Fli-1 and EKLF Gene Expression in Patients with MDS 5q- Syndrome.
Authors: Neuwirtova Radana , Fuchs Ota , Provaznikova Dana , Cermak Jaroslav , Siskova Magda , Jonasova Anna , Michalova Kyra 
Published: October 2019

Abstract
Abstract 2788
Poster Board II-764

Introduction.
Patients with MDS-5q- syndrome have macrocytic anemia often with hypoplastic erythropoiesis and on the contrary thrombocythemia with effective though dysplastic megakaryopoiesis. Megakaryocytes and erythroid cells are thought to share a common progenitor MEP (T.P.McDonald et al., Exp. Hematology 1993). There are two key transcription factors which together with other transcription factors and relevant cytokines and receptors determine the hemopoietic differentiation of the common stem cell: erythroid Krüppel-like factor (EKLF) for erythroid lineage and Friend leukemia virus integration 1 (FLi-1) for megakaryopoiesis (Pilar Frontelo et al., Blood 2007; G.A.Blobel, Blood 2007; F.Bouilloux et al., Blood 2008). There is functional cross antagonism between FLi-1 and EKLF (J.Starck et al., Mol. Cel. Biology 2003). FLi-1 is active only if dephosphorylated (H.Huang et al., ASH Abstracts 2008). The question is whether both factors play any role in 5q- syndrome.

Methods.
FLi-1 and EKLF gene expressions were determined in mononuclear cells isolated from the whole blood or bone marrow using Ficoll-Paque PLUS. Expression of both factors was measured by quantitative real-time PCR. RT-PCR products were verified by electrophoresis and direct sequencing. The assays were performed for sample in duplicate. Glyceraldehyd-3-phosphate dehydrogenase (GAPDH), FLi-1 and EKLF were amplified in 25 μl reaction mixture containing 12.5 μl SYBR Green JumpStart Taq Ready Mix, 2.5 μl 2 μM FLi-1 or EKLF forward and reverse primers, 0,25 μl internal reference dye and 1 μl cDNA. Relative levels of FLi-1 and EKLF mRNAs were calculated to the level of housekeeping GAPDH mRNA.

Results.
FLi-1 and EKLF were measured in blood mononuclear cells of 8 patients fulfilling all criterias of 5q- syndrome. FLi1mRNA/GAPDHmRNA was higher in all samples, average value was 0.0930 (0.0242-0.4274) compared to control value 0.0194. FLi1mRNA/GAPDHmRNA in bone marrow mononuclear cells of 7 patients with 5q- syndrome was higher in all samples but one. The average value was 0.0827 (0.0070-0.2554) compared to healthy controls 0.0044.
EKLF gives very low values in the majority of patients′ blood and bone marrow samples as well as in healthy controls. The evaluation is therefore less reliable then FLi-1 assessment. EKLFmRNA/GAPDHmRNA in blood was 0.0004 (0.0-0.0023) compared to the control 0.0222. The results of EKLF in 5 bone marrow samples are inconsistent. Three are lower than the control (0.0068), 1 of remaining 2 samples is extremely high (0.3491). It is interesting that this patient is the only one who responded to erythropoietin and is transfusion independent.

Summary.
Our preliminary results with FLi-1 and EKLF gene expression measurement are in agreement with expected findings: increased FLi-1 expression corresponds to thrombocytemia in 5q- syndrome patients and expression of EKLF, lower than in controls would correspond to anemia in these patients. However, EKLF values are less reliable because of very low values in patients as well as in controls and because of inconsistent results in bone marrow samples. We prepare to follow both factors in 5q- patients after the treatment with lenalidomide. Lenalidomide improves anemia in 5q- syndrome patients and temporarily causes decrease of thrombocytes (A.List et al., N.Engl.J.Med. 2005, 2006). Inhibition of phosphatases by lenalidomide (S.Wei et al., Proc.Natl.Acad.Sci.USA 2009) can stop FLi-1 dephosphorylation which leads to FLi-1 inactivation. Hypotetically inactive FLi-1 would enable EKLF to induce MEP into erythroid lineage.
Supported by MSM 0021620808

Disclosures:
No relevant conflicts of interest to declare.