A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication

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Abstract

Types 1 and 2 diabetes affect some 380 million people worldwide. Both ultimately result from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with a peak percentage (2%) engaged in the cell cycle in the first year of life1,2,3,4. In embryonic life and after early childhood, beta cell replication is barely detectable. Whereas beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts1,2,3,4,5,6,7,8. Hence, there remains an urgent need for antidiabetic therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, using a high-throughput small-molecule screen (HTS), we find that analogs of the small molecule harmine function as a new class of human beta cell mitogenic compounds. We also define dual-specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine and the nuclear factors of activated T cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation and differentiation. Using three different mouse and human islet in vivo–based models, we show that harmine is able to induce beta cell proliferation, increase islet mass and improve glycemic control. These observations suggest that harmine analogs may have unique therapeutic promise for human diabetes therapy. Enhancing the potency and beta cell specificity of these compounds are important future challenges.

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Figure 1: High-throughput screening reveals harmine family members as agonists of beta cell proliferation.
Figure 2: Structure-activity relationship analysis of harmine analogs on beta cell proliferation.
Figure 3: Calcineurin-NFAT-Dyrk1a signaling is implicated in harmine-induced beta cell proliferation.
Figure 4: Effects of harmine in three in vivo models of beta cell replication and regeneration.

References

  1. 1

    Kassem, S.A., Ariel, I., Thornton, P.S., Scheimberg, I. & Glaser, B. Beta cell proliferation and apoptosis in the developing normal human pancreas and in hyperinsulinism of infancy. Diabetes 49, 1325–1333 (2000).

    CAS  PubMed  Google Scholar 

  2. 2

    Meier, J.J. et al. Beta cell replication is the primary mechanism subserving the postnatal expansion of beta cell mass in humans. Diabetes 57, 1584–1594 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Köhler, C.U. et al. Cell cycle control of beta cell replication in the prenatal and postnatal human pancreas. Am. J. Physiol. Endocrinol. Metab. 300, E221–E230 (2011).

    PubMed  Google Scholar 

  4. 4

    Gregg, B.E. et al. Formation of a human beta cell population within pancreatic islets is set early in life. J. Clin. Endocrinol. Metab. 97, 3197–3206 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Butler, A.E. et al. Beta cell deficit and increased beta cell apoptosis in humans with diabetes. Diabetes 52, 102–110 (2003).

    CAS  PubMed  Google Scholar 

  6. 6

    Saisho, Y. et al. Beta cell mass and turnover in humans: effects of obesity and aging. Diabetes Care 36, 111–117 (2013).

    Google Scholar 

  7. 7

    Kulkarni, R.N., Bernal-Mizrachi, E., Garcia-Ocaña, A. & Stewart, A.F. Human β-cell proliferation and intracellular signaling: driving in the dark without a roadmap. Diabetes 61, 2205–2213 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Bernal-Mizrachi, E., Kulkarni, R.N., Stewart, A.F. & Garcia-Ocaña, A. Human β-Cell proliferation and intracellular signaling part 2: still driving in the dark without a roadmap. Diabetes 63, 819–831 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Soucek, L. & Evan, G.I. The ups and downs of Myc biology. Curr. Opin. Genet. Dev. 20, 91–95 (2010).

    CAS  PubMed  Google Scholar 

  10. 10

    Wierstra, I. & Alves, J. The c-myc promoter: still MysterY and Challenge. Adv. Cancer Res. 99, 113–333 (2008).

    PubMed  Google Scholar 

  11. 11

    Bretones, G., Delgado, M.D. & Leon, J. Myc and cell cycle control. Biochim. Biophys. Acta doi:10.1016/j.bbagrm.2014.03.013 (2014).

  12. 12

    Pelengaris, S., Kahn, M. & Evan, G.I. Suppression of myc-induced apoptosis in beta cells exposes multiple oncogenic properties of myc and triggers carcinogenic progression. Cell 109, 321–334 (2002).

    CAS  PubMed  Google Scholar 

  13. 13

    Pelengaris, S. & Khan, M. Oncogenic co-operation in β-cell tumorigenesis. Endocr. Relat. Cancer 8, 307–314 (2001).

    CAS  PubMed  Google Scholar 

  14. 14

    Finch, A. et al. Bcl-XL gain of function and p19ARF loss of function cooperate oncogenically with Myc in vivo by distinct mechanisms. Cancer Cell 10, 113–120 (2006).

