Multiple myeloma (MM) is an incurable plasma cell malignancy predominantly negative for the surface protein tyrosine phosphatase CD45. Published data show that an increased proportion of CD45-ve cells present at diagnosis correlates with poorer patient outcome and increased risk of extra-medullary disease. The aim of this study was to investigate whether the identification of biological differences associated with variable CD45 expression might explain this apparent adverse clinical impact.
The U266 human myeloma cell line (HMCL) comprises both CD45+ve and CD45-ve populations and was used as an isogenic model to characterise the biology of each subset. Gene expression profiling (GEP) was performed on sorted cells (U266 CD45+ve vs U266 CD45-ve populations) using the Illumina HT12-V2 array. GeneGo analysis identified the hypoxia-induced epithelial-mesenchymal transition (EMT) pathway as being the most differentially active pathway between the CD45-ve (EMT on) and CD45+ve (EMT off) populations. EMT enables vital cellular changes during embryogenesis and wound healing and has been shown to be utilised by a range of carcinomas as a mechanism that promotes disease progression and metastasis.
The GEP findings were validated with qRT-PCR that confirmed the increased expression of SNAI1 (SNAIL), CTGF and HES1 in the CD45-ve subset. There was, however, no difference in the level of expression of hypoxia-inducible genes including HIF1B, HIF2B, CREBBP, HYOU1 or VEGFA. NOTCH pathway activation was confirmed within the CD45-ve population by a high HES1:DTX1 transcriptional ratio and intra-cellular NICD protein, again consistent with high EMT activity. Interestingly the GEP data, again confirmed with qRT-PCR, also demonstrated increased expression of a range of tumour suppressor genes prone to promoter hypermethylation within the CD45+ve subset while the CD45-ve cells preferentially expressed a range of oncogenes known to be associated with a more aggressive clinical phenotype in solid tumours but not known to be associated with MM.
Biological characterisation of the two populations demonstrated distinct cellular phenