Changes In Cell Cycle Progression Research Articles

Exploring the therapeutic potential of simvastatin in pancreatic neuroendocrine neoplasms: insights into cell cycle regulation and apoptosis.

Pancreatic neuroendocrine neoplasm (pNEN) poses significant challenges in clinical management due to their heterogeneity and limited treatment options. In this study, we investigated the potential of simvastatin (SIM) as an anti-tumor agent in pNEN. We conducted cell culture experiments using QGP-1 and BON-1 cell lines and assessed cell viability, proliferation, migration, and invasion following SIM treatment. To further validate our findings, we performed in vivo experiments using a mouse xenograft model. Additionally, we investigated the underlying molecular mechanisms by analyzing changes in cell cycle progression, apoptosis, and signaling pathways. SIM treatment suppresses pNEN growth both in vitro and in vivo, and led to G1 phase arrest in QGP-1 cells. In contrast, SIM affected both the G1-S and G2-M phase transitions in the BON-1 cell line and induced apoptosis, indicating diverse mechanisms of action. Furthermore, SIM treatment resulted in decreased expression of mutant p53 (mutp53) in BON-1 cells, suggesting a potential therapeutic strategy targeting mutp53. Modulation of the MAPK pathway was also implicated in QGP-1 cells. Our study highlights SIM as a promising candidate for pNEN treatment by inducing cell cycle arrest or apoptosis, potentially through the p53 and MAPK pathways. Further research is warranted to fully elucidate SIM's mechanisms of action and evaluate its therapeutic potential in clinical settings.

The Imidazacridine Derivative LPSF/AC-05 Induces Apoptosis, Cell Cycle Arrest, and Topoisomerase II Inhibition in Breast Cancer, Leukemia, and Lymphoma.

Cancer is the major cause of morbidity and mortality worldwide. Current treatments for both solid and hematological tumors are associated with severe adverse effects and drug resistance, necessitating the development of novel selective antineoplastic drugs. The present study describes the antitumor activity of the imidazacridine derivative 5-acridin-9-ylmethylidene-2-thioxoimidazolidin-4-one (LPSF/AC05) in breast cancer, leuke-mia, and lymphoma cells. Cytotoxicity assays were performed in PBMC and in breast cancer, leukemia, and lymphoma cell lines using the MTT method. Changes in cell cycle progression and apoptosis were assessed using flow cytometry. Moreover, topoisomerase II inhibition as-says were performed. LPSF/AC05 exhibited cytotoxicity in six of the nine cell lines tested. The best results for leukemia and lymphoma were observed in the Toledo, Jurkat, and Raji cell lines (IC50 = 27.18, 31.04, and 33.36 M, respectively). For breast cancer, the best re-sults were observed in the triple-negative cell line MDA-MB-231 (IC50 = 27.54 μM). The compound showed excellent selectivity, with no toxicity to normal human cells (IC50 > 100M; selectivity index > 3). Cell death was primarily induced by apoptosis in all cell lines. Furthermore, LPSF/AC05 treatmentinduced cell cycle arrest at the G0/G1 phase in leuke-mia/lymphoma and at the G2/M phase in breast cancer. Finally, topoisomerase II was inhibited. These results indicate the potential ap-plication of LPSF/AC05 in cancer therapy.

Potential Effects of Photobiomodulation Therapy on Human Dental Pulp Stem Cells

Background: A significant clinical technology enabling the use of stem cells in dentistry is Photobiomodulation (PBM). The current study aimed to investigate various PBM treatment variables on human dental pulp stem cell proliferation, survival, and changes in cell cycle progression. Methods: Human third molar samples were collected, and human Dental Pulp Stem Cells (hDPSCs) were isolated, expanded, seeded, and cultured in media containing 0, 1, 5, or 10% FBS. PBM treatments using an 810 nm diode laser at various doses were performed 24, 48, and 72 h after seeding. Cell proliferation and apoptosis were assessed. A two-way ANOVA test was performed among all treatment groups, followed by multiple comparison tests (Tukey’s test) performed between each treated group and its control untreated group. Results: After 24 h, only cells cultured in 10% FBS showed a significant (p < 0.005) increase in the proliferation rate of cells treated with T7 (0.4 W × 10 s). After 48 h, hDPSCs cultured in both 5% and 10% FBS showed a significant (p < 0.005) increase in the proliferation rate of cells treated with T7 (0.4 W × 10 s) as compared to the untreated control. After 72 h, only cells cultured in 10% FBS showed a significant increase in the proliferation rate of the cells treated with T9 (1 W × 3 s) (p < 0.005) and T7 (0.4 W × 10 s) (p < 0.00005). Conclusions: Low-power laser therapy at a wavelength of 810 nm induced hDPSC proliferation at the following parameters: power output 0.4 W, irradiance 0.5 W/cm2, fluence 0.416 J/cm2, exposure time 10 s.

