Specific Role Research Articles

Abstract 2903: CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts)

Abstract Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein that contributes to the progression of various cancers, including pancreatic cancer. The higher expression of CTHRC1 in tumor tissues is associated with poorer survival outcomes. However, its specific roles in tumor extracellular matrix (ECM) remodeling remain unclear. Our study aims to investigate the influences of CTHRC1 on pancreatic stellate cells (PSCs), a main source of ECM production in pancreatic cancer. The analyses of the publicly available pancreatic cancer patient data revealed that CTHRC1 is mainly expressed in cancer stroma and highly correlated with ECM-related genes. An in vitro study showed that more than 40% of these genes can be upregulated by CTHRC1. CTHRC1 specifically activated PSC into myofibroblast-like cancer-associated fibroblasts (myCAFs), which are characterized by a significantly upregulated POSTN gene expression. Periostin (coded by the POSTN gene) has a central role in the CTHRC1-PSCs-cancer metastasis axis. Furthermore, CTHRC1 promoted pancreatic cancer cell proliferation through PSC activation to a greater extent than via direct stimulation. Proof-of-concept experiments showed that the long-term (4-week) inhibition of CTHRC1 led to significant tumor suppression and ECM reduction, and also resulted in an unexpected shift in the CAF subtype from myCAFs to inflammatory CAFs (iCAFs). PSC activation was demonstrated to be the key molecular mechanism responsible for the tumor-promoting effects of CTHRC1, and CTHRC1 has a critical role in CAF subtype differentiation and tumor microenvironment (TME) remodeling. The inhibition of CTHRC1 as a therapeutic strategy for the treatment of pancreatic cancer warrants further investigation. Citation Format: Jin Gyeong Lee, Min Kyung Kang, Fen Jiang, Kyoungjin Ryu, Juwon Kim, Yun-Yong Park, Sang Seok Koh. CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2903.

Abstract 3401: Uncovering virus induced dysregulated processes in cancer and their impact on outcomes

Abstract The National Cancer Institute has designated several viruses such as EBV, HBV, HCV, HIV, HPV, and HTLV as agents that either cause cancer or increase the risk of developing cancer. However, extensive analyses of tumor genome and transcriptome datasets have identified numerous additional virus types, the functional implications of which remain largely unknown. Viral infection often dysregulates unique molecular processes within tumor cells and alters the composition of immune cells in the tumor microenvironment. For instance, HPV-positive tumors have been shown to exhibit a significantly elevated expression of the DNA editing enzyme APOBEC3B compared to HPV-negative tumors. Furthermore, HPV positive tumors display and increased number of C-to-T mutations induced by this protein. The genome and transcriptome of tumor tissue biopsies contain a wealth of information about these virus-associated signatures. However, advanced signal processing approaches are needed to extract these viral signatures from complex tumor datasets, and elucidate their specific roles in cancer. Here, we developed a quantitative approach to investigate the link between viral infection and dysregulation of a seven-membered A3 enzyme family (A3A/B/C/D/F/G/H). The primary role of these enzymes is to restrict viral infections by inducing C-to-U mutations in viral genomes. However, at least three members of this enzyme family (A3A/B/H) can also induce mutations in cellular DNA, and drive tumor formation and evolution. We used NMF (Nonnegative Matrix Factorization) to deconvolute the expression profiles of A3 enzymes in >10,000 bulk RNAseq datasets from diverse TCGA tumors. This analysis revealed the expression profiles of A3 enzymes within tumor and tumor microenvironment cells including various immune cells. Next, we conducted an association analysis to identify the link between the presence of viral infection and the proportion of each of these tumor and tumor microenvironment expression profiles. As expected, our analyses revealed strong associations between tumor-specific A3 expression profiles and infections by oncolytic viruses such as HPV. Notably, and somewhat unexpectedly, our analyses also revealed associations with viruses about which little is known in the field of cancer. For example, in certain tumors, we identified a robust association between bacteriophages and a tumor-specific A3 expression profile characterized by A3B upregulation. Taken together, our analyses provide evidence for the link between A3 dysregulation and infection by viruses including bacteriophages. Citation Format: Mohadeseh Soleimanpour moghadam, Azad Khosh, Diako Ebrahimi. Uncovering virus induced dysregulated processes in cancer and their impact on outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3401.

Abstract 103: Prostate cancer bone metastasis: Therapeutic targeting of tumor associated macrophages

Abstract Introduction: Castration-resistant bone metastatic prostate cancer is a morbid and deadly disease with an exceedingly low response rates to available immunotherapies. Understanding the mechanisms behind immune evasion and tumor progression is critical to improving therapy. Historically, bone metastases have been challenging to model in the laboratory. Our foundational work was built upon fresh patient samples taken directly from the operating room for analysis across multiple platforms: IHC, single cell expression profiling, and spatial transcriptomics. We compared tumors from patients with bone metastatic disease to tumors from patients with disease localized to the prostate. Our work (Cancer Cell, 2021 and Nature Communications, 2023) has consistently pointed towards dysregulated myeloid populations within the tumor microenvironment, particularly TREM2+ tumor associated macrophages (TAMs), as key drivers of disease progression. Methods: Our patient samples and murine pre-clinical models of prostate cancer highlighted several specifically upregulated molecules and pathways in TAMs. Here we focus on TAMs in vitro and in vivo using a new and genetically tractable myeloid model to interrogate the mechanistic role of key pathways, and how they may contribute to T-cell immune suppression. The ER-Hoxb8 system of conditional myeloid differentiation permitted the generation of isogenic cell lines to interrogate the roles of key molecules TREM2, MSR1, APOE, and MERTK through overexpression or CRISPR/Cas9 knockout. Results: Using a system of co-culture in vitro, we quantified the role of these proteins in suppressing T-cell proliferation and cytokine production, as well as their role in TAM differentiation and function. In vivo, these lines were co-injected subcutaneously with syngeneic RM1-BM3 tumor cells to quantify their effect on tumor growth and T-cell suppression in vivo. In addition to localized subcutaneous tumors, bone metastatic disease was established by intracardiac injection. Furthermore, established tumors were treated with TAM-directed small molecule therapy (e.g., MERTK inhibition) as well as immune checkpoint inhibition (anti-PD1) alone, and in combination. Conclusions: TAMs are immune suppressive myeloid cells that are not found in normal tissues. They are specifically recruited to tumors and highly enriched in bone metastatic disease. Transcriptomic analyses highlighted signaling pathways potentially amenable to productive therapeutic targeting. The ER-Hoxb8 system was highly effective in generating a genetically tractable model of TAM for study in vitro and in vivo. Our work supports the hypothesis that the specific targeting of TAMs, by focusing on critical signaling pathways, can be effective in the treatment of pre-clinical models of prostate cancer. This new approach merits further study as a potential strategy for the treatment of patients with bone metastases. Citation Format: Hanyu Zhang, Shenglin Mei, Nathan E. Jeffries, Douglas M. Dahl, Chin-Lee Wu, Philip J. Saylor, David B. Sykes. Prostate cancer bone metastasis: Therapeutic targeting of tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 103.

Abstract 4633: Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is always diagnosed as stage IV with a 5 year survival rate of less than 3% due to its aggressive metastatic phenotype. ATC lacks differentiation and loses its thyroid-like functions making it a difficult cancer to treat due to its lack of targetable genetic lesions and unresponsiveness to conventional treatments. Therefore, we investigated genes that had aberrant expression in ATC in relation to differentiation. Using NCBI Gene expression omnibus (GSE33630, GSE85457), which provides publicly available transcriptomic datasets, and bioinformatics software, GEO2R, we found that Homeobox D4 (HOXD4) has 12.5FC higher expression in ATC vs. normal tissue (p=0.000552) and 4.36FC higher in ATC vs. papillary thyroid cancer (PTC) in human patients (p=6.69e-08). HOXD4 is a transcription factor that plays a crucial role in cell differentiation and has been associated with a worse prognosis in cancers such as ovarian serous carcinoma. As loss of differentiation is essential to ATC’s refractory nature and therapeutic resistance, we are exploring modulation of differentiation by all-trans retinoic acid (ATRA). ATRA was used to induce differentiation in ATC cell line T238 therefore abrogating the aggressive phenotype of this cancer. T238 treated with ATRA had a 26% decrease in proliferation at 24 hours, 46% at 48 hours, and 87% at 72 hours measured using a trypan blue exclusion assay. T238 treated with ATRA also had a 44% decrease in migratory capacity at 24 hours and 24% at 48 hours measured by a cell scratch wound assay. Immortalized normal thyroid cell line NTHY-ori-3-1 did not have a significant decrease in either proliferation or migratory capacity when treated with ATRA. T238 cells treated with ATRA for 24 hours had a 30% decrease in HOXD4 protein levels. Lastly, immunohistochemistry staining of HOXD4 in human patient tissue samples confirmed the over-expression of HOXD4 in ATC (19.5% staining) vs. normal tissue (2.5%) and PTC (7.5%). Overall, we hypothesize that the aberrant expression of HOXD4 in ATC plays a crucial role in the aggressive metastatic phenotype and therefore differentiation agents such as ATRA can target this biomarker to modulate the ATC phenotype. Therefore, HOXD4 can be used as a potential prognostic indicator for early detection of ATC. Future studies will involve CRISPRi based knockdown of HOXD4 in vitro and validation of these results in animal in vivo models. Citation Format: Kaci Kopec, Tara Jarboe, Nicole R. DeSouza, Humayun K. Islam, Jan Geliebter, Raj K. Tiwari. Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4633.

The Role of H3K27me3-Mediated Th17 Differentiation in Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a common chronic progressive inflammatory autoimmune disease. T helper 17 (Th17) cells are the major effector cells mediating AS inflammation. Histone 3 Lys 27 trimethylation (H3K27me3) is an inhibitory histone modification that silences gene transcription and plays an important role in Th17 differentiation. The objective of this study was to investigate the expression of H3K27me3 in patients with AS and to explore its epigenetic regulation mechanism of Th17 differentiation during AS inflammation. We collected serum samples from 45 patients with AS at various stages and 10 healthy controls to measure their Interleukin-17 (IL-17) levels using ELISA. A quantitative polymerase chain reaction was used to quantify the mRNA levels of RORc and the signaling molecules of the JAK2/STAT3 pathway, JMJD3, and EZH2. Additionally, Western blot analysis was performed to quantify the protein levels of H3K27me3, RORγt, JAK2, STAT3, JMJD3, and EZH2 in cell protein extracts. The results showed that H3K27me3 expression in peripheral blood mononuclear cells (PBMCs) was significantly lower in patients with active AS compared to both the normal control groups and those with stable AS. Moreover, a significant negative correlation was observed between H3K27me3 expression and the characteristic transcription factor of Th17 differentiation, RORγt. We also discovered that patients with active AS exhibited significantly higher levels of JMJD3, an inhibitor of H3K27 demethylase, compared to the normal control group and patients with stable AS, while the expression of H3K27 methyltransferase (EZH2) was significantly lower. These findings suggest that H3K27me3 may be a dynamic and important epigenetic modification in AS inflammation, and JMJD3/EZH2 regulates the methylation level of H3K27me3, which may be one of the key regulatory factors in the pathogenesis of AS. These findings contribute to our understanding of the role of epigenetics in AS and may have implications for the development of novel therapeutic strategies for AS.

Abstract 129: Understanding the molecular drivers that regulate Ewing sarcoma cell fate

Abstract Like other translocation-positive sarcomas, Ewing sarcoma (ES) harbors a pathognomonic fusion protein (FP) capable of widespread epigenetic reprogramming that can lead cells to acquire a high-grade, undifferentiated phenotypic state. To better understand how this might occur, we employed a CRISPR knockout (CRISPR-KO) model of A673, targeting exon 4 of the N-terminus EWSR1 portion of the FP and generated a clonal pool (EWSR1 CRISPR KO). We used single-cell RNA and ATAC sequencing (scMultiome) to identify the transcriptional and chromatin changes after the FP knockout (FP KO). We identified several clusters of ES cell heterogeneity. Since the EWSR1-KO is a pooled population, we used known FP-regulated genes to delineate cells bearing the FP KO from wild-type (FP WT) cells. We observed robust expression of FP-activated genes, PRKCB, and LIPI, in many clusters suggesting regular FP function (FP WT). Conversely, FP-repressed genes, LOX, and IGFBP3 were elevated in a single cluster containing only EWSR1-KO cells. This suggests the FP was successfully knocked out in this cluster (FP KO). That FP KO cluster also lost transcriptional resemblance to ES. A granular identification with cell lines confirmed that all clusters except FP KO were identified as A673 cells. However, the FP KO resembled cell lines with a fibroblastic phenotype. We analyzed enriched motifs and observed no significant difference between the FP KO and FP WT for EWSR1::FLI1. However, the FP KO cells were enriched for motifs in the Fos/Jun family and Smad2/3, which have roles in EMT and mesenchymal differentiation. The FP-KO cells significantly expressed the Fos/Jun family of proteins. Next, we identified peak-gene associations within a ± 500 kb window around the TSS. When we analyzed peaks linked to genes expressed in FP-WT, we found enrichment of the Fos/Jun protein family (p < 1e-34), Ets family, and GGAA-microsatellites. However, the FP-KO were strongly enriched for Fos/Jun protein family (p < 1e-540) with no change in Ets family motif. Knocking out the FP in the A673 cell line significantly shifted the ES lineage. We observed peaks linked to genes positively associated or regulated by the FP were enriched for Fos/Jun family, Ets family, and GGAA-microsatellites. There may be a cooperative interaction between these TFs that affects the genes linked to the FP. The fact that FP loss enriched the Fos/Jun family motifs, but not Ets family or GGAA-microsatellites, suggests that FP loss activates FP-repressed genes and enables chromatin remodeling to open previously inaccessible DNA binding sites. Citation Format: Danh Truong, David McCall, Clement Agyemang, Sandhya Krishnan, Alexander Lazar, Joseph Ludwig. Understanding the molecular drivers that regulate Ewing sarcoma cell fate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 129.

Abstract 5770: Inhibition of KRAS G12D mutant with small molecules

Abstract Background: RAS mutations are occurring at a high rate in colorectal cancer, lung cancer, and pancreatic cancer. RAS is family of small GTPase proteins with important roles in cell growth, differentiation, proliferation, and survival. There are three types of RAS genes: HRAS, KRAS, and NRAS. The The RAS gene is the most commonly found oncogene in human cancer. KRAS exhibits a highest mutation rate compared to those of HRAS and NRAS in various cancer types. Methods: Colorectal cell line LIM1215, SW48 and NSCLC cell line NCI-H1975, NCI-H838 were used for MTT assays. Apoptosis assay was performed using the annexin V-FITC apoptosis detection kit (BioBud, Cat. LS-02-100). Colony forming assay was performed with 6-well culture plates by seeding 500 cells/well. The expression levels of proteins involved in Q2a-induced signaling pathway were determined by Western blot. In vivo study was performed with tumor xenografts. HT-29 cells were implanted into the right flank of 5-week-old BALB/c nude mice. Tumor size was determined by measuring the diameter (width, length) of the tumor every two days with a digital caliper. All group mice sacrificed at day 19. Results: Patients with cancer involving KRAS mutations are resistant to therapeutics that target epidermal growth factor receptor and show poor outcomes. In addition, effective treatments that target G12D in KRAS have not been developed. Thus, therapeutic agents targeting the G12D mutation in KRAS are needed. In this study, we identified the Q2a compound, a quinazoline-based derivative that specifically acts on the KRAS G12D mutation. This compound induced cell apoptosis by reducing phosphorylation of AKT in the PI3K pathway downstream of KRAS. Q2a compound specifically inhibited the growth of KRAS G12D mutant cells. In addition, cells with the KRAS G12D mutation were more sensitive to Q2a compound compared to the sensitivity of wild-type cells and those with other KRAS mutations, resulting in apoptosis. Treatment with the Q2a compound also decreased C-RAF phosphorylation in the MAPK pathway, which is downstream of KRAS. In KRAS G12D-mutated colorectal cancer xenografts, Q2a compound inhibited tumor production without significant liver toxicity. Conclusion: Q2a compounds show potential as therapeutic agents that target KRAS G12D mutations in colorectal and pancreatic cancers. Kyewords: Anti-Cancer Drug, KRAS G12D Mutant, apoptosis, colon cancerJi Yoon Lee, Dae-Hee Lee*Department of Marine Bio Food Science, Gangneung-Wonju national University Citation Format: Dae-Hee Lee, Ji Yoon Lee. Inhibition of KRAS G12D mutant with small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5770.

Theoretical Studies of DNA Microarray Present Potential Molecular and Cellular Interconnectivity of Signaling Pathways in Immune System Dysregulation.

Autoimmunity is defined as the inability to regulate immunological activities in the body, especially in response to external triggers, leading to the attack of the tissues and organs of the host. Outcomes include the onset of autoimmune diseases whose effects are primarily due to dysregulated immune responses. In past years, there have been cases that show an increased susceptibility to other autoimmune disorders in patients who are already experiencing the same type of disease. Research in this field has started analyzing the potential molecular and cellular causes of this interconnectedness, bearing in mind the possibility of advancing drugs and therapies for the treatment of autoimmunity. With that, this study aimed to determine the correlation of four autoimmune diseases, which are type 1 diabetes (T1D), psoriasis (PSR), systemic sclerosis (SSc), and systemic lupus erythematosus (SLE), by identifying highly preserved co-expressed genes among datasets using WGCNA. Functional annotation was then employed to characterize these sets of genes based on their systemic relationship as a whole to elucidate the biological processes, cellular components, and molecular functions of the pathways they are involved in. Lastly, drug repurposing analysis was performed to screen candidate drugs for repositioning that could regulate the abnormal expression of genes among the diseases. A total of thirteen modules were obtained from the analysis, the majority of which were associated with transcriptional, post-transcriptional, and post-translational modification processes. Also, the evaluation based on KEGG suggested the possible role of TH17 differentiation in the simultaneous onset of the four diseases. Furthermore, clomiphene was the top drug candidate for regulating overexpressed hub genes; meanwhile, prilocaine was the top drug for regulating under-expressed hub genes. This study was geared towards utilizing transcriptomics approaches for the assessment of microarray data, which is different from the use of traditional genomic analyses. Such a research design for investigating correlations among autoimmune diseases may be the first of its kind.

SSR Marker–Based Genetic Diversity and Relationship Analyses of Stephania tetrandra S. Moore

AbstractStephania tetrandra S. Moore (family: Menispermaceae), a dioecious herbaceous vine and the only species in the subgenus Botryodiscia of the genus Stephania of the family Menispermaceae, is mainly distributed in hilly areas south of the Huaihe River in China and found in many provinces of China, showing a high genetic diversity. This paper aimed to study genetic diversity of and genetic relationship among individuals of S. tetrandra within China to provide a basis for evaluation, exploitation, and utilization of S. tetrandra by using simple sequence repeat (SSR) molecular markers. Our results show that effective products were amplified from the 26 screened SSR gene loci, a total of 183 alleles amplified (2–16 alleles amplified by each pair of primers). Among the 26 loci, 16 had a PIC value higher than 0.5, indicating a high level of polymorphism. For most of the loci, the number of effective alleles was lower than that of the observed alleles, and the observed heterozygosity was lower than the expected heterozygosity. The genetic differentiation coefficient (0.021–0.547) was lower than 0.05 (low level of genetic differentiation) for 7 loci and higher than 0.25 (very high level of genetic differentiation) for 2 loci, and had a value representing a medium level of genetic differentiation for the remaining 17 loci. The intra-population inbreeding coefficient had a positive value for 21 loci, suggesting the presence of inbreeding and homozygous excess. The gene flow value was bigger than 1, indicating that genetic drift and natural selection played an unimportant role in population genetic differentiation of S. tetrandra. Based on discriminant analysis of principal components and Bayesian Information Criterion, K-means clustering was performed on 620 samples. These samples were divided into 9 genetic clusters, whose similarity coefficients and genetic distances were 0.755–0.918 and 0.067–0.280, respectively, indicating that these clusters were highly similar and short-distanced. The Bayesian clustering analysis was implemented in the STRUCTURE software to analyze the genetic structure of S. tetrandra and it was found that the 620 samples could be clustered into 5 ancestor groups; the 9 clusters and 40 natural populations inherited genes from the 5 groups to varying degrees, but the proportion of genes inherited from the 5 groups by each cluster and natural population differed. S. tetrandra was characterized by the presence of population structure and pronounced genetic subdivision, which, together with the presence of gene flow, may indicate a relatively stable recent state of these populations.

Abstract 1438: Investigating the roles of EMT in small cell lung cancer tumorigenesis and chemoradiation response

Abstract In this study, we investigated the contributions of the epithelial-mesenchymal transition (EMT) program to small cell lung cancer (SCLC) tumorigenesis and chemoradiation (CRT) response. SCLC is a highly aggressive and deadly neuroendocrine malignancy. Two major factors that contribute to the high mortality of SCLC are (1) early metastasis and (2) rapid development of therapy resistance. Recent research suggests upregulation of EMT and the EMT transcription factor Twist1 correlated with accelerated tumor progression and CRT resistance in SCLC. Due to the lack of suitable animal models, the causal relationship between the EMT program and SCLC tumorigenesis and therapy response has not been rigorously investigated. To this end, we have generated a novel genetically engineered mouse model (GEMM) termed RPGT (Rb1Flox; Trp53Flox; ROSA26LSL-rtTA-IRES-EGFP; Twist1-TetO7-Luc). The RPGT GEMM enables the generation of autochthonous SCLC tumors after induction with Cre recombinase adenovirus. By withdrawing or providing doxycycline to mice, we can control Twist1 expression to activate or inactivate EMT in tumors. Upon characterizing mouse tumor samples with histological and molecular analyses, we observed that both control (no Twist1 overexpression) and Twist1-overexpressing RPGT mice developed neuroendocrine SCLC tumors. While SCLC-A was the predominant subtype in both cohorts, RPGT tumors exhibited more heterogeneity. Importantly, although no difference in tumorigenesis was observed, Twist1 overexpression dramatically increased the metastatic incidence in RPGT (87-100%) compared to control (25-50%) animals, indicating an important role of EMT in SCLC dissemination. Furthermore, transcriptomic profiling of primary tumors and matching metastases in RPGT mice revealed downregulation of Twist1 and EMT in metastases, suggesting that EMT suppression was necessary for metastatic outgrowth. To evaluate the impact of EMT activation on SCLC sensitivity to CRT, we performed in vitro viability assays on primary tumor cell lines established from RPGT tumors. We found that repressing Twist1 expression enhanced SCLC susceptibility to CRT. To validate these results in vivo, we treated RPGT mice with vs. without Twist1 overexpression with CRT. Consistent with our in vitro results, our in vivo data indicated that Twist1 overexpression promoted CRT resistance in SCLC tumors. Overall, our data suggest that the EMT program plays an important role in SCLC differentiation, metastasis, and resistance against chemo-radiotherapy. Citation Format: Triet Nguyen, Jinhee Chang, Kathleen Gabrielson, Amol Shetty, Yang Song, Audrey Lafargue, Shreya Jagtap, Danielle Council, Aaron Chan, Dipanwita Dutta Chowdhury, Muhammad Ajmal Khan, Nick Connis, Francesca Carrieri, Eddie Imada, Daniel Sforza, Eric Gardner, Christopher McFarland, Luigi Marchionni, Mohammad Rezaee, Christine Hann, Phuoc Tran. Investigating the roles of EMT in small cell lung cancer tumorigenesis and chemoradiation response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1438.

