Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Is a New Biomarker for Mantle Cell Lymphoma: Expression, Localization, and Phosphorylation Study

Mantle cell lymphoma (MCL) is a relatively distinct B-cell non-Hodgkin lymphoma subtype with aggressive and often recurrent clinical course. At diagnosis, MCL often manifests with leukemization, a feature more common to chronic lymphocytic leukemia (CLL). Common features and differences between MCL and CLL were not yet explored by comprehensive global approaches, despite such understanding potentially being very neat for deciphering pathogenesis and tailoring therapies of these clinically distinct diseases.

In our study, we have compared MCL(n=10), CLL(n=10) and normal control(n=8) B-cell samples using the Affymetrix Human Genome HG-U133 Plus 2.0 Array. We studied different mRNA levels of ~47.000 transcripts represented on the array. The comparative analyses identified a set of 892 differentially expressed genes between MCL and NBC; and 774 differentially expressed genes between CLL and NBC. In order to find MCL/CLL-specific biomarkers we focused on the intersection of differently expressed genes in both groups (CLL vs NBC and MCL vs NBC). There were 222 mRNAs in the intersection, 216 of them were deregulated in the same direction in both groups while 6 mRNAs were deregulated in the opposite direction. This set of 6 disease-specific mRNAs contained previously reported biomarkers (CD200, LEF1), and also the Myristoylated alanine-rich C-kinase substrate (MARCKS) that has not yet been studied in MCL. Thus we utilized the validation patient groups (N^MCL=6, N^CLL=8) and confirmed differential expression of MARCKS on protein levels by flow cytometry and immunofluorescence. As MARCKS was previously shown to either bound to the cell membrane, to reside in the cytosol, or alternatively become transmitted to nuclei, we investigated subcellular localization of MARCKS using immunofluorescence (IF). The cytoplasmic MARCKS signal in MCL was significantly higher than in CLL while the opposite was observed for the nuclear IF signal. The ratio between cytoplasmic and nuclear signal was 2.5 for MCL and 0.8 for CLL (p < 0,0001). The active forms of MARCKS were shown to become phosphorylated on serineresidues and this prompted us to study the phosphorylation forms of MARCKS in MCL. Indeed, one of the residues, Ser159/163, was hyperphosphorylated in the MCL cytoplasm and its level and distribution markedly differed from CLL or NBC. We next searched for regulatory mechanisms upstream of the MARCKS expression in MCL vs CLL. MARCKS is a predicted target of several microRNAs (according to DIANA-TarBase v7.0), among them also of miR-155 (that is differentially expressed between MCL and CLL). To further investigate the regulatory relationship between mir-155 and MARCKS we utilized a CLL cell line MEC-1 and using the CRISPR/Cas9 technology we prepared individual cell clones that were mutated within the mature miR-155 sequence that recognizes MARCKS mRNA. As expected, the miR-155-MEC-1 mutants expressed markedly higher level of MARCKS compared to the control MEC-1 cells.

In conclusion, our work identified a set of six differentially expressed mRNAs when comparing MCL and CLL, among them, MARCKS. We further showed that MARCKS is differentially expressed, localized, and phosphorylated between MCL and CLL, and that MARCKS is partly controlled by oncogenic microRNA miR-155. MARCKS may play an important role in MCL pathogenesis and can serve as useful MCL biomarker.

Grant support: GAČR 16-05649S & P305/12/1033, AZV: 16-27790A and 16-31586A. Institutional support: CZ.1.05/1.1.00/02.0109, UNCE 204021, LH15170, PRVOUK P24, LQ1604.