Researchers at the Dana-Farber Cancer Institute have identified a glutamine metabolism pathway that is activated by oncogenic K-Ras in pancreatic cancers.1 Moreover, the team found three different enzyme targets within the pathway that could be blocked to specifically inhibit proliferation of the malignant cells.

Cancer cells have higher metabolic demands than normal cells due to their high levels of growth and proliferation. This increased rate of glycolysis, dubbed the Warburg effect, helps sustain tumor growth.

Recent studies have shown that some oncoproteins, including c-Myc (MYC) and K-Ras, which are known to activate genes involved in cancer cell proliferation, also act as drivers for cancer metabolism.

In a 2012 paper published in Cell, a Dana-Farber team led by Alec Kimmelman identified two K-Ras-activated glucose metabolism pathways in pancreatic cancer cells. Inhibiting the pathways helped stop cancer cell proliferation.2

The K-Ras oncogene is expressed in various cancer types, including about 90% of pancreatic cancers. It is associated with poor prognosis and causes resistance to cancer drugs including epidermal growth factor receptor (EGFR) inhibitors. Attempts to target the oncogene directly have not been successful due to the complex biology and interactions of the enzyme's mutant form.

Building on their previous work, Kimmelman and colleagues found that K-Ras activated an anabolic glutamine metabolic pathway specifically in the cancer cells that not only generated energy and building blocks for protein synthesis but also regulated redox homeostasis to allow cancer cell growth.

Kimmelman is an assistant professor of radiation oncology at Harvard Medical School and Dana-Farber. The paper also included researchers from the Beth Israel Deaconess Medical Center, The University of Texas MD Anderson Cancer Center, Weill Cornell Medical College and Massachusetts General Hospital.

Healthy cells employ a glutamine metabolic pathway that converts glutamine-derived glutamate to a-ketoglutarate (a-KG) using the enzyme glutamate dehydrogenase 1 (GLUD1). a-KG is then used to fuel the tricarboxylic acid (TCA) cycle to create energy. In healthy cells, glutamine also can be processed by transaminases in an anabolic pathway that results in both a-KG and the synthesis of nonessential amino acids (NEAAs).

The researchers wanted to probe whether cancer cells were dependent on either of these pathways (see "Glutamine metabolism").

In pancreatic ductal adenocarcinoma (PDAC) cells, glutamine deprivation or inhibition of glutaminase (GLS), the enzyme that converts glutamine to glutamate early in metabolic processing, suppressed cell growth. Addition of NEAAs plus a-KG, but not a-KG alone, restored cell growth in the glutamine-deprived cells.

These findings suggest that the cancer cells depend on the alternative transaminase-dependent glutamine metabolism pathway for proliferation.

For confirmation, the team showed that a nonspecific transaminase inhibitor or a specific inhibitor of the aspartate transaminase glutamic-oxaloacetic transaminase 1 (GOT1) decreased cell growth compared with vehicle. Inhibition of GLUD1 or other transaminases did not affect cell growth.

Genetic knockout studies further showed that GOT1 and the malic enzymes malate dehydrogenase 1 (MDH1) and NADP-dependent malic enzyme (ME1) catalyzed a series of downstream reactions in the pathway that ultimately resulted in the conversion of NADP+ to NADPH by pyruvate.

The full picture shows that the pathway maintains low levels of oxidative stress and reactive oxygen species (ROS) within tumors. Indeed, knockdown of pathway enzymes increased ROS levels and inhibited proliferation, whereas treatment with an antioxidant restored cell proliferation.

In mice with human PDAC xenografts, expression of small hairpin RNAs targeting GOT1, MDH1 or ME1 suppressed tumor growth, whereas control shRNA or shRNA targeting GLUD1 did not. In healthy human pancreatic ductal cells or diploid fibroblasts, inhibition of the enzymes did not alter cell growth.

Finally, the team drew a connection between oncogenic K-Ras expression and the anabolic glutamine metabolism pathway in PDAC. K-Ras knockdown in the cells decreased levels of GOT1 and increased levels of GLUD1, suggesting that the oncogene upregulates enzymes involved in the alternative pathway.

Safety and specificity

"Based on our work, GOT1, MDH1 and ME1 are all potential therapeutic targets," Kimmelman told SciBX. The team now plans to develop inhibitors of the new targets.

Paul Bingham, VP of research at Cornerstone Pharmaceuticals Inc., said, "The most important thing to do next is to find out if you can attack the malic enzymes and transaminases safely. They have identified really clear targets, but they are still a long way from showing that you can attack these enzymes in an intact animal and not cause unwanted toxicities."