Ion channels as biomarkers of altered myogenesis in myofiber precursors of Duchenne muscular dystrophy.

Myogenesis is essential for skeletal muscle formation, growth, and regeneration and can be altered in Duchenne muscular dystrophy (DMD), an X-linked disorder due to the absence of the cytoskeletal protein dystrophin. Ion channels play a pivotal role in muscle differentiation and interact with the dystrophin complex. To investigate ion channel involvement in myogenesis in dystrophic settings, we performed electrophysiological characterization of two immortalized mouse cell lines, wild-type (WT) H2K-2B4 and the dystrophic (DYS) H2K-SF1, and measured gene expression of differentiation markers and ion channels. Inward and outward currents/density increased as differentiation progressed in both WT and DYS cells. However, day-11 DYS cells showed higher (27%) inward current density with an increased expression ratio of Scn5a/Scn4a and decreased (48%) barium-sensitive outward current compared to WT. Furthermore, day-11 DYS cells showed more positive resting membrane potential (+10mV) and lower membrane capacitance (50%) compared to WT. DYS cells also had reduced Myog and Myf5 expression at days 6 and 11. Overall, ion channel profile and myogenesis appeared altered in DYS cells. These results are a first step in validating ion channels as potential drug targets to ameliorate muscle degeneration in DMD settings and as differentiation biomarkers in innovative platforms.

Abstract 172: Targeting the poliovirus receptor to activate T cells and induce myeloid-derived suppressor cells to differentiate to M1-like macrophages via the IFNγ-pSTAT1-IRF8 axis in cancer therapy

Abstract T cell immunoglobulin and ITIM domain (TIGIT) is one of the most important immune checkpoints expressed on lymphocytes, and poliovirus receptor (PVR, also CD155) serves as the most crucial ligand for TIGIT, harboring an important function in cancer cells and influencing the tumor microenvironment (TME). While it's well-established that TIGIT blockade could reverse immunosuppression, the question of whether direct inhibition of PVR yields comparable results remains to be fully elucidated. In this study, we generated a mouse gene Pvr knock out (KO) Lewis lung cancer (LLC) cell line and found that the Pvr-KO tumors tended to develop a pro-inflammatory TME, as reflected by a downregulation of monocytic-myeloid derived suppressor cells (M-MDSCs) and an upregulation of CD8+ central memory T cells. Through bulk RNA-seq and multi-cytokines measurement, we quantified the transcription and protein levels of immunostimulatory cytokines and chemokines and found they were significantly upregulated in Pvr-KO TME, including GM-CSF, IFN-γ, TNF-α, IL-2/4/6/10, CCLs (including CCL2/3/4/5) and CXCLs (including CXCL1/2/5). We further employed single-cell RNA sequencing (scRNA-seq) on 20,026 intra-tumoral CD45+ immune cells. Using a conserved MDSCs gene set and the 'AddModuleScore' function of Seurat package, we identified the clusters of MDSCs and found the differentiation ability of M-MDSCs in Pvr-KO TME was enhanced. Through pseudo-temporal ordering, we found the M-MDSCs in the Pvr-KO TME have a trend to differentiate towards M1-like macrophages, which could play an essential positive role in anti-tumor immunity. We further focused on the scRNA-seq of T cell subsets and found the enhanced function of cytotoxic CD8+ T cells, leading to increased production of IFN-γ. In the intercellular communications analysis, a significant upregulation of the IFNG-IFNR signal between T cells: MDSCs in the Pvr-KO group was observed. Moreover, we found that Irf8, a key factor in myeloid cell differentiation, was significantly upregulated in the M-MDSCs of Pvr-KO tumor. We then employed CUT & Tag sequencing and confirmed that IFN-γ induces phosphate Stat1, which then binds on the promoter of Irf8. These findings suggest that the IFN-γ-Stat1-Irf8 axis plays a crucial role in MDSCs differentiation and may be a potential target for therapeutic intervention in cancer. Next, an anti-PVR nanobody was synthesized and its efficacy was evaluated in the context of LLC and 4T1 tumor grafts. When in combination with anti-PD-1 therapy, anti-PVR exhibited a remarkable capacity to reduce tumor size in both tumor models with no notable indications of toxicity observed. Our findings provide new insights into the role of PVR in modulating the TME and offer potential strategies for enhancing the effectiveness of immunotherapy. Citation Format: Mingyang Feng, Sirui Tan, Qizhi Ma, Benxia Zhang, Yue Chen, Qiu Li, Yongsheng Wang. Targeting the poliovirus receptor to activate T cells and induce myeloid-derived suppressor cells to differentiate to M1-like macrophages via the IFNγ-pSTAT1-IRF8 axis in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 172.