    CAS  PubMed  Google Scholar 

  15. 15

    Laybutt, D.R. et al. Overexpression of c-myc in beta cells of transgenic mice causes proliferation and apoptosis, downregulation of insulin gene expression and diabetes. Diabetes 51, 1793–1804 (2002).

    CAS  PubMed  Google Scholar 

  16. 16

    Cano, D.A. et al. Regulated beta-cell regeneration in the adult mouse pancreas. Diabetes 57, 958–966 (2008).

    CAS  PubMed  Google Scholar 

  17. 17

    Karslioglu, E. et al. cMyc is the principal upstream driver of beta cell proliferation in rat insulinoma cell lines and Is an effective mediator of human beta cell replication. Mol. Endocrinol. 25, 1760–1772 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Chung, N. et al. Median absolute deviation to improve hit selection for genome-scale RNAi screens. J. Biomol. Screen. 13, 149–158 (2008).

    CAS  PubMed  Google Scholar 

  19. 19

    Goktug, A.N., Chai, S.C.C. & Chen, T. Data analysis approaches in high throughput screening. in Drug Discovery Ch. 7, doi:10.5772/52508 (2013).

    Google Scholar 

  20. 20

    Becker, W. & Sippl, W. Activation, regulation and inhibition of Dyrk1a. FEBS J. 278, 246–256 (2011).

    CAS  PubMed  Google Scholar 

  21. 21

    Ogawa, Y. et al. Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1a. Nat. Commun. 1, 86 10.1038/ncomms1090 (2010).

    PubMed  Google Scholar 

  22. 22

    Tahtouh, T. et al. Selectivity, co-crystal structures and neuroprotective properties of leucettines, a family of protein kinase inhibitors derived from the marine sponge alkaloid leucettamine B. J. Med. Chem. 55, 9312–9330 (2012).

    CAS  PubMed  Google Scholar 

  23. 23

    Walte, A. et al. Mechanism of dual specificity kinase activity of Dyrk1a. FEBS J. 280, 4495–4511 (2013).

    CAS  PubMed  Google Scholar 

  24. 24

    Jain, P. et al. Human cdc2-like kinase 1 (CLK1): a novel target for Alzheimer's disease. Curr. Drug Targets 15, 539–550 (2014).

    CAS  PubMed  Google Scholar 

  25. 25

    Shen, W. et al. Small molecule inducer of beta cell proliferation identified by high-throughput screening. J. Am. Chem. Soc. 135, 1669–1672 (2013).

    CAS  PubMed  Google Scholar 

  26. 26

    Gallo, E.M., Cante-Barrett, K. & Crabtree, G.R. Lymphocyte calcium signaling from membrane to nucleus. Nat. Immunol. 7, 25–32 (2006).

    CAS  PubMed  Google Scholar 

  27. 27

    Heit, J.J. et al. Calcineurin/NFAT signaling regulates pancreatic β-cell growth and function. Nature 443, 345–349 (2006).

    CAS  PubMed  Google Scholar 

  28. 28

    Goodyer, W.R. et al. Neonatal beta cell development in mice and humans is regulated by calcineurin/NFaT. Dev. Cell 23, 21–34 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Demozay, D., Tsunekawa, S., Briaud, I., Shah, R. & Rhodes, C.J. Specific glucose-induced control of insulin receptor-supstrate-2 expression is mediated by Ca2+-dependent calcineurin-NFAT signaling in primary pancreatic islet β-cells. Diabetes 60, 2892–2902 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Nica, A.C. et al. Cell-type, allelic and genetic signatures in the human pancreatic beta cell transcriptome. Genome Res. 23, 1554–1562 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Wang, H. et al. Improved low molecular weight Myc-Max inhibitors. Mol. Cancer Ther. 6, 2399–2408 (2007).

    CAS  PubMed  Google Scholar 

  32. 32

    de Alboran, I.M. et al. Analysis of cMYC function in normal cells via conditional gene-targeted mutation. Immunity 14, 45–55 (2001).

    CAS  PubMed  Google Scholar 

  33. 33

    Fotaki, V. et al. Dyrk1a haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice. Mol. Cell. Biol. 22, 6636–6647 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Rachdi, L. et al. Dyrk1a haploinsufficiency induces diabetes in mice through decreased pancreatic beta cell mass. Diabetologia 57, 960–969 (2014).

    CAS  PubMed  Google Scholar 

  35. 35

    Rachdi, L. et al. Dyrk1a induces pancreatic beta cell mass expansion and improves glucose tolerance. Cell Cycle 13, 2221–2229 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Brierley, D.I. & Davidson, C. Developments in harmine pharmacology – implications for ayahuasca use and drug-dependence treatment. Prog. Neuropsychopharmacol. Biol. Psychiatry 39, 263–272 (2012).

