Expression Of Transcription Factor FOXO1 Research Articles

Mitochondrial Reprogramming By Bcl-2 Inhibitor Venetoclax Enhances αCD19 CAR-T Cell Fitness and Anti-Tumor Efficacy

Introduction Patients undergoing anti-CD19 chimeric antigen receptor T cell (CAR T) therapy for the treatment of aggressive B-cell non-Hodgkin lymphomas (B-NHL) can expect a complete response rate of merely 43-68%. Insubstantial clinical responses to CAR T therapy and a small window of survival for patients facing relapsed/refractory (R/R) disease highlights the critical need for improvement of CAR T therapy in aggressive B-NHL cases. Recent findings suggest that enriching for a CD8 + T Stem Cell Memory (T SCM) population within adoptive cell therapies translates to enhanced antitumor function and persistence in vivo, and thus produces favorable clinical outcomes. T SCM cells display heightened self-renewal, low mitochondrial membrane potential (∆Ψm), and are metabolically dependent upon oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO). Selection of CD8 + T SCM subsets is therefore necessary to ensure a robust antitumor response. Previously, we revealed synergy between the Bcl-2 inhibitor, Venetoclax, and CAR T cells in the context of B-NHL. Venetoclax exposure increased CD8 + T SCM cell frequency, decreased ∆Ψm, reduced T Regulatory (T REG) cell frequency, and bolstered effector cytokine production. Though the role of Bcl-2 inhibition within B-NHLs is well characterized (FDA-approved for frontline and relapsed/refractory chronic lymphocytic leukemia [CLL] treatment), further investigation regarding the impact of Venetoclax on the patient T cell compartment is needed. In this study, we demonstrate a role for Venetoclax as a qualitative enhancer of bulk CAR T cell product using both healthy-donor and B-NHL patient-derived T cells, and implicate modulation of mitochondrial pathways as a likely mechanism of action. Methods Whole peripheral blood mononuclear cells were collected from consenting healthy human donors or CLL patients undergoing ramp-up with Venetoclax (BDR 164122) at Roswell Park Comprehensive Cancer Center, and activated with αCD3/αCD28. CLL samples were collected at baseline, 1 day, 7 days, and 1-month timepoints. Transduction was performed with lentiviral vectors encoding CD28 or 4-1BB-based α-CD19 CAR constructs 24 hours post-activation. Transduced cells were cultured in RPMI with hIL-2 and hIL-7. In vivo studies were performed on 7-week-old SCID mice using Raji-Luc, Rec-1, and U2932. Venetoclax was given daily [100mg/kg] via oral gavage. CAR T cells were delivered by single infusion via tail-vein injection at a dose of either 1x10 6 or 3x10 6 cells total.Mice were imaged via IVIS spectrum. Western blot was performed on stimulated untransduced T cells or CAR T cells exposed to Venetoclax. Total protein was quantified, and membranes were probed for apoptotic and mitochondrial protein expression. Patient sample immunophenotyping was performed on CLL patient samples using panels interrogating memory differentiation, exhaustion, and T REG populations. Results Consistent with our hypothesis, a significant increase in mitochondrial proteins associated with FAO and OXPHOS - ACC, PDH, and AMPKα - was observed in exposed CAR T cells, as well increased expression of transcription factor FOXO1 - an essential driver of CD8 + memory differentiation. Voltage-dependent anion channel (VDAC) expression increased, suggesting heightened efflux of respiratory substrates into the tricarboxylic acid cycle. Hexokinase-I, a critical glycolytic enzyme, was significantly downregulated. We next investigated our combination therapy in vivo and observed extended survival outcome by 15 days. Further in vivo studies investigating preclinical models of mantle cell lymphoma and activated B-cell diffuse large B-cell lymphoma are currently ongoing. Within our CLL patient samples, 24-hour and 7-day timepoints reflected a favorable population increase in CD8 + T SCM cells as compared to baseline samples, and marginally restored CD8 + T cell frequencies. Conclusion Metabolic properties guide T cell memory differentiation and fitness. There exists potential to manipulate this mechanism and select the most metabolically fit T cell population for adoptive cell therapies. In addition to answering fundamental questions of CAR T cell biology, the combination of Venetoclax and CAR T cell therapy may provide a solution to the observed clinical gap, in which CAR T therapy as a single agent does not afford favorable response rates in the context of aggressive and R/R B-NHLs.

