Targeting Compensatory Metabolic Pathways: Novel Approaches To Overcome Resistance To Glutaminase Inhibition In Nf1 Driven Malignant Peripheral Nerve Sheath Tumors

Acquired therapeutic resistance to targeted therapies has garnered a lot of attention owing to its clinical relevance in cancer research. In addition to acquired mutations as part of the resistant mechanism, key metabolic pathways are often dysregulated in resistant cells. One such important metabolic pathway mediated by glutamine, is critical for the survival and proliferation of many cancer cells. In this study, we studied the mechanism of development of acquired resistance to glutaminase (GLS1) inhibitor, CB-839, in NF1 driven MPNST cell lines. While NF1-null cell lines ST8814 and S462 were sensitive to CB-839, wild-type NF1 bearing cell line, STS26T did not show inhibition of proliferation by CB-839. To develop therapeutic resistance, we treated NF1-null S462 cell line by chronic exposure to CB-839 in a dose escalating manner. Cell viability assay and western blot analysis confirmed abrogation of CB-839 mediated inhibition of proliferation and induction of apoptosis in resistant cell lines. Arginosuccinate synthetase (ASS1), a key metabolic enzyme, has previously been shown to be upregulated in response to chronic pharmacologic arginine deprivation. Western blot analysis in CB-839 sensitive versus resistant S462 cell lines showed upregulation of ASS1 expression. Wild type NF1 carrying cell line STS26T also showed high levels of ASS1 expression. To test whether upregulated ASS1 levels contribute to the development of resistance, we carried out siRNA mediated knockdown of ASS1 in both CB-839 resistant S462 cell line and STS26T cell line. Knockdown of ASS1 resulted in inhibition of proliferation in STS26T cell line as well as restoring sensitivity of resistant S462 cell line to CB-839. Furthermore, overexpression of ASS1 in parental CB-839 sensitive S462 cell line resulted in diminished response to CB-839 mediated inhibition of proliferation. Metabolic profiling of parental versus resistant S462 cell lines showed drastically reduced levels of succinate, a key metabolic intermediate in the TCA cycle, suggesting that upregulation of ASS1 in the resistant cell line results in a cross talk between glutamine and arginine pathways thereby feeding the glutamine into urea cycle rather than TCA cycle. Global histone profiling in CB-839 resistant S462 cell line showed a marked decrease in the repressive methylation marks H3K9me3 as well as H3K27me3. Rescue experiment using exogenous succinate indeed resulted in restoring suppressed H3K9me3 signal in the CB-839 resistant cell line. Taken together, our data strongly suggests that compensatory metabolic pathways such as ASS1 can be used as a means of developing therapeutic resistance to glutaminase inhibition. Targeting of such compensatory pathways is a novel potential approach that needs to be evaluated further in the treatment of drug resistant NF1 driven MPNST tumors. Citation Format: Tahir N. Sheikh, Chao Lu, Gary K. Schwartz. Targeting compensatory metabolic pathways: Novel approaches to overcome resistance to glutaminase inhibition in NF1 driven malignant peripheral nerve sheath tumors [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 248.