Decreased Cell Growth Research Articles

Decreased mitochondrial transcription factor A and mitochondrial DNA copy number promote cyclin-dependent kinase inhibitor 1A expression and reduce tumorigenic properties of colorectal cancer cells

PurposeColorectal cancer is one of the most common and deadliest cancer types worldwide. In the last years, changes in the mitochondrial DNA (mtDNA) copy number have been described to correlate with the prognostic outcome for colorectal cancer patients by impacting different tumorigenic properties. One key regulator of mtDNA is the mitochondrial transcription factor A (TFAM) that acts as a limiting factor of mtDNA copy number. Here, we investigated the effect of TFAM deficiency on mtDNA and tumorigenic properties in the human colorectal cancer cell line SW480.MethodsTFAM expression was stably downregulated in the colorectal cancer cell line SW480 using the CRISPR-Cas9 approach. To dissect the molecular alterations induced by deletion of TFAM, RNA sequencing and gene set enrichment analysis was performed on TFAM-wild-type and TFAM-deficient SW480 cells. Functional consequences of TFAM downregulation were assessed in cellular assays.ResultsWe showed that TFAM deficiency leads to decreased mtDNA copy number and reduced expression of mtDNA-encoded genes. TFAM-deficient cells also revealed higher activity of senescence-associated β-galactosidase and decreased cell growth parameters. Moreover, RNA sequencing showed that the expression of cyclin dependent kinase inhibitor 1A (CDKN1A/p21) is significantly increased in TFAM-deficient cells.ConclusionOur results suggest that TFAM-induced changes of the mitochondrial genome lead to upregulated CDKN1A/p21 expression in colorectal cancer cells identifying p21 as a new possible linker between mitochondria and nucleus.

Abstract A022: Disrupting redox homeostasis initiates anti-tumor immune responses in pancreatic cancer

Abstract Although immune therapy has made impressive progress in cancer treatment, pancreatic ductal adenocarcinoma (PDAC) remains an exception, likely due to its immunosuppressive tumor microenvironment that prevents immune responses against the tumor. Our current work aims to reactivate the suppressive immune infiltrate using radiation therapy and oxidative stress to potentiate antitumor immune responses in PDAC. Our lab previously found that PDAC cells are dependent on the antioxidant peroxiredoxin-4 (PRDX4) expression. PRDX4 prevents excessive levels of reactive oxygen species in cells and while high PRDX4 expression is associated with a more aggressive disease, it is dispensable in normal tissue. As such, disrupting redox homeostasis by inhibiting PRDX4 represents a targetable vulnerability in cancer cells. To investigate the radio-sensitizing potential of PRDX4, we knocked-down (KD) PRDX4 in human pancreatic tumor cells as well as in mouse cancer cells obtained from the spontaneous KPC model (driven by the KRas and P53 mutations), in combination with radiation. Presence and quantification of DNA damage were performed by immunofluorescence, and impaired cell survival was analyzed by clonogenic assays. Changes in immune secretion were assessed by RT-qPCR and Elisa and in vitro macrophage phenotyping was performed upon exposure to tumor cell conditioned media. Our data so far show an accumulation of cytosolic DNA upon PRDX4 KD in PDAC cells, with the presence of micronuclei as well as less aggregated DNA. Interestingly, targeting PRDX4 before radiation amplifies the release of radiation-induced cytosolic DNA while significantly decreasing cell growth and survival, compared to radiation alone. Cytosolic DNA has been shown to trigger innate immune signaling in tumor cells. Consistently, PRDX4 loss induces the upregulation of many pro-inflammatory genes, including CCL5, CCL20, and CXCL10, in a STING-dependent manner. Combining PRDX4 KD with radiation leads to a higher gene upregulation of these immune stimulatory chemokines, as well as an increased protein secretion within tumor cell media. These findings support an amplified immunomodulatory effect of radiation when combined with PRDX4 loss in PDAC cells. To assess whether targeting PRDX4 can remodel the tumor microenvironment, we applied tumor conditioned media from cancer cells on isolated naive mouse CD45+/CD11c+/F4/80+ macrophages. Media from cells treated with PRDX4 KD + radiation strongly increased the migration ability of macrophages while modulating the expression of a subset of chemokine-encoding genes in immune cells. Using the spontaneous KPC model depleted for PRDX4, along with syngeneic orthotopic models, we are now aiming to confirm this radio-sensitizing and immune-modulatory potential of PRDX4 in vivo. The aggressiveness of PDAC requires research towards novel treatment strategies and our data strongly support evidence for targeting cancer metabolism, particularly antioxidant systems, in synergy with existing cancer therapies, like radiation and immune therapy. Citation Format: Lucie Malbeteau, Emily Poulton, Vishal Pandya, Marianne Koritzinsky. Disrupting redox homeostasis initiates anti-tumor immune responses in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A022.

