The Myeloma Cells Escape from Bone Marrow to Skin Extramedullary Localization upon Bortezomib Resistance: Role of CXCR4

The malignant plasma cells (PC) dependence on the bone marrow (BM) microenvironment is a main feature of multiple myeloma (MM), mainly due to high expression of cell adhesion molecules including CD44, CD54 (ICAM1), CD56, CD49d and chemokines receptors such as CXCR4. At the end stage of the disease, a rare MM complication, that may occurs, is a secondary extramedullary involvement without a leukemic phase and, among the extramedullary localizations, the skin is one of the possible sites, due to the physiological homing of PCs. However, the mechanisms behind the extramedullary spread are not completely understood.

In a patient with refractory resistant MM, who has developed a cutaneous localization after 16 months from the diagnosis under Bortezomib (BOR) treatment, we analyzed the gene expression profiles (GEPs) by GeneChip U133 Plus 2.0 (Affymetrix), the immunophenotypic and immunohistochemistry (IHC) profiles of MM cells, across the course of the disease. To confirm the results, IHC profiles on selected molecules were then analyzed in other six MM patients with secondary skin involvement without PC leukemia.

On BM CD138⁺ PCs at diagnosis and BM relapse, by GEP analysis, 742 genes resulted differentially expressed. Moreover, a more stringent analysis identified 19 down-regulated and 42 up-regulated genes; data were confirmed by Real Time PCR on selected genes mainly involved in PC homing. At the BM relapse, we found that MM cells expressed CXCR4 and were negative for CD56; moreover, a significant down-regulation of ICAM1 (CD54) and ALCAM was observed together with the up-regulation of MAGE family genes, DKK1 and SEMA3A in the BM relapse sample compared to diagnosis one. At the cutaneous involvement development, 4 months after BM relapse, by IHC analysis, the MM cells localized in the skin showed the expression of both CD56 and CD54. On the other hand, the BM MM cells immunophenotype confirmed the presence of CD56 but showed the lack of CXCR4 expression. IHC for CD54, CD56 and CXCR4 expression was then performed on both skin and bone biopsies of the other patients analyzed. To go further inside on the role of CXCR4 we treated with BOR (5-10nM) for 24-48h human myeloma cell lines (HMCLs) RPMI-8226 and U266, known to be resistant to BOR, evaluating the CXCR4 expression at both mRNA and protein level, by real-time PCR and by cytofluorimetry, respectively. CXCR4 resulted downregulated after BOR treatment in BOR resistant HMCLs. Consistently, the loss of CXCR4 expression was recently associated to BOR resistance and extramedullary disease in a mouse model of MM suggesting that CXCR4 loss can be correlated with BOR resistance and PCs mobilization from BM, leading to an extramedullary disease.

In conclusion, our data indicate that the loss of CXCR4 expression by MM cells across the disease progression, together with CD54 and CD56 down-regulation and their re-acquisition at the extramedullary site, are involved driving the escape of PCs from BM to the extramedullary localization in the skin in the context of Bortezomib resistance.