STEM CELLS HOMEOSTASIS AND AGING

Abstract Adult (tissue) stem cells support tissue homeostasis and repair throughout the life of an individual. Numerous changes occur with age that result in altered stem cell behavior and reduced tissue maintenance and regeneration. Changes can be cell autonomous including changes in cell cycle progression, decreased bioenergetic efficiency, increased DNA damage, and epigenetic alterations. In addition, poorly understood changes to the local and systemic environments occur that result in decreased stem cell activity or alterations in commitment or differentiation potential. This symposium will cover the impact of aging on multiple adult stem cell systems, including hematopoietic stem cells (HSCs), intestinal stem cells (ISCs), and neural stem cells.

Navtemadlin, a Novel MDM2 Inhibitor, Potentiated Venetoclax-Induced Antitumor Efficacy in TP53 Wild-Type Acute Myeloid Leukemia (AML)

Introduction: The tumor suppressor p53 ( TP53) maintains genomic integrity, and nearly 50% of cancers are characterized by a loss or mutation in the TP53 gene. Murine double minute 2 (MDM2) negatively regulates p53 expression and promotes oncogenesis in TP53 wild-type ( TP53 WT) tumors. Navtemadlin (nvtm) is a potent, selective, orally available MDM2 inhibitor that overcomes MDM2 dysregulation by restoring p53 activity and inducing apoptosis in TP53 WT tumors. AML is a heterogeneous hematological malignancy resulting from the transformation of stem cells and is characterized by poor therapy response. Thus, combination therapies are required to avoid tumor escape mechanisms, such as alterations in apoptotic machinery and metabolism. Venetoclax (ven), a BCL-2 inhibitor, is a promising therapy in AML. Overexpression of anti-apoptotic members of the BCL-2 family, including myeloid cell leukemia-1 (MCL-1) limits ven response, thus requiring MCL-1 inhibitors to further improve sensitivity. A proof-of-concept study with idasanutlin (idasa), a first-generation MDM2 inhibitor and ven demonstrated manageable safety and encouraging efficacy in relapsed/refractory AML patients (Daver et al. Blood 2023). Here, we present a preclinical evaluation of nvtm alone and in combination with ven in the setting of TP53 WT AML, with a focus on metabolic pathways, pro-survival signaling, and bone marrow (BM) niche. Methods: AML-derived cell line, MOLM-13 ( TP53 WT, FLT3-ITD) was cultured alone, with HS5 AML stromal cells or with cytokines. Cultures were treated with DMSO, nvtm (0.1-1.5 μM), or ven (0.5 μM) for short-term (4-8 hrs) or long-term (48-72 hrs) and analyzed for changes in cell cycle progression, viability, clonogenicity potential, and metabolism (glycolysis and mitochondrial oxidative phosphorylation by analyzing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) respectively). Primary AML BM mononuclear cells (MNCs) were cultured in cytokine support media. Results: In MOLM-13 cells, short-termnvtm (0.5 µM) treatment rapidly restored p53 expression and phosphorylation, with greater efficacy than idasa. Long-term treatment significantly induced apoptosis in a dose-dependent manner from 0.25 μM (p<0.01) with 80% cytotoxicity at 0.75 μM (p<0.001). Cell cycle analysis at 48 hr showed that nvtm significantly arrested cells in the sub-G0-G1 phase. Clonogenicity potential was significantly lowered in nvtm-treated cells. Metabolic alterations in the Mito Stress and Glycostress tests showed nvtm significantly lowered EACR, OCR, and mitochondrial membrane potential, as compared to idasa. We next evaluated the effects of nvtm in ven-treated MOLM-13 cells. Combination treatment significantly lowered the metabolism, cell viability, and clonogenicity potential of MOLM-13 cells, compared to nvtm or ven treatment. Immunoblotting analysis revealed a reduction in key anti-apoptosis proteins (BCL-2, MCL-1, PARP). Strikingly, nvtm downregulated ven-induced MCL-1 expression in a dose-dependent manner. Culturing MOLM-13 cells in HS5-conditioned media, transwells overlying HS5 cells, or with HS5 cells increased MOLM-13 cell resistance to nvtm. However, in HS5 co-cultures, a combination of nvtm and ven strongly re-sensitized MOLM-13 cells for apoptosis. Nvtm also significantly blocked the cytoprotective effects of AML-relevant cytokines (IL-6, SCF, FLT3 ligand, TPO, and GM-SCF). In cultures of primary AML BM-MNCs (n=11), nvtm alone at clinically relevant concentrations reduced viability (14-40%), and a combination nvtm and ven significantly enhanced AML cytotoxicity (30-52%). Conclusions: Nvtm, a potent second-generation MDM2 inhibitor, rapidly restored p53 function, induced apoptosis in p53 WT AML MOLM-13 by impeding cell cycle, viability, clonogenicity potential, and reducing both glycolysis and oxidative phosphorylation. Nvtm combined with ven significantly enhanced these effects. Importantly, this combination blocked stromal cell-mediated (contact and soluble factors) cytoprotection. These encouraging results provide a mechanistic basis for the evaluation of this combination in vivo and as a therapeutic strategy to overcome documented ven resistance mechanisms in TP53 WT AML patients.

GSK3α Regulates Temporally Dynamic Changes in Ribosomal Proteins upon Amino Acid Starvation in Cancer Cells.

Amino acid availability is crucial for cancer cells' survivability. Leukemia and colorectal cancer cells have been shown to resist asparagine depletion by utilizing GSK3-dependent proteasomal degradation, termed the Wnt-dependent stabilization of proteins (Wnt/STOP), to replenish their amino acid pool. The inhibition of GSK3α halts the sourcing of amino acids, which subsequently leads to cancer cell vulnerability toward asparaginase therapy. However, resistance toward GSK3α-mediated protein breakdown can occur, whose underlying mechanism is poorly understood. Here, we set out to define the mechanisms driving dependence toward this degradation machinery upon asparagine starvation in cancer cells. We show the independence of known stress response pathways including the integrated stress response mediated with GCN2. Additionally, we demonstrate the independence of changes in cell cycle progression and expression levels of the asparagine-synthesizing enzyme ASNS. Instead, RNA sequencing revealed that GSK3α inhibition and asparagine starvation leads to the temporally dynamic downregulation of distinct ribosomal proteins, which have been shown to display anti-proliferative functions. Using a CRISPR/Cas9 viability screen, we demonstrate that the downregulation of these specific ribosomal proteins can rescue cell death upon GSK3α inhibition and asparagine starvation. Thus, our findings suggest the vital role of the previously unrecognized regulation of ribosomal proteins in bridging GSK3α activity and tolerance of asparagine starvation.

Cell cycle arrest and apoptosis are early events in radiosensitization of EWS::FLI1+ Ewing sarcoma cells by Mithramycin A

Purpose The oncogenic fusion protein EWS::FLI1 is an attractive therapeutic target in Ewing sarcoma (ES). Mithramycin A (MithA) is a potent and specific inhibitor of EWS::FLI1 that can selectively radiosensitize ES cells through transcriptional inhibition of DNA double-strand break (DSB) repair. Here, we evaluate temporal changes in cell cycle progression and apoptosis in ES cells treated with MithA and/or ionizing radiation (RTx), testing the hypothesis that combining MithA with ionizing radiation would synergistically impair cell cycle progression and enhance apoptotic elimination to a greater extent than either agent alone. Materials and Methods Four EWS::FLI1 + ES cell lines TC-71, RD-ES, SK-ES-1, and A673, and one EWS::ERG cell line (CHLA-25) were exposed to 10nM MithA or vehicle and followed 24 h later by exposure to 2 Gy x-radiation or sham irradiation. Reactive oxygen species (ROS) activity was evaluated by cytometric assay, and assay of antioxidant gene expression by RT-qPCR. Cell cycle changes were evaluated by flow cytometry of nuclei stained with propidium iodide. Apoptosis was assessed by cytometric assessment of Caspase-3/7 activity and by immunoblotting of PARP-1 cleavage. Radiosensitization was evaluated by clonogenic survival assay. Proliferation (EdU) and apoptosis (TUNEL) were evaluated in SK-ES-1 xenograft tumors following pretreatment with 1 mg/kg MithA, followed 24 h later by a single 4 Gy fraction of x-radiation. Results MithA-treated cells showed reduced levels of ROS, and were associated with increased expression of antioxidant genes SOD1, SOD2, and CAT. It nonetheless induced persistent G0/G1 arrest and a progressive increase of the sub-G1 fraction, suggesting apoptotic degeneration. In vitro assays of Caspase-3/7 activity and immunoblotting of Caspase-3/7 dependent cleavage of PARP-1 indicated that apoptosis began as early as 24 h after MithA exposure, reducing clonogenic survival. Tumors from xenograft mice treated with either radiation alone, or in combination with MithA showed a significant reduction of tumor cell proliferation, while apoptosis was significantly increased in the group receiving the combination of MithA and RTx. Conclusions Taken together, our data show that the anti-proliferative and cytotoxic effects of MithA are the prominent components of radiosensitization of EWS::FLI1+ ES, rather than the result of acutely enhanced ROS levels.