He added that the three new enzyme targets upregulated in tumors are wild-type and thus would be present in some quantities in healthy cells.

Cornerstone's CPI-613, an analog of a-lipoic acid that targets pyruvate dehydrogenase (PDH) and a-KG dehydrogenase, is in
Phase I/II testing with gemcitabine to treat pancreatic cancer. Bingham said the analog targets multiple activities of the cancer mitochondrial metabolic pathway including redox metabolism.

Neil Jones, senior principal scientist at Cancer Research UK's Cancer Research Technology Ltd. commercial arm, cautioned that "historically, perturbation of the glutamine pathway has caused some concerns in terms of potential toxicity, especially in the brain, so it would be important to assess these liabilities as early as possible in development of potential therapies. The fact that normal cell line models were not affected by glutamine pathway modulations goes some way toward addressing this."

According to Susan Critchlow, associate director of innovative medicines and oncology at AstraZeneca plc, "Future studies could evaluate whether small molecule inhibitors of the glutamine metabolism pathway inhibit the growth of established xenograft models and [could] confirm the expected metabolomics profile is desired."

She noted that the validation studies in the paper only used genetic knockdown approaches in small tumors.

AstraZeneca and Cancer Research Technology are identifying cancer metabolism targets and developing therapeutics under a three-year deal. The partners recently extended the deal for two more years through the beginning of 2015.

Critchlow thinks inhibitors of the enzymes identified in the paper could combine well with standard of care for pancreatic cancer, which involves chemotherapy and radiation. The reason, she said, is that enzyme inhibition disrupts the ability of cancer cells to cope with oxidative stress, and standard of care increases intracellular oxidative stress.

She added, "GOT1 shRNA-mediated knockdown gives rise to a cytostatic effect in vivo, suggesting that combination with standard of care may be required to induce tumor regression."

Kimmelman agreed. "Our results suggest that since this pathway is critical for redox balance, there could be synergy with available therapies that generate reactive oxygen species," he said.

Beyond the pancreas

The link between K-Ras and the glutamine metabolism pathway could mean that the three new targets are at work in multiple tumor types.

"Future studies could investigate whether alternative K-Ras-driven tumors, such as non-small cell lung cancers, display this altered glutamine metabolism pathway, opening up the opportunity to extend this therapeutic opportunity into alternative disease segments," said Critchlow.

Jones agreed. "Defining in more detail the potential patient populations and ascertaining the strengths of the K-Ras lineage against these targets and whether they would help other K-Ras-driven tumor types would also help position these targets," he said.

Bingham was less sanguine about the broad applicability of the findings.

"The metabolic implications of oncogenic K-Ras are different in different cell lines. K-Ras may not be activating this particular pathway in other tumor types," he said. "Even if this strategy proves safe, it might only work for this subset of pancreatic tumors. However, this is a nasty group of tumors, so it would be no small accomplishment."

Kimmelman told SciBX that his team has not tested other cancer types. "However, it is possible that since oncogenic K-Ras is responsible for regulating expression of key enzymes in this pathway, that other tumors that have K-Ras mutations may also rely on it for growth," he said. "This will need to be studied."

He said Dana-Farber has filed a patent application covering the work. The IP is available for licensing.

Martz, L. SciBX 6(13); doi:10.1038/scibx.2013.302 Published online April 4, 2013


1.   Son, J. et al. Nat. Med.; published online March 27, 2013; doi:10.1038/nature12040 Contact: Lewis C. Cantley, Weill Cornell Medical College, New York, N.Y. e-mail: Contact: Alec C. Kimmelman, Dana-Farber Cancer Institute, Boston, Mass. e-mail:

2.   Ying, H. et al. Cell 149, 656-670 (2012)


      AstraZeneca plc (LSE:AZN; NYSE:AZN), London, U.K.

      Beth Israel Deaconess Medical Center, Boston, Mass.

      Cancer Research Technology Ltd., London, U.K.

      Cancer Research UK, London, U.K.

      Cornerstone Pharmaceuticals Inc., Cranbury, N.J.

      Dana-Farber Cancer Institute, Boston, Mass.

      Harvard Medical School, Boston, Mass.

      Massachusetts General Hospital, Boston, Mass.

      The University of Texas MD Anderson Cancer Center, Houston, Texas

      Weill Cornell Medical College, New York, N.Y.