Acute myeloid leukemia (AML) is a devastating disease, and clinical outcomes are still far from satisfactory. Here, to identify novel targets for AML therapy, we performed a genome-wide CRISPR/Cas9 screen using AML cell lines, followed by a second screen in vivo. We show that PAICS, an enzyme involved in de novo purine biosynthesis, is a potential target for AML therapy. AML cells expressing shRNA-PAICS exhibited a proliferative disadvantage, indicating a toxic effect of shRNA-PAICS. Treatment of human AML cells with a PAICS inhibitor suppressed their proliferation by inhibiting DNA synthesis and promoting apoptosis and had anti-leukemic effects in AML PDX models. Furthermore, CRISPR/Cas9 screens using AML cells in the presence of the inhibitor revealed genes mediating resistance or synthetic lethal to PAICS inhibition. Our findings identify PAICS as a novel therapeutic target for AML and further define components of de novo purine synthesis pathway and its downstream effectors essential for AML cell survival.
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Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, et al. Genomic classification and prognosis in acute myeloid leukemia. N. Engl J Med. 2016;374:2209–21. https://doi.org/10.1056/NEJMoa1516192
Döhner H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Büchner T, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129:424–47. https://doi.org/10.1182/blood-2016-08-733196
Short NJ, Rytting ME, Cortes JE. Acute myeloid leukaemia. Lancet. 2018;392:593–606. https://doi.org/10.1016/S0140-6736(18)31041-9
Welch JS, Ley TJ, Link DC, Miller CA, Larson DE, Koboldt DC, et al. The origin and evolution of mutations in acute myeloid leukemia. Cell. 2012;150:264–78. https://doi.org/10.1016/j.cell.2012.06.023
Lawrence MS, Stojanov P, Mermel CH, Robinson JT, Garraway LA, Golub TR, et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature. 2014;505:495–501. https://doi.org/10.1038/nature12912
Garraway LA, Lander ES. Lessons from the cancer genome. Cell. 2013;153:17–37. https://doi.org/10.1016/j.cell.2013.03.002
Boehm JS, Hahn WC. Towards systematic functional characterization of cancer genomes. Nat Rev Genet. 2011;12:487–98. https://doi.org/10.1038/nrg3013
Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31:230–2. https://doi.org/10.1038/nbt.2507
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23. https://doi.org/10.1126/science.1231143
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21. https://doi.org/10.1126/science.1225829
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6. https://doi.org/10.1126/science.1232033
Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera MeC, Yusa K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol. 2014;32:267–73. https://doi.org/10.1038/nbt.2800
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343:84–7. https://doi.org/10.1126/science.1247005
Shi J, Wang E, Milazzo JP, Wang Z, Kinney JB, Vakoc CR. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat Biotechnol. 2015;33:661–7. https://doi.org/10.1038/nbt.3235
Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014;343:80–4. https://doi.org/10.1126/science.1246981
Yamauchi T, Masuda T, Canver MC, Seiler M, Semba Y, Shboul M, et al. Genome-wide CRISPR-Cas9 Screen Identifies Leukemia-Specific Dependence on a Pre-mRNA Metabolic Pathway Regulated by DCPS. Cancer Cell. 2018;33:386–400.e5. https://doi.org/10.1016/j.ccell.2018.01.012
Hinze L, Pfirrmann M, Karim S, Degar J, McGuckin C, Vinjamur D, et al. Synthetic lethality of wnt pathway activation and asparaginase in drug-resistant acute leukemias. Cancer Cell. 2019;35:664–76.e7. https://doi.org/10.1016/j.ccell.2019.03.004
Han K, Jeng EE, Hess GT, Morgens DW, Li A, Bassik MC. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions. Nat Biotechnol. 2017;35:463–74. https://doi.org/10.1038/nbt.3834
Stone RM, Mandrekar SJ, Sanford BL, Laumann K, Geyer S, Bloomfield CD, et al. Midostaurin plus chemotherapy for acute myeloid leukemia with a FLT3 mutation. N. Engl J Med. 2017;377:454–64. https://doi.org/10.1056/NEJMoa1614359
Perl AE, Martinelli G, Cortes JE, Neubauer A, Berman E, Paolini S, et al. Gilteritinib or chemotherapy for relapsed or refractory. N. Engl J Med. 2019;381:1728–40. https://doi.org/10.1056/NEJMoa1902688
Stein EM, DiNardo CD, Pollyea DA, Fathi AT, Roboz GJ, Altman JK, et al. Enasidenib in mutant. Blood. 2017;130:722–31. https://doi.org/10.1182/blood-2017-04-779405
DiNardo CD, Stein EM, de Botton S, Roboz GJ, Altman JK, Mims AS, et al. Durable remissions with Ivosidenib in IDH1-mutated relapsed or refractory AML. N. Engl J Med. 2018;378:2386–98.
