DNA methylation and hypomethylating agents in high-risk myelodysplastic syndromes and acute myeloid leukemia

Epigenetic alterations are common in cancer. One example is aberrant hypermethylation of the promoters of tumor suppressor genes and hence silencing of gene expression. Azacitidine, a DNA hypomethylating drug, has been shown to prolong survival in patients with high-risk myelodysplastic syndromes (MDS) compared to conventional care regimens, and is now recommended as first-line therapy for patients not eligible for allogeneic stem cell transplantation.

Azacitidine has DNA hypomethylating properties and has been shown to re-induce expression of aberrantly silenced genes in various cell lines. However, its mechanism of action in primary hematopoietic progenitors in vivo is relatively unknown.

This thesis aimed to assess the prognostic value of DNA methylation in high-risk MDS and in de novo acute myeloid leukemia (AML), to evaluate the effect and feasibility of maintenance treatment with azacitidine in patients in complete remission (CR) after induction chemotherapy, and to study mechanisms of action of azacitidine in primary MDS and normal bone marrow (NBM) progenitors.

We show, for the first time, a correlation between promoter methylation patterns and outcome of induction chemotherapy in a cohort of 60 patients with high-risk MDS or AML following MDS (MDS-AML). Patients who were hypermethylated in the E-cadherin (CDH) promoter had lower CR rates than those without methylation (P=0.008). CDH methylation was also associated with shorter survival (P=0.003).

By contrast, in a material of 107 patients with de novo AML CDH methylation had no impact on survival or on outcome of induction chemotherapy. In fact, promoter hypermethylation of P15ink4b, previously reported as a poor prognostic marker in MDS and MDS-AML, as well as genome wide promoter methylation corresponded to a better survival (P=0.001 and 0.005, respectively). Another novel finding was that de novo AML patients with a low degree of global DNA methylation had a poorer response to induction chemotherapy (P=0.005). These differences in the prognostic value of methylation status in MDS/MDS-AML and de novo AML suggest important differences in disease biology and response to treatment between the two entities.

Several mechanisms of action for azacitidine have been suggested, including induction of apoptosis, differentiation of blasts, histone modification, immunomodulation and DNA and RNA demethylation. However, the majority of data results from cell line experiments or from sequential bone marrow sampling during azacitidine treatment. In paper IV of this thesis we exposed primary MDS and normal bone marrow (NBM) progenitors to azacitidine in vitro.

Interestingly, azacytidine caused marked up-regulation of gene expression in MDS but not in NBM CD34+ marrow cells. Compared to cell line experiments, induction of apoptosis as well as global and gene-specific hypomethylation was less pronounced in primary cells. Interestingly, azacitidine in doses up to 5 μM had no negative effect on proliferation in suspension cultures, and doses up to 0.5 μM even had a positive effect on colony growth. This is a useful finding since it may support the use of the drug for patients with low-risk MDS, and as maintenance after allogeneic stem cell transplantation.

List of scientific papers

I. Negative impact of DNA hypermethylation on the outcome of induction chemotherapy in older patients with high-risk myelodysplastic syndromes and acute myeloid leukemia following myelodysplastic syndrome. M Grövdal, R Khan, A Aggerholm, P Antunovic, J Astermark, P Bernell, LM Engström, L Kjeldsen, O Linder, L Nilsson, A Olsson, J Wallvik, JM Tangen, G Öberg, SE Jacobsen, P Hokland, A Porwit and E Hellström-Lindberg. Clinical Cancer Research. 2007;13(23):7107-12.
https://doi.org/10.1158/1078-0432.CCR-07-1193

II. Maintenance treatment with azacytidine for patients with high-risk myelodysplastic syndromes (MDS) or acute myeloid leukemia following MDS in complete remission after induction chemotherapy. M Grövdal, M Karimi, R Khan, A Aggerholm, P Antunovic, J Astermark, P Bernell, LM Engström, L Kjeldsen, O Linder, L Nilsson, M Skov Holm, JM Tangen, G Öberg, SE Jacobsen, P Hokland, A Porwit and E Hellström-Lindberg. British Journal of Haematology. 2010;150(3):293-302.
https://doi.org/10.1111/j.1365-2141.2010.08235.x

III. Gene-specific and global methylation patterns predict outcome in patients with acute myeloid leukemia. S Deneberg, M Grövdal, M Karimi, M Jansson, H Nahi, A Corbacioglu, V Gaidzik, K Döhner, C Paul, TJ Ekström, E Hellström-Lindberg and S Lehmann. Leukemia. 2010;24(5):1-10.
https://doi.org/10.1038/leu.2010.41

IV. Complex effects of azacitidine in primary myelodysplastic and normal bone marrow cell cultures. M Grövdal, M Karimi, M Nikpour, L McGovern, K Luttropp, M Jansson, AM Forsblom and E Hellström-Lindberg. [Manuscript]