Abstract 447: Genetic and pharmacological inhibition of TUT4 and TUT7 sensitizes AML cells to BCL-2 inhibition

Abstract The terminal uridyltransferases TUT4/7 (ZCCHC11/6) are important mediators of RNA stability, RNA decay, histone mRNA regulation, viral RNA sensing, and miRNA biogenesis. Regulation of RNA processing pathways can be mediated by a number of RNA-binding proteins (RBPs), and dysfunctional RNA processing has implications as a therapeutic target in leukemia and lymphoma. We performed genetic and pharmacological perturbation of the activity of terminal uridyltransferases TUT4/7 (ZCCHC11/6) in MLL-rearranged acute myeloid leukemia (AML). A combination of catalytic inactivation and knockout by CRISPR/Cas9 of TUT4 and TUT7, respectively, were used to abrogate uridylation in THP-1 cells, with enzyme function measured by a cellular miRNA uridylation assay. Inhibition of uridylation in THP-1 cells reduced proliferation, induced changes in cell cycle progression, and enhanced sensitivity to the BCL-2 inhibitor, venetoclax, alone and in combination with azacitidine. BCL-2 expression was found to be reduced at the mRNA and protein levels, while increases in expression of BCL-2 regulating miRNAs miR139-5p and miR494-3p were observed. GSEA performed on uridylation deficient cells highlighted changes in cell cycle regulation, mitochondrial metabolism, and cell death pathways relative to unedited controls. These effects could be recapitulated with pharmacological inhibition using sub-inhibitory doses of a novel, selective inhibitor of TUT4/7, TS-002266, while the weakly active enantiomer TS-002455 did not show combination activity. Treatment of primary AML with combination of TS-002266 and venetoclax improved activity over venetoclax alone in a number of primary AML samples. Thus, we propose that TUT4/7 and BCL-2 inactivation may be a therapeutic strategy in leukemia. Citation Format: Corey Fyfe, Gabriella Fiorentino, Michael J. Steinbaugh, Joana Campos, Hannah Lawson, Jozef Durko, Donal O'Carroll, Kamil Kranc, Darren Harvey, Shomir Ghosh, Robert M. Campbell. Genetic and pharmacological inhibition of TUT4 and TUT7 sensitizes AML cells to BCL-2 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 447.

Forskolin affects proliferation, migration and Paclitaxel-mediated cytotoxicity in non-small-cell lung cancer cell lines via adenylyl cyclase/cAMP axis

Non-Small-Cell Lung Cancer (NSCLC) is considered one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths worldwide. Despite the undoubted therapeutic advances that have occurred in clinical practice over time, due to its high degree in both heterogeneity and resistance, NSCLC remains largely incurable. As a natural cAMP elevating agent, Forskolin has shown anti-cancer properties in different tumor types, thus supposing its possible usage in treating malignancies. In this study, we investigated the Forskolin outcome in H1299 and A549 NSCLC cell lines, either alone or in combination with Paclitaxel. We proved that Forskolin impairs cell growth and migration ability of these cells, concurrently. Albeit with a different extent between H1299 and A549, changes in cell-cycle progression and epithelial-mesenchymal markers were observed in response to Forskolin administration. Interestingly, comparable cell growth impairment was also obtained with the cAMP phosphodiesterase inhibitor IBMX, while the employment of adenylyl cyclase inhibitor SQ22536 counteracted, at least in part, the Forskolin-mediated anticancer effects. Besides as a single agent, we also demonstrated that Forskolin strongly enhances Paclitaxel-induced cytotoxicity, affecting cell death mainly via apoptosis induction. Notably, H89-mediated protein kinase A (PKA) inhibition further deteriorated the combination outcome. Altogether, our data designate Forskolin as a possible anticancer molecule in NSCLC, and recognize the adenylyl cyclase/cAMP axis as one of the pathways involved in. Although achieved at preclinical stage, our findings encourage the design of future studies aimed at further exploring the Forskolin employment in NSCLC treatment.

Genetic and Pharmacological Inhibition of TUT4/TUT7 Sensitizes AML Cells to BCL-2 Inhibition

The terminal uridyltransferases TUT4/7 (ZCCHC11/6) are important mediators of RNA stability, RNA decay, histone mRNA regulation, viral RNA sensing, and miRNA biogenesis. Regulation of RNA processing pathways can be mediated by a number of RNA-binding proteins (RBPs), and dysfunctional RNA processing has implications as a therapeutic target in leukemia and lymphoma. We performed genetic and pharmacological perturbation of the activity of terminal uridyltransferases TUT4/7 (ZCCHC11/6) in MLL-rearranged acute myeloid leukemia (AML). A combination of catalytic inactivation and knockout by CRISPR/Cas9 of TUT4 and TUT7, respectively, were used to abrogate uridylation in THP-1 cells, with enzyme function measured by a cellular miRNA uridylation assay. Inhibition of uridylation in THP-1 cells reduced proliferation, induced changes in cell cycle progression, and enhanced sensitivity to the BCL-2 inhibitor, venetoclax, alone and in combination with azacitidine. BCL-2 expression was found to be reduced at the mRNA and protein levels, while increases in expression of BCL-2 regulating miRNAs miR139-5p and miR494-3p were observed. GSEA performed on uridylation deficient cells highlighted changes in cell cycle regulation, mitochondrial metabolism, and cell death pathways relative to unedited controls. These effects could be recapitulated with pharmacological inhibition using sub-inhibitory doses of a novel, selective inhibitor of TUT4/7, TS-002266, while the weakly active enantiomer TS-002455 did not show combination activity. Treatment of primary AML with combination of TS-002266 and venetoclax improved activity over venetoclax alone in a number of primary AML samples. Thus, we propose that TUT4/7 and BCL-2 inactivation may be a therapeutic strategy in leukemia.

