HIGH LEVELS OF CEREBLON MESSENGER RNA ARE THE CHARACTERISTIC FEATURE OF LOWER RISK MYELODYSPLASTIC SYNDROMES WITH 5Q DELETION AND ARE CONNECTED WITH THE EFFICACY OF LENALIDOMIDE

Background:

Cereblon (CRBN) was named according to its putative role in cerebral development, especially in memory and learning. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), cullin-4 (CUL4) and regulator of cullin 1 (ROC1). This complex regulates DNA repair, DNA replication and transcription. CRBN is a primary target of thalidomide teratogenicity (Ito et al., Science 2010; 327:1345-1350). The binding of immunomodulatory drugs (IMiDs), including lenalidomide, to CRBN is associated with cytotoxicity of IMiDs and is used to treat multiple myeloma, myelodysplastic syndromes (MDS) and lymphomas. Down-regulation of the CRBN expression is associated with the development of marked IMiDs resistance in human multiple myeloma cells (Zhu et al., Blood 2011; 118: 4771-4779; Lopez Girona et al., Leukemia 2012; 26: 2326-2335). Therefore, the CRBN expression is required for the antimyeloma activity of IMiDs.

Aims:

To gain insight into the role of CRBN in lower risk MDS with 5q deletion or with normal chromosome 5 and into the mechanisms of lenalidomide action, we studied the CRBN expression in these two groups of lower risk MDS patients and in healthy controls.

Methods:

Informed consent was obtained from all patients and healthy controls. Mononuclear cells were isolated from bone marrow [19 MDS patients with 5qsyndrome, 28 patients with low-risk MDS with normal chromosome 5 (non5q- ) and 15 healthy controls] and from peripheral blood [23 MDS patients with 5qsyndrome, 19 non5q-patients and 11 healthy controls] by Ficoll-Paque PLUS gradient separation, washed with phosphate-buffered saline and rest red cells were lysed. Total RNA was isolated using RNA isolation solvent and complementary DNA was synthesized from total RNA using SuperScript II reverse transcriptase. Relative levels of the CRBN mRNA were determined by Taq- Man-based quantitative real-time PCR and by calculation to the level of housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The experiments were performed in duplicate. Evaluation of 2-ddCt indicates the fold change in gene expression relative to the control.

Results:

The median of CRBN mRNA levels was the highest (3.6) in total RNA isolated from peripheral blood mononuclear cells of lower risk myelodysplastic syndromes with 5q deletion, the lower (2.2) in non5q- MDS and the lowest (1.0) in healthy controls. The similar results were obtained in bone marrow blood mononuclear cells (medians 3.2 in 5q- syndrome, 2.2 in non5q- and 1.2 in healthy controls). The differences between all groups were statistically significant (P<0.05, Mann-Whitney test). We analysed CRBN mRNA levels before and in the course of the treatment of 5q- syndrome with lenalidomide. High level of CRBN mRNA before and during the treatment correlated with sustained response to lenalidomide. In one 5q- syndrome patient who relapsed after the discontinuation of successful treatment the level of CRBN mRNA sharply decreased. This patient did not respond to the second course of lenalidomide and progressed to RAEB II.

Summary / Conclusion:

Low risk MDS patients with 5q deletion have the highest levels of CRBN mRNA in comparison to lower risk MDS with normal chromosome 5 or healthy controls. High levels of CRBN mRNA in 5q- syndrome are necessary for the efficacy of treatment by lenalidomide. Great decrease of CRBN levels during the treatment by lenalidomide is connected with the absence of response and the disease progression.

Supported by IGA MH CR, the research grant NT/13836-4/2012.

Abstrakta v časopise Haematologica 2013, Suppl1

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