<b><i>Background:</i></b> Tumorigenesis is associated with deregulation of nutritional requirements, intermediary metabolites production, and microenvironment interactions. Unlike their normal cell counterparts, tumor cells rely on aerobic glycolysis, through the Warburg effect. <b><i>Summary:</i></b> The pentose phosphate pathway (PPP) is a major glucose metabolic shunt that is upregulated in cancer cells. The PPP comprises an oxidative and a nonoxidative phase and is essential for nucleotide synthesis of rapidly dividing cells. The PPP also generates nicotinamide adenine dinucleotide phosphate, which is required for reductive metabolism and to counteract oxidative stress in tumor cells. This article reviews the regulation of the PPP and discusses inhibitors that target its main pathways. <b><i>Key Message:</i></b> Exploiting the metabolic vulnerability of the PPP offers potential novel therapeutic opportunities and improves patients’ response to cancer therapy.

Colorectal cancer is the third most common neoplasia and the second cause of cancer-related deaths worldwide. Unlike normal cells, tumor cells reprogram metabolic pathways to meet their bioenergetics, redox, biosynthetic demands and rely mostly on aerobic glycolysis (Warburg effect). Colorectal cancer cells upregulate the pentose phosphate pathway (PPP), and p53 is a crucial regulator. The PPP is a significant route for glucose catabolism and is required for DNA synthesis of rapidly-proliferating cells. Its oxidative phase (oxPPP), catalyzed by the rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD), provides the cell with nicotinamide adenine dinucleotide phosphate (NADPH) that is needed for biosynthetic and detoxifying processes. For the past six decades, 5-Fluorouracil (5-FU) has been the treatment of choice in colorectal cancer. However, 5-FU exhibits high toxicity and drug resistance in patients. Therefore, we hypothesized that targeting the PPP might offer novel therapeutic opportunities in colorectal cancer and improve the response to 5-FU at lower and safer doses. Accordingly, we aim to investigate and characterize the anti-tumor effect of the G6PD inhibitor, 6-aminonicotinamide (6-AN), alone, or in combination with 5-FU on colorectal cancer cells. We explored the effects of oxPPP inhibition on colorectal cancer cell growth, reactive oxygen species (ROS) production, cell cycle regulation, and senescence. We have shown that 6-AN arrested cell growth in HCT116 and HCT116 p53−/− colorectal cancer cells while spared normal-like colon cells (NCM460D). Moreover, 6-AN induced S-phase arrest and early senescence in HCT116 cells as demonstrated by cell cycle analysis using flow cytometry and senescence-associated beta-galactosidase assay, respectively. We also tested the effects of 6-AN/5-FU combination treatments on cellular viability using MTT assay and validated our results using SRB and trypan blue exclusion assays. Interestingly, combination treatments of 6-AN with 5-FU resulted in synergistic effects as estimated using Compusyn software. We used HCT116 colorectal cancer cells resistant to 5-FU (HCT116 5FU-R) or lacking p53, as models for tumor resistance. Importantly, the combination treatment sensitized both HCT116 p53−/− and HCT116 5FU-R colorectal cancer cells to 5-FU and was accompanied by a reduction in G6PD activity and increased ROS using NBT reduction assay. Our findings indicate that combining 6-AN with 5-FU may decrease resistance and further sensitize colorectal cancer cells to 5-FU treatment independently of p53 and drug resistance status. Exploiting this metabolic vulnerability may offer a novel clinical approach and improve patient's therapy. Citation Format: Noorhan Ghanem, Chirine El Baba, Lara Al Saleh, Berthe Hayar, Patrick Aouad, Marwa Al Hassan, Riyad El-Khoury, Julnar Usta, Nadine Darwiche. Therapeutic targeting of the pentose phosphate pathway in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 236.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and represents a highly heterogeneous and aggressive disease. Up to 40% of all DLBCL patients experience treatment failure after the standard immunochemotherapy R-CHOP. Defining novel therapeutic strategies is therefore a pressing need. Reduced acetylation at histone 3 lysine 27 (H3K27ac) represents a pathogenetic hallmark of DLBCL and selective histone deacetylase 3 inhibition (HDAC3i) has recently emerged as a viable strategy to reverse abnormal epigenetic signatures and induce strong anti-tumor effects by increasing H3K27ac. Despite this compelling rationale, the overall response remains suboptimal, suggesting unrecognized mechanisms of resistance. To unbiasedly uncover sensitizers, we performed a genome-wide CRISPR screen and identified knockout (KO) of the top candidate GNAS (encoding G-protein α subunit, Gαs) for sensitizing resistant DLBCL cells to HDAC3i. The sensitizing effects on reduced viable cell number were only observed in GNAS KO cells treated with HDAC3i, but not in the control cells (“synthetic lethality”). We have validated this sensitizing phenotype in multiple resistant cell lines including three human DLBCL cell lines and one mouse B-cell lymphoma cell line. Interestingly, we showed that the sensitization was not related to the canonical G-protein functions but to novel regulatory mechanisms in viral mimicry activation and chromatin modification. Compared with the control conditions, the interaction between GNAS KO and HDAC3i has been linked with differential chromatin accessibility and endogenous retrovirus expression, upregulated double-stranded RNA expression, and increased phosphorylation of TBK1 whose activation plays a vital role in antiviral immune responses. Strikingly, a selective TBK1 degrader completely and specifically rescued the sensitizing phenotype in GNAS KO cells treated with HDAC3i, confirming the critical role of TBK1 in inducing sensitization. Furthermore, we identified potential immune interactions by exploring the clinical data from tumor samples of 309 DLBCL patients that low GNAS expression was correlated with cytokine and inflammatory signaling as well as stromal cell interactions in the tumor microenvironment, altogether associated with longer overall survival. These observations were aligned with our results in the mouse xenograft and allograft tumor experiments that GNAS KO with/without HDAC3i led to differential phenotypes. Overall, our research uncovers novel immunomodulating activities for potentiating the efficacy of epigenetic agents in non-Hodgkin lymphoma. This expands our understanding of novel interactions between immune and epigenetic regulation in lymphoma biology and provides new therapeutic opportunities for patients with resistant lymphoma who have poor prognoses and limited treatment options. Citation Format: Michael Y. He, Kit I. Tong, Mehran Bakhtiari, Ryder Whittaker Hawkins, Ting Liu, Yong Zeng, Noorhan Ghanem, Housheng Hansen He, Robert Kridel. Characterizing novel immunomodulating interactions for potentiating HDAC3 inhibition in non-Hodgkin lymphoma [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 1757.