A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

The human SH-SY5Y neuroblastoma cell line is widely used in neuroscience research as a neuronal cell model. Following differentiation to a neuron-like state, SH-SY5Y cells become more morphologically similar to neurons and form functional synapses. Previous studies have managed to differentiate SH-SY5Y cells towards cholinergic, dopaminergic and adrenergic fates. However, their application in disease modeling remains limited as other neuronal subtypes (e.g., glutamatergic, GABAergic) are also implicated in neurological disorders, and no current protocols exist to generate these subtypes of differentiated SH-SY5Y cells. Our study aimed to evaluate the use of a xeno-free version of B-27, a supplement commonly used in neuronal culture, for SH-SY5Y maintenance and differentiation. To evaluate the proliferative capacity of SH-SY5Y cells cultured in B-27, we performed growth curve analyses, immunocytochemical staining for Ki-67 and qRT-PCR to track changes in cell cycle progression. SH-SY5Y cells cultured in FBS or under serum-starved conditions were used as controls. We observed that SH-SY5Y cells show reduced growth and proliferation rates accompanied by decreased CDK6 and CDK1 expression following 4-day exposure to B-27, suggesting B-27 induces a quiescent state in SH-SY5Y cells. Importantly, this reduced growth rate was not due to increased apoptosis. As cell cycle exit is associated with differentiation, we next sought to determine the fate of SH-SY5Y cells cultured in B-27. B-27-cultured SH-SY5Y cells show changes in cell morphology, adopting pyramidal shapes and extending neurites, and upregulation of neuronal differentiation markers (GAP43, TUBB3, and SYP). B-27-cultured SH-SY5Y cells also show increased expression of glutamatergic markers (GLUL and GLS). These findings suggest that B-27 may be a non-toxic inducer of glutamatergic SH-SY5Y differentiation. Our study demonstrates a novel way of using B-27 to obtain populations of glutamatergic SH-SY5Y cells. As dysregulated glutamatergic signaling is associated with a variety of neuropsychiatric and neurodegenerative disorders, the capability to generate glutamatergic neuron-like SH-SY5Y cells creates endless disease modeling opportunities. The ease of SH-SY5Y culture allows researchers to generate large-scale cultures for high-throughput pharmacological or toxicity studies. Also compatible with the growing popularity of animal-component-free studies, this xeno-free B-27/SH-SY5Y culture system will be a valuable tool to boost the translational potential of preliminary studies requiring glutamatergic neuronal cells of human origin.

MicroRNA-4521 targets hepatoma up-regulated protein (HURP) to inhibit the malignant progression of breast cancer

ABSTRACT Breast cancer (BC) is a common malignancy among women, and microRNAs (miRNAs) play a role in its progression. Reportedly, microRNA-4521 (miR-4521) participates in regulating the progression of some carcinomas. In the present work, miR-4521 and hepatoma up-regulated protein (HURP) mRNA expressions in BC tissues and cell lines were examined by qRT-PCR. After miR-4521 was overexpressed or knocked down in BC cells, CCK-8 experiment and EdU experiment were employed to detect BC cell growth. Moreover, the Transwell assay was adopted to detect the migration and invasion. Subsequently, flow cytometry was executed to evaluate the changes in cell cycle progression. KEGG analysis was utilized to predict the signaling pathways related with the target genes of miR-524-5p. The binding relationship between miR-4521 and HURP 3ʹUTR was validated by dual-luciferase reporter gene experiment. HURP and NF-kB p65 protein expression in BC cells was detected by Western blot. It was revealed that, miR-4521 was lowly expressed in BC tissues, and its expression was associated with tumor grade, lymph node metastasis and clinical stage of the patients. Overexpression of miR-4521 suppressed the growth, migration, invasion and cell cycle progression of BC cells and restrained p65 expression; downregulation of miR-4521 in BC cells showed opposite biological effects. Additionally, HURP was a downstream target gene of miR-4521, and overexpression of HURP reversed the above effects of miR-4521 overexpression. In short, miR-4521 can inhibit BC progression by modulating the HURP/NF-κB pathway.

