu Sang , Chronis Rhonda , McClellan Tre , Hwang Jae , Yang Ryan , Baker Heather , Cai Danying , Keck James , Shopland Lindsay
Published: April 2023

Cytokine Release Syndrome (CRS) is a systemic inflammatory response that is caused by the large, rapid release of cytokines in the blood by immune cells affected by infection or immunotherapy and remains a prominent challenge for T cell-based therapeutics. Bispecific T cell engagers (BiTEs) are fusion proteins that are generated by linking the targeting region of two antibodies: one arm binds to the tumor-associated antigen, and the other arm binds to T cells to promote T cell-mediated cytotoxicity in an MHC-independent manner. A CD19xCD3 BiTE has been approved by the FDA for the treatment of CD19-positive B-cell lymphoma. Despite having great clinical efficacy, CRS remains a major concern for BiTE therapy. This necessitates the development of a translational model that could predict CRS toxicity more accurately. Various in-vitro models proposed to assess BiTE-related CRS have several shortcomings as they do not provide details about the systemic immune response, off-target effects, neurotoxicity, tissue damage, and organ failure. Here we propose an in-vivo approach to concomitantly assess the efficacy and CRS toxicity of CD19xCD3 BiTE either alone or in combination with Rituximab (monoclonal antibody targeting CD20) in the Raji luciferase xenograft model of Burkitt’s lymphoma in PBMC humanized NSG-MHC Class I/II knockout mice. Five days post human PBMC engraftment (Study Day- SD 5), the mice were injected with Raji-luciferase cells. On SD 6, the mice were treated with BiTE, Rituximab, or the combination of BiTE and Rituximab. Additional control groups included OKT3 and Anti-CD28, the anti-T cell antibodies that failed clinically due to severe CRS. Efficacy was assessed through in-vivo imaging on SD5, SD8, and SD11. We observed tumor growth inhibition with BiTE, Rituximab, and the combination treatment. Clinical observations were recorded daily post treatment. Following cytokines were measured 6 hours after dosing and at study termination - IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ. Elevated cytokine levels were observed in the BiTE/combination group 6 hours post dosing but not in the Rituximab alone group. Together, the data suggest that the model could be used to assess the preclinical efficacy and CRS toxicity of BiTE.

Citation Format: Deep S. Shah, Catalina Simion, Sang hu, Rhonda Chronis, Tre McClellan, Jae Hwang, Ryan Yang, Heather Baker, Danying Cai, James Keck, Lindsay Shopland. Novel preclinical model for the evaluation of cytokine release syndrome in response to CD19xCD3 bispecific antibody in humanized mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4150.
Abstract 4154: mRNA-based immunotherapies to treat women's cancers
Authors: Tse Dan
Published: April 2023

With its ability to potentially code for any given protein, peptide and fragments, synthetic mRNA has landed itself a broad range of cancer immunotherapy applications, including costimulatory receptors, therapeutic antibodies, vaccines, and cytokines able to change the tumor microenvironment.We have been able to leverage our mRNA platform to generate a vaccine for HPV-associated cervical cancer (CC), as well as an immune-stimulatory cytokine for triple negative breast cancer (TNBC), both diseases that disproportionately affects minorities who do not participate in routine medical screenings and contribute to disparity in mortality beyond individual risk factors.IL-12 is a potent pro-inflammatory type 1 cytokine with potential to enter the clinical practice as immunotherapy for cancer. Its use in the form of recombinant protein and/or DNA plasmid has been hampered by issues with systemic toxicity or protein bioavailability within the tumor. To overcome these roadblocks, we developed a novel secreted single chain IL-12p70 mRNA. Intra-tumoral dosing of this mRNA induced tumor regression in a syngeneic and orthotopic mouse model of TNBC. Almost all cervical cancers are HPV (human papilloma virus) associated. We have developed a therapeutic vaccine, based on a single mRNA coding for two de-risked antigens, that is able to induce a T cell response against the oncogenic proteins E6 and E7 of the most common serotype (HPV16). When injected intramuscularly in mice bearing C3.43 masses, this vaccine suppressed tumor growth and generated immunological memory.Currently, these women’s cancers do not have a cure or an effective standard of care. With an efficacy that relies on a few injections and non-invasive routes of administration, we believe our innovative RNA-based pharmaceuticals might close some treatment gaps.

Citation Format: Dan Tse. mRNA-based immunotherapies to treat women's cancers. [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 4154.
Abstract 4202: Kidney function and risk of renal cell carcinoma
Authors: Alcala Karine , Alcala Nicolas , Martin Richard , Brennan Paul , Muller David , Robbins Hilary A. , Johansson Mattias
Published: April 2023

Background: The relationship between kidney function and risk of renal cell carcinoma (RCC) is not well understood. In this study, we evaluated the association between estimated glomerular filtration rate (eGFR) and risk of incident RCC, and assessed whether this association depends on time between eGFR measurement and RCC diagnosis. We also sought to evaluate if eGFR may be useful to predict RCC risk.

Methods: We conducted this study in the UK Biobank cohort based on 440,983 participants of whom 984 were diagnosed with RCC during 4,552,747 person-years of follow-up. The temporal relation between kidney function and RCC was evaluated with flexible parametric survival models for eGFR calculated from creatinine, cystatin C and both, adjusted for C-reactive protein (CRP) and common RCC risk factors. We also assessed the benefit of combining CRP and eGFR with a published RCC risk prediction model by estimating calibration and discrimination using a resampling algorithm as internal validation.

Results: We found that a lower eGFR - an indication of poor kidney function - was associated with higher RCC risk when measured up to five years prior to diagnosis. We estimated the RCC hazard ratio per standard deviation decrease in eGFR when measured one year before diagnosis at 1.22 (95% confidence interval [95% CI]: 1.11-1.34), and at 1.14 (95% CI: 1.05-1.19) when measured five years before diagnosis. The corresponding RCC HR for eGFR measured ten years before diagnosis was 1.03 (95% CI: 0.95-1.12). Adding eGFR to the RCC risk model provided a small improvement in risk discrimination 2 years before diagnosis with a C-index of 0.76 (95% CI: 0.71-0.81) compared to the published model (0.73, 95% CI: 0.68-0.79).

Conclusion: This study demonstrated that markers of kidney function are robustly associated with RCC risk when measured within the last five years leading up to diagnosis. However, kidney function markers do not seem to provide important improvements in RCC risk discrimination beyond established risk factors.

Citation Format: Karine Alcala, Nicolas Alcala, Richard Martin, Paul Brennan, David Muller, Hilary A. Robbins, Mattias Johansson. Kidney function and risk of renal 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 4202.