Robust detection of oncometabolic aberrations by¹H-¹³C heteronuclear single quantum correlation in live cells and intact tumorsex-vivo

Abstract

Extensive efforts have been made to use non-invasive 1 H 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 2hydroxyglutarate (2-HG) in tumors harboring isocitrate dehydrogenase 1 (IDH1) mutations. As many metabolites have similar chemical shifts, the resulting 1 H spectra of intact biological material are highly convoluted, limiting the application of 1 H MR to high abundance metabolites. Hydrogen-Carbon Heteronuclear single quantum correlation 1 H-13 C HSQC is routinely employed in organic synthesis to resolve complex spectra but has received limited attention for biological studies. Here, we show that 1 H-13 C HSQC offers a dramatic improvement in sensitivity compared to one-dimensional (1D) 13 C NMR and dramatic signal deconvolution compared to 1D 1 H spectra in an intact biological setting.Using a standard NMR spectroscope without specialized signal enhancements features such as magic angle spinning, metabolite extractions or 13 C-isotopic enrichment, we obtain well-resolved 2D 1 H-13 C 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 1 H-13 C HSQC as a non-invasive diagnostic tool for metabolic precision oncology.IDH1/2 mutations have been the foci of a number of experimental and clinical studies 4 .Both cytoplasmic IDH1 and mitochondrial IDH2 enzymes catalyze the conversion of isocitrate to ⍺-ketoglutarate 5 . Recurrent mutations in the active site of these enzymes result in a neomorphic activity that enables NADPH-dependent reduction of ⍺ketoglutarate to the oncometabolite R-2-hydroxyglurate (2-HG) 6,7 . Thus, the accumulation of 2-HG inside IDH1/2 mutant tumors can reach millimolar level and is the molecular hallmark of this mutation. In glioma, patients harboring IDH mutations have better prognoses compared to the IDH-wildtype group 8,9 , and would be the candidate for targeted therapies such as IDH1 mutant inhibitor, AGI-120 (NCT03564821).The demand driving the development of drugs targeting tumor metabolism extends beyond IDH mutations. An emerging metabolic vulnerability-based precision oncology is collateral lethality 10,11 . Homozygous deletion of major tumor suppressor genes can result in the collateral deletion of chromosomal neighboring metabolic housekeeping genes such as the pentose phosphate shunt enzyme 6-phosphogluconate dehydrogenase (PGD) at the 1p36 locus 10-12 . PGD enzyme catalyzes the oxidative conversion of 6phosphogluconate (6-PG) in the oxidative arm of the pentose phosphate pathway, and its deletion results in >100-fold increased accumulation of 6-phosphogluconate and its hydrolysis product gluconate 12 . ...