Rising Stars of Cancer Metabolism and Signaling

Cancer metabolism is influenced both by cell-intrinsic factors and cell-extrinsic factors. There has been substantial effort to understand how cell-intrinsic factors, such as oncogenic lesions, alter cellular metabolism. However, much less is known about how microenvironmental conditions alter cancer metabolism. Nutrient availability is a cell-extrinsic factor that substantially influences cellular metabolism, yet we have relatively little information regarding nutrient availability in tumors and how this impacts cancer metabolism. To address this, we developed a quantitative metabolomics approach to measure nutrient levels in the interstitial fluid (IF) of tumors, providing insight into the metabolic substrates available to cells in their local microenvironment. To determine how local nutrient availability alters cancer cell metabolism, we have developed a cell culture medium based on observed IF nutrient levels in murine pancreatic cancers. Pancreatic cancer cells, as well tumor stromal cells, can be grown in this IF-based medium to study how cells utilize metabolism to support homeostasis and function when constrained by microenvironmental nutrient levels. I will discuss metabolic adaptations that pancreatic cancer cells require to grow under such microenvironmental nutrient constraints that we have identified using this approach. In conclusion, we provide new tools determine how nutrient availability impacts cellular metabolism in healthy and diseased mammalian tissues.

Cells and organisms must coordinate their metabolic activity with changes in their nutrient environment. Our lab focuses on the regulation of nucleotide metabolism by the cell signaling pathways. Nucleotides play a central role in metabolism at a fundamental and cellular level. Purine and pyrimidine bases can be synthesized de novo or recycled through the salvage pathways. Nucleotides are essential for the synthesis of nucleic acids, proteins and cell membranes. Our goal is to decipher the molecular mechanisms by which the growth signaling pathways impinge on the metabolic pathways contributing to nucleotide synthesis. I will discuss our ongoing research on how signaling pathways modulate cellular metabolism to mobilize building blocks for macromolecular synthesis. We anticipate that our studies will yield new insights into how cellular metabolism, particular nucleic acid metabolism, is regulated in normal cells and pathological settings.

Most patients diagnosed with acute myeloid leukemia (AML) who receive intensive chemotherapy achieve a significant clinical response; however, the majority will relapse and succumb to their disease. Relapsed disease in AML has been shown to be caused by the inability of current therapies to completely eradicate leukemia stem cells (LSCs). Thus, novel therapies specifically designed to target LSCs in relapsed AML patients are urgently needed. Previously, we and others have shown that LSCs can be targeted by perturbing energy metabolism without harming normal stem cells. Therefore, the goal of our current studies is to identify and target metabolic dependencies of relapsed LSCs, with the objective to improve outcomes for relapsed AML patients. To achieve this objective, we measure metabolic differences in LSCs isolated from newly diagnosis AML patients and following chemotherapy relapse. This analysis revealed that the relapsed LSCs are metabolically distinct from diagnosis LSCs. We have shown that some of the metabolic differences, including nicotinamide metabolism, are targetable and decrease the function of relapsed LSCs and not diagnosis LSCs or normal stem cells. Further, we have identified that a subset of metabolites that are increased at relapse confer resistance to chemotherapy as well as other AML therapies. Overall, these data suggest that metabolic changes that occur during disease progression, may represent therapeutic targets and influence therapy response in AML.

Lung cancer is the leading cause of cancer deaths in the United States in both men and women. Immunotherapy has recently emerged as a promising strategy for this devastating disease, but its efficacy is modest, prolonging progression-free survival only 4-16 months in a subset of patients with advanced disease. Obesity – a risk factor for the occurrence and progression of more than a dozen tumor types – appears to enhance the response to checkpoint inhibitors. However, the reasons for the effect of obesity to improve outcomes in lung cancer patients treated with immunotherapy remain unclear. In the current study, we replicated the clinical findings of slower tumor growth and improved efficacy of checkpoint inhibitor with obesity: Lewis lung carcinoma tumor volume reached 1699±120 mm3 in regular chow fed mice five weeks after implantation, and 916±73 mm3 in mice fed a high-fat, high-carbohydrate Western diet (P=0.0005), while in mice treated with an anti-PD-1 antibody, tumor volume reached 410±115 mm3 in lean mice and 56±17 mm3 in Western diet fed mice (P=0.02). Our data indicate that the impact of obesity to improve the efficacy of immunotherapy in this murine model of lung cancer results from increased gluconeogenesis, which promotes T cell activation, and increased lipolysis, which results in protection against T cell exhaustion. When we inhibited gluconeogenesis by treatment with an antisense oligonucleotide to knock down pyruvate carboxylase, this resulted in a partial reversal of the protective effect of obesity (tumor volume 269±53 mm3, P=0.005 vs. Western diet fed mice). We measured mRNA expression of the activation marker CD25 in tumor-infiltrating CD3 T cells of mice in which pyruvate carboxylase was knocked down, and found that CD25 expression was reduced by 55% (P=0.006) in mice in which gluconeogenesis was inhibited, consistent with an effect of glucose from obesity-associated increases in gluconeogenesis promoting T cell activation.

