Mouse Cell Lines Research Articles

Abstract 5957: Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer

Abstract Introduction Transcription regulators, such as cyclin-dependent kinase 9 (CDK9), have been implicated in the super-enhancer driven transcriptional control of oncogenes in multiple cancers including pancreatic cancer. CDK9 has been shown to be employed by cancer cells for the constant production of short-lived oncoproteins such as c-Myc to maintain their survival. Due to its critical role in regulating transcription in cancer cells, CDK9 has emerged as a potential therapeutic target. Here, we report the antitumor activity of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, in preclinical models for pancreatic cancer. Experimental Procedures Fragment-based MolecuLern driven AI workflow methods were used to design a series of ATP-competitive CDK9 specific kinase inhibitors. BLX-3030 was selected as one of the most potent leads with an IC50 of 1.1 nM in a cell-free CDK9 kinase assay. A Sulforhodamine B (SRB) assay was used to evaluate the cell growth inhibitory activity of BLX-3030 in a panel of 10 pancreatic ductal adenocarcinoma (PDAC) and 2 pancreatic neuroendocrine tumor (pNET) cell lines. Western blotting was used to determine the effects of BLX-3030 treatment on c-Myc expression in cancer cells. The combination effects between BLX-3030 and standard of care regimen, gemcitabine (GEM) and nab-paclitaxel (NP), was assessed using the SRB assay. Finally, the in vivo antitumor activity of BLX-3030 alone or in combination with GEM and NP was evaluated in a mouse cell line xenograft model for pancreatic cancer. Results BLX-3030 exhibited potent cell growth inhibitory activity in the panel of pancreatic cancer cell lines with IC50 values ranging from 133.5 nM to 508.1 nM. The potency of BLX-3030 in the cell lines appears to correlate with the expression level of c-Myc in the cell lines. BLX-3030 showed similar activities in PDAC and pNET cell lines with mean IC50 values of 244.5 nM and 194.6 nM, respectively. However, treatment with BLX-3030 for 48 hours did not seem to significantly reduce c-Myc expression in PSN-1 PDAC cells. When combined with GEM and NP, BLX-3030 demonstrated mostly additive effects in PSN-1 cells. Preliminary data from the in vivo study show that BLX-3030 improves the antitumor activity of GEM and NP. Conclusion In summary, BLX-3030 potently inhibited the growth of both PDAC and pNET cells in vitro. The growth inhibitory activity of BLX-3030 in PDAC cell lines correlates with their c-Myc expression levels. BLX-3030 showed an additive effect when combined with standard of care drugs (GEM and NP) in PDAC cell lines. Overall, the novel CDK9 inhibitor, BLX-3030, demonstrated potent activity in pancreatic cancer cell line models and presents a promising preclinical lead for pancreatic cancer. (This work was supported in part by Biolexis Therapeutics and the Seena Magowitz Foundation). Citation Format: Yesenia Barrera-Millan, Zhaoliang Li, Hariprasad Vankayalapati, David J. Bearss, Daniel D. Von Hoff, Haiyong Han. Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic 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 5957.

