Effects of a cancer metabolite on immune response

Immunotherapy is currently considered as one of the most exciting areas in cancer therapy, however it still needs
further improvement. The failure to prevent, control and completely eradicate malignancies by infiltrating immune cells
can be exacerbated by the metabolic flexibility of neoplastic cells. All forms of immunotherapy, including immune
checkpoint inhibitors (ICIs), CAR-T and CAR-NK cells are likely to be impacted by the build-up of specific metabolites and
signaling molecules in the tumor microenvironment (TME). Transcriptomics analysis of TCGA data revealed that in many
cancer types high expression of glutaminase (GLS) is usually associated with poor survival rate. GLS is an enzyme that,
together with glutamate dehydrogenase, metabolize glutamine into alpha-ketoglutarate and 2 molecules of ammonia.
Ammonia, a waste byproduct of amino-acid metabolism, tends to accumulate in the TME at supra-physiological
concentrations, 50-100x higher than the level of ammonia in plasma. Therefore, I hypothesized that a TME rich in
ammonia would be more immunosuppressive. My preliminary data support the idea that ammonia is an
immunosuppressive factor since it strongly inhibits the cytotoxic activity of NK cells toward target cancer cells and may
ultimately impact all forms of cancer immunotherapies. The research proposal will tackle the following questions:
1) How could immune effector cells, such as NK cells and cytotoxic T cells (CTLs) sense ammonia?
2) How can we prevent the accumulation of ammonia in TME and recover the cytotoxic function of immune cells?
3) Will the reduction in ammonia production stimulate anti-tumor immunity and lead to tumor growth inhibition?
In order to uncover the ammonia’s mechanism of action, I have already tested if ammonia affects the release of
cytolytic granules by effectors cells. I found that degranulation process is not inhibited by ammonia. Therefore, we plan
to carry out a proteome and secretome analysis of ammonia-treated cytotoxic NK and T cells. We may find changes in
the levels of activating or inhibitory receptors, lytic enzymes or secreted cytokines, since they all are necessary for
efficient killing of target cancer cells. Additionally, ammonia was reported to induce autophagy in cancer cells. Indeed, I
found that ammonia upregulates the level of Sequestosome-1/p62, a cargo receptor for the autophagic degradation of
proteins. Therefore, we plan to confirm the hypothesis that ammonia inhibits cytotoxicity function of NK and CTLs by
inducing autophagy. Recent reports also showed that function of CTLs can be suppressed by increased concentration of
extracellular potassium (K+) ion, due to autophagy. Thus, we plan to measure extracellular K+ concentration,
accompanied with metabolome analysis of ammonia-treated NK and CTLs.
In order to inhibit the build-up of ammonia in TME, we will test several pharmacological metabolism modulators that:
i) inhibit GLS and glutamine transporters SLC1A5 and SLC7A5 in cancer cells, previously screened for high expression of
GLS and ammonia production;
ii) activate the carbamoyl phosphate synthase-1 to trap ammonia in the urea cycle using carglumic acid, an FDAapproved
treatment for hyperammonemia.
The efficacy of these drugs on the production of ammonia and the cytotoxic activity of NK cells and CTLs will be
evaluated. Lastly, we will evaluate in vivo efficacy of the most efficient metabolism modulators that were able to reduce
the ammonia production by cancer cells. Their efficacy in reducing ammonia levels in tumor interstitial fluids, tumor
infiltration by CTL and NK and tumor growth will be tested in the syngeneic mouse model. The selected compound, that
achieved the best control of tumor growth will be next combined with immune checkpoint inhibitors (ICIs), such as anti-
PD1 or anti-CTLA4, to test whether the inhibition of ammonia build-up in tumors enhances the response of the tumors
to immunotherapy.
Finally, the implementation of the project will provide the necessary evidence regarding the role of ammonia in inducing
immunosuppression and resistance to immunotherapeutics. The results will demonstrate its mechanism of action and
provide proof of concept that abrogation of ammonia accumulation in the TME could be a beneficial approach for
effective immunotherapy.