Abstract

Chronic lymphocytic leukemia (CLL) is characterized by progressive accumulation of small, mature B-lymphocytes aberrantly expressing CD5. One of the crucial factors in B-CLL development is the decreased apoptosis of malignant B-Cells.The chemokine receptor CXCR4 and the MHC class II invariant chain (Ii) CD74, are expressed on CLL B-cells and play a role in cells' migration and survival. CXCL12, the cognate ligand of CXCR4, promotes homing of CLL cells into the bone marrow (BM) and survival within the niche. Furthermore, CXCR4 is activated by macrophage migration inhibitory factor (MIF), a chemokine-like inflammatory cytokine and non-cognate ligand of CXCR4. MIF and its homolog MIF2 are bona fide ligands of CD74.CD74 is highly expressed on CLL cells and expression is correlated with the adversely prognostic factor ZAP 70. Interestingly, MIF plays a role in the survival of CLL cells by binding to CD74. Furthermore, increased MIF levels are found in the plasma of CLL patients and genetic Mif deficiency leads to delayed development of CLL in the Eµ-TCL1 mouse model. Finally, the aim of our study was to investigate the expression and interaction between CXCR4 and CD74 as well as their potential contribution to B-Cell survival in B-CLL.

An initial cohort of 350 patients with different backgrounds of hematological malignancies was analyzed for CXCR4 and CD74 expression on mononuclear cells. Interestingly, the highest coexpression of both receptors was observed in B-CLL (n=10) by flow cytometry (FCM), where cells were stained for CD3+, CD14+ and CD19+/CD5+ and compared to cells from healthy donors (n=20). For in vivo and in vitro studies cell lines and a mice model were chosen, which express both receptors CXCR4 and CD74 on the surface of B-Cells.The human CLL line JVM3 and primary CLL cells from aged leukemic Eμ-TCL1 mice were used to study the cross-talk of CXCR4 and CD74 in regulation of CLL survival after incubation with rhMIF, rhCXCL12, or rhMIF-2 in conjunction with Bcl2-inhibitor treatment (Venetoclax, 30µM) or pharmacological blockade of CXCR4 and/or CD74 (AMD3100 30µM, neutralizing CD74 antibody LN2 5µg/ml). Potential direct CXCR4/CD74 interactions were studied by confocal laser scanning microscopy (CLSM) and coimmunoprecipitation (CoIP). Chemotaxis towards CXCL12, MIF and MIF-2 were analyzed to understand homing behavior, which contributes to B-Cell survival.

CXCR4 and CD74 were found significantly coexpressed on B-cells from CLL patients compared to controls (p< 0.0001), suggesting a functional interaction between these receptors. Colocalization was confirmed by CLSM and CoIP detected receptor complexes between CD74 and CXCR4 on B- Cells from the JVM3 and OSU CLL cell lineFunctionally, inhibition of either receptor substantially impaired survival rates that did not differ from a simultaneous co-blockade of CD74 and CXCR4, indicating a crosstalk and/or merging of downstream signaling pathways. Apoptosis assays indicated that inhibition of CXCR4 and/or CD74 triggered substantial cell death (78,17 +/- 1,84%). Of note, single vs. double blockade experiments confirmed that CD74 and CXCR4 functionally interact to control CLL survival. Inversely, pre-incubation of JVM3 cells with CXCL12, MIF and MIF-2 decreased apoptosis by 43, 36 % (MIF) and 51, 50 % (CXCL12) compared to the control.The ligands MIF, MIF2 and CXCL12 promoted chemotactic migration of JVM3 B-cells that contributes to the B-Cell survival mechanism.

Together, our results suggest an important role of MIF proteins in the survival and homing of malignant CLL cells that is mediated through a functional interaction between the CXCR4 and CD74 survival receptors. In conclusion, high levels of MIF attract B-Cells with the coexpression of CXCR4 and CD74, which build a complex after binding to their ligands and activate survival pathways to inhibit cell death. The MIF/CXCR4/CD74 axis could represent a novel target for therapeutic intervention.

Disclosures

Brümmendorf: Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Koschmieder: Roche: Other: Clinical Trial participation; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.