Abstract 2680: Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis

Abstract Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic subtype of breast cancer. Lack of known targets, patient-to-patient variations of target antigens and resistance to immune checkpoint inhibitors make TNBC one of the most challenging cancers to treat. TNBC is enriched in tumor-infiltrating neutrophils (TINs). TINs are key contributors to pro-tumorigenic processes linked to “immunologically cold” tumors, in part by preventing recruitment of T-lymphocytes (TILs). Prior studies identified the Enhancer of zeste homolog 2 (EZH2) as a prominent methyltransferase expressed in primary and metastatic TNBC cells. EZH2 is an upstream regulator of the stimulator of interferon genes (STING), a critical innate sensor mediating anti-tumor responses that is inhibited in cold tumors. To date, EZH2/STING signaling in TINs is relatively unknown. Here, we hypothesized that EZH2 inhibition and subsequent STING activation could exert anti-tumor activity by modulating TINs. We derived EZH2-knockout (KO) and EZH2-overexpressed (OX) cell lines from the well-established 4T1 TNBC mouse cell line. We demonstrate a 10-fold decrease in lung metastasis and significant reduction of 4T1 EZH2 KO tumor growth compared to the parent 4T1 cell line. Concomitantly, the TIN:TIL ratio was significantly reduced in 4T1 EZH2 KO vs. control tumors. Overexpression of EZH2 in 4T1 TNBC cells did not affect the primary tumor growth or lung metastasis. A therapeutic study using the repeated injection of the STING agonist MSA-2 after tumor engraftment in vivo also demonstrated a decrease in lung metastasis and tumor growth in MSA-2 vs. vehicle injection with both 4T1 parent and EZH2 KO cell lines. Together, our findings suggest a key role for STING pathway modulation and TIN/TIL poise in primary TNBC progression and lung metastasis. Citation Format: Lenore Monterroza, Maria Parrilla, Sarah Samaranayake, Dormarie Rivera, Brian Dobosh, Periasamy Selvaraj, Rabindra M. Tirouvanziam. Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis [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 2680.

Abstract 5926: YB-1 inhibition as an effective approach for the treatment of osteosarcoma

Abstract Background: Osteosarcoma (OS) is a bone cancer primarily affecting adolescents and young adults, with about 20% of patients having detectable metastatic disease at the time of diagnosis. Despite a large number of clinical trial efforts, metastatic sarcoma remains a lethal disease, and there have been no meaningful advances in therapy or improvements in patient outcomes for decades. Y-box binding protein 1 (YB-1) is a multifunctional cold-shock protein that translationally activates diverse stress response factors with pro-metastatic activities in many cancer types. Importantly, YB-1 is strongly associated with poor outcomes in sarcoma patients and is a major metastatic driver in high-risk sarcomas. Methods: We recently described a novel compound, CET056, that inhibits cancer progression via YB-1 inhibition. We hypothesized that CET056 had the potential to suppress the progression of OS and could provide a new treatment for this disease. We screened osteosarcoma human and mouse cell lines for YB-1 protein expression. OS cells were functionally characterized for responses to CET056 in vitro and in vivo. Results: YB-1 expression was seen in nearly all cell lines tested. CET056 treatment of OS cells resulted in reduced cell viability and YB-1 protein expression in a dose- and time- dependent manner, as well as reduced formation of colonies and spheroids. In vivo efficacy studies, which employed a syngeneic subcutaneous tumor model, revealed that CET056 effectively inhibited tumor progression and enhanced overall survival in contrast to vehicle treated control mice. Conclusion: These results support our hypothesis and illustrate that YB-1 inhibition by CET056 is a promising candidate for further development as a therapy for OS. Citation Format: Kirsten Stefan, Arpit Dheeraj, Dhanir Tailor, Lara E. Davis, Sanjay V. Malhotra. YB-1 inhibition as an effective approach for the treatment of osteosarcoma [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 5926.

