2-Deoxyglucose Glycolysis Inhibitor Augment Oncolytic Virotherapy to Induce Oxidative Stress and Apoptosis in Breast Cancer (Part Ⅲ)

One of the "hallmarks of cancer" is altered energy metabolism, which is increased glycolysis in cancer cells, the primary source of energy that uses this metabolic pathway to generate ATP. Oncolytic virotherapy with aerobic glycolysis inhibitor smart therapeutic approach to induce apoptosis in cancer cells. The current study aimed to use the 2-Deoxyglucose (2DG), a specific glycolysis inhibitor, to enhance the Newcastle disease virus (NDV). In this study, a mouse model of breast cancer allograft with mammary adenocarcinoma tumor cells (AN3) was used and treated with 2DG, NDV, and a combination of both. Anti-tumor efficacy and glycolysis analysis (hexokinase -1 (HK-1), pyruvate, and ATP) were determined. The induction of oxidative stress was investigated by reactive oxygen species (ROS) and total glutathione assay examination. Apoptosis induction was investigated using immunohistochemistry (cleaved Caspase-3) and histopathology. The result showed that combination therapy enhances anti-tumor efficacy (decrease in relative tumor volume and increase in tumor growth inhibition) of NDV against breast cancer. This effect was accompanied by a reduction in HK-1 concentration, pyruvate, and ATP (glycolysis products). Moreover, NDV+2DG therapy induces oxidative stress (decreases total glutathione and increases ROS). Immunohistochemistry and histopathological examination showed the apoptotic area in tumor tissues in treated groups. In conclusion, the present study found that the combination therapy could be considered as an effective cancer therapy through induction of glycolysis inhibition, oxidative stress, and apoptosis selectively in cancer cells.

ONC201/TIC10 Is Empowered by 2-Deoxyglucose and Causes Metabolic Reprogramming in Medulloblastoma Cells in Vitro Independent of C-Myc Expression

The purpose of this study was to examine whether the imipridone ONC201/TIC10 affects the metabolic and proliferative activity of medulloblastoma cells in vitro. Preclinical drug testing including extracellular flux analyses (agilent seahorse), MTT assays and Western blot analyses were performed in high and low c-myc-expressing medulloblastoma cells. Our data show that treatment with the imipridone ONC201/TIC10 leads to a significant inihibitory effect on the cellular viability of different medulloblastoma cells independent of c-myc expression. This effect is enhanced by glucose starvation. While ONC201/TIC10 decreases the oxidative consumption rates in D458 (c-myc high) and DAOY (c-myc low) cells extracellular acidification rates experienced an increase in D458 and a decrease in DAOY cells. Combined treatment with ONC201/TIC10 and the glycolysis inhibitor 2-Deoxyglucose led to a synergistic inhibitory effect on the cellular viability of medulloblastoma cells including spheroid models. In conclusion, our data suggest that ONC201/TIC10 has a profound anti-proliferative activity against medulloblastoma cells independent of c-myc expression. Metabolic targeting of medulloblastoma cells by ONC201/TIC10 can be significantly enhanced by an additional treatment with the glycolysis inhibitor 2-Deoxyglucose. Further investigations are warranted.

EXTH-68. DUAL METABOLIC REPROGRAMMING BY ONC201/TIC10 AND 2-DEOXYGLUCOSE HAS A STRONG ANTIPROLIFERATIVE EFFECT ON MEDULLOBLASTOMA CELLS

BACKGROUND Medulloblastoma represents one of the most common brain tumors in children. While the understanding of the molecular characteristics of this disease has very much advanced, more efficient and less toxic therapeutics are still in high demand. In this study we examined whether the imipridone ONC201/TIC10 affects the metabolic and proliferative activity of medulloblastoma cells alone and in combination with 2-Deoxyglucose in vitro. METHODS Extracellular flux (agilent seahorse) and Western blot analyses were performed to assess effects on tumor cell metabolism and the expression of proteins of the respiratory chain in established medulloblastoma cells. MTT assays and spheroid assays were performed to examine anti-proliferative effects in a 2-D and 3-D setting. RESULTS Treatment with ONC201/TIC10 has a strong inihibitory effect on the cellular viability of different medulloblastoma cells independent of c-Myc expression. While ONC201/TIC10 decreases the oxidative consumption rates in D458 (c-Myc high) and DAOY (c-Myc low) cells, extracellular acidification rates experienced an increase in D458 and a decrease in DAOY cells. Treatment with ONC201/TIC10 in combination with the glycolysis inhibitor 2-Deoxyglucose synergistically inhibited the cellular viability of medulloblastoma cells and impaired the growth of spheroids. CONCLUSION Overall, ONC201/TIC10 profoundly inhibits the proliferative activity of medulloblastoma cells in a c-Myc-independent manner. Additional treatment with the glycolysis inhibitor 2-Deoxyglucose synergistically enhances the anti-medulloblastoma activity of ONC201/TIC10. This promising approach warrants further investigations.

