Guanosine triphosphate links MYC-dependent metabolic and ribosome programs in small-cell lung cancer
Fang Huang, Kenneth E. Huffman, Zixi Wang, Xun Wang, Kailong Li, Feng Cai, Chendong Yang, Ling Cai, Terry S. Shih, Lauren G. Zacharias, Andrew Chung, Qian Yang, Milind D. Chalishazar, Abbie S. Ireland, C. Allison Stewart, Kasey Cargill, Luc Girard, Yi Liu, Min Ni, Jian Xu, Xudong Wu, Hao Zhu, Benjamin Drapkin, Lauren A. Byers, Trudy G. Oliver, Adi F. Gazdar, John D. Minna, Ralph J. DeBerardinis
Fang Huang, Kenneth E. Huffman, Zixi Wang, Xun Wang, Kailong Li, Feng Cai, Chendong Yang, Ling Cai, Terry S. Shih, Lauren G. Zacharias, Andrew Chung, Qian Yang, Milind D. Chalishazar, Abbie S. Ireland, C. Allison Stewart, Kasey Cargill, Luc Girard, Yi Liu, Min Ni, Jian Xu, Xudong Wu, Hao Zhu, Benjamin Drapkin, Lauren A. Byers, Trudy G. Oliver, Adi F. Gazdar, John D. Minna, Ralph J. DeBerardinis
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Research Article Metabolism Oncology

Guanosine triphosphate links MYC-dependent metabolic and ribosome programs in small-cell lung cancer

Abstract

MYC stimulates both metabolism and protein synthesis, but how cells coordinate these complementary programs is unknown. Previous work reported that, in a subset of small-cell lung cancer (SCLC) cell lines, MYC activates guanosine triphosphate (GTP) synthesis and results in sensitivity to inhibitors of the GTP synthesis enzyme inosine monophosphate dehydrogenase (IMPDH). Here, we demonstrated that primary MYChi human SCLC tumors also contained abundant guanosine nucleotides. We also found that elevated MYC in SCLCs with acquired chemoresistance rendered these otherwise recalcitrant tumors dependent on IMPDH. Unexpectedly, our data indicated that IMPDH linked the metabolic and protein synthesis outputs of oncogenic MYC. Coexpression analysis placed IMPDH within the MYC-driven ribosome program, and GTP depletion prevented RNA polymerase I (Pol I) from localizing to ribosomal DNA. Furthermore, the GTPases GPN1 and GPN3 were upregulated by MYC and directed Pol I to ribosomal DNA. Constitutively GTP-bound GPN1/3 mutants mitigated the effect of GTP depletion on Pol I, protecting chemoresistant SCLC cells from IMPDH inhibition. GTP therefore functioned as a metabolic gate tethering MYC-dependent ribosome biogenesis to nucleotide sufficiency through GPN1 and GPN3. IMPDH dependence is a targetable vulnerability in chemoresistant MYChi SCLC.

Authors

Fang Huang, Kenneth E. Huffman, Zixi Wang, Xun Wang, Kailong Li, Feng Cai, Chendong Yang, Ling Cai, Terry S. Shih, Lauren G. Zacharias, Andrew Chung, Qian Yang, Milind D. Chalishazar, Abbie S. Ireland, C. Allison Stewart, Kasey Cargill, Luc Girard, Yi Liu, Min Ni, Jian Xu, Xudong Wu, Hao Zhu, Benjamin Drapkin, Lauren A. Byers, Trudy G. Oliver, Adi F. Gazdar, John D. Minna, Ralph J. DeBerardinis

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Figure 6

GTP abundance regulates Pol I function in Mychi cells in part through GPN1 and GPN3.

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GTP abundance regulates Pol I function in Mychi cells in part through GP...
(A) Gene sets correlated with GPN3 or GPN1 mRNA from 9,879 tumors in the pan-cancer TCGA database. Gene set enrichment analysis was performed on the top 2% of genes positively correlated with GPN3 or GPN1. (B) mRNA abundance of Myc, Gpn1, and Gpn3 in SCLCs obtained from genetically engineered mouse models with mutant Trp53, Rb1, and Rbl2 (RPR2) and tumors with mutant Trp53 and Rb1 plus transgenic MycT58A (RPM). *P < 0.05, ****P < 0.0001. (C) GPN1 and GPN3 mRNA abundance in DMS53 and DMS53-CR. **P < 0.01, ***P < 0.001. (D) Abundance of GPN1 and GPN3 in DMS53 and DMS53-CR. (E) Abundance of MYC, IMPDH2, GPN1, and GPN3 in H1436 cells expressing dox-inducible empty vector (EV) or MYC treated with doxycycline at the indicated doses for 6 days. (F) OP-Puro signal in pooled H82 cells expressing an EV or with CRISPR/Cas9-mediated knockout of GPN3 or GPN1. **P < 0.01. (G) Proliferation of H82 pools shown in F. **P < 0.01, ****P < 0.0001. (H) Immunoprecipitation with anti-Myc-tag or mouse IgG followed by Western blot for GPN1 or Myc in H82 cells with CRISPR/Cas9-mediated GPN3 knockout and reexpression of wild-type GPN3. (I) GTP pulldown for wild-type or mutant isoforms of GPN3-Myc or GPN1-Myc. GTP was used to compete for binding of GPN1/3 to GTP-agarose beads. (J) GTP pulldown for GPN3, GPN1, RAS, or RRN3 using H82 lysates with increasing concentrations of GTP to compete for binding to GTP-agarose beads. Data are shown as mean and SD (B, C, F, and G). Statistical significance was assessed using a 2-tailed Student’s t test (B and C), 1-way ANOVA with Tukey’s multiple-comparison test (F and G). All experiments were repeated twice or more.