Abstract 2871: Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical cancer

Abstract Purpose Statement: Alterations in the PI3K/AKT signaling pathway are common in cervical cancer. The most commonly observed mutations are in PIK3CA (26%) and PTEN (8%). However, the biological and metabolic implications of acquiring these mutations in the cervical cancer genome are poorly characterized. Materials/Methods: Isogenic cell lines with PIK3CA E545K and PTEN null mutations were generated using Crispr/CAS9. Cell viability and clonogenic cell survival assays were performed in the presence and absence of inhibitors of glutamine and asparagine metabolism. Glucose and glutamine metabolism was evaluated by stable isotope labeling with U13C glucose and13C5 glutamine in presence of glutamine antagonist DON. RNA-seq was employed to evaluate transcriptomic alterations. Results: Introduction of the PIK3CA E545K mutation and PTEN mutation in cervical tumor cells leads to an increase in AKT signaling compared to the parental (wild-type) cells. Notably, RNASeq and mass spectrometric analysis of both the tumor cells and their culture media revealed heightened dependencies on glucose and glutamine in the tumor cells carrying the PIK3CA E545K and PTEN mutations as compared to the wild-type cells. Treatment with glutamine antagonists (DON, JHU-083) and GLS inhibitors (CB-839 and IACS-6274), resulted in reduced cell viability and clonogenic survival in tumor cells with PI3K/PTEN mutation compared to wild-type. Additionally, we observed a metabolic reliance on the amino acid asparagine in tumors with PI3K/PTEN alterations when treated with DON. Asparaginase in combination with DON effectively inhibited the proliferation of human and mouse cell lines derived from primary tumors with PTEN mutations. Furthermore, the incorporation of a glutamine antagonist and asparaginase into conventional chemoirradiation further enhanced the therapeutic efficacy in cervical tumors with PI3K/PTEN alterations which are otherwise treatment resistant. Conclusions: Orthogonal evaluations using glutamine agonists, GLS inhibitors, and mass spectrometric point to a significant role played by PI3K/PTEN mutations in altering cervix tumor cell metabolism towards a more glutamine dependent phenotype. This study suggests a potential strategy for improving the treatment of PI3K-AKT-altered cervical tumors by targeting glutamine and asparagine metabolism in combination with standard therapies. These treatment strategies are currently being evaluated in vivo using a patient derived xenograft model with PIK3CA E545K mutation as well as an immunocompetent cervical tumor model in mice with PTEN deletion. Citation Format: Nishanth Noel Gabriel, Naoshad Muhammad, Kevin Cho, Kay Jayachandran, Jin Zhang, Gary Patti, Julie Schwarz. Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical 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 2871.

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.

The molecular signature of BCR::ABLP210 and BCR::ABLT315I in a Drosophila melanogaster chronic myeloid leukemia model

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder resulting from a balanced translocation leading to BCR::ABL1 oncogene with increased tyrosine kinase activity. Despite the advancements in the development of tyrosine kinase inhibitors (TKIs), the T315I gatekeeper point mutation in the BCR::ABL1 gene remains a challenge. We have previously reported in a Drosophila CML model an increased hemocyte count and disruption in sessile hemocyte patterns upon expression of BCR::ABL1p210 and BCR::ABL1T315I in the hemolymph. In this study, we performed RNA sequencing to determine if there is a distinct gene expression that distinguishes BCR::ABL1p210 and BCR::ABL1T315I. We identified six genes that were consistently upregulated in the fly CML model and validated in adult and pediatric CML patients and in a mouse cell line expressing BCR::ABL1T315I. This study provides a comprehensive analysis of gene signatures in BCR::ABL1p210 and BCR::ABL1T315I, laying the groundwork for targeted investigations into the role of these genes in CML pathogenesis.

MTORC2-AKT signaling to PFKFB2 activates glycolysis that enhances stemness and tumorigenicity of intestinal epithelial cells.

Although elevated glycolysis has been widely recognized as a hallmark for highly proliferating cells like stem cells and cancer, its regulatory mechanisms are still being updated. Here, we found a previously unappreciated mechanism of mammalian target of rapamycin complex 2 (mTORC2) in regulating glycolysis in intestinal stem cell maintenance and cancer progression. mTORC2 key subunits expression levels and its kinase activity were specifically upregulated in intestinal stem cells, mouse intestinal tumors, and human colorectal cancer (CRC) tissues. Genetic ablation of its key scaffolding protein Rictor in both mouse models and cell lines revealed that mTORC2 played an important role in promoting intestinal stem cell proliferation and self-renewal. Moreover, utilizing mouse models and organoid culture, mTORC2 loss of function was shown to impair growth of gut adenoma and tumor organoids. Based on these findings, we performed RNA-seq and noticed significant metabolic reprogramming in Rictor conditional knockout mice. Among all the pathways, carbohydrate metabolism was most profoundly altered, and further studies demonstrated that mTORC2 promoted glycolysis in intestinal epithelial cells. Most importantly, we showed that a rate-limiting enzyme in regulating glycolysis, 6-phosphofructo-2-kinase (PFKFB2), was a direct target for the mTORC2-AKT signaling. PFKFB2 was phosphorylated upon mTORC2 activation, but not mTORC1, and this process was AKT-dependent. Together, this study has identified a novel mechanism underlying mTORC2 activated glycolysis, offering potential therapeutic targets for treating CRC.