SIPA1 boosts migration and proliferation, and blocks apoptosis of glioma by activating the phosphorylation of the FAK signaling pathway.

BackgroundWe aimed to analyze the regulatory effects of SIPA1 (signal-induced proliferation-associated protein 1) on glioma progression and the dominant signaling pathway.MethodsDifferential level of SIPA1 in glioma and normal tissues and cells was determined. Migratory, proliferative, apoptotic and cell cycle progression changes in A172 cells with overexpression or knockdown of SIPA1 were examined. Finally, protein levels of phosphorylated FAKs in A172 cells intervened by SIPA1, and the FAK inhibitor PF562271 were detected.ResultsSIPA1 was upregulated in glioma cases. Knock-down of SIPA1 reduced migratory and proliferative rates of glioma cells, increased apoptotic cell rate, and declined cell ratio in the S phase. The knockdown of SIPA1 also downregulated cell cycle proteins. In addition, SIPA1 upregulated phosphorylated FAKs in A172 cells and thus boosted malignant phenotypes of glioma.ConclusionsSIPA1 is upregulated in glioma that boosts migratory and proliferative potentials of glioma cells by activating the phosphorylation of the FAK signaling pathway.

Bone Marrow Stromal Cell Mediated Resistance to Mertk Inhibition in Acute Myeloid Leukemia

MERTK is a receptor tyrosine kinase of the TAM family (TYRO-3, AXL, MERTK) that is aberrantly expressed in 80-100% of pediatric and adult primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by small molecule tyrosine kinase inhibitors (TKIs), UNC2025 or MRX-2843, altered cell cycle progression and induced apoptosis in leukemia cell cultures. Moreover, treatment with these MERTK TKIs prolonged survival in mouse xenograft models of acute leukemia, but was not curative. In these models, treatment with the TKI effectively reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance.To evaluate the role of the bone marrow stromal niche in mediating resistance to MERTK inhibition by MRX-2843, AML cell lines were cultured in the presence of a fibroblast-like cell line (HS27) and induction of cell death and apoptosis was determined by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with the HS27 cell line significantly reduced cell death in Kasumi-1 AML cell cultures in response to treatment with 200nM MRX-2843 compared to leukemia cells alone (31.9% versus 61.2%, p<0.05). Similar results were observed in NOMO-1 and OCI-AML5 AML cell cultures. Additionally, cell cycle profiles were determined by flow cytometry in the presence or absence of stromal cells and MRX-2843. Treatment of Kasumi-1 cell cultures with 200nM MRX-2843 in the absence of stromal cells resulted in a significant accumulation of cells in G2 phase relative to vehicle-treated cultures (8.9% versus 14.8%, p<0.01) and was accompanied by an increase in γH2AX expression detected by immunoblot. In contrast, there was no change in cell cycle progression or γH2AX expression in cultures treated with MRX-2843 when co-cultured with the stromal cell line. NOMO-1 cells also accumulated in G2 phase in response to treatment with MRX-2843 and were protected from changes in cell cycle progression by co-culture with HS27 cells. Additionally, MERTK inhibition resulted in induction of polyploidy in NOMO-1 cell cultures, but this effect was abrogated in the presence of stromal cells. Collectively these results indicate abrogation of cell cycle progression in leukemia cells in response to treatment with MRX-2843 and demonstrate protection from this effect mediated by stromal cell interactions.The MERTK ligand, GAS6, mediates resistance to cytotoxic chemotherapy in leukemia cells and is a poor prognostic marker in AML. To evaluate the role of the MERTK ligands GAS6 and PROS1 in resistance to MERTK inhibition, their expression in stromal cells was determined by immunoblot. GAS6, but not PROS1, was expressed in HS27 cells; however, MRX-2843 retained its ability to inhibit MERTK phosphorylation in stromal cell co-cultures. We hypothesized that upregulation of the other TAM kinases, AXL and/or TYRO3, may mediate resistance as they share common ligands. To test this hypothesis, Kasumi-1 cells were treated with UNC4241, a small molecule pan-TAM inhibitor, in the presence of stromal cells. While co-culture protected Kasumi-1 cells from MRX-2843 induced apoptosis, treatment with UNC4241 induced apoptosis in the presence of stromal cells (32.7% versus 20.4% in vehicle control, p<0.01).To further evaluate the interactions between stromal cells and leukemia cells, Kasumi-1 cells were cultured in HS27-conditioned medium in the presence or absence of MRX-2843. Conditioned medium was not sufficient to provide protection from MRX-2843 induced apoptosis (74.0% vs. 69.8% in unconditioned medium, p>0.05). Similarly, addition of 500nM exogenous GAS6 or PROS1 did not protect leukemia cells from apoptosis in the presence of MRX-2843. Together, these results suggest that direct cell-cell contact between leukemia and stromal cells is necessary to provide protection.Together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from MRX-2843 induced apoptosis and cell cycle alterations and suggest that bone marrow mobilizing agents may be effective to overcome resistance to MERTK inhibitors. DisclosuresWang:Meryx: Equity Ownership; University of North Carolina: Patents & Royalties. Frye:University of North Carolina: Patents & Royalties; Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Earp:Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. DeRyckere:Meryx: Equity Ownership. Graham:Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Zinc oxide nanofluids: The influence of modality combinations on prostate cancer DU145 cells.