Targeting the Pentose Phosphate Pathway for SARS-CoV-2 Therapy

SARS-CoV-2 is causing the coronavirus disease 2019 (COVID-19) pandemic, for which effective pharmacological therapies are needed. SARS-CoV-2 induces a shift of the host cell metabolism towards glycolysis, and the glycolysis inhibitor 2-deoxy-d-glucose (2DG), which interferes with SARS-CoV-2 infection, is under development for the treatment of COVID-19 patients. The glycolytic pathway generates intermediates that supply the non-oxidative branch of the pentose phosphate pathway (PPP). In this study, the analysis of proteomics data indicated increased transketolase (TKT) levels in SARS-CoV-2-infected cells, suggesting that a role is played by the non-oxidative PPP. In agreement, the TKT inhibitor benfooxythiamine (BOT) inhibited SARS-CoV-2 replication and increased the anti-SARS-CoV-2 activity of 2DG. In conclusion, SARS-CoV-2 infection is associated with changes in the regulation of the PPP. The TKT inhibitor BOT inhibited SARS-CoV-2 replication and increased the activity of the glycolysis inhibitor 2DG. Notably, metabolic drugs like BOT and 2DG may also interfere with COVID-19-associated immunopathology by modifying the metabolism of immune cells in addition to inhibiting SARS-CoV-2 replication. Hence, they may improve COVID-19 therapy outcomes by exerting antiviral and immunomodulatory effects.

Aliphatic Amines are Viable Pro-drug Moieties in Phosphonoamidate Drugs

ABSTRACTPhosphate and phosphonates containing a single P-N bond are frequently used pro-drug motifs to improve cell permeability of these otherwise anionic moieties. Upon entry into the cell, the P-N bond is cleaved by phosphoramidases to release the active agent. Here, we apply a novel mono-amidation strategy to our laboratory’s phosphonate-containing glycolysis inhibitor and show that a diverse panel of phosphonoamidates may be rapidly generated for in vitro screening. We show that, in contrast to the canonical L-alanine or benzylamine moieties which have previously been reported as efficacious pro-drug moieties, small aliphatic amines demonstrate greater drug release efficacy for our phosphonate inhibitor. These results expand the scope of possible amine pro-drugs that can be used as second pro-drug leave groups for phosphate or phosphonate-containing drugs.Abstract Figure

Regulation of the Elevated T Cell Glycolysis May Alleviate Acute Graft-Versus-Host Disease Post-Allotransplant

Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Metabolism not only provide energy and substrates for T cell growth and survival, but also instruct effector functions, differentiation, and gene expression of T cells. In this regard, the metabolic profile of T cells was reported to play a critical role in the occurrence and development of many immunological disorders such as systemic lupus erythematosus and rheumatoid arthritis. Murine studies found that alloreactive T cells use aerobic glycolysis as the predominant metabolic process to meet activation and proliferation demand after allo-HSCT. However, the metabolic profile of T cells and the approach for regulating T cell metabolism in aGVHD patients remains to be elucidated. Aims: To determine the metabolic state in T cells of patients with aGVHD. Moreover, to investigate the effect of the novel approach targeting the abnormal metabolism in T cells of aGVHD patients, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT. Methods: In this prospective case-control study, a total of 25 patients with aGVHD and 25 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. T cell subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. Th1, Th2, Th17, and Treg cells were identified as CD4+IFN-γ+, CD4+IL-4+, CD4+IL17A+, and CD4+CD25+Foxp3+, respectively. Tc1 and Tc2 cells were identified as CD8+IFN-γ+ and CD8+IL-4+, respectively. In order to determine the metabolic state in T cells of patients with aGVHD and non-aGVHD, the metabolic profile was determined using a Seahorse XF96 Analyzer. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. The mitochondrial mass, the mitochondrial membrane potential, the protein expressions for the lipid metabolism enzyme CTP1a and the glycolytic activator PFKFB3 were measured by flow cytometry. To further understand the metabolic state of T cells in aGVHD and non-aGVHD patients and investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of T cells. Subsequently, to explore the potential way of targeting the abnormal metabolism in T cells, the glycolysis inhibitor 3-PO was administrated to T cells from aGVHD patients. Results: When compared with T cells in non-aGVHD patients, T cells in aGVHD patients were polarized towards pro-inflammatory T cells, characterized by an elevated proportion of Tc1, Th1 and Th17. Furthermore, T cells isolated from aGVHD patients exhibited higher extracellular acidification rate, as well as the increased glucose consumption rate and lactate production rate compared to those in non-aGVHD patients. Moreover, elevated expression of PFKFB3 was observed in T cells, especially in naïve T cells of aGVHD patients, but oxygen consumption rate, CPT1A, mitochondrial mass or membrane potential showed no significant differences in T cells between aGVHD and non-aGVHD patients. These results implied higher glycolytic activity of T cells in aGVHD patients when compared with those in non-aGVHD patients. Consistent with the increased glycolytic activity observed in T cells from aGVHD patients, the mRNA levels of genes involved in the glycolytic pathway were substantially elevated in T cells of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vitro treatment with glycolysis inhibitor 3-PO improved the activity of T cells derived from aGVHD patients through down-regulating glycolytic activity of T cells. Summary/Conclusion: The current study demonstrated that T cells in aGVHD patients preferentially depend on glycolysis to meet activation and proliferation demands. Furthermore, the activity of T cells from aGVHD patients could be ameliorated by glycolysis inhibitor 3-PO in vitro. Although further validation is required, T cell glycolysis promises to be a novel therapeutic target for aGVHD patients after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Glycolysis Restoration Attenuates Damaged Bone Marrow Endothelial Cells