Abstract B084: Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer

Abstract Background: Over 20,000 women are diagnosed with ovarian cancer annually, and more than half will die within 5 years. This rate has changed little in the last 30 years, highlighting the need for therapy innovation. Although immunotherapy has revolutionized cancer treatment, efforts to harness endogenous patient immune responses have yielded limited therapeutic activity in ovarian cancer patients. T cells engineered to express a T cell receptor (TCR) targeting proteins uniquely overexpressed in tumors have the potential to control tumor growth without toxicity. Mesothelin (Msln) is over-expressed in ovarian cancer, contributes to the malignant and invasive phenotype, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors. Methods: The ID8VEGF mouse cell line was used to evaluate if T cells engineered to express a mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill ovarian cancer. Tumor-bearing mice were treated with TCRMsln T cells plus anti-PD-1, anti-Tim-3 or anti-Lag-3 checkpoint-blocking antibodies alone or in combination, ultimately targeting up to three inhibitory receptors simultaneously. Single-cell RNA-sequencing (scRNAseq) was used to profile the impact of combination checkpoint blockade on engineered T cells and the tumor microenvironment (TME). Results: In a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated in established tumors, delayed ovarian tumor growth, and prolonged mouse survival. However, elements in the TME limited engineered T cell persistence and cytolytic function. Triple checkpoint blockade, but not single- or double-agent treatment, dramatically increased antitumor function by intratumoral TCRMsln T cells. scRNAseq of tumor-infiltrating cells revealed distinct transcriptome changes in engineered and endogenous T cells and myeloid-derived cells. Engineered T cells, when combined with triple checkpoint blockade, increased expression of genes associated with effector and memory gene signatures, including proliferation and metabolic function, and reduced expression of genes associated with exhaustion. Moreover, combining adoptive immunotherapy with triple checkpoint blockade significantly prolonged survival in the cohort of treated tumor-bearing mice, relative to mice that received TCRMsln T cells alone or with anti-PD1 or double-agent treatments. Conclusions: Inhibitory receptor/ligand interactions within the TME can dramatically reduce T cell function, suggesting tumor cells may upregulate the ligands for PD-1, Tim-3 and Lag-3 for protection from tumor-infiltrating lymphocytes. In an advanced ovarian cancer model, triple checkpoint blockade significantly improved engineered T cell function and outcomes in mice in a setting where single checkpoint blockade had no significant activity. These results suggest that disrupting multiple inhibitory pathways simultaneously, which can be more safely pursed in a cell intrinsic form through genetic engineering, may be necessary for improved efficacy in patients. Citation Format: Kristin G. Anderson, Yapeng Su, Madison G. Burnett, Breanna M. Bates, Magdalia L. Rodgers Suarez, Susan L. Ruskin, Valentin Voillet, Raphael Gottardo, Philip D. Greenberg. Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B084.

An integrative platform for detection of RNA 2′-O-methylation reveals its broad distribution on mRNA

Ribose 2'-O-methylation is involved in critical biological processes, but its biological functions and significance in mRNAs remain underexplored. We have developed NJU-seq, a sensitive method for unbiased 2'-O-methylation (Nm) profiling, and Nm-VAQ, a site-specific quantification tool. Using these tools in tandem, we identified thousands of Nm sites on mRNAs of human and mouse cell lines, of which 68 of 84 selected sites were further validated to be more than 1% 2'-O-methylated. Unlike rRNA, most mRNA Nm sites were from 1% to 30% methylated. In addition, mRNA Nm was dynamic, changing according to the circumstance. Furthermore, we show that fibrillarin is involved as a methyltransferase. By mimicking the detected Nm sites and the context sequence, the RNA fragments could be 2'-O-methylated and demonstrated higher stability but lower translation efficiency. Last, profiling of Nm sites in lung surgery samples revealed common signatures of lung cancer pathogenesis, providing potential new diagnostic markers.