Abstract 5959: Targeting TRIM11 is a potential therapeutic strategy for malignant gliomas

Abstract Background: TRIM11 (tripartite motif-containing protein 11) belongs to the TRIM/RBCC (the RING B-box coiled-coil) family of E3 ubiquitin ligases. Emerging evidence has demonstrated that TRIM11 is a novel target against cancer and neurodegenerative diseases as it plays vital role in cellular proliferation, differentiation, tumor progression, and apoptosis. Our previous work demonstrated that TRIM11 is over-expressed in high-grade gliomas and promotes proliferation, invasion, migration and tumor growth, suggesting that TRIM11 may be used as a target for malignant glioma treatment. The tumor suppressor p53 plays critical roles in tumor prevention, and deregulated p53 pathway has been found in a majority of glioblastomas (GBMs). Recent studies have shown that TRIM proteins are involved in the regulation of p53, and TRIM proteins are also regulated by p53. Methods: To evaluate the effect of TRIM11 on GBM progression in vivo, we intracranially implanted GBM cells over-expressing TRIM11 in immunocompromised mice, and the animal survival was monitored. We have synthesized a series of TRIM11 inhibitors and evaluated their anti-GBM potential in vitro. Furthermore, we tested the crosstalk between TRIM11 and p53 by knocking down TP53 through RNA interference. Results: Our in vivo data showed that the control group mice (GL261 parent line) survived longer than the mice bearing TRIM11 over-expressing GL261 tumors (p<0.05), suggesting TRIM11 enhances tumor progression in vivo. In vitro data showed that the oncogenic effect of TRIM11 may be related to its influence on the apoptosis pathway since a robust induction of poly (ADP-ribose) polymerase (PARP) was observed in TRIM11 over-expressing GBM cells. When treated with Temozolomide (TMZ), TRIM11 over-expressing cells were more resistant and showed a higher survival rate compared to parental cells. We screened a small library of TRIM11 inhibitors (~ 25 compounds), and identified several compounds which significantly inhibited the growth of both established and primary GBM cells in a dose-dependent manner, implicating that TRIM11 inhibitors may serve as novel agents for GBM treatment. We found that inactivation of p53 by siRNA increased the expression of TRIM11 and modulated the sensitivities of GBM cells to chemotherapy drugs. Conclusion: Despite remarkable advances in the prognosis and multidisciplinary treatment strategies, GBM is still the most lethal primary brain tumor, and there is a pressing need for newer anti-GBM agents with novel targets and mode of action. Our preclinical studies demonstrate a significant anti-tumor effect of TRIM11 inhibitors on malignant glioma cells. Our finding that TRIM11 may be modulated by p53 adds a new layer of regulation to this complicated signaling pathway. A better understanding of the crosstalk between p53 and TRIM11 will benefit the clinical application of targeting p53 and TRIM11 signaling pathways for therapeutical indications in GBM. Citation Format: Kaijun Di, Bhaskar C. Das, Daniela Alexandru Abrams, Daniela A. Bota. Targeting TRIM11 is a potential therapeutic strategy for malignant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5959.

Abstract 1389: Understanding the differential role of an IL-6 family cytokine in varying subtypes of breast cancer patients for novel therapeutic intervention

Abstract In the United States, it is estimated that over 295,000 women will be diagnosed with breast cancer, and over 43,000 breast cancer deaths in 2023. Mortality in breast cancer is due in large part due to distant metastasis to organs such as the bone, lung, liver, and brain. Our laboratory has identified that the IL-6-family cytokine oncostatin M (OSM) plays a crucial role in initiating metastasis by secreting pro-metastatic molecules such as VEGF, LOXL2 and IL-6, and increasing circulating tumor cell numbers and metastases to bone and lung in vivo. More recent work within our lab has identified unique roles for OSM in varying subtypes of breast cancer patients, particularly between estrogen receptor positive (ER+) and ER- patients. It is well known that patients with ER- breast cancer, specifically triple negative breast cancer (TNBC; ER- PR- HER2-), have limited therapeutic options. However, the divergent role OSM plays in ER+ and ER- breast cancer and the mechanism by which this occurs has yet to be fully understood. Thus, we aimed to fully elucidate the difference between OSM in ER+ versus ER- breast cancer. RNA sequencing analysis on OSM-treated human ER+ cells and ER- TNBC cells discovered significant differences in gene expression between both cell lines upon treatment with OSM, including alteration in DNA-replication and cell cycle pathway genes. Afterwards to evaluate the correlation between OSM and DNA synthesis and this role on metastasis, we evaluated the role of a novel anti-OSM therapeutic and its correlation with decreased metastasis. This research aims to elucidate the unique role OSM plays in ER+ and ER- breast cancer and will provide a molecular understanding of how this occurs and will provide detailed information on which patients will benefit most from anti-OSM therapeutics. Citation Format: Cody L. Wolf, Ken Tawara, Cheryl L. Jorcyk. Understanding the differential role of an IL-6 family cytokine in varying subtypes of breast cancer patients for novel therapeutic intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1389.