Synthesis of rice husk-zinc oxide-chitosan-based sponge: A green and eco-friendly nanocomposite for enhanced antibacterial, hemostasis, and wound healing applications

Wounds are a serious health concern that is often made worse by conditions including bacterial infections, poor blood circulation, and decreased cell growth. This work presents a novel sustainable wound dressing sponge made of zinc oxide nanoparticles (ZnO-NPs), rice husk (RH), and chitosan (CS). CS functions as a biocompatible matrix that induces cell adhesion and proliferation, while ZnO-NPs provide their well-documented antibacterial activity, which is critical for wound infection prevention. RH, a natural and renewable resource, strengthens the structural integrity of the composite and offers further antioxidant advantages. CS/RH/ZnO nanocomposite was synthesized by the freeze-drying method, and its evaluation was done through various tests, including wound healing assays, antibacterial tests, and histological analysis. Antibacterial tests revealed substantial inhibition zones against Pseudomonas aeruginosa (24 mm) and Staphylococcus aureus (22 mm), which is comparable to other commonly used wound care materials, validating the material’s effectiveness in preventing infections. Within 14 days, in vivo testing showed a 75 % wound closure rate, which was far higher than the control group's 50 % closure rate. Histopathological analysis showed marked re-epithelialization and active tissue regeneration, evidenced by pronounced acanthosis in the epidermis. In addition to improving therapeutic effectiveness, this multipurpose approach to wound care lessens dependency on non-renewable resources. This study demonstrated that the development of a sponge wound dressing from rice husk, a natural agricultural byproduct, represents a sustainable approach to wound care. By reducing reliance on non-renewable resources and minimizing the environmental impact of medical waste, it offers an eco-friendly solution that enhances wound management.

Evaluating STING Agonist Impact on Kaposi’s Sarcoma-Associated Virus-related Cancers

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiological agent of multiple malignancies associated with compromised immunity, such as Kaposi’s sarcoma (KS) and Primary Effusion Lymphoma (PEL). KSHV infection is lifelong with no available treatment or vaccines and poses a high risk to immunocompromised individuals. The virus is transmitted via the oral and sexual route, and has a biphasic life cycle with a short lytic replication cycle followed by long-term latency, with the expression of few genes making detection difficult. Reactivation from latency results in a cascade of viral gene expression and pathogenesis. Critical to controlling KSHV infection is the cGAS-STING DNA-sensing pathway (STING), known to activate the type I interferon response and induce cancer cell death or growth inhibition. STING exhibits both anti-viral and anti-tumor responses, showing promise as a protein of interest to study for the development of a therapeutic treatment. By examining the in vitro effectiveness of STING agonists in inhibiting KSHV oncogenesis in PEL, the study aimed to provide the foundation for a potential novel therapeutic approach. Six patient-derived PEL cell lines were treated with the STING agonist diaminobenzamidazole (diABZI) in a dose-dependent manner. Four of the PEL cell lines exhibited decreased cell growth, viability, and colony formation, while two PEL cell lines were resistant to treatment, correlating with the presence or absence of STING expression. RNA-sequencing was performed and discovered upregulation of multiple interferon-stimulated genes (ISG) in responsive cell lines. Further investigations will explore STING agonists’ inhibition of PEL oncogenesis in vitro via colony formation and in vivo using a PEL xenograft model with NSG mice. Successful completion of this project will offer insight into potential clinical treatments for targeting KSHV infection and reactivation.

Glucose Transporter 1 Inhibitors Induce Autophagy and Synergize With Lenvatinib in Thyroid Cancer Cells.

Less differentiated thyroid cancer may upregulate the expression of glucose transporter 1 (GLUT1) and increase glycolytic activity. However, it is uncertain whether GLUT1 can be used as a target for therapy. Thyroid cancer cell lines were treated with two different GLUT1 inhibitors, STF-31 and BAY-876. Functional assays were conducted to evaluate the effects of these inhibitors on cell biology. GLUT1 inhibitors dose-dependently decreased cell growth and clonogenicity of thyroid cancer cells. Cell cycle analysis showed that these inhibitors caused G2/M arrest instead of apoptosis. Additionally, treatment with GLUT1 inhibitors led to the activation of autophagy. In both the Transwell and spheroid models, GLUT1 inhibitors significantly suppressed cell invasiveness. Moreover, GLUT1 inhibitors demonstrated synergistic interactions when combined with lenvatinib. Treatment with GLUT1 inhibitors activates autophagy and provokes cell cycle arrest, accompanied by a decrease in colony formation and invasive capacity in thyroid cancer cells.