DiNardo CD, Pratz K, Pullarkat V, Jonas BA, Arellano M, Becker PS, et al. Venetoclax combined with decitabine or azacitidine in treatment-naive, elderly patients with acute myeloid leukemia. Blood. 2019;133:7–17.
Yamauchi T, Takenaka K, Urata S, Shima T, Kikushige Y, Tokuyama T, et al. Polymorphic Sirpa is the genetic determinant for NOD-based mouse lines to achieve efficient human cell engraftment. Blood. 2013;121:1316–25.
Canver MC, Lessard S, Pinello L, Wu Y, Ilboudo Y, Stern EN, et al. Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nat Genet. 2017;49:625–34.
Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature. 2015;527:192–7.
Hasunuma T, Kikuyama F, Matsuda M, Aikawa S, Izumi Y, Kondo A. Dynamic metabolic profiling of cyanobacterial glycogen biosynthesis under conditions of nitrate depletion. J Exp Bot. 2013;64:2943–54.
Fushimi T, Izumi Y, Takahashi M, Hata K, Murano Y, Bamba T. Dynamic metabolome analysis reveals the metabolic fate of medium-chain fatty acids in AML12 cells. J Agric Food Chem. 2020;68:11997–2010.
Martinez Molina D, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science. 2013;341:84–7.
Xu H, Gopalsamy A, Hett EC, Salter S, Aulabaugh A, Kyne RE, et al. Cellular thermal shift and clickable chemical probe assays for the determination of drug-target engagement in live cells. Org Biomol Chem. 2016;14:6179–83.
Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15:554.
Meyers RM, Bryan JG, McFarland JM, Weir BA, Sizemore AE, Xu H, et al. Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells. Nat Genet. 2017;49:1779–84.
Tyner JW, Tognon CE, Bottomly D, Wilmot B, Kurtz SE, Savage SL, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562:526–31.
Hoxhaj G, Hughes-Hallett J, Timson RC, Ilagan E, Yuan M, Asara JM, et al. The mTORC1 signaling network senses changes in cellular purine nucleotide levels. Cell Rep. 2017;21:1331–46.
Agarwal S, Chakravarthi BVSK, Behring M, Kim HG, Chandrashekar DS, Gupta N, et al. PAICS, a purine nucleotide metabolic enzyme, is involved in tumor growth and the metastasis of colorectal cancer. Cancers. 2020;12. https://doi.org/10.3390/cancers12040772.
Colic M, Wang G, Zimmermann M, Mascall K, McLaughlin M, Bertolet L, et al. Identifying chemogenetic interactions from CRISPR screens with drugZ. Genome Med. 2019;11:52.
Oakes SA, Papa FR. The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol. 2015;10:173–94.
Li SX, Tong YP, Xie XC, Wang QH, Zhou HN, Han Y, et al. Octameric structure of the human bifunctional enzyme PAICS in purine biosynthesis. J Mol Biol. 2007;366:1603–14.
HARTMAN SC, BUCHANAN JM. Biosynthesis of the purines. XXVI. The identification of the formyl donors of the transformylation reactions. J Biol Chem. 1959;234:1812–6.
LUKENS LN, BUCHANAN JM. Biosynthesis of the purines. XXIV. The enzymatic synthesis of 5-amino-1-ribosyl-4-imidazolecarboxylic acid 5’-phosphate from 5-amino-1-ribosylimidazole 5’-phosphate and carbon dioxide. J Biol Chem. 1959;234:1799–805.
Chabner BA, Roberts TG. Timeline: chemotherapy and the war on cancer. Nat Rev Cancer. 2005;5:65–72.
Jackson RC, Harkrader RJ. Synergistic and antagonistic interactions of methotrexate and 1-beta-D-arabinofuranosylcytosine in hepatoma cells. The modulating effect of purines. Biochem Pharmacol. 1981;30:223–9.
Batova A, Diccianni MB, Omura-Minamisawa M, Yu J, Carrera CJ, Bridgeman LJ, et al. Use of alanosine as a methylthioadenosine phosphorylase-selective therapy for T-cell acute lymphoblastic leukemia in vitro. Cancer Res. 1999;59:1492–7.
