ANALYSIS OF CHANGES IN ABNORMAL DNA METHYLATION IN THE PROCESS OF COMPLEX TREATMENT IN ACUTE MYELOID LEUKEMIA IN CHILDREN

An aberrant DNA methylation distribution is a functional event in the process of leukemogenesis and a target of epigenetic therapy. To identify DNA methylation markers most common for any molecular subtype of pediatric acute myeloid leukemia (AML) we have applied a method of unbiased differential methylation screening of the genomes and designed  multiplex MS-PCR system of DNA methylation markers belonging to the promoter regions of the genes  EGFLAM, TMEM176A/176B, GSG1L, CLDN7, CXCL14 and SOX8. The system has a sensitivity of 90—91% for determining the malignant process. We have studied bone marrow samples  from 39 children with AML. All patients were treated by decitabine and ATRA in complex with chemotherapy (CT). Methylation index (MI) was 0.197 ± 0.181 for patients with a myeloblasts content less than 40%, and 0.514 ± 0.222 for patients with myeloblasts content more than 40% (p = 0.000736). Methylation of the CLDN7, GSGL1 and EGFLAM genes is absent  in the group with low MI. Patients with the initial content of myeloblasts less than 40% demonstrate absence of methylation on the 15th day after the start of the CT. The average MI decreases in the group with the initially high content of myeloblasts due to decrease in the frequencies of methylation of the genes CXCL14, TMEM176A/176B, GSGL1 and SOX8. The 5-day course  of demethylation therapy is accompanied by an increase in the content of blast cells and an equalization of the methylation profile. With the marker system developed it is possible to evaluate the malignant progression of blast cells, which are considered morphologically normal after CT, demonstrating at the same time the abnormal methylation profile of tumor cells.

1. Li S, Mason CE, Melnick A. Genetic and epigenetic heterogeneity in acute myeloid leukemia. Curr Opin Genet Dev. 2016. 36:100-106. doi: 10.1016/j.gde.2016.03.011.

2. Swerdlow SH, Campo E, Harris NL et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon, France. 2008.

3. Gaidzik V, Dohner K. Prognostic implications of gene mutations in acute myeloid leukemia with normal cytogenetics. Semin. Oncol. 2008. 35:346-355. doi: 10.1053/j.seminoncol.2008.04.005.

4. Valerio DG, Katsman-Kuipers JE, Jansen JH et a l. Mapping epigenetic regulator gene mutations in cytogenetically normal pediatric acute myeloid leukemia. Haematologica. 2014. 99(8):e130 — e132. doi:10.3324/haematol.2013.094565.

5. Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 1999. 99:247 — 257.

6. Herman JG, Civin CI, Issa J.-P.J et al. Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies. Cancer Res. 1997. 57(5): 837-41.

7. Kelly TK, De Carvalho DD, Jones PA. Epigenetic modifications as therapeutic targets. Nature Biotechnology. 2010. 28:1069-1078. doi: 10.1038/nbt.1678.

8. Simmons D. Epigenetic influence and disease. Nature Education. 2008. 1(1):6.

9. Boultwood J, Wainscoat JS. Gene silencing by DNA methylation in haematological malignancies. Br J Haematol. 2007. 138:3-11. DOI:10.1111/j.1365-2141.2007.06604.x.

10. Ehrlich M. DNA hypomethylation in cancer cells. Epigenomics. 2009. doi: 10.2217/epi.09.33.

11. Stresemann C, Lyko F. Modes of action of the DNA methyl-transferase inhibitors azacytidine and decitabine. Int J Cancer. 2008. 123(1):8-13. doi: 10.1002/ijc.23607.

12. Tanas AS, Rudenko (Shkarupo) VV, Kuznetsova EB, Zaletayev DV, Strelnikov VV. Epigenomics. 2010. 2(2):325-33. doi: 10.2217/epi.10.3.

13. Руденко В.В., Немировченко В.С., Tанас А.С., Попа А.В., Казакова С.А., Кузнецова Е.Б., Залетаев Д.В., Стрельников В.В. Новые маркеры аномального метилирования ДНК при остром миелоидном лейкозе у детей, идентифицированные непредвзятым скринингом дифференциального метилирования геномов. Медицинская генетика. 2015. 14(1): 36-44.

14. Rius M, Lyko F. Epigenetic cancer therapy: rationales, targets and drugs. Oncogene. 2012. 31(39):4257-65. doi: 10.1038/onc.2011.601.

15. Navada SC, Steinmann J, Lubbert M, Silverman LR. Clinical development of demethylating agents in hematology. J Clin Invest. 2014. 124(1): 40-6. doi: 10.1172/JCI69739.

16. West AC, Johnstone RW. New and emerging HDAC inhibitors for cancer treatment. J Clin Invest. 2014. 124(1):30-9. doi: 10.1172/JCI69738.

17. Ahuja N, Easwaran H, Baylin SB. Harnessing the potential of epigenetic therapy to target solid tumors. J Clin Invest. 2014. 124(1):56-63. doi: 10.1172/JCI69736.

18. Lakshmikuttyamma A, Scott SA, DeCoteau JF, Geyer CR. Reexpression of epigenetically silenced AML tumor suppressor genes by SUV39H1 inhibition. Oncogene. 2010. 29:576-588. doi: 10.1038/onc.2009.361.

19. The cancer genome atlas research network. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013. 368(22):2059-2074. DOI: 10.1056/NE-JMoa1301689.

20. Paschka P, Schlenk RF, Gaidzik VI, Habdank M, Krînke J, Bullinger L, Spàth D, Kayser S, Zucknick M, Gîtze K, Horst HA, Germing U, Dîhner H, Dîhner K. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication. Journal of Clinical Oncology. 2010. 28(22): 3636-3643. doi: 10.1200/JCO.2010.28.3762.

21. Lund K, John J Cole JJ, VanderKraats ND, McBryan T, Pchelintsev NA, Clark W, Copland M, John R Edwards JR, Peter D Adams PD. DNMT inhibitors reverse a specific signature of aberrant promoter DNA methylation and associated gene silencing in AML. Genome Biolog. 2014. 15:406. doi: 10.1186/s13059-014-0406-2.