MAD2L1 supports MYC-driven liver carcinogenesis in mice and predicts poor prognosis in human hepatocarcinoma.

Mitotic arrest deficient 2 like 1 (MAD2L1) is a component of the mitotic spindle assembly checkpoint implicated in cancer cell proliferation and tumorigenesis. The functional role of MAD2L1 in hepatocellular carcinoma (HCC) has not been adequately investigated, especially in vivo. In the current manuscript, we sought to address the function of MAD2L1 in hepatocarcinogenesis. We found that MAD2L1 expression is upregulated in human HCCs, where its expression is associated with higher aggressive tumor grade, elevated proliferative activity, and poor prognosis. In human HCC cell lines, MAD2L1 knockdown led to decreased cell growth. Moreover, RNA-seq results demonstrated that MAD2L1 silencing induces the expression of genes associated with cell cycle, DNA replication, and various cancer-related pathways, supporting the critical role of MAD2L1 during HCC growth and differentiation. In a c-MYC-induced mouse HCC model, we revealed an increased expression of Mad2l1. Furthermore, Mad2l1 CRIPSR-mediated silencing prevented c-MYC-driven mouse liver development. Altogether, our study suggests that MAD2L1 plays a crucial role in hepatocarcinogenesis, and that its suppression could be a promising therapeutic strategy for treating human HCC.

BRD9 promotes the progression of gallbladder cancer via CST1 upregulation and interaction with FOXP1 through the PI3K/AKT pathway and represents a therapeutic target

Gallbladder cancer (GBC) is highly aggressive and has poor prognosis, with most patients only diagnosed at an advanced stage. Furthermore, treatment options are limited, and their effect is unsatisfactory. Bromodomain-containing protein (BRD) is an epigenetic regulator that plays a carcinogenic role in several tumors, including squamous cell lung cancer, acute myeloid leukemia, synovial sarcoma, and malignant rhabdomyosarcoma. However, the expression, biological function, and molecular mechanisms of action of BRD9 in GBC are still unknown. Kaplan–Meier analysis, qRT-PCR, and analysis of clinical features were used to assess the clinical significance of BRD9 in GBC. Cell Counting Kit-8 and colony formation assays were performed to determine the effects of BRD9 on cell growth. The functional role of BRD9 in GBC was explored using qRT-PCR, western blotting, siRNA, and CHIP-qPCR. mRNA sequencing was performed to explore the underlying mechanisms of BRD9, and a nude mouse model of GBC was established to explore the anti-tumor effects of the BRD9 inhibitor I-BRD9 in vivo. BRD9 expression was elevated in GBC tissues compared with adjacent non-tumor tissues, and high BRD9 expression was associated with poor prognosis in patients with GBC. BRD9 knockdown by siRNA significantly decreased cell growth. Targeting BRD9 with I-BRD9 inhibited the proliferation of GBC cells without significant toxic effects. Additionally, I-BRD9 treatment suppressed CST1 expression in GBC cell lines, thereby inhibiting the PI3K-AKT pathway. The transcription factor FOXP1 was found to interact with BRD9 to regulate CST1 expression. Collectively, these results suggest that BRD9 may be a promising biomarker and therapeutic target for GBC.