Abstract A010: Mitochondrial depletion and metabolic reprogramming is a novel phenotype of Lynch syndrome-related endometrial carcinogenesis

Abstract Background: Lynch syndrome (LS), defined by mutations in DNA mismatch repair genes including MSH2, carries a 60% lifetime risk of developing endometrial cancer (EC). Our recent studies in Msh2-deficient mice revealed mitochondrial dysfunction in EC pathogenesis. We aimed to define mechanisms of mitochondrial dysfunction and impact on metabolism to identify targets for EC prevention. Methods: We assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2flox/flox, abbreviated Msh2KO), primary cell lines from this model, and human EC cells with isogenic MSH2-knockdown. mtDNA damage was measured using RT-PCR. Mitochondrial content was quantified in vitro by immunofluorescence (IF) for TOM20 and in vivo by anti-TOM20 immunohistochemistry (IHC). Mitochondrial stress tests (MSTs) assessed mitochondrial function in vitro. Metabolite profiling via high resolution ion chromatography mass spectrometry was conducted on Msh2KO EC and MSH2-intact EC from the Pten +/− mouse model. Glycolysis was suppressed in vitro by replacing glucose with galactose in culture media. Results: Baseline mtDNA damage was elevated in MSH2-deficient MFE280 and RL95-2 EC cell lines by 4.8- to 7.9-fold compared to MSH2-intact KLE and Hec50 cells (p<0.001). MSH2 knockdown increased mtDNA damage in KLE and Hec50 cells by 2.5- to 5.7-fold (p<0.05). MSH2-deficient mouse and human cell lines had lower mitochondrial content compared to MSH2-intact counterparts as indicated by TOM20 staining, with over 60% reductions in mitochondrial volume in MSH2-deficient mouse cell lines and over 95% reductions in MSH2-deficient human EC cells compared to respective controls (p<0.0001). IHC showed reduced mitochondrial content during EC development in Msh2KO mice and in human LS EC tissues. MSTs revealed decreased baseline and induced mitochondrial function in Msh2KO cells relative to controls and in MSH2-deficient human cells (MFE280, RL95-2, and Hec59) compared to MSH2-intact human cells (KLE, Hec1a, Hec50), with p<0.0001. Within the top 10 differentially modulated metabolites in Msh2KO EC compared to Pten +/− EC were glucose 6-phosphate (log2 fold-change (FC) -1.79), mannose 6-phosphate (log2FC -1.77), fructose 6-phosphate (log2FC -1.54), and lactate (log2FC 0.68), padj<0.05. Galactose reduced viability in MSH2-deficient mouse and human cells more than in their MSH2-intact counterparts (p<0.01). Conclusions: We identified mitochondrial depletion as a novel phenotype of MSH2-deficient EC. mtDNA damage in MSH2-deficient cells suggests a role for MSH2 in mtDNA integrity. MSH2-deficient EC is characterized by mitochondrial DNA damage, mitochondrial content reduction, and decreased mitochondrial function. Decreased mitochondrial respiration for energy production facilitates metabolic reprogramming toward glycolysis, signified by increased usage of glycolytic intermediates and dependence on glycolysis for survival. Mitochondrial and metabolic aberrations could be evaluated as novel biomarkers for EC development and targets for prevention in women with LS. Citation Format: Mikayla B. Bowen, Brenda Melendez, Anna Zal, Collene Jeter, Wai Kin Chan, Lin Tan, Philip Lorenzi, Diana Moreno, Leah Peralta, Qian Zhang, Nisha Gokul, Karen Lu, Hyun-Eui Kim, Melinda S. Yates. Mitochondrial depletion and metabolic reprogramming is a novel phenotype of Lynch syndrome-related endometrial carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A010.

Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo

Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur invivo. Using denaturing purification of invivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.