Duval N, Luhrs K, Wilkinson TG, Baresova V, Skopova V, Kmoch S, et al. Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: cellular models of de novo purine biosynthesis deficiency disorders. Mol Genet Metab. 2013;108:178–89.
Zhou S, Yan Y, Chen X, Wang X, Zeng S, Qian L, et al. Roles of highly expressed PAICS in lung adenocarcinoma. Gene. 2019;692:1–8.
Chakravarthi BVSK, Rodriguez Pena MDC, Agarwal S, Chandrashekar DS, Hodigere Balasubramanya SA, Jabboure FJ, et al. A role for de novo purine metabolic enzyme PAICS in bladder cancer progression. Neoplasia. 2018;20:894–904. https://doi.org/10.1016/j.neo.2018.07.006
Meng M, Chen Y, Jia J, Li L, Yang S. Knockdown of PAICS inhibits malignant proliferation of human breast cancer cell lines. Biol Res. 2018;51:24.
Chakravarthi BVSK, Goswami MT, Pathi SS, Dodson M, Chandrashekar DS, Agarwal S, et al. Expression and role of PAICS, a de novo purine biosynthetic gene in prostate cancer. Prostate. 2018;78:693–4. https://doi.org/10.1002/pros.23533
HENDERSON JF, KHOO KY. On the mechanism of feedback inhibition of purine biosynthesis de novo in ehrlich ascites tumor cells in vitro. J Biol Chem. 1965;240:3104–9.
HARTMAN SC, BUCHANAN JM. The biosynthesis of the purines. Ergeb Physiol. 1959;50:75–121.
Sant ME, Lyons SD, Phillips L, Christopherson RI. Antifolates induce inhibition of amido phosphoribosyltransferase in leukemia cells. J Biol Chem. 1992;267:11038–45.
Pelet A, Skopova V, Steuerwald U, Baresova V, Zarhrate M, Plaza JM, et al. PAICS deficiency, a new defect of de novo purine synthesis resulting in multiple congenital anomalies and fatal outcome. Hum Mol Genet. 2019;28:3805–14.
Schuhmacher M, Kohlhuber F, Hölzel M, Kaiser C, Burtscher H, Jarsch M, et al. The transcriptional program of a human B cell line in response to Myc. Nucleic Acids Res. 2001;29:397–406.
Kim J, Lee JH, Iyer VR. Global identification of Myc target genes reveals its direct role in mitochondrial biogenesis and its E-box usage in vivo. PLoS One. 2008;3:e1798.
Barfeld SJ, Fazli L, Persson M, Marjavaara L, Urbanucci A, Kaukoniemi KM, et al. Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer. Oncotarget. 2015;6:12587–602.
Furukawa J, Inoue K, Maeda J, Yasujima T, Ohta K, Kanai Y, et al. Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals. Sci Rep. 2015;5:15057.
Takenaka R, Yasujima T, Furukawa J, Hishikawa Y, Yamashiro T, Ohta K, et al. Functional Analysis of the Role of Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) in Adenine Transport in HepG2 Cells. J Pharm Sci. 2020;109:2622–2628.
Townsend EC, Murakami MA, Christodoulou A, Christie AL, Köster J, DeSouza TA, et al. The public repository of xenografts enables discovery and randomized phase II-like trials in mice. Cancer Cell. 2016;30:183.
We thank the members of the Department of Medicine and Biosystemic Science at Kyushu University for assistance, advice, and helpful discussion and Simon Osborne, Craig Southern, Debra Taylor, and Kevin Buchan from LifeArc for providing MRT00252040, and Elise Lamar for critical reading of the manuscript. This work is supported in part by a Grant-in-Aid for Young Scientists (19K17859), Research Grant of KANAE Foundation, MSD Life Science Foundation, The Yasuda Medical Foundation, Mochida Memorial Foundation for medical and pharmaceutical research, The Shinnihon Foundation of Advanced Medical Treatment Research, Takeda Science Foundation (to TY), a Grant-in-Aid for Scientific Research (S)(16H06391)(to KA) and an American Society of Hematology Bridge Grant, a Grant-in-Aid for Scientific Research (A) (17H01567), Grant-in-Aid for Scientific Research (S) (20H05699), and AMED under grant number 18063889 (to TM).
The authors declare no competing interests.
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Yamauchi, T., Miyawaki, K., Semba, Y. et al. Targeting leukemia-specific dependence on the de novo purine synthesis pathway. Leukemia 36, 383–393 (2022). https://doi.org/10.1038/s41375-021-01369-0