Abstract A079: SB-216 inhibits oncogenic beta-tubulin subtypes in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies, with only 12% of patients living longer than five years after diagnosis. Recent studies have shown that inhibiting microtubule dynamics may be a promising therapeutic avenue for treating patients with this cancer. Microtubules are critical in cancer cell growth and division, as well as intracellular cytoskeletal support and internal trafficking. In PDAC, βIII- and βIVb-tubulin are highly upregulated in contrast to normal pancreas cells. Decreased expression of βIII- and βIVb-tubulin, through silencing or inhibition, correlates with reduced PDAC cell growth, tumorigenic potential, metastases in xenograft models, and chemoresistance. We hypothesized that a novel colchicine binding site beta-tubulin inhibitor, SB-216, will decrease cell growth while also decreasing the expression of oncogenic βIII- and βIVb-tubulin. To examine the anti-proliferative effects, PDCL110, a PDAC patient-derived cell line, cells were seeded (6,000 cells/well), and after 24 hours, treated with 0.1% DMSO (control), 2 nM, 5 nM, 10 nM, or 15 nM SB-216. Plates were analyzed on the IncuCyte Live-Cell Analysis System for six days, where phase contrast images of confluency were captured every four hours. To characterize the mRNA expression of βIII- and βIVb-tubulin, Panc-1 and PDCL110 cells were treated with 0.1% DMSO and 5 nM SB-216 for 48 hours. Next, total RNA was extracted using a TRIzol reagent. cDNAs were prepared using the SYBR Green RNA Reverse Transcription kit, and mRNA expression of beta-tubulin isotypes was quantified using the QuantStudio Real-Time PCR system. The expression of tubulin isotypes was normalized with the internal control ACTIN. Cell growth curves illustrated a significant (p<0.006) decrease in proliferation after 48 hours of treatment at all tested concentrations of SB-216. After 72 hours, cell growth inhibition became more significant (p<0.0001) in a dose-dependent manner. SB-216 treatment of a commercially available PDAC cell line (Panc-1) led to decreased mRNA expression of TUBB3 (class βIII, p<0.05) and TUBB4B (class βIVb, p<0.05) compared to DMSO control. Similarly, TUBB4B expression was significantly (p<0.05) decreased in the PDCL110 cells upon SB-216 treatment. These results enhance our confidence in utilizing SB-216 as a therapeutic agent against PDAC. Future studies are directed at elucidating the effects of SB-216 on metastases, chemoresistance, and mitochondrial function. Through an improved understanding of the mechanisms behind microtubule inhibition, we can meet the urgent clinical need of finding targeted treatments for this complex cancer. Citation Format: Lauren C Gattie, Chun Cai, Rui Wang, Wei Li, Evan Glazer. SB-216 inhibits oncogenic beta-tubulin subtypes in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A079.

TRIM47 promotes the Warburg effect and reduces ferroptosis in prostate cancer by FBP1 and FOXO1.

Prostate cancer (PC), a malignant tumor occurring in the male prostate tissue, has a high incidence rate. In this study, we explored the role of tripartite motif 47 (TRIM47) in the progression of PC and its underlying mechanism. PC and paracancerous tissues were collected from Shenzhen Peoples's Hospital. The following methods were employed in this experiment: quantitative polymerase chain reaction (qPCR), immunofluorescent staining, cell counting kit-8 (CCK-8), ethynyl deoxyuridine (EdU), and Western blot. The expression levels of TRIM47 were up-regulated in patients with PC. TRIM47 was found to promote cell growth and induce the Warburg effect, while also reducing ferroptosis in PC cells. Conversely, the knockdown of TRIM47 [small interfering RNA, (si)-TRIM47] decreased cell growth and the Warburg effect, while promoting ferroptosis in PC cells. Additionally, TRIM47 was observed to induce the protein expression levels of fructose-1,6-bisphosphatase 1 (FBP1) and forkhead box protein O1 (FOXO1) in PC cells. Further, TRIM47 protein was found to interact with both the FBP1 and FOXO1 proteins in the PC cells. The inhibition of FBP1 attenuated the effects of TRIM47 on the Warburg effect in PC cells, while the inhibition of FOXO1 diminished the effects of TRIM47 on ferroptosis in PC cells. Our findings suggest that TRIM47 promotes the Warburg effect of PC by inducing FBP1 and FOXO1. Thus, our findings suggest that targeting TRIM47 could serve as a viable therapeutic strategy for the treatment of PC.

P53 Status Influences the Anti-proliferative Effect Induced by IFITM1 Inhibition in Estrogen Receptor-positive Breast Cancer Cells.

