Genome-wide CRISPR screen identifies synthetic lethality between DOCK1 inhibition and metformin in liver cancer

Abstract

Metformin is currently a strong candidate anti-tumor agent in multiple cancers. However, its anti-tumor effectiveness varies among different cancers or subpopulations, potentially due to tumor heterogeneity. It thus remains unclear which hepatocellular carcinoma (HCC) patient subpopulation(s) can benefit from metformin treatment. Here, through a genome-wide CRISPR-Cas9-based knockout screen, we find that DOCK1 levels determine the anti-tumor effects of metformin and that DOCK1 is a synthetic lethal target of metformin in HCC. Mechanistically, metformin promotes DOCK1 phosphorylation, which activates RAC1 to facilitate cell survival, leading to metformin resistance. The DOCK1-selective inhibitor, TBOPP, potentiates antitumor activity by metformin in vitro in liver cancer cell lines and patient-derived HCC organoids, and in vivo in xenografted liver cancer cells and immunocompetent mouse liver cancer models. Notably, metformin improves overall survival of HCC patients with low DOCK1 levels but not among patients with high DOCK1 expression. This study shows that metformin effectiveness depends on DOCK1 levels and that combining metformin with DOCK1 inhibition may provide a promising personalized therapeutic strategy for metformin-resistant HCC patients.

Key words： CRISPR screen; DOCK1; hepatocellular carcinoma; metformin; small GTPase

References

1 Ally A, Balasundaram M, Carlsen R, Chuah E, Clarke A, Dhalla N, Holt RA, Jones SJM, Lee D, Ma Y et al (2017) Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell 169 (7): 1327- 1341.e23

2 Ben Sahra I, Regazzetti C, Robert G, Laurent K, Le Marchand-Brustel Y, Auberger P, Tanti J-F, Giorgetti-Peraldi S, Bost F (2011) Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1. Can Res 71: 4366- 4372

3 Bonnefont-Rousselot D, Raji B, Walrand S, Gardès-Albert M, Jore D, Legrand A, Peynet J, Vasson MP (2003) An intracellular modulation of free radical production could contribute to the beneficial effects of metformin towards oxidative stress. Metabolism 52: 586- 589

4 Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68: 394- 424

5 Bredenoord AL, Clevers H, Knoblich JA (2017) Human tissues in a dish: The research and ethical implications of organoid technology. Science (New York, NY) 355

6 Broutier L, Mastrogiovanni G, Verstegen MM, Francies HE, Gavarró LM, Bradshaw CR, Allen GE, Arnes-Benito R, Sidorova O, Gaspersz MP et al (2017) Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Nat Med 23: 1424- 1435

7 Brugnera E, Haney L, Grimsley C, Lu M, Walk SF, Tosello-Trampont A-C, Macara IG, Madhani H, Fink GR, Ravichandran KS (2002) Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex. Nat Cell Biol 4: 574- 582

8 Carlson CM, Frandsen JL, Kirchhof N, McIvor RS, Largaespada DA (2005) Somatic integration of an oncogene-harboring Sleeping Beauty transposon models liver tumor development in the mouse. Proc Natl Acad Sci USA 102: 17059- 17064

9 Chen H-P, Shieh J-J, Chang C-C, Chen T-T, Lin J-T, Wu M-S, Lin J-H, Wu C-Y (2013) Metformin decreases hepatocellular carcinoma risk in a dose-dependent manner: population-based and in vitro studies. Gut 62: 606- 615

10 Côté J-F, Vuori K (2002) Identification of an evolutionarily conserved superfamily of DOCK180-related proteins with guanine nucleotide exchange activity. J Cell Sci 115: 4901- 4913