The combination of phototherapy and chemotherapy (chemophototherapy), presents a promising multimodal method for comprehensive cancer treatment. The aim of this study is to investigate the influence of low doses of zinc oxide (ZnO) nanofluids and ultraviolet A (UVA) irradiation on the cytotoxicity and cellular uptake of doxorubicin (DOX) on human prostate cancer DU145 cells. ZnO nanoparticles were prepared by the solvothermal method and 10% bovine serum albumin was used as the dispersant. The cytotoxic effect of DOX alone and in combination with different concentrations of ZnO nanofluids (0.95-15.6 μg/ml) in the presence and absence of UVA irradiation on DU145 cells was evaluated by -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. DOX residue inside and outside of DU145 cells was explored by fluorescence microscopy and UV-Vis absorption spectroscopy, respectively. The role of ZnO nanofluids and UVA irradiation in DOX-induced apoptosis and cell cycle arrest were evaluated by DAPI staining, comet assay, and flow cytometry. The results revealed that low dose of ZnO nanofluids (0.95 μg/ml) accompanied with irradiation enhanced the cytotoxicity and intracellular delivery of DOX in DU145 cells. The percentage of chromatin fragmentation/condensation and DNA tail of DU145 cells treated simultaneously with DOX and ZnO nanofluids was increased after UVA irradiation, whereas no significant changes in cell cycle progression were observed. The results indicate that ZnO nanofluids in the presence of UVA irradiation could increase DOX efficiency in DU145 cells, suggesting such modality combinations as a promising approach in cancer treatment.

New insights into cytotoxic mechanisms of bozepinib against glioblastoma

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults and the current treatments only have a modest effect on patient survival. Recent studies show that bozepinib (BZP), a purine derivative, has potential applications in cancer treatment. The aim of this study was to evaluate the effect of BZP against GBM cells, specially concerning the purinergic system. Thus, GBM cells (C6 and U138 cell lines) were treated with BZP and cell viability, cell cycle, and annexin/PI assays, and active caspase-3 measurements were carried out. Besides, the effect of BZP over the purinergic system was also evaluated in silico and in vitro. Finally, we evaluate the action of BZP against important markers related to cancer progression, such as Akt, NF-κB, and CD133. We demonstrate here that BZP reduces GBM cell viability (IC50 = 5.7 ± 0.3 µM and 12.7 ± 1.5 µM, in C6 and U138 cells, respectively), inducing cell death through caspase-dependent apoptosis, autophagosome formation, activation of NF-κB, without any change in cell cycle progression or on the Akt pathway. Also, BZP modulates the purinergic system, inducing an increase in CD39 enzyme expression and activity, while inhibiting CD73 activity and adenosine formation, without altering CD73 enzyme expression. Curiously, one cycle of treatment resulted in enrichment of GBM cells expressing NF-κB and CD133+, suggesting resistant cells selection. However, after another treatment round, the resistant cells were eliminated. Altogether, BZP presented in vitro anti-glioma activity, encouraging further in vivo studies in order to better understand its mechanism of action.

BIMG-18. ELEVATED CYSTATHIONINE IN MEDULLOBLASTOMA DEMONSTRATES TUMOR-SPECIFIC METHIONINE METABOLISM

We investigated tumor-specific metabolism medulloblastoma using a non-biased MS-imaging screen and identified a pattern of methionine flux that may present a therapeutic opportunity. We studied brain tumors that form in mice genetically engineered to develop Sonic Hedgehog (SHH)-driven medulloblastoma. We subjected sagittal sections including brain and medulloblastoma to MS-imaging, generating concentration maps for hundreds of metabolites MW 100–400. We then confirmed results by analyzing tumor, brain and blood by LC-MS/MS, high-resolution NMR and 2D NMR-TOCSY, and used immunohistochemistry to determine the cellular localization of implicated enzymes. MS imaging, accomplished by matrix-assisted laser desorption electrospray ionization (MALDESI), detected cystathionine at an order of magnitude higher concentration in medulloblastomas compared to adjacent brain. No other metabolite showed such a strong, tumor-specific localization. LC-MS/MS and NMR methods confirmed cystathionine elevation. As cystathionine is the product of homocysteine and serine, catalyzed by cystathionine beta-synthase (CBS), we investigated CBS expression by IHC. Consistent with prior studies, we found that only astrocytes expressed CBS, both in the normal brain and within the tumors. ScRNA-seq confirmed Cbs only in astrocytes, and showed tumor cells express methionine-metabolizing enzymes Mat2a, Dnmt1, Ancy and Mtr. Together, these findings show that tumor cells generate and export homocysteine, which astrocytes convert to cystathionine. Tumor cystathionine generation responded to changes in methionine- cycle metabolites. In vivo, systemic administration of homocysteine increased tumor cystathionine which decreased in response to systemic folate, the methyl donor for homocysteine methyltransferase. Cystathionine itself was inert in tumors as tumor cells cultured in up to 8 mM cystathionine showed no change in cell cycle progression. Our studies show that medulloblastomas utilize methionine and generate homocysteine, but avoid folate-dependent homocysteine-methionine recycling by exporting homocysteine for detoxification by local astrocytes. This model suggests that treatments that impose methionine scarcity, folate scarcity or CBS inhibition may produce anti-tumor effects in medulloblastoma.

