1037 - Activation of S100-A9/EMMPRIN axis triggers survival/proliferation pathways in leukemic cells. A novel target for chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a malignancy of CD5+ B-cells characterized by a defect in apoptosis in which leukemic cells are continuously supported by their microenvironment and frequently guided to proliferation. The tumor microenvironment (TME) of lymphoid tissues affects immune cell function playing an important role in the pathogenesis of hematological cancers by providing protective and supportive niches for malignant cells. CLL is an example of this TME dependence where progression occurs as the consequence of specific microenvironmental signals that support an imbalance between reduced cell turnover and excessive proliferation of leukemic cells. Antigen stimulation and inflammation are key events, commonly involved in the initiation and progression of CLL. Supporting this idea, our group proposed a direct link between inflammation and a progressive disease (Prieto, Sotelo and Seija., et al., Blood, 2017). Our study described that pro-inflammatory S100-A9 protein is aberrantly expressed in CLL cells of progressive patients, exported to the serum in exosomes, and responsible of NF-kβ activation originating a positive activation loop during tumor progression. To gain deeper insights into the origins of the molecular mechanism underlying this observation and to better understand how inflammation may impact disease progression, we initiated this work by characterizing the expression levels of previously described receptors of S100-A9 (CD33, TLR4, receptor for advanced glycation end products –RAGE- and Extracellular Matrix Metalloprotease Inducer –EMMPRIN-) in CLL cells. Our results show that leukemic cells exhibit high expression of EMMPRIN and RAGE receptors, and low expression of TLR4 and CD33. Additionally, proteomic analysis of serum exosomes of progressive CLL identified the presence of S100-A9 protein but not S100A8, indicating that the active form in these patients is likely the homodimer of S100-A9 (main ligand of EMMPRIN receptor), and not the heterotetrametric form S100A8/S100-A9 (main ligand of RAGE receptor). Since EMMPRIN has been linked with metastasis and tumor progression and, in CLL associated with stroma interaction and NF-kβ activation, we focused on characterize its expression in CLL cells associating it with clinical evolution. Our results show that leukemic clone expresses EMMPRIN in an N-glycosylated form with significantly higher expression in unmutated patients compared with mutated CLLs and healthy donors (n=36; n=28 and n=12, respectively, p=***, Unpaired t test). Interestingly, this higher expression also correlates with earlier treatment requirement (n=25, r=-0.61; p=**, Spearman rank-test), suggesting a potential role for EMMPRIN receptor during disease evolution. Since we previously demonstrated that S100-A9/EMMPRIN axis activates NF-kβ pathway, we wonder if other survival/proliferative pathways also depend on this interaction. Thus, we analyze the phosphorylation of key residues involved in activation of Pi3K/AKT and MAPK/JNK pathways. Our findings demonstrate that stimulation with recombinant S100-A9 significantly increases the phosphorylation state of Ser176/180 in IKK, Ser473 and Thr308 in AKT, and Thr183/185 in JNK molecules, (p=****; p=****; p=*** and p=**, respectively, Unpaired t test), in CLL cells from 10 patients. Interestingly, these patients needed treatment earlier and depict high levels of EMMPRIN expression. In contrast, not significant differences were found after S100-A9 incubation of CLL cells from indolent patients who depict lower EMMPRIN expression. These findings further support our previous data about that S100-A9/EMMPRIN axis have a major role during the disease progression status of progressive CLLs. To confirm these results we evaluated different downstream molecules involved in cell survival/proliferation known to be molecular targets of these pathways. After S100-A9 stimulation progressive patients depict significant changes confirming up-regulation of Mcl1, Bcl2, pRb-1 and Survivine proteins; up-regulation of CCL3 and CCL4 mRNAs, and down-modulation of p27 mRNA, a clear marker of quiescent cell status. Finally, we also confirm the specificity of our results using specific inhibitors either for S100-A9 (Tasquinimod and Paquinimod) or EMMPRIN (blocking antibody, -clon/UM-8D6-). Our findings show that S100-A9/EMMPRIN axis is engaged in NF-kβ and Pi3K/AKT activation, two key pathways strongly associated with tumor progression.

CLL remains an incurable disease, underscoring the critical need for a deeper understanding of the mechanisms promoting tumor progression and/or refractoriness. This work describes for the first time a new survival/proliferation axis initiated by S100-A9/EMMPRIN interaction, which appears to be active in CLL patients during disease progression. Overall our results raise new questions about the role of inflammation during CLL evolution and propose new targets to be explored within the heterogeneous biological and clinical scenario of CLL.