Similarly, when we inhibited systemic lipolysis using a small molecule inhibitor of the rate-limiting lipolytic enzyme adipose triglyceride lipase (ATGL), we found that while checkpoint inhibition initially led to tumor regression, tumors ultimately grew to a similar volume to those of regular chow fed mice (tumor volume 396±41 mm3, P<0.0001 vs. Western diet fed mice), suggesting an effect of obesity-associated increases in lipolysis to prevent T cell exhaustion. This was confirmed by assessment of T cell PD-1 expression: in PD-1 antibody treated mice, T cell PD-1 expression was higher in regular chow fed mice, consistent with T cell exhaustion (2.48±0.42 [expression in relative units] in lean mice vs. 1.00±0.29 in Western diet fed mice, P=0.02). However, ATGL inhibition led to an increase in PD-1 expression in Western diet fed mice (2.61±0.34, P=0.02 vs. chow fed mice), with PD-1 mRNA levels in atglistatin-treated Western diet fed mice similar to those of regular chow fed mice.

Having observed that the increase in gluconeogenesis observed with obesity promotes T cell activation, while the increase in lipolysis protects against T cell exhaustion, we next wanted to understand whether either glucose or fatty acids has a direct effect on Lewis lung carcinoma cell division. However, physiologic increases in glucose (5 to 7 mM), fatty acids (0.2 to 1.0 mM), and insulin (100 to 250 pM) similar to those observed in obesity did not have a significant effect on cell division in vitro. These data suggest that obesity is protective against lung cancer and improves the efficacy of immunotherapy due to its effect to boost anti-tumor immune function DocuSign Envelope ID: ECE41E31-09E4-4CD6-884F-241CD55DFED9 while not increasing tumor cell division as a result of increased glucose and/or insulin as is seen in obesity-associated tumor types. Treating tumor-bearing mice with kaempferol, a small molecule agent which increases lipolysis, improved the effect of anti-PD-1 immunotherapy to slow tumor growth in lean mice (tumor volume 84±16 mm3 with kaempferol vs. 435±52 mm3 without, P=0.0002). These data identify kaempferol or other small molecule activators of lipolysis as deserving of further exploration to enhance the efficacy of checkpoint inhibitors in lean subjects. Such a strategy, informed by mechanistic insights into how obesity improves the efficacy of immunotherapy, could allow patients to benefit from the improvements in anti-cancer immune function resulting from obesity without subjecting them to the many deleterious consequences of obesity.

The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.

Therapy resistance is a major driver of poor outcomes in colorectal cancer (CRC), contributing to the second highest rate of cancer related deaths worldwide. The antimetabolite 5-Fluorouracil (5FU) remains the backbone of CRC chemotherapy treatments, but only 10-15% of patients respond, hence a better understanding of the mechanisms driving 5FU resistance in CRC is vital.

Metabolic reprogramming in cancers can alter therapeutic sensitivity, and changes to multiple metabolic programs have been reported for Kras mutant CRC, however the importance of this in contributing to therapy resistance remains unclear. We postulated that response to 5FU may be mediated by distinct metabolic programs in Kras mutant CRC, and a more thorough understanding of the metabolic impact of 5FU may help us predict 5FU resistance clinically.

Using a multi-omics approach we map response to 5FU-based treatments in CRC models, and uncover a significant role for oxidative mitochondrial metabolism in mediating 5FU response that promotes cell survival and facilitates resistant outgrowth. We demonstrate that oxidative phosphorylation levels can be used to predict 5FU resistance clinically, aiding patient stratification. Importantly, this induced metabolic dependency can be therapeutically exploited to improve 5FU responses, and presents an interesting avenue for novel therapeutic combination strategies in CRC that warrant further exploration.

Antioxidants are used by cancers to quench oxidative stress and survive. The most abundant cellular antioxidant is glutathione (GSH). Synthesis of GSH is non-redundantly controlled by glutamate-cysteine ligase catalytic subunit (GCLC). The requirement for GSH in vivo is unclear. To interrogate this, we developed Gclcf/f CreERT2 mice with the ability to induce ablation of GSH synthesis in vivo. Here, we show that while systemic GSH synthesis is required for cancer development, tumor-intrinsic GSH is dispensable. Deletion of GCLC in non-tumor bearing mice resulted in weight loss, depletion of adipose tissue and lower levels of circulating triglycerides. Newly synthesized circulating lipids are produced by the liver. Surprisingly, the liver tissue showed no signs of pathology, but instead displayed a strong induction of genes associated with the antioxidant transcription factor NRF2. Intriguingly, NRF2 activity is also associated with suppressed transcription of lipogenic enzymes, and expression of lipogenic enzymes was reduced in GSH-depleted livers. Further, GCLC KO tumors also showed reduced expression of lipogenic enzymes and a nearcompleted depletion of triglycerides. Finally, we show that combined inhibition of GCLC and de novo lipid synthesis synergistic kills cancer cells. These findings suggest an important function for GSH in the maintenance of lipid homeostasis both in malignant and normal tissues.

We recently determined that concurrent inhibition of autophagy, using the lysosomal inhibitor chloroquine (CQ), and of ERK, using a small molecule ERK inhibitor (ERKi), synergistically suppressed the growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and patient xenograft-derived (PDX) organoids in vitro and PDX tumors in vivo (Bryant et al., 2019, Nat Med 25:628). Our findings, together with similar observations by McMahon, Kinsey and colleagues (Kinsey et al., 2019, Nat Med 25:620), provided the rationale for our initiation of a Phase I clinical trial evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996) with hydroxychloroquine (HCQ) in PDAC. We have extended these findings and determined that combined inhibition of ERK and autophagy is effective in additional cancers driven by KRAS/NRAS, BRAF, or EGFR. Additionally, we have also shown that the combined inhibition of RASG12C and autophagy was effective in KRASG12C-mutant cancers. Our ongoing studies are centered on developing additional combinations for targeting autophagy. First, we are evaluating inhibitors of the ULK kinases, key initiators of autophagy, in combination with ERKi. Second, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers as well as mediators of resistance to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment.