Abstract A69: Mutagenicity of urea cycle dysregulation and its implications for cancer immunotherapy
Authors: Lee Joo Sang , Carmel Narin , Karathia Hiren , Auslander Noam , Rabinovich Shiran , Keshet Rom , Stettner Noa , Silberman Alon , Agemy Lilach , Helbling Daniel , Eilam Raya , Sun Qin , Brandis Alexander , Weiss Hila , Dimmock David , Stern-Ginossar Noam , Scherz Avigdor , Ulitsky Igor , Nagamani Sandesh CS , Elhasid Ronit , Hannenhalli Sridhar , Ruppin Eytan , Erez Ayelet 
Published: February 2019

Abstract
Immune checkpoint therapy leads to durable clinical responses in many cancer patients, but fails in others. To improve the clinical response to immunotherapy, it is highly important to identify predictive biomarkers. While checkpoint genes’ expression levels, tumor neo-antigen load and microsatellite instability (MSI) have been associated with enhanced response to checkpoint immunotherapies, they yet provide only a modest predictive signal and hence there is a need to identify additional predictive factors. Specifically, while there is growing evidence that metabolic alterations can affect the tumor and modulate the immune response, the potential effects of altered cancer metabolism on tumor mutagenesis and immunotherapy remain unexplored.
The urea cycle (UC) converts excess nitrogen derived from the breakdown of nitrogen-containing molecules (e.g., ammonia) to urea, a relatively non-toxic and disposable nitrogenous compound. We and others have shown that silencing of the UC enzyme ASS1 promotes cancer proliferation by diverting its substrate aspartate toward CAD enzyme, which mediates the first three reactions in the pyrimidine synthesis pathway. We now demonstrate, by analysis of the TCGA data, tumor samples and cancer cell line experiments, that UC dysregulation (UCD) is a much wider common metabolic phenomenon that maximizes nitrogen utilization in cancer, favoring pyrimidine synthesis over urea disposal. Of note, while UCD is significantly associated with decreased cancer patient survival, the overall mutational load is not.
Remarkably, we find that the UCD changes the 1:1 purine (R)-to-pyrimidine (Y) ratio in favor of pyrimidine in cancer cells. Moreover, in analysis of both TCGA data and UC perturbed cancer cells we find that: (a) UCD is significantly associated with a novel and unique pattern of purine-to-pyrimidine transversion mutational bias across many cancer types at the DNA coding (sense) strand, and (b) this trend becomes stronger and more significant at both the mRNA and protein levels, testifying to its functional implications. Notably, the overall mutational load in cancer is negatively correlated with UCD, testifying to their independence.
To test whether the mutational bias is associated with better immunotherapy response, we analyze published data of three large melanoma cohorts. We find that responders of both anti-PD1 and anti-CTLA4 therapy exhibit significantly higher UCD and R-&gt;Y mutational bias than non-responders. We further observe that the peptides carrying transverse R-&gt;Y mutations are preferentially presented as neoantigens in responders independent of mutational load, and this trend becomes significant for more clonal neoantigens, promoting UCD as a potential biomarker for the success of immunotherapy.
Finally, as nitrogen metabolites are excreted in the urine, we hypothesize that these changes may be detectable in urine of UCD-cancers. We observe increased levels of pyrimidine derived metabolites in the urine of mice bearing colon tumors associated with UCD in the tumors compared to normal intestine. In an analogous manner, we find significantly higher levels pyrimidine derived metabolites in the urine of human patients with prostate cancer compared to controls.
Collectively, these results support our hypothesis that UCD is a prevalent metabolic phenomenon in cancer, generating mutational biased neo-peptides, worsening patients’ prognosis and yet enhancing the response to immune therapy independent of mutational load and MSI. Taken together, our findings point to the important role of UCD in a broad spectrum of cancers, to the potential use of UCD related metabolites as cancer biomarkers, and last but not least, to the role of UCD in predicting response to immune check point therapy. Broadly, our results suggest future therapeutic interventions aiming to increase UCD levels to enhance the coverage and efficiency of cancer immunotherapy.
Citation Format: Joo Sang Lee, Narin Carmel, Hiren Karathia, Noam Auslander, Shiran Rabinovich, Rom Keshet, Noa Stettner, Alon Silberman, Lilach Agemy, Daniel Helbling, Raya Eilam, Qin Sun, Alexander Brandis, Hila Weiss, David Dimmock, Noam Stern-Ginossar, Avigdor Scherz, Igor Ulitsky, Sandesh CS Nagamani, Ronit Elhasid, Sridhar Hannenhalli, Eytan Ruppin, Ayelet Erez. Mutagenicity of urea cycle dysregulation and its implications for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A69.