    CAS  PubMed  Google Scholar 

  37. 37

    Waki, H. et al. The small molecule harmine is an antidiabetic cell-type specific regulator of PPARγ expression. Cell Metab. 5, 357–370 (2007).

    CAS  PubMed  Google Scholar 

  38. 38

    Purwana, I. et al. GABA promotes human beta-cell proliferation and modulates glucose homeostasis. Diabetes 63, 4197–4205 (2014).

    CAS  PubMed  Google Scholar 

  39. 39

    Wang, W. et al. Identification of small molecule inducers of pancreatic beta cell proliferation. Proc. Natl. Acad. Sci. USA 106, 1427–1432 (2009).

    CAS  PubMed  Google Scholar 

  40. 40

    Chamberlain, C.E. et al. Menin determines K-Ras proliferative outputs in endocrine cells. J. Clin. Invest. 124, 4093–4101 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    He, T.C. et al. Identification of cMyc as a target of the APC pathway. Science 281, 1509–1512 (1998).

    CAS  PubMed  Google Scholar 

  42. 42

    Zhang, J.H., Chung, T. & Oldenburg, K. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

    CAS  PubMed  Google Scholar 

  43. 43

    Lipinski, C.A., Lombardo, F., Dominy, B.W. & Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3–26 (2001).

    CAS  PubMed  Google Scholar 

  44. 44

    Ricordi, C. & Rastellini, C. Methods in pancreatic islet separation. in Methods in Cell Transplantation (ed. Ricordi, C.) 433–438 (R.G. Landes Co, Austin, Texas), (2000).

  45. 45

    Cozar-Castellano, I. et al. Lessons from the first comprehensive molecular characterization of cell cycle control in rodent insulinoma cell lines. Diabetes 57, 3056–3068 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Metukuri, M.R. et al. ChREBP mediates glucose-stimulated pancreatic beta cell proliferation. Diabetes 61, 2004–2015 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Fiaschi-Taesch, N.M. et al. Hepatocyte growth factor (HGF) enhances engraftment and function of non-human primate islets. Diabetes 57, 2745–2754 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Fiaschi-Taesch, N.M. et al. A survey of the human pancreatic beta cell G1/S proteome reveals a potential therapeutic role for cdk-6 and cyclin D1 in enhancing human beta cell replication and function in vivo. Diabetes 58, 882–893 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Fiaschi-Taesch, N. et al. Induction of human beta cell proliferation and engraftment using a single G1/S regulatory molecule, cdk6. Diabetes 59, 1926–1936 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Peshavaria, M. et al. Regulation of pancreatic beta cell regeneration in the normoglycemic 60% pancreatectomy mouse. Diabetes 55, 3289–3298 (2006).

    CAS  PubMed  Google Scholar 

  51. 51

    Alvarez-Perez, J.C. et al. Hepatocyte growth factor/c-Met signaling is required for β-cell regeneration. Diabetes 63, 216–223 (2014).

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors wish to thank R. Vasavada, N. Fiaschi-Taesch, H. Chen, K. Takane, M. Ohlmeyer, R. DeVita, E. Schadt, C. Argmann, B. Losic, D. Lebeche, S. Kim and B. Wagner for their many helpful discussions during this study. We thank the NIDDK-supported Integrated Islet Distribution Program (IIDP), T. Kin at the University of Alberta in Edmonton and P. Witkowski at the University of Chicago for providing human islets. Ad.GFP and Ad.NFATC1 were provided by D. Lebeche (Icahn School of Medicine at Mount Sinai). This work was supported by grants from the National Institutes of Health (R-01 DK55023 (A.F.S.), U-01 DK089538 (A.F.S.), R-01 DK065149 (D.K.S.), R-01 DK067351 (A.G.-O.) and R-01 DK077096 (A.G.-O.)), the JDRF (17-2011-598 and 1-2011-603 (A.F.S.)) and the American Diabetes Association (1-14-BS-059) (A.G.-O.).

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P.W., J.-C.A.-P., D.P.F., H.L., S.S., A.B., A.K., R.S., D.K.S., A.G.-O. and A.F.S. designed and performed experiments. P.W., A.G.-O. and A.F.S. wrote the paper.

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Correspondence to Andrew F Stewart.

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Wang, P., Alvarez-Perez, JC., Felsenfeld, D. et al. A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication. Nat Med 21, 383–388 (2015). https://doi.org/10.1038/nm.3820

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