Antrodia cinnamomea, a Treasured Medicinal Mushroom, Induces Growth Arrest in Breast Cancer Cells, T47D Cells: New Mechanisms Emerge.

Reported cases of breast cancer have skyrocketed in the last decades with recent advances in examination techniques. Brest cancer has become the second leading cause of mortality among women worldwide, urging the scientific community to develop or find new drugs from natural sources with potent activity and a reasonable safety profile to tackle this ailment. Antrodia cinnamomea (AC) is a treasured medicinal fungus which has attracted attention due to its potent hepatoprotective and cytotoxic activities. We evaluated the antiproliferative activity of the ethanol extract of artificially cultured AC (EEAC) on breast cancer cells (T47D cells) in vivo and in vitro. Ethanol extract of artificially cultured AC inhibited T47D cells’ proliferation mediated by cell cycle arrest at G1 phase as well induced autophagy. Immunoblotting assay confirmed that EEAC not only decreased the expression of the cell-cycle-related proteins but also increased the expression of transcription factor FOXO1, autophagic marker LC3 II, and p62. Ethanol extract of artificially cultured AC mediated endoplasmic reticulum stress by promoting the expression of IRE1 (inositol-requiring enzyme 1α), GRP78/Bip (glucose regulating protein 78), and CHOP (C/EBP homologous protein). Apart from previous studies, HDACs (histone deacetylases) activity was inhibited as demonstrated by a cell-free system, immunoblotting, and immunofluorescence assays following EEAC treatment. The in vivo studies demonstrated that EEAC decreased tumor volume and inhibited tumor growth without any significant side effects. High performance liquid chromatography profile demonstrated similar triterpenoids compared to the profile of wild AC ethanol extract. The multiple targets of EEAC on breast cancer cells suggested that this extract may be developed as a potential dietary supplement targeting this debilitating disease.

Heat therapy promotes the expression of angiogenic regulators in human skeletal muscle.

Heat therapy has been shown to promote capillary growth in skeletal muscle and in the heart in several animal models, but the effects of this therapy on angiogenic signaling in humans are unknown. We evaluated the acute effect of lower body heating (LBH) and unilateral thigh heating (TH) on the expression of angiogenic regulators and heat shock proteins (HSPs) in healthy young individuals. Exposure to LBH (n = 18) increased core temperature (Tc) from 36.9 ± 0.1 to 37.4 ± 0.1°C (P < 0.01) and average leg skin temperature (Tleg) from 33.1 ± 0.1 to 39.6 ± 0.1°C (P < 0.01), but did not alter the levels of circulating angiogenic cytokines and bone marrow-derived proangiogenic cells (CD34(+)CD133(+)). In skeletal muscle, the change in mRNA expression from baseline of vascular endothelial growth factor (VEGF), angiopoietin 2 (ANGPT2), chemokines CCL2 and CX3CL1, platelet factor-4 (PF4), and several members of the HSP family was higher 30 min after the intervention in the individuals exposed to LBH (n = 11) compared with the control group (n = 12). LBH also reduced the expression of transcription factor FOXO1 (P = 0.03). Exposure to TH (n = 14) increased Tleg from 32.8 ± 0.2 to 40.3 ± 0.1°C (P < 0.05) but Tc remained unaltered (36.8 ± 0.1°C at baseline and 36.9 ± 0.1°C at 90 min). This intervention upregulated the expression of VEGF, ANGPT1, ANGPT2, CCL2, and HSPs in skeletal muscle but did not affect the levels of CX3CL1, FOXO-1, and PF4. These findings suggest that both LBH and TH increase the expression of factors associated with capillary growth in human skeletal muscle.