Musashi-2 potentiates colorectal cancer immune infiltration by regulating the post-translational modifications of HMGB1 to promote DCs maturation and migration

Post-translational modifications (PTMs) of the non-histone protein high-mobility group protein B1 (HMGB1) are involved in modulating inflammation and immune responses. Recent studies have implicated that the RNA-binding protein (RBP) Musashi-2 (MSI2) regulates multiple critical biological metabolic and immunoregulatory functions. However, the precise role of MSI2 in regulating PTMs and tumor immunity in colorectal cancer (CRC) remains unclear. Here, we present data indicating that MSI2 potentiates CRC immunopathology in colitis-associated colon cancer (CAC) mouse models, cell lines and clinical specimens, specifically via HMGB1-mediated dendritic cell (DC) maturation and migration, further contributes to the infiltration of CD4+ and CD8+ T cells and inflammatory responses. Under stress conditions, MSI2 can exacerbate the production, nucleocytoplasmic transport and extracellular release of damage-associated molecular patterns (DAMPs)-HMGB1 in CRC cells. Mechanistically, MSI2 mainly enhances the disulfide HMGB1 production and protein translation via direct binding to nucleotides 1403–1409 in the HMGB1 3′ UTR, and interacts with the cytoplasmic acetyltransferase P300 to upregulate its expression, further promoting the acetylation of K29 residue in HMGB1, thus leading to K29-HMGB1 nucleocytoplasmic translocation and extracellular release. Furthermore, blocking HMGB1 activity with glycyrrhizic acid (Gly) attenuates MSI2-mediated immunopathology and immune infiltration in CRC in vitro and in vivo. Collectively, this study suggests that MSI2 may improve the prognosis of CRC patients by reprogramming the tumor immune microenvironment (TIME) through HMGB1-mediated PTMs, which might be a novel therapeutic option for CRC immunotherapy.

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines aremodified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assayprovides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

Abstract B026: Targeting wild type and ESR1 mutations as neoantigens in hormone receptor positive breast cancer

Abstract Hormone receptor positive (HR+) metastatic breast cancer remains a difficult clinical problem. Endocrine Therapies (ET) have remained the mainstay of therapy for decades. However, a large percent of these patients become resistant to the therapies and succumb to the disease. One of the most common resistance mechanisms is the ER alpha type I (ESR1) point mutations with amino acid substitutions such that they become constitutively active. About 40-50% of metastatic HR+ patients develop these mutations. Hence, there is a need to develop strategies that target WT and mutant ESR1 in order to achieve robust anti-tumor immune responses. Type I polarized dendritic cell (DC1) vaccine developed by our group has shown to be a powerful immunotherapeutic tool to treat breast cancer by boosting the CD4 Th1 responses against oncodrivers like HER2 and HER3. This is facilitated by identification of immunogenic human peptides using a novel peptide screening method targeting these oncodrivers and breaching the tolerance. Having strong evidence of the effectiveness of DC1 vaccines, our study focusses on testing efficacy of DC1 pulsed WT ESR1 peptides (DC1-WT ESR1) and clinically prevalent ESR1 mutations/neoantigens (DC1-Mutant ESR1) in pre-clinical mouse model. In this study, we tested the homology between human and murine WT and mutated ESR1 peptides and also assessed the efficacy of DC1-WT ESR1 and DC1-Mutant ESR1 vaccines in mice to generate CD4 Th1 response (IFNγ production). To develop pre-clinical ESR1 mouse tumor models, two different cell lines were validated for nuclear ESR1 expression. We tested the efficacy of DC1-WT ESR1 vaccine in orthotopic ESR1+ tumor bearing mice by administrating six DC1 vaccines intratumorally. Our results show around 90% and above homology between human and murine WT and mutated ESR1 peptides generating immunogenic responses in mice. In vitro treatment of ESR1+ mouse cell lines with IFNγ resulted in reduced proliferation. This effect was in line with the mouse tumor model, wherein we observed reduced tumor burden post administration of six DC1-WT ESR1 vaccines intratumorally in ESR1+ pre-clinical mouse models. In addition, we observed about a 12% of complete regression in these mice. Upon using combinatorial approaches such as fulvestrant and DC1-WT ESR1, we observed about a 14% of complete regression, however, the DC1 alone mice showed 62% responders and did a lot better than the fulvestrant alone. Overall, these findings suggest that DC1 vaccines targeting ESR1 could be a promising tool to treat aggressive ESR1+ breast cancer. We now aim to use combinatorial approaches such as CDK4/6 inhibitors and DC1-WT ESR1 to boost the anti-tumor responses and further test the efficacy of DC1-Mutant ESR1 vaccines using pre-clinical ESR1+ mouse models. Citation Format: Namrata Gautam, Gabriella Albert, Colin Synder, Brian Czerniecki. Targeting wild type and ESR1 mutations as neoantigens in hormone receptor positive breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B026.

