l-2-Hydroxyglutarate remodeling of the epigenome and epitranscriptome creates a metabolic vulnerability in kidney cancer models
Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Richard Kirkman, Madhuparna Pandit, EunHee Shim, Hayley Widden, Juan Liu, Yasaman Heidarian, Nader H. Mahmoudzadeh, Alexander J. Fitt, Devin Absher, Han-Fei Ding, David K. Crossman, William J. Placzek, Jason W. Locasale, Dinesh Rakheja, Jonathan E. McConathy, Rekha Ramachandran, Sejong Bae, Jason M. Tennessen, Sunil Sudarshan
Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Richard Kirkman, Madhuparna Pandit, EunHee Shim, Hayley Widden, Juan Liu, Yasaman Heidarian, Nader H. Mahmoudzadeh, Alexander J. Fitt, Devin Absher, Han-Fei Ding, David K. Crossman, William J. Placzek, Jason W. Locasale, Dinesh Rakheja, Jonathan E. McConathy, Rekha Ramachandran, Sejong Bae, Jason M. Tennessen, Sunil Sudarshan
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Research Article Metabolism Oncology

l-2-Hydroxyglutarate remodeling of the epigenome and epitranscriptome creates a metabolic vulnerability in kidney cancer models

Abstract

Tumor cells are known to undergo considerable metabolic reprogramming to meet their unique demands and drive tumor growth. At the same time, this reprogramming may come at a cost with resultant metabolic vulnerabilities. The small molecule l-2-hydroxyglutarate (l-2HG) is elevated in the most common histology of renal cancer. Similarly to other oncometabolites, l-2HG has the potential to profoundly impact gene expression. Here, we demonstrate that l-2HG remodels amino acid metabolism in renal cancer cells through combined effects on histone methylation and RNA N6-methyladenosine. The combined effects of l-2HG result in a metabolic liability that renders tumors cells reliant on exogenous serine to support proliferation, redox homeostasis, and tumor growth. In concert with these data, high–l-2HG kidney cancers demonstrate reduced expression of multiple serine biosynthetic enzymes. Collectively, our data indicate that high–l-2HG renal tumors could be specifically targeted by strategies that limit serine availability to tumors.

Authors

Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Richard Kirkman, Madhuparna Pandit, EunHee Shim, Hayley Widden, Juan Liu, Yasaman Heidarian, Nader H. Mahmoudzadeh, Alexander J. Fitt, Devin Absher, Han-Fei Ding, David K. Crossman, William J. Placzek, Jason W. Locasale, Dinesh Rakheja, Jonathan E. McConathy, Rekha Ramachandran, Sejong Bae, Jason M. Tennessen, Sunil Sudarshan

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Figure 4

Regulation of PSAT1 expression through l-2HG–induced m6A.

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Regulation of PSAT1 expression through l-2HG–induced m6A. (A and B) Immu...
(A and B) Immunoblot of METTL3 and PSAT1 from 769p (A) and 786-O (B) cells stably expressing the indicated shRNA (shC, control shRNA). Actin and Ponceau serve as loading control. Relative PSAT1 was normalized to Ponceau. (C) RT-qPCR analysis of PSAT1 mRNA expression (normalized to RPLPO) from 786-O and 769p cells stably expressing control shRNA (shC) or METTL3-targeting shRNA. Data are represented as mean ± SD from n = 3 biological replicates. *P < 0.05, **P <0.005. (D) Immunoblot of ALKBH5 and PSAT1 from HK-2 renal epithelial cells transiently transfected (52 hours) with scramble siRNA (Scr) or siRNAs targeting ALKBH5 (#1, #2, #3). Relative PSAT1 was determined by normalizing to Ponceau loading control. (E and F) Immunoblot of ALKBH5 and PSAT1 from 786-O/L2HGDH (E) and 769p/L2HGDH (F) cells transiently transfected (52 hours) with the indicated siRNA Scr, scramble siRNA. Relative PSAT1 was normalized to Ponceau. (G) Immunoblot of FTO and PSAT1 from 769p/L2HGDH cells transiently transfected (52 hours) with the indicated siRNA. Relative PSAT1 was normalized to Ponceau. (H–J) Relative m6A by SELECT at the GGACT site within the PSAT1 3′-UTR. (H) Relative m6A in 786-O cells transduced with control shRNA shC or shMETTL3. (I and J) Relative m6A levels in 786-O (I) and 769p (J) cells transduced with vector control or L2HGDH. Data are shown as mean ± SD from n = 3 biological replicates. (K and L) 769p cells were stably transduced with FLAG-tagged PSAT1 (WT) construct. Cells were transiently transfected (52 hours) with the indicated siRNA followed by immunoblotting (K). (L) Quantitative densitometry of FLAG levels normalized to actin. Data are represented as mean ± SD from n = 3 biological replicates. ANOVA was used, and Tukey’s post hoc P values are shown. (M) 769p cells were stably transduced with FLAG-tagged PSAT1 (MUT) construct. Cells were transiently transfected (52 hours) with the indicated siRNA followed by immunoblotting.