Accumulation of cis- and trans-regulatory variations is associated with phenotypic divergence of a complex trait between yeast species.

Gene regulatory variations accumulate during evolution and alter gene expression. While the importance of expression variation in phenotypic evolution is well established, the molecular basis remains largely unknown. Here, we examine two closely related yeast species, Saccharomyces cerevisiae and Saccharomyces paradoxus, which show phenotypical differences in morphology and cell cycle progression when grown in the same environment. By profiling the cell cycle transcriptome and binding of key transcription factors (TFs) in the two species and their hybrid, we show that changes in expression levels and dynamics of oscillating genes are dominated by upstream trans-variations. We find that multiple cell cycle regulators show both cis- and trans-regulatory variations, which alters their expression in favor of the different cell cycle phenotypes. Moreover, we show that variations in the cell cycle TFs, Fkh1, and Fkh2 affect both the expression of target genes, and the binding specificity of an interacting TF, Ace2. Our study reveals how multiple variations accumulate and propagate through the gene regulatory network, alter TFs binding, contributing to phenotypic changes in cell cycle progression.

Anti-tumour activity of deer growing antlers and its potential applications in the treatment of malignant gliomas

A recent study showed that antlers have evolved a high rate of growth due to the expression of proto-oncogenes and that they have also evolved to express several tumour suppressor genes to control the risk of cancer. This may explain why deer antler velvet (DAV) extract shows anti-tumour activity. The fast growth of antler innervation through the velvet in close association to blood vessels provides a unique environment to study the fast but non-cancerous proliferation of heterogeneous cell populations. We set out to study the anti-cancer effect of DAV in glioblastoma (GB) cell lines in comparison with temozolomide, a chemotherapeutic drug used to treat high-grade brain tumours. Here we report, for the first time, that DAV extract from the tip, but not from mid-parts of the antler, exhibits an anti-tumour effect in GB cell lines (T98G and A172) while being non-toxic in non-cancerous cell lines (HEK293 and HACAT). In T98G cells, DAV treatment showed reduced proliferation (37.5%) and colony-formation capacity (84%), inhibited migration (39%), induced changes in cell cycle progression, and promoted apoptosis. The anticancer activity of DAV extract as demonstrated by these results may provide a new therapeutic strategy for GB treatment.

Effect of Proline on Cell Death, Cell Cycle, and Oxidative Stress in C6 Glioma Cell Line.

Since proline metabolism has been implicated to play an underlying role in apoptotic signaling and cancer, and hyperprolinemic patients present susceptibility to tumors development, this study investigated the effect of proline on cell death, cell cycle, antioxidant enzymes activities, and immunocontent/activity of proteins involved in cell death/survival signaling pathways in C6 glioma cells. C6 cells were incubated with proline (0-5mM) for 1h, 24h, 48h, 72h, or 7days. Proline in high concentrations slightly decreased LDH release, and no cytotoxic effect was seen by Annexin-PI staining. Superoxide dismutase and catalase activities were increased by proline (1mM) after 72h, suggesting an increase in reactive species levels. Acetylcholinesterase activity was inhibited by proline at 1, 3, and 5mM. The cell cycle progression was not altered. Results from Western blot analyses showed that proline at 1mM after 72h increased p-NF-ĸB and decreased acetylcholinesterase immunocontent but did not altered AKT, p-AKT, GSK3β, and p-GSK3β. Taken together, the data suggest that high proline levels seems to favor the signaling pathways towards cell proliferation, since acetylcholinesterase, which may act as tumor suppressor, is inhibited by proline. Also, p-NF-κB is increased by proline treatment and its activation is related to tumor cell proliferation and cellular response to oxidants. Proline also induced oxidative stress, but it appears to be insufficient to induce a significant change in cell cycle progression. These data may be related, at least in part, to the increased susceptibility to tumor development in hyperprolinemic individuals.