Interferon-induced trans-membrane protein 1 (IFITM1) is known to be involved in breast cancer progression. We aimed to investigate its role in estrogen receptor (ER)-positive breast cancer cells with wild-type p53 and tamoxifen-resistant breast cancer cells. The ER-positive breast cancer cell lines, MCF-7 with wild-type p53 and T47D with mutant p53, were used. We established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with 4-hydroxytamoxifen. IFITM1 inhibition in MCF-7 cells significantly decreased cell growth and migration. MCF-7 cells with suppression of IFITM1 using siRNA or ruxolitinib showed reduced cell viability after tamoxifen treatment compared with that in the control MCF-7 cells. Unexpectedly, mRNA and protein levels of IFITM1 were decreased in TamR cells compared with those in MCF-7 cells. TamR cells with suppression of IFITM1 using siRNA or ruxolitinib showed no change in cell viability after treatment with tamoxifen. P53 knockdown using siRNA reduced the mRNA levels of IRF9 and increased mRNA and protein levels of SOCS3 in MCF-7 cells, suggesting that loss or mutation of p53 can affect the induction of IFITM1 via the JAK/STAT signaling pathway in breast cancer. Furthermore, MCF-7 cells with p53 knockdown using siRNA showed no decrease in cell viability after tamoxifen treatment or IFITM1 inhibition, indicating that p53 status may be important for cell death after tamoxifen treatment or IFITM1 inhibition. IFITM1 inhibition may enhance the sensitivity to tamoxifen based on p53-dependent enhancement of IFN signaling in wild-type p53, ER-positive breast cancer cells.

Glutathione overproduction mediates lymphoma initiating cells survival and has a sex-dependent effect on lymphomagenesis

Lymphoid tumor patients often exhibit resistance to standard therapies or experience relapse post-remission. Relapse is driven by Tumor Initiating Cells (TICs), a subset of tumor cells capable of regrowing the tumor and highly resistant to therapy. Growing cells in 3D gels is a method to discern tumorigenic cells because it strongly correlates with tumorigenicity. The finding that TICs, rather than differentiated tumor cells, grow in 3D gels offers a unique opportunity to unveil TIC-specific signaling pathways and therapeutic targets common to various cancer types. Here, we show that culturing lymphoid cells in 3D gels triggers reactive oxygen species (ROS) production, leading to non-tumor lymphoid cell death while enabling the survival and proliferation of a subset of lymphoma/leukemia cells, TICs or TIC-like cells. Treatment with the antioxidant N-acetylcysteine inhibits this lethality and promotes the growth of primary non-tumor lymphoid cells in 3D gels. A subset of lymphoma cells, characterized by an increased abundance of the antioxidant glutathione, escape ROS-induced lethality, a response not seen in non-tumor cells. Reducing glutathione production in lymphoma cells, either through pharmacological inhibition of glutamate cysteine ligase (GCL), the enzyme catalyzing the rate-limiting step in glutathione biosynthesis, or via knockdown of GCLC, the GCL catalytic subunit, sharply decreased cell growth in 3D gels and xenografts. Tumor cells from B-cell lymphoma/leukemia patients and λ-MYC mice, a B-cell lymphoma mouse model, overproduce glutathione. Importantly, pharmacological GCL inhibition hindered lymphoma growth in female λ-MYC mice, suggesting that this treatment holds promise as a therapeutic strategy for female lymphoma/leukemia patients.

Targeting HMGCS1 restores chemotherapy sensitivity in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a common hematological malignancy with overall poor prognosis. Exploring novel targets is urgent and necessary to improve the clinical outcome of relapsed and refractory (RR) AML patients. Through clinical specimens, animal models and cell-level studies, we explored the specific mechanism of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMGCS1) in AML and the mechanism of targeting HMGCS1 to attenuate cell proliferation, increase chemotherapy sensitivity and improve the occurrence and development of AML. Here, we reveal that HMGCS1 is overexpressed in RR patients and negatively related to overall survival (OS). Knocking out HMGCS1 in AML cells attenuated cell proliferation and increased chemotherapy sensitivity, while stable overexpression of HMGCS1 had the opposite effects. Mechanistically, we identified that knockout of HMGCS1 suppressed mitogen-activated protein kinase (MAPK) pathway activity, while overexpression of HMGCS1 could remarkably enhance the pathway. U0126, a MEK1 inhibitor, offset the effects of HMGCS1 overexpression, indicating that HMGCS1 promotes RR AML through the MAPK pathway. Further, we verified that hymeglusin, a specific inhibitor of HMGCS1, decreases cell growth both in AML cell lines and primary bone marrow cells of AML patients. Furthermore, combination of hymeglusin and the common chemotherapeutic drug cytarabine and adriamycin (ADR) had synergistic toxic effects on AML cells. Our study demonstrates the important role of HMGCS1 in AML, and targeting this protein is promising for the treatment of RR AML.

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1 (IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6β and WFS1, are rational engineering targets. Although both ATF6β and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of ATF6β decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6β and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6β improved the UPR specifically later in fed-batch leading to increased overall productivity.

Preclinical efficacy of BRG1/BRM ATPase inhibitor in B lymphoblastic leukemia.