11 Dang CV, Reddy EP, Shokat KM, Soucek L (2017) Drugging the “undruggable” cancer targets. Nat Rev Cancer 17: 502- 508

12 Feng H, Hu B, Liu K-W, Li Y, Lu X, Cheng T, Yiin J-J, Lu S, Keezer S, Fenton T et al (2011) Activation of Rac1 by Src-dependent phosphorylation of Dock 180(Y1811) mediates PDGFRα-stimulated glioma tumorigenesis in mice and humans. J Clin Invest 121: 4670- 4684

13 Feng H, Hu B, Jarzynka MJ, Li Y, Keezer S, Johns TG, Tang CK, Hamilton RL, Vuori K, Nishikawa R et al (2012) Phosphorylation of dedicator of cytokinesis 1 (Dock180) at tyrosine residue Y722 by Src family kinases mediates EGFRvIII-driven glioblastoma tumorigenesis. Proc Natl Acad Sci USA 109: 3018- 3023

14 Fukui Y, Hashimoto O, Sanui T, Oono T, Koga H, Abe M, Inayoshi A, Noda M, Oike M, Shirai T, et al (2001) Haematopoietic cellspecific CDM family protein DOCK2 is essential for lymphocyte migration. Nature 412: 826- 831

15 Galdieri L, Gatla H, Vancurova I, Vancura A (2016) Activation of AMP-activated protein kinase by metformin induces protein acetylation in prostate and ovarian cancer cells. J Biol Chem 291: 25154- 25166

16 Guo X, Zhao Y, Yan H, Yang Y, Shen S, Dai X, Ji X, Ji F, Gong X-G, Li L et al (2017) Single tumor-initiating cells evade immune clearance by recruiting type II macrophages. Genes Dev 31: 247- 259

17 Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE, Shaw RJ (2008) AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 30: 214- 226

18 Inoki K, Zhu T, Guan K-L (2003) TSC2 mediates cellular energy response to control cell growth and survival. Cell 115: 577- 590

19 Jiang Y, Sun A, Zhao Y, Ying W, Sun H, Yang X, Xing B, Sun W, Ren L, Hu B et al (2019) Proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma. Nature 567: 257- 261

20 Jin H, Shi Y, Lv Y, Yuan S, Ramirez CFA, Lieftink C, Wang L, Wang S, Wang C, Dias MH et al (2021) EGFR activation limits the response of liver cancer to lenvatinib. Nature 595: 730- 734

21 Kiyokawa E, Hashimoto Y, Kobayashi S, Sugimura H, Kurata T, Matsuda M (1998) Activation of Rac1 by a Crk SH3-binding protein, DOCK180. Genes Dev 12: 3331- 3336

22 Kudo M, Finn RS, Qin S, Han K-H, Ikeda K, Piscaglia F, Baron A, Park J-W, Han G, Jassem J et al (2018) Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. The Lancet 391: 1163- 1173

23 Laurin M, Côté J-F (2014) Insights into the biological functions of Dock family guanine nucleotide exchange factors. Genes Dev 28: 533- 547

24 Laurin M, Huber J, Pelletier A, Houalla T, Park M, Fukui Y, HaibeKains B, Muller WJ, Côté J-F (2013) Rac-specific guanine nucleotide exchange factor DOCK1 is a critical regulator of HER2-mediated breast cancer metastasis. Proc Natl Acad Sci USA 110: 7434- 7439

25 Lee J, Yesilkanal AE, Wynne JP, Frankenberger C, Liu J, Yan J, Elbaz M, Rabe DC, Rustandy FD, Tiwari P et al (2019) Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism. Nature 568: 254- 258

26 Li Y, Xu S, Mihaylova MM, Zheng B, Hou X, Jiang B, Park O, Luo Z, Lefai E, Shyy JYJ et al (2011) AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice. Cell Metab 13: 376- 388

27 Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc J-F, de Oliveira AC, Santoro A, Raoul J-L, Forner A et al (2008) Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359: 378- 390