Background: Bone marrow(BM) endothelial cells(ECs), a key component of BM microenvironment, is essential for the physiology and regeneration of hematopoietic stem cells (HSCs). The damage of ECs is recognized by us and other researchers as a mainstay in the pathophysiology of a serious of life-threatening complications after chemoradiotherapy and myeloablative hematopoietic cell transplantation(HSCT), including poor graft function (PGF) (2013BBMT, 2015BMT, 2016Blood, 2019Blood Advances). Despite numerous researches focused on the BM ECs contributing to HSC regeneration following myelotoxicity, the mechanisms underlying the injured BM ECs itself remain to be elucidated. Under physiological conditions, energy metabolism plays an instrumental role in maintaining EC function, and markedly perturbed of EC metabolism is linked to many pathologies, like cancer and diabetes. However, little is known about the metabolism state and its role in impaired BM ECs. Aims: The current study was performed to investigate the metabolism status in BM ECs after chemotherapy-induced injury. Moreover, we evaluated the metabolic state and its role in BM ECs of PGF patients post-allotransplant. Finally, we evaluated the therapeutical potential of anti-metabolic drugs to the dysfunctional BM ECs derived from PGF patients. Methods: Two EC injury models in vitro were established with the cultivated human BM ECs treated by 5-Fluorouracil (5-FU) and hydrogen peroxide. The findings from the above models were further validated by a prospective case-control study enrolled 15 patients with PGF, 30 matched patients with good graft function (GGF) and 15 healthy donors (HD). To determine the metabolic status of BM ECs, the expression of metabolism regulating genes was analyzed by qRT-PCR (mRNA level) and flow cytometry (protein level). Glucose metabolism levels were measured by glucose consumption and lactate production assays. To evaluate the functions of BM ECs, apoptosis, migration and tube formation assays were performed. To investigate the effect of anti-metabolic drugs to injured BM ECs, the glycolysis inhibitor 3PO and PPARd agonist GW501516 were administrated to the cultivated BM ECs treated by 5-FU , hydrogen peroxide or derived from PGF. Results: We demonstrated that the glycolysis in BM ECs could be induced by the treatment with either 5-FU or hydrogen peroxide in vitro, consistent with the dysfunction(impaired migration, angiogenesis, and higher level of apoptosis) of BM ECs, which could be attenuated by glycolysis restoration. Mechanistically, we revealed that the aberrant glycolysis and dysfunction of BM ECs could be triggered by PPARd knockdown in vitro, while the PPARd were down-regulated by either 5-FU or hydrogen peroxide treatment in vitro, Furthermore, PPARd agonist GW501516 treatment attenuated the perturbed function and number of injured BM ECs treated by either 5-FU or hydrogen peroxide. Subsequently, the prospective case-control study demonstrated elevated expressions of the glycolytic activator PFKFB3 and decreased PPARd were observed in BM ECs of PGF patients, when compared with those of GGF patients and HD, indicating that BM ECs of PGF patients have a hyper-glycolytic metabolism. Moreover, either glycolysis (PFKFB3) inhibitor 3PO or PPARd agonist GW501516 treatment reduced the aberrant glycolysis and improved the number and function of BM ECs derived from patients with PGF in vitro, revealing the critical role of defective glycolysis in the impaired BM ECs of PGF. Summary / Conclusions: These findings reveal that hyper-glycolysis mediated by PPARd inhibition is involved in the dysfunction of BM ECs after injury. Defective glycolysis may contribute to the pathobiology of BM ECs of PGF patients, which could be attenuated by glycolysis inhibitor 3PO or PPARd agonist GW501516 in vitro. Our findings might merit further consideration of targeting BM ECs glycolysis or PPARd as a promising therapeutic approach for PGF patients post-allotransplant in the future. Disclosures No relevant conflicts of interest to declare.