10030 Background: Acute lymphoblastic leukemia (ALL) is the most common blood cancer in children and young adults. While ALL has a high cure rate, approximately 20% of patients who initially respond to treatment relapse and prognosis post relapse remains poor. New therapeutic treatments are needed to reduce relapse rates in B-ALL pediatric patients and to minimize the toxic side effects seen with current therapies. We and others have previously shown that BRG1/BRM ATPase inhibitors have efficacy in targeting acute myeloid leukemia (AML) and one, FHD-286, is currently being evaluated for its use in AML in an ongoing clinical trial (NCT04891757). BRG1 and BRM are the ATP-dependent catalytic subunits of the SWI/SNF chromatin remodeling complex which has a critical role in controlling gene expression, cell growth and survival of AML cells. Our prior work revealed that BRG1/BRM ATPase inhibition leads to selective transient reduction of normal B cells. Further, data from Depmap also nominates SMARCA4, the gene encoding BRG1, as a dependency in B-ALL cell lines, leading us to hypothesize that BRG1/BRM inhibition may be an effective therapy for B-ALL. Methods: We treated B-ALL cell lines with varying genetic lesions ( KMT2A-rearranged: RS4;11, KOPN-8, SEM; ETV6::RUNX1 fusion: REH and DUX4-rearranged: Nalm6) with dose titrations of FHD-286 and measured cell growth over the course of six days. In addition, at 48- and 72-hours post-treatment, we assayed cells for viability, cell cycle kinetics, and apoptosis induction via flow cytometry to provide insight into FHD-286’s mechanism of action in B-ALL. Results: We found that FHD-286 treatment resulted in decreased cell growth in a dose-dependent manner across all B-ALL cell lines. While all cell lines were displayed reduced proliferation at nanomolar doses, sensitivity varied. Nalm6, REH and SEM cells were the most sensitive with low nanomolar IC50s (2-7nM). In contrast, two of the KMT2Ar B-ALL lines, RS4;11 and KOPN-8, required higher doses to achieve growth inhibition (40-70nM). To better understand the mechanism by which FHD-286 is affecting cell growth, we evaluate whether FHD-286 is targeting cell growth through the apoptotic pathway or through cell cycle arrest. Our preliminary data indicates cell growth inhibition is only slightly attributable to cell cycle arrest and apoptosis induction, leading us to explore terminal differentiation as a mechanism of slowed proliferation. Conclusions: Our results show FHD-286 can strongly inhibit B-ALL cell growth in vitro, indicating FHD-286 as a potential therapeutic agent to treat B-ALL. In vivo validation is ongoing. Further directions include defining the effect of FHD-286 on chromatin remodeling, gene expression, and exploring efficacy in combination therapy, which has been shown to increase FHD-286 efficacy against AML. Our data may ultimately justify inclusion of B-ALL patients in clinical studies of BRG1/BRM inhibitors.

An exploratory study on the effect of kynurenine metabolites on sEnd-2 endothelioma cells.

Cancer is the second leading cause of mortality worldwide. The development of anticancer therapy plays a crucial role in mitigating tumour progression and metastasis. Epithelioid hemangioendothelioma is a very rare cancer, however, with a high systemic involvement. Kynurenine metabolites which includel-kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and quinolinic acid have been shown to inhibit T-cell proliferation resulting in a decrease in cell growth of natural killercells and T cells. Furthermore, metabolites such as l-kynurenine have been shown to inhibit proliferation of melanoma cells in vitro. Considering these metabolite properties, the present study aimed to explore the in vitro effects of l-kynurenine, quinolinic acid and kynurenic acid on endothelioma sEnd-2 cells and on endothelial (EA. hy926 cells) (control cell line). The in vitro effect at 24, 48, and 72 h exposure to a range of 1-4 mM of the respective kynurenine metabolites on the two cell lines in terms of cell morphology, cell cycle progression and induction of apoptosis was assessed. The half inhibitory concentration (IC50), as determined using nonlinear regression, for l-kynurenine, quinolinic acid and kynurenic acid was 9.17, 15.56, and 535.40 mM, respectively. Optical transmitted light differential interference contrast and hematoxylin and eosin staining revealed cells blocked in metaphase, formation of apoptotic bodies and compromised cell density in l-kynurenine-treated cells. A statistically significant increase in the number of cells present in the sub-G1 phase was observed in l-kynurenine-treated sample. To our knowledge, this was the first in vitro study conducted to investigate the mechanism of action of kynurenine metabolites on endothelioma sEnd-2 cells. It can be concluded that l-kynurenine exerts an antiproliferative effect on the endothelioma sEnd-2 cell line by decreasing cell growth and proliferation as well as a metaphase block. These hallmarks suggest cell death via apoptosis. Further research will be conducted on l-kynurenine to assess the effect on cell adhesion in vitro and in vivo as cell-cell adhesion has been shown to increase metastasis to distant organs therefore, the inhibition of adhesion may lead to a decrease in metastasis.

