Barekatain, Y. | Search bioRxiv

bioRxiv2019

Robust detection of oncometabolic aberrations by 1H-13C heteronuclear single quantum correlation in live cells and intact tumors ex-vivo

Extensive efforts have been made to use non-invasive 1H magnetic resonance (MR) spectroscopy to quantify metabolites that are diagnostic of specific disease states. Within the realm of precision oncology, these efforts have largely centered on quantifying 2-hydroxyglutarate (2-HG) in tumors harboring isocitrate dehydrogenase 1 (IDH1) mutations. As many metabolites have similar chemical shifts, the resulting 1H spectra of intact biological material are highly convoluted, limiting the application of 1H MR to high abundance metabolites. Hydrogen-Carbon Heteronuclear single quantum correlation 1H-13C HSQC is routinely employed in organic synthesis to resolve complex spectra but has received limited attention for biological studies. Here, we show that 1H-13C HSQC offers a dramatic improvement in sensitivity compared to one-dimensional (1D) 13C NMR and dramatic signal deconvolution compared to 1D 1H spectra in an intact biological setting. Using a standard NMR spectroscope without specialized signal enhancements features such as magic angle spinning, metabolite extractions or 13C-isotopic enrichment, we obtain well-resolved 2D 1H-13C HSQC spectra in live cancer cells, in ex-vivo freshly dissected xenografted tumors and resected primary tumors. We demonstrate that this method can readily identify tumors with specific genetic-driven oncometabolite alterations such as IDH mutations with elevation of 2-HG as well as PGD-homozygously deleted tumors with elevation of gluconate. These data support the potential of 1H-13C HSQC as a non-invasive diagnostic tool for metabolic precision oncology.

biophysics↗

bioRxiv2019

Methylthioadenosine is Not Dramatically Elevated in MTAP-Homozygous Deleted Primary Glioblastomas

In BriefThe co-deletion of MTAP in the CDKN2A locus is a frequent event in diverse cancers including glioblastoma. Recent publications report that significant accumulations of the MTAP substrate, methylthioadenosine (MTA), can sensitize MTAP-deleted cancer cells to novel inhibitors of PRMT5 and MAT2A for targeted therapy against tumors with this particular genetic alteration. In this work, using comprehensive metabolomic profiling, we show that MTA is primarily secreted, resulting in exceedingly high levels of extracellular MTA in vitro. We further show that primary human glioblastoma tumors minimally accumulate MTA in vivo, which is likely explained by the metabolism of MTA by MTAP-competent stromal cells. Together, these data challenge whether the metabolic conditions required for therapies to exploit vulnerabilities associated MTAP deletions are present in primary human tumors, questioning their translational efficacy in the clinic.\n\nHighlightsO_LIMethylthioadenosine (MTA) is elevated in MTAP-deleted cancer cells in vitro, which provides a selective vulnerability to PRMT5 and MAT2A inhibitors\nC_LIO_LIAccumulation of MTA in MTAP-deleted cancer cells is predominately extracellular, suggesting active secretion of MTA.\nC_LIO_LIMTAP-deleted primary human glioblastoma tumors show minimal intratumoral elevations of MTA, which is likely explained by secretion and metabolism by MTAP-competent stromal cells.\nC_LI\n\nSUMMARYHomozygous deletion of the CDK2NA locus frequently results in co-deletion of methylthioadenosine phosphorylase (MTAP) in many fatal cancers such as glioblastoma multiforme (GBM), resulting in elevations of the substrate metabolite, methylthioadenosine (MTA). To capitalize on such accumulations, therapeutic targeting of protein arginine methyltransferase 5 (PRMT5) and methionine adenosyl transferase (MAT2A) are ongoing. While extensively corroborated in vitro, the clinical efficacy of these strategies ultimately relies on equally significant accumulations of MTA in human tumors. Here, we show that in vitro accumulation of MTA is a predominately extracellular phenomenon, indicating secretion of MTA from MTAP-deleted cells. In primary human GBMs, we find that MTA levels are not significantly higher in MTAP-deleted compared to MTAP-intact tumors or normal brain tissue. Together, these findings highlight the metabolic discrepancies between in vitro models and primary human tumors and should thus be carefully considered in the development of the precision therapies targeting MTAP-homozygous deleted GBM.

cancer biology↗