Abstract 1773: Deciphering the roles of germline predisposition variants and somatic mutations on breast cancer cells and the tumor microenvironment

Abstract While great progress has been made in the understanding and treatment of cancer, tumor heterogeneity remains a diagnostic and prognostic challenge. Single-cell sequencing techniques and high-resolution cellular imaging now afford researchers the ability to investigate the effects of germline variants (especially when compared to somatic mutations) on molecular characteristics of cancer cells and non-cancer cells in the tumor microenvironment (TME). Here, we investigated a breast cancer (BRCA) cohort of over 100 individuals, for which we have generated sc/sn RNA-seq, snATAC-seq, and CODEX imaging data as well as bulk whole exome sequencing and RNA-seq data. We identified 21 BRCA cases carrying pathogenic and likely pathogenic rare germline variants in known cancer predisposition genes, such as BRCA2, BRIP1, ATM, and TP53. These predisposition genes showed diverse expression patterns across stromal and immune cell types, indicating roles in cell types beyond breast epithelia. For cases carrying pathogenic germline variants in such genes, we use snRNA-seq/snATAC-seq to assess gene expression changes and pathway alterations (e.g.,HRD and apoptosis) across all cell types and investigate phenotypic changes associated with pathogenic germline variants in cancer cells compared to stroma and immune cells in the TME. By evaluating chromatin accessibility changes and allele-specific expression, we reveal the connection between epigenetic regulators and differential roles of germline variants across cell types. Finally, the integrated analysis of germline variants and somatic mutations from predisposition genes at single cell resolution broadened our understanding of the role of germline variants in TME during tumorigenesis. Citation Format: Fernanda Martins Rodrigues, Kelsey Gallant, Reyka Jayasinghe, Michael Iglesia, Andrew Houston, Siqi Chen, Preet Lal, Ryan Fields, William Gillanders, Feng Chen, Li Ding. Deciphering the roles of germline predisposition variants and somatic mutations on breast cancer cells and the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1773.

Abstract 5099: Prevalence of fibroblast growth factor receptor (FGFR) alterations (alts) and programmed death-ligand 1 (PD-L1) expression (exp) in Chinese solid tumor patients (pts)

Abstract Background: PD-L1 expression drives anti-PD(L)-1 treatment decisions across many cancer indications. FGFR inhibitors have been approved for the treatment of metastatic urothelial cancer (UC) and cholangiocarcinoma with FGFR alts. BALVERSA (erdafitinib), a selective pan-FGFR kinase inhibitor, has shown clinical activity against FGFR altered solid tumors in pts who exhausted standard treatment options. It is important to understand the prevalence of FGFR alts and relationship between FGFR alts and PD-L1 expression levels in different cancer types. Methods: In this real-world study, 9937 Chinese solid tumor pts with the PD-L1 (22C3) immunohistochemistry and OrigiMed 450-gene next-generation sequencing panel results were selected. Chi-square test and Fisher test were used to explore the association between the FGFR alts and PD-L1 expression. Results: FGFR1-4 somatic alts were found in 9.0% of Chinese cancer pts evaluated. FGFR1 amplification (amp) was the most prevalent alts identified (1.9%). There were no significant differences in frequency of FGFR alts between the Chinese cohort and MSK-IMPACT cohort in each cancer type tested. Up to 40.6% of pts evaluated were PD-L1 positive (CPS ≥1 or TPS ≥1%). The PD-L1 positive rate (TPS ≥1%) in SCLC (12.4%, n=121) and NSCLC adenocarcinoma tumors (LUAD) (26.1%, n=2902) was relatively low. In few tumor types, higher expression of PD-L1 in FGFR altered samples was observed compared with FGFR unaltered samples in pan-tumor (mean TPS 14.8% vs.8.6%, P=6.0*E-9; mean CPS 6.3 vs. 6.1, P=3*E-4). Those includes LUAD (mean TPS 14.2% vs. 7.4%, P = 1.7*E-16), colorectal tumor (mean CPS 4.6 vs. 3.9, P = 4.6*E-8), and uterine tumor (mean CPS 8.1 vs. 6.5, P = 9.6*E-5). While PD-L1 expression is lower in FGFR altered samples than FGFR unaltered samples in UC (n=154; mean CPS 3.9 vs. 6.8, P = 0.004). No relationship was observed between FGFR alts and PD-L1 expression in cholangiocarcinoma (n=610). Specific FGFR alts eligible for erdafitinib UC treatment were further investigated in UC and observed with rate 10.4%, including FGFR3 short variants (9.7%) as main alts. Specific FGFR altered tumors showed similar trend with lower PD-L1 expression than FGFR unaltered tumors in UC. The inverse relationship between FGFR alts and PD-L1 expression in UC compared to other solid tumors may explain variable role of FGFRs since the predominant FGFR alt types vary among different cancer types (FGFR3 short variants enriched in UC vs no such predominant FGFR alt types in other cancer types). Conclusion: The large Chinese solid tumor cohort analysis showed comparable FGFR alts prevalence between Chinese and Western population. Differences in relationship between FGFR alts and PD-L1 expression across tumor types reflect the differential role of predominant FGFR types in each tumor type. Citation Format: Min Qing, Fei Yang, Xiaowei Chen, Xuesong Lyu, Aodi Wang, Yanfei Yu, Shibu Thomas, Longen Zhou. Prevalence of fibroblast growth factor receptor (FGFR) alterations (alts) and programmed death-ligand 1 (PD-L1) expression (exp) in Chinese solid tumor patients (pts) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5099.