28 Lord SR, Cheng W-C, Liu D, Gaude E, Haider S, Metcalf T, Patel N, Teoh EJ, Gleeson F, Bradley K et al (2018) Integrated pharmacodynamic analysis identifies two metabolic adaption pathways to metformin in breast cancer. Cell Metab 28: 679

29 Nathan DM, Buse JB, Davidson MB, Ferrannini E, Holman RR, Sherwin R, Zinman B (2009) Medical management of hyperglycemia in type 2 diabetes: a consensus algorithm for the initiation and adjustment of therapy: a consensus statement of the American diabetes association and the european association for the study of diabetes. Diabetes Care 32: 193- 203

30 Nuciforo S, Fofana I, Matter MS, Blumer T, Calabrese D, Boldanova T, Piscuoglio S, Wieland S, Ringnalda F, Schwank G et al (2018) Organoid models of human liver cancers derived from tumor needle biopsies. Cell Rep 24: 1363- 1376

31 Pernicova I, Korbonits M (2014) Metformin-mode of action and clinical implications for diabetes and cancer. Nat Rev Endocrinol 10: 143- 156

32 Pollak MN (2012) Investigating metformin for cancer prevention and treatment: the end of the beginning. Cancer Discov 2: 778- 790

33 Qiu Z, Li H, Zhang Z, Zhu Z, He S, Wang X, Wang P, Qin J, Zhuang L, Wang W et al (2019) A Pharmacogenomic Landscape in Human Liver Cancers. Cancer Cell 36: 179- 193

34 Quinn BJ, Kitagawa H, Memmott RM, Gills JJ, Dennis PA (2013) Repositioning metformin for cancer prevention and treatment. Trends Endocrinol Metab 24: 469- 480

35 Rossman KL, Der CJ, Sondek J (2005) GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6: 167- 180

36 Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11: 783- 784

37 Sun X, Niu X, Chen R, He W, Chen D, Kang R, Tang D (2016) Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis. Hepatology 64: 488- 500

38 Tajiri H, Uruno T, Shirai T, Takaya D, Matsunaga S, Setoyama D, Watanabe M, Kukimoto-Niino M, Oisaki K, Ushijima M et al (2017) Targeting ras-driven cancer cell survival and invasion through selective inhibition of DOCK1. Cell Rep 19: 969- 980

39 Vancura A, Bu P, Bhagwat M, Zeng J, Vancurova I (2018) Metformin as an anticancer agent. Trends Pharmacol Sci 39: 867- 878

40 Wang B, Wang M, Zhang W, Xiao T, Chen CH, Wu A, Wu F, Traugh N, Wang X, Li Z et al (2019) Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute. Nat Protoc 14: 756- 780

41 Wei L, Lee D, Law C-T, Zhang MS, Shen J, Chin DW-C, Zhang A, Tsang FH-C, Wong CL-S, Ng IO-L et al (2019) Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC. Nat Commun 10: 4681

42 Wiesner SM, Decker SA, Larson JD, Ericson K, Forster C, Gallardo JL, Long C, Demorest ZL, Zamora EA, Low WC et al (2009) De novo induction of genetically engineered brain tumors in mice using plasmid DNA. Can Res 69: 431- 439

43 Xie J, Xia L, Xiang W, He W, Yin H, Wang F, Gao T, Qi W, Yang Z, Yang X et al (2020) Metformin selectively inhibits metastatic colorectal cancer with the KRAS mutation by intracellular accumulation through silencing MATE1. Proc Natl Acad Sci U S A 117: 13012- 13022

44 Xu X, Barry DC, Settleman J, Schwartz MA, Bokoch GM (1994) Differing structural requirements for GTPase-activating protein responsiveness and NADPH oxidase activation by Rac. J Biol Chem 269: 23569- 23574

45 Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N et al (2001) Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108: 1167- 1174

46 Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, Benner C, Chanda SK (2019) Metascape provides a biologistoriented resource for the analysis of systems-level datasets. Nat Commun 10: 1523