The Significance Of Fli1 and p53 For The Effective Megakaryopoiesis In 5q- Syndrome and The Effect Of Lenalidomide Treatment
Authors: Neuwirtova Radana , Stritesky Jan , Fuchs Ota , Vostry Martin , Kostecka Arnost , Belickova Monika , Siskova Magdalena , Michalova, prof. Kyra , Cermak Jaroslav , Prof. Ass. , Jonasova Anna T 
Published: October 2019

Abstract

Introduction
Friend leukemia virus integration 1 (Fli1) is one of the factors involved in the differentiation of megakaryocytic and erythroid progenitor (MEP). Fli1 plays an important role in the development of megakaryocytes (MGC) (Doré and Crispino 2011). We demonstrated the increased level of Fli1 mRNA in bone marrow mononuclear cells of MDS patients (pts) with 5q- syndrome (Ann Hematol 2013). Kumar et al. (2011) explained the Fli1 increase as a consequence of miR-145 haploinsufficiency in 5q- syndrome. Fli1 is one of the target genes of miR-145. Our presumption is that the interaction between Fli1, MDM2 and p53 is important for the magakaryopoiesis in 5q- syndrome. Truong et al. (2005) demonstrated that Fli1 activates E3-ubiquitin ligase MDM2, which plays an important role in p53 degradation in proteasome. Therefore Fli1 probably protects MGC against ribosomal stress contrary to erythroid cells in 5q- syndrome pts. However, Fli1 and p53 mRNA levels, analysed by us in these patients need not mirror protein levels, therefore we decided to analyse Fli1 and p53 protein levels by imunohistochemistry with monoclonal antibodies.

Patients and methods
We examined bone marrow histological specimens from 21 5q- syndrome pts, 14 MDS low risk pts with normal chromosome 5 (non5q) and 10 normal bone marrow controls. Fli1 was examined with rabbit monoclonal antibody (clone BV4-Diagnostic BioSystems), p53 with mouse monoclonal antibody clone DO-1, Immunotech). Detection system used was N-Histofine (Nichirei Bio). In five 5q- syndrome pts immunohistochemistry was repeated after lenalidomide treatment. In 2 pts p53 mutation was assessed by next-generation sequencing (GS Junior, Roche). Fli1 and p53 mRNA levels in total mononuclear cell RNA were determined by TaqMan-based quantitative real-time polymerase chain reaction (PCR). Eight 5q- syndrome pts were treated with lenalidomide, bone marrow was reexamined in five pts after the treatment in six pts.

Results
We did not find any difference of Fli1 positive MGC, nor in the intensity of Fli1 in nucleai staining (42 - 94 % of Fli1 positive MGC) in both, MDS groups and in controls. p53 was in the majority of MGC negative or only weakly positive (in less than 20% of MGC). In order to quantify p53 staining in other types of mononuclear cells beside MGC, double staining is needed to identify the relevant (erythroid?) type of cells. However, there was qualitatively significant difference of p53 positivity, which was strong in cells of 5q- syndrome pts, weak in non5q MDS pts and very weak in controls. In four successfully treated pts with lenalidomide 20%-50% decrease of Flil positive MGC and marked reduction of p53 positivity in other cells was observed. One patient responded temporarily to lenalidomid, but in 15 months she transformed to RAEB II. In this patient two p53 mutations [p.G245S (23.9%) /p.F134L (3,5%)] were found. In our group of 5q- syndrome pts another woman had p53 mutation [V173M (49.6%)]. She transformed after 3 years in RAEB II. Both pts express strong positivity of p53 staining in non-MGC cells regardless of being now treated by azacitidine. Concerning the comparison of our findings of Fli mRNA and of p53 mRNA levels in mononuclear bone marrow cells with immunohistochemical data, we found partial agreement only in the case of p53. We did not confirm the increase of Fli1mRNA level in bone marrow mononuclear cells (3.5 x in comparison to non5q MDS and controls) of 5q- syndrome pts. Nevertheless, our presumption of the protective Fli1 function against MGC apoptosis remains focus of our further study.

Conclusion
Contrary to our findings of the increased Fli1 mRNA level in mononuclear bone marrow cells of 5q- syndrome pts, the protein Fli1 levels did not differ from controls either in 5q- syndrome or in non5q MDS pts. p53 as the protein was negative or weakly positive in MGC in all pts and controls. The protein p53 was weakly positive in mononuclear bone marrow ( erythroid?) cells in controls, slightly positive in non5q MDS pts and differently strong positive in 5q- syndrome pts. The strongest p53 positivity was observed in two 5q- syndrome pts with mutated TP53 clone. Lenalidomide treatment of 5q- syndrome patients substantially decreased both Fli1 and p53 protein levels. In purpose to specify the type of bone marrow mononuclear cells we introduce double staining of these cells.
Supported by the research grant NT/13836-4/2012 from MH CR and by PRVOUK-P27/LF1/2

Disclosures:
No relevant conflicts of interest to declare.