Wdr17 Regulates Cell Proliferation, Cell Cycle Progression and Apoptosis in Mouse Spermatocyte Cell Line.

We identified Wdr17 as a highly expressed gene in pachytene spermatocytes by transcriptomic analysis of mouse testis. Germ cell-deficient infertile mouse models had significantly reduced Wdr17 expression. We performed gene interference and overexpression in the mouse spermatocyte cell line GC-2spd(ts) and investigated how Wdr17 affects spermatocyte growth and development. Our results showed that Wdr17 suppression significantly decreased cell growth rate and increased cell apoptosis in GC-2spd(ts) cells. Wdr17 suppression also arrested the cell cycle at the G1 phase. On the contrary, Wdr17 overexpression significantly promoted cell proliferation and inhibited cell apoptosis in GC-2spd(ts) cells. More cells were enriched at the S stage with a concomitant reduction of cells at the G1 stage. Wdr17 promotes mouse spermatocyte proliferation by advancing cell cycle progression and inhibiting cell apoptosis, indicating its potential role in regulating spermatogenesis in the mouse.

Exploring the role of casein kinase 1α splice variants across cancer cell lines

Casein kinase 1α (CK1α) is a serine/threonine protein kinase that acts in various cellular processes affecting cell division and signal transduction. CK1α is present as multiple splice variants that are distinguished by the presence or absence of a long insert (L-insert) and a short carboxyl-terminal insert (S-insert). When overexpressed, zebrafish CK1α splice variants exhibit different biological properties, such as subcellular localization and catalytic activity. However, whether endogenous, alternatively spliced CK1α gene products also differ in their biological functions has yet to be elucidated. Here, we identify a panel of splice variant specific CK1α antibodies and use them to show that four CK1α splice variants are expressed in mammals. We subsequently show that the relative abundance of CK1α splice variants varies across distinct mouse tissues and between various cancer cell lines. Furthermore, we identify pathways whose expression is noticeably altered in cell lines enriched with select splice variants of CK1α. Finally, we show that the S-insert of CK1α promotes the growth of HCT 116 cells as cells engineered to lack the S-insert display decreased cell growth. Together, we provide tools and methods to identify individual CK1α splice variants, which we use to begin to uncover the differential biological properties driven by specific splice variants of mammalian CK1α.

Effects of down-regulated carbonic anhydrase 8 on cell survival and glucose metabolism in human colorectal cancer cell lines.

Carbonic anhydrase 8 (CA8) is a member of the α-carbonic anhydrase family but does not catalyze the reversible hydration of carbon dioxide. In the present study, we examined the effects of CA8 on two human colon cancer cell lines, SW480 and SW620, by suppressing CA8 expression through shRNA knockdown. Our results showed that knockdown of CA8 decreased cell growth and cell mobility in SW620 cells, but not in SW480 cells. In addition, downregulated CA8 resulted in a significant decrease of glucose uptake in both SW480 and SW620 cells. Interestingly, stable downregulation of CA8 decreased phosphofructokinase-1 expression but increased glucose transporter 3 (GLUT3) levels in SW620 cells. However, transient downregulation of CA8 fails to up-regulate GLUT3 expression, indicating that the increased GLUT3 observed in SW620-shCA8 cells is a compensatory effect. In addition, the interaction between CA8 and GLUT3 was evidenced by pull-down and IP assays. On the other hand, we showed that metformin, a first-line drug for type II diabetes patients, significantly inhibited cell migration of SW620 cells, depending on the expressions of CA8 and focal adhesion kinase. Taken together, our data demonstrate that when compared to primary colon cancer SW480 cells, metastatic colon cancer SW620 cells respond differently to downregulated CA8, indicating that CA8 in more aggressive cancer cells may play a more important role in controlling cell survival and metformin response. CA8 may affect glucose metabolism- and cell invasion-related molecules in colon cancer, suggesting that CA8 may be a potential target in future cancer therapy.

