Glioblastoma (GBM) represents an aggressive and immune-resistant cancer. Preclinical investigations have identified anti-tumor activity of a ketogenic diet (KD) potentially being used to target GBM’s glycolytic phenotype. Since immune cells in the microenvironment have a similar reliance upon nutrients to perform their individual functions, we sought to determine if KD influenced the immune landscape of GBM. Consistent with previous publications, KD improved survival in GBM in an immune-competent murine model. Immunophenotyping of tumors identified KD-influenced macrophage polarization, with a paradoxical 50% increase in immune-suppressive M2-like-macrophages and a decrease in pro-inflammatory M1-like-macrophages. We recapitulated KD in vitro using a modified cell culture based on metabolomic profiling of serum in KD-fed mice, mechanistically linking the observed changes in macrophage polarization to PPARγ-activation. We hypothesized that parallel increases in M2-macrophage polarization tempered the therapeutic benefit of KD in GBM. To test this, we performed investigations combining KD with the CSF-1R inhibitor (BLZ945), which influences macrophage polarization. The combination demonstrated a striking improvement in survival and correlative studies confirmed BLZ945 normalized KD-induced changes in macrophage polarization. Overall, KD demonstrates antitumor activity in GBM; however, its efficacy is attenuated by promoting an immunosuppressive phenotype in macrophages. Combinatorial strategies designed to modulate macrophage polarization represent a rational approach to improve the anti-tumor activity of KD in GBM.

Pancreatic cancer is the third leading cause of cancer-related mortality in the United States, with rising incidence and mortality. The receptor for advanced glycation end products (RAGE) and its ligands significantly contribute to pancreatic cancer progression by enhancing cell proliferation, fostering treatment resistance, and promoting a pro-tumor microenvironment via activation of the nuclear factor-kappa B (NF-κB) signaling pathways. This study validated pathway activation in human pancreatic cancer and evaluated the therapeutic efficacy of TTP488 (Azeliragon), a small-molecule RAGE inhibitor, alone and in combination with radiation therapy (RT) in preclinical models of pancreatic cancer. Human (Panc1) and murine (Pan02) pancreatic cancer cell lines exhibited elevated levels of RAGE and its ligands compared to normal pancreatic tissue. In vitro, Azeliragon inhibited RAGE-mediated NF-κB activation and ligand-mediated cell proliferation in pancreatic cancer cell lines. Target engagement of Azeliragon was confirmed in vivo, as determined by decreased NF-κB activation. Azeliragon demonstrated significant growth delay in mouse models of pancreatic cancer and additive effects when combined with RT. Additionally, Azeliragon modulated the immune suppressive tumor microenvironment in pancreatic cancer by reducing immunosuppressive cells, including M2 macrophages, regulatory T cells, and myeloid-derived suppressor cells, while enhancing CD8+ T cell infiltration. These findings suggest that Azeliragon, by inhibiting RAGE-mediated signaling and modulating immune response, may serve as an effective anti-cancer agent in pancreatic cancer.

Severely damaged adult zebrafish extraocular muscles (EOMs) regenerate through dedifferentiation of residual myocytes involving a muscle-to-mesenchyme transition. Members of the Twist family of basic helix-loop-helix transcription factors (TFs) are key regulators of the epithelial-mesenchymal transition (EMT) and are also involved in craniofacial development in humans and animal models. During zebrafish embryogenesis, twist family members (twist1a, twist1b, twist2, and twist3) function to regulate craniofacial skeletal development. Because of their roles as master regulators of stem cell biology, we hypothesized that twist TFs regulate adult EOM repair and regeneration. In this study, utilizing an adult zebrafish EOM regeneration model, we demonstrate that inhibiting twist3 function using translation-blocking morpholino oligonucleotides (MOs) impairs muscle regeneration by reducing myocyte dedifferentiation and proliferation in the regenerating muscle. This supports our hypothesis that twist TFs are involved in the early steps of dedifferentiation and highlights the importance of twist3 during EOM regeneration.

Enhanced lipid metabolism, which involves the active import, storage, and utilization of fatty acids from the tumor microenvironment, plays a contributory role in malignant glioma transformation, thereby serving as an important gain of function. In this work, through studies initially designed to understand and reconcile possible mechanisms underlying the antitumor activity of a high-fat ketogenic diet, we discovered that this phenotype of enhanced lipid metabolism observed in glioblastoma may also serve as a metabolic vulnerability to diet modification. Specifically, exogenous polyunsaturated fatty acids (PUFAs) demonstrate the unique ability of short-circuiting lipid homeostasis in glioblastoma cells. This leads to lipolysis-mediated lipid droplet breakdown, an accumulation of intracellular free fatty acids, and lipid peroxidation–mediated cytotoxicity, which was potentiated when combined with radiation therapy. Leveraging these data, we formulated a PUFA-rich modified diet that does not require carbohydrate restriction, which would likely improve long-term adherence when compared with a ketogenic diet. The modified PUFA-rich diet demonstrated both antitumor activity and potent synergy when combined with radiation therapy in mouse glioblastoma models. Collectively, this work offers both a mechanistic understanding and a potentially translatable approach of targeting this metabolic phenotype in glioblastoma through diet modification and/or nutritional supplementation that may be readily integrated into clinical practice.

Reprogramming of metabolic pathways and abnormal acetylation of DNA are two common features of cancer cells, including prostate cancer (PCa). Our results show that polyherbal mixtures of botanicals, like Zyflamend®, can modify lipogenesis in CWR22Rv1 cells (a castrate‐resistant human PCa cell line) by enhancing the activity of 5'adenosine monophosphate (AMP)‐ activated protein kinase (AMPK), a potential tumor suppressor protein that functions as a central energy sensor within the cell. By increasing the phosphorylation of AMPK, Zyflamend® decreases de novo lipid biosynthesis by decreasing the activity of acetyl‐CoA carboxylase (ACC) and the expression of FASN and SREBP1c. Also via AMPK, Zyflamend® inhibits the activity of mTORC1 complex by directly phosphorylating Raptor, decreasing the activity of P70 (S6K, the marker for protein synthesis). All of these effects can be mimicked by treatment of AICAR (activator of AMPK). Furthermore, Zyflamend® increases the acetylation of DNA via mechanisms that appear to involve ATP‐citrate lyase (ACLY). Thus, our results suggest that botanicals, via AMPK, inhibit castrate‐resistant prostate cancer via mechanisms that involve lipid metabolism that may reshuttle acetyl CoAs for histone acetylation, and inhibition of mTOR mediated cell proliferation. Grant Funding Source : This research was supported in part by Tennessee Agricultural Experiment Station