Dramatic Down-Regulation Of MNDA Expression In CP-CML Cells From The LSC Compartment
Authors: Bourgne Céline , Janel Alexandre , Berger Juliette , Guerci Agnès , Jamot Caroline , Chaudagne Nathalie , Deurveilher Sylvie , Pigeon Pascale , Hermet Eric , Boiret-Dupré Nathalie , Verrelle Pierre , Arnaud Philippe , Milot Eric , Berger Marc G 
Published: October 2019

Abstract

Introduction
Chronic Phase - Chronic Myeloid Leukemia (CP-CML) is a myeloproliferative disorder characterized by malignant proliferation of the granulocytic lineage without the arrest of cell differentiation. Tyrosine Kinase Inhibitors (TKI) have revolutionized CML treatment but several studies showed that a combination of TKI and Interferon alpha (IFNα) provides better clinical response.
Myeloid Nuclear Differentiation Antigen (MNDA), which belongs to the hematopoietic interferon-inducible nuclear proteins with the 200-amino-acid repeat (HIN200) gene family, encodes a protein expressed in myeloid cells but whose function remains poorly understood. Because of its high expression in polymorphonuclear cells, its involvement in cell differentiation and apoptosis, and its induction by IFNα, we evaluated MNDA expression in CML cells and its modulation after incubation with IFNα.

Material and methods
We tested MNDA expression in several cell lines (K562, KCL22, LAMA84, TF1 and U937 (positive control)), in polymorphonuclear cells from healthy donors (HD-PMN, n=13) and in primary cells from patients with CP-CML at diagnosis (CP-CML; n=17). The relative expression of the MNDA transcript was analyzed using the 2-ΔΔCt method and was normalized to the endogenous reference gene GAPDH. HD-PMN were used as calibrator. We developed a multiparametric flow cytometry assay (CD45-V500/CD14-APC-H7/CD15-PerCpCy5.5/CD34-PC7/CD38-V450/MNDA-FITC) to detect MNDA protein in the different cell subsets, particularly in CD34+cells.

Results
As previously described, MNDA was poorly expressed in the K562 cell line. Similarly, mRNA was detected at low levels in two other CML cell lines (KCL22, LAMA84) and in TF1 cells, but at a high level in the U937 cell line, used as a positive control. In each cell line, the transcript expression was correlated to the protein level, as evaluated by flow cytometry (MFI ratio: 2.04±0.21, 2.36±0.24, 1.59±0.14, 1.88±0.11 and 8.77±0.54 for K562, KCL22, LAMA84, TF1 and U937, respectively (n=3)). In CP-CML primary cells, MNDA expression was greatly diminished as compared with HD-PMN in both mRNA (0.20±0.08 (n=17) vs. 1.32±0.21 (n=10); p=1.52x10-6) and protein (MFI ratio: 6.9±0.98 vs. 16.31±1.25, p=0.001).
After having verified that IFNα (2000 U/ml, 16 hours) induced MNDA expression in HD mononuclear cells but not in PMN, we observed that induction of MNDA was moderate in CML cell lines K562 and LAMA84 (2-fold increase, n=3) whereas the level of MNDA mRNA was significantly increased in TF1 cells (28-fold increase, n=4). Induction in primary CML cells was variable (3/5 patients).
Aiming to evaluate the expression of MNDA in leukemic stem cells (LSC), we first analyzed MNDA expression in CD34+ and CD34+/CD38- cells from HD. We observed that MNDA is down-regulated in healthy CD34+ and CD34+/CD38- cells compared to mature cells (mRNA: about 4 logs, protein: 8-10 fold lower, n=4), but we always detected a significant signal in CD34+cells (MFI ratio: 2.76±0.46, n=3). However, MNDA was not expressed by CML cells from the LSC compartment (n=4). This inhibition does not seem to be antagonized by nilotinib or IFNα (n=2).

Discussion/Conclusion
MNDA expression appears to be clearly down-regulated in CP-CML cells and dramatically so in the LSC compartment. In some patients, we observed sustained sensitivity to IFNα, but only in the compartment of more mature cells. This suggests early deregulation of MNDA expression which seems to be only partially dependant on differentiation. The mechanisms involved in this down-regulation remain to be elucidated but could be independent to TK activity of BCR-ABL protein and resistant to IFNα in the LSC compartment. This marked deregulation of MNDA in the LSC compartment is an additional argument in favor of intrinsic changes specific to primitive cells.

Disclosures:
No relevant conflicts of interest to declare.