Abstract 3055: Characterizing the role of HNF4α isoforms in human PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly and recalcitrant malignancy with a 5-year survival rate of 12%. Transcriptomic studies have classified PDAC into a classical or basal-like subtype. The classical subtype has a better prognosis, response to chemotherapy, and express higher levels of endodermal lineage specifiers compared to the basal-like subtype. However, the field lacks a comprehensive understanding of the key subtype-specific regulators. Functional analysis of transcription factors associated with each subtype are needed to elucidate their roles in regulation of PDAC subtypes and to identify therapeutically targetable vulnerabilities. Previous work in our lab demonstrated that HNF4α is a key regulator of the classical subtype. Using genetically engineered mouse models, our lab showed that loss of HNF4α leads to an increase in tumor burden, a shift from glandular to poorly differentiated histology, and a decrease in the classical subtype score. However, while murine PDAC only express one set of HNF4α isoforms, human PDAC express both sets of HNF4α isoforms, suggesting murine PDAC may not fully recapitulate human disease. There are 2 subsets of HNF4α isoforms driven by expression from 2 distinct promoters, P1 and P2. P1 and P2 isoforms are differentially expressed within the gastrointestinal (GI) tract and play dichotomous roles in GI malignancies. In HNF4α-positive human PDAC, P2 isoforms are always expressed but there is variable expression of P1 isoforms. Based on the differential role of HNF4α isoforms in both normal tissue and cancer, we hypothesize that P1 isoforms will restrain tumor growth and promote greater classical/GI differentiation compared to P2 isoforms. We utilized CRISPRi to selectively inhibit transcription at the P1 or P2 promoter in HNF4α-positive, human cell lines expressing both isoforms. To test for sufficiency, we exogenously expressed P1 or P2 isoforms in HNF4α-negative, human cell lines. We then performed Incucyte, CRISPRi competition, and anchorage-independent growth assays. We performed RNA-seq and ChIP-seq on both exogenous and endogenous HNF4α isoforms. Functional assays revealed that HNF4α isoforms regulated growth in a context dependent manner. Exogenous P1 isoforms significantly inhibited growth while loss of endogenous P1 isoforms was dispensable for growth. Exogenous P2 isoforms inhibited growth to a lesser degree while loss of endogenous P2 isoforms led to a significant reduction in growth. Despite ChIP-seq analysis revealing very similar binding profiles, RNAseq analysis revealed that P1 isoforms are significantly more transcriptionally active than P2 isoforms and are stronger inducers of GI differentiation. Taken together, our data demonstrates that P2 isoforms are more compatible with growth in PDAC compared to P1 isoforms, which promote a more differentiated cell state. Mechanistically, this may be due to the increased transcriptional activity of P1 isoforms. Citation Format: Pengshu Fang, Acramul Kabir, Gabriela Fort, Eric L. Snyder. Characterizing the role of HNF4α isoforms in human PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3055.

Elucidating the role of ZRF1 in monocyte-to-macrophage differentiation, cell proliferation and cell cycle in THP-1 cells

Abstract Objectives ZRF1 (Zuotin-related factor 1) is a versatile protein engaged in protein folding, gene regulation, cellular differentiation, DNA damage response, and immune system and cancer development regulation. This study investigates the role of ZRF1 in monocyte-to-macrophage transformation, and its effects on cell proliferation and the cell cycle. Methods We generated ZRF1-depleted THP-1 cells and induced macrophage differentiation using phorbol 12-myristate 13-acetate (PMA). Differentiation was assessed via microscopy and flow cytometry, while cell proliferation was quantified with the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) assay, and the cell cycle was analyzed through flow cytometry using propidium iodide staining. Results ZRF1-depleted THP-1 cells exhibited notable morphological changes. Flow cytometry post-PMA treatment indicated these cells were smaller and less granular than controls. Proliferation rates of ZRF1-depleted monocytes and macrophages were significantly higher than controls, particularly over longer durations. Cell cycle analysis showed ZRF1 depletion notably affected the G0-G1 phase, highlighting its significant role in macrophage differentiation. Conclusions The findings provide important insights into ZRF1’s role in monocyte-to-macrophage differentiation and its impact on cell proliferation and the cell cycle. This research not only supports existing knowledge about ZRF1 but also enhances our understanding of its multifaceted roles in cellular processes.

MTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis.

Metabolism participates in the control of stem cell function and subsequent maintenance of tissue homeostasis. How this is achieved in the context of adult stem cell niches in coordination with other local and intrinsic signaling cues is not completely understood. The Target of Rapamycin (TOR) pathway is a master regulator of metabolism and plays essential roles in stem cell maintenance and differentiation. In the Drosophila male germline, mTORC1 is active in germline stem cells (GSCs) and early germ cells. Targeted RNAi-mediated downregulation of mTor in early germ cells causes a block and/or a delay in differentiation, resulting in an accumulation of germ cells with GSC-like features. These early germ cells also contain unusually large and dysfunctional autolysosomes. In addition, downregulation of mTor in adult male GSCs and early germ cells causes non-autonomous activation of mTORC1 in neighboring cyst cells, which correlates with a disruption in the coordination of germline and somatic differentiation. Our study identifies a previously uncharacterized role of the TOR pathway in regulating male germline differentiation.

Rapid Volumetric Bioprinting of Decellularized Extracellular Matrix Bioinks.

Decellularized extracellular matrix (dECM)-based hydrogels are widely applied to additive biomanufacturing strategies for relevant applications. The extracellular matrix components and growth factors of dECM play crucial roles in cell adhesion, growth, and differentiation. However, the generally poor mechanical properties and printability have remained as major limitations for dECM-based materials. In this study, heart-derived dECM (h-dECM) and meniscus-derived dECM (Ms-dECM) bioinks in their pristine, unmodified state supplemented with the photoinitiator system of tris(2,2-bipyridyl) dichlororuthenium(II) hexahydrate and sodium persulfate, demonstrate cytocompatibility with volumetric bioprinting processes. This recently developed bioprinting modality illuminates a dynamically evolving light pattern into a rotating volume of the bioink, and thus decouples the requirement of mechanical strengths of bioprinted hydrogel constructs with printability, allowing for the fabrication of sophisticated shapes and architectures with low-concentration dECM materials that set within tens of seconds. As exemplary applications, cardiac tissues are volumetrically bioprinted using the cardiomyocyte-laden h-dECM bioink showing favorable cell proliferation, expansion, spreading, biomarker expressions, and synchronized contractions; whereas the volumetrically bioprinted Ms-dECM meniscus structures embedded with human mesenchymal stem cells present appropriate chondrogenic differentiation outcomes. This study supplies expanded bioink libraries for volumetric bioprinting and broadens utilities of dECM toward tissue engineering and regenerative medicine.