Comparative Cytotoxic Effect Of Methanolic Extract Of Cinnamon Zeylanicum Bark With Commercial Trans Cinnamaldehyde In Animal Cell Culture

A comparison of the cytotoxic effects of methanolic cinnamon extract (MCE) from the bark of Cinnamon zeylanicum L. (Lauraceae) and commercially available cinnamaldehyde was carried out using mouse cell line (C2C12). The bark of Cinnamon zeylanicum was extracted in 50% methanol and trans cinnamaldehyde was procured commercially. Methanolic cinnamon extract (MCE) is highly concentrated as a result showing 50% cell cytotoxicity to C2C12 cells at concentration 100 μg/ml than commercial cinnamaldehyde at 66.08 μg/ml.

Preclinical models of breast cancer: B6BC, a transplantable hormone receptor-positive C57BL/6 mouse cell line.

Preclinical models of breast cancer: B6BC, a transplantable hormone receptor-positive C57BL/6 mouse cell line.

PSIP1/LEDGF reduces R-loops at transcription sites to maintain genome integrity

R-loops that accumulate at transcription sites pose a persistent threat to genome integrity. PSIP1 is a chromatin protein associated with transcriptional elongation complex, possesses histone chaperone activity, and is implicated in recruiting RNA processing and DNA repair factors to transcription sites. Here, we show that PSIP1 interacts with R-loops and other proteins involved in R-loop homeostasis, including PARP1. Genome-wide mapping of PSIP1, R-loops and γ-H2AX in PSIP1-depleted human and mouse cell lines revealed an accumulation of R-loops and DNA damage at gene promoters in the absence of PSIP1. R-loop accumulation causes local transcriptional arrest and transcription-replication conflict, leading to DNA damage. PSIP1 depletion increases 53BP1 foci and reduces RAD51 foci, suggesting altered DNA repair choice. Furthermore, PSIP1 depletion increases the sensitivity of cancer cells to PARP1 inhibitors and DNA-damaging agents that induce R-loop-induced DNA damage. These findings provide insights into the mechanism through which PSIP1 maintains genome integrity at the site of transcription.

Quantitative catalogue of mammalian mitotic chromosome-associated RNAs

The faithful transmission of a cell’s identity and functionality to its daughters during mitosis requires the proper assembly of mitotic chromosomes from interphase chromatin in a process that involves significant changes in the genome-bound material, including the RNA. However, our understanding of the RNA that is associated with the mitotic chromosome is presently limited. Here, we present complete and quantitative characterizations of the full-length mitotic chromosome-associated RNAs (mCARs) for 3 human cell lines, a monkey cell line, and a mouse cell line derived from high-depth RNA sequencing (3 replicates, 47 M mapped read pairs for each replicate). Overall, we identify, on average, more than 20,400 mCAR species per cell-type (including isoforms), more than 5,200 of which are enriched on the chromosome. Notably, overall, more than 2,700 of these mCARs were previously unknown, which thus also expands the annotated genome of these species. We anticipate that these datasets will provide an essential resource for future studies to better understand the functioning of mCARs on the mitotic chromosome and in the cell.