TNF receptor 2 knockout mouse had reduced lung cancer growth and schizophrenia-like behavior through a decrease in TrkB-dependent BDNF level

The relationship between schizophrenia (SCZ) and cancer development remains controversial. Based on the disease-gene association platform, it has been revealed that tumor necrosis factor receptor (TNFR) could be an important mediatory factor in both cancer and SCZ development. TNF-α also increases the expression of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) in the development of SCZ and tumor, but the role of TNFR in mediating the association between the two diseases remains unclear. We studied the vital roles of TNFR2 in the progression of tumor and SCZ-like behavior using A549 lung cancer cell xenografted TNFR2 knockout mice. TNFR2 knockout mice showed significantly decreased tumor size and weight as well as schizophrenia-like behaviors compared to wild-type mice. Consistent with the reduced tumor growth and SCZ-like behaviors, the levels of TrkB and BDNF expression were significantly decreased in the lung tumor tissues and pre-frontal cortex of TNFR2 knockout mice. However, intravenous injection of BDNF (160 μg/kg) to TNFR2 knockout mice for 4 weeks increased tumor growth and SCZ-like behaviors as well as TrkB expression. In in vitro study, significantly decreased cell growth and expression of TrkB and BDNF by siTNFR2 transfection were found in A549 lung cancer cells. However, the addition of BDNF (100 ng/ml) into TNFR2 siRNA transfected A549 lung cancer cells recovered cell growth and the expression of TrkB. These results suggest that TNFR2 could be an important factor in mediating the comorbidity between lung tumor growth and SCZ development through increased TrkB-dependent BDNF levels.

Abstract 7197: Elacridar sensitizes liver cancer to the PKC-inhibitor ICA-1S

Abstract Liver cancer is a disease in which cancer cells form in the tissues of the liver. Globally, liver cancer is one of the most frequent fatal malignancies; in the United States, liver cancer is the sixth leading cause of cancer death. Generally, there is a poor prognosis for liver cancer. Chemotherapy and immunotherapy are the best available treatment options. This research aims to provide an alternative treatment option with drugs that specifically attack liver cancer cells. Protein Kinase C (PKCs) are signaling molecules that have been reported to be associated with multiple cellular processes and cancer. PKC activity reportedly is elevated in colorectal cancer cells and metastasizes to the liver. 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) is a PKC-ι specific inhibitor we have previously shown to be effective against some cancer cell lines by decreasing cell growth and inducing apoptosis. In this study, we investigated the effectiveness of ICA-1S against liver cancer cell lines; HEPG2 and PLC5/PRF/5. An equal number of HEPG2 and PLC5/PRF/5 cell lines were seeded into 6 well plates, treated with different concentrations (0.1µM, 0.5µM, 1µM, 5µM, and 10µM) of ICA-1s every 24 hours for three days after which the media was discarded, and cells were counted. The percentage inhibition of ICA-1S on HEPG2 and PLC5/PRF/5 cell lines was calculated. To investigate the resistance inflection, the proliferation assay was also conducted with a combination of an efflux pump inhibitor, Elacridar(10µM), and different concentrations (0.1µM to 10µM) of ICA-1S. The expression of PKC-ι was investigated across all treatments. The dose-response result for treatment with ICA-1S for both HEPG2 and PLC5/PRF/5 cell lines showed a resistance inflection point at 0.5µM and 1µM, where the proliferation rate of the cells declined by 50% and 79% for HEPG2, and 41% and 54% for PLC5/PRF/5 respectively; beyond these points, the cell proliferation rate significantly rises. The results of dose-response for treatment with a combination of ICA-1S and Elacridar showed a continuous decline in cell proliferation rate up to 78% and 84% for HEPG2 and PLC5/PRF/5 respectively as the concentrations of ICA-1S increased from 0.1µM to 10µM. The PKC-ι level was downregulated in the combination treatment. We have shown in this study that liver cancer cells exhibit resistance to PKC inhibitor, ICA-1S, at high concentrations. This resistance inflection may be due to several factors including the activation of drug-efflux proteins. Thus, we proved that the introduction of an efflux pump inhibitor, Elacridar, sensitizes the liver cancer cells to ICA-1S, offsets the resistance of the liver cancer cells to ICA-1S at high concentrations, and enables ICA-1S to be effective in downregulating PKC-ι and decreasing cell growth of liver cancer cells. Citation Format: Abigail Oluwafisayo Olatunji, Mahfuza Marzan, Mildred Acevedo-Duncan. Elacridar sensitizes liver cancer to the PKC-inhibitor ICA-1S [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 7197.