RNA-seq analysis reveals modulation of inflammatory pathways by an enriched-triterpene natural extract in mouse and human macrophage cell lines

Chronic inflammation is crucial in developing insulin resistance and type 2 diabetes. Previous studies have shown that a leaf extract of Eucalyptus tereticornis, with ursolic acid (UA), oleanolic acid (OA), and ursolic acid lactone (UAL) as the main molecules (78 %) mixed with unknown minor metabolites (22 %), provided superior anti-inflammatory, hypoglycemic, and hypolipidemic effects than reconstituted triterpenoid mixtures in macrophage cell lines and a pre-diabetic mouse model. Further identification of the molecular mechanisms of action of this mixture of triterpenes is required. This study aims to analyse the RNA expression profiles of mouse and human macrophage cell lines treated with the natural extract and its components. Activated macrophage cell lines were treated with the natural extract, UA, OA, UAL or a triterpene mixture (M1). RNA was extracted and sequenced using the DNBseq platform and the EnrichR software to perform gene enrichment analysis using the Gene Ontology database, Kyoto Encyclopedia of Genes and Genomes, and Reactome. To conduct clustering analysis, we standardised the normalised counts of each gene and applied k-means clustering. The combination of molecules in the natural extract has an additive or synergic effect that affects the expression of up-regulated genes by macrophage activation. Triterpenes (M1) regulated 76 % of human and 68 % of mouse genes, while uncharacterised minority molecules could regulate 24 % of human and 32 % of mouse genes. The extract inhibited the expression of many cytokines (IL6, IL1, OSM), chemokines (CXCL3), inflammatory mediators (MMP8 and MMP13) and the JAK-STAT signalling pathway in both models. The natural extract has a more powerful immunomodulatory effect than the triterpene mixture, increasing the number of genes regulated in mouse and human models. Our study shows that Eucalyptus tereticornis extract is a promising option for breaking the link between inflammation and insulin resistance.

New Pd(II)-pincer type complexes as potential antitumor drugs: synthesis, nucleophilic substitution reactions, DNA/HSA interaction, molecular docking study and cytotoxic activity.

Two new complexes of Pd(II), [Pd(L1)Cl]Cl (Pd1) and [Pd(L2)Cl]Cl (Pd2), (where L1 = N2,N6-bis(5-methylthiazol-2-yl)pyridine-2,6-dicarboxamide and L2 = N2,N6-di(benzo[d]thiazol-2-yl)pyridine-2.6-dicarboxamide) were synthesized. Characterization of the complexes was performed using elemental analysis, IR, 1H NMR spectroscopy and MALDI-TOF mass spectrometry. The nucleophilic substitution reactions of complexes with L-Methionine (L-Met), L-Cysteine (L-Cys) and guanosine-5'-monophosphate (5'-GMP) were studied by stopped-flow method at physiological conditions (pH = 7.2 and 37 °C). Complex Pd1 was more reactive than Pd2 in all studied reactions, while the order of reactivity of the selected ligands was: L-Met > L-Cys > 5'-GMP. The interaction of complexes with calf thymus-DNA (CT-DNA) was studied by Uv-Vis absorption and fluorescence emission spectroscopy. Competitive binding studies with intercalative agent ethidium bromide (EB) and minor groove binder Hoechst 33258 were performed as well. Both complexes interacted with DNA through intercalation and minor groove binding, where the latter was preferred. Additionally, the interaction of Pd1 and Pd2 complexes with human serum albumin (HSA) was studied employing fluorescence quenching spectroscopy. The results indicate a moderate binding affinity of complexes, with slightly stronger binding of the Pd1. Fluorescence competition experiments with site-markers (eosin Y and ibuprofen) for HSA were used to locate the binding site of Pd1 to the HSA. Additionally, the interaction with DNA and HSA was studied by molecular docking and the revealed results were in good agreement with the experimentally obtained ones. Pd1 complex exhibited cytotoxicity toward human (HCT116) and mouse cell lines (CT26) of colorectal cancer, mouse (4T1) and human (MDA-MB468) breast cancer lines and non-cancerous mouse mesenchymal stem cells (mMSC). In addition, Pd1 complex demonstrated significant selectivity towards cancer cells over non-cancerous mMSC, indicating a high potential to eliminate malignant cells without affecting normal cells. It induced apoptosis in CT26 cells, effectively arrested the cell cycle in the S phase, and selectively down-regulated cyclin D and cyclin E. Moreover, it can alter the expression of cell cycle regulators by increasing p21 and decreasing p-AKT. These findings confirm its ability to disrupt key tumor cell survival signals and suggest that the Pd1 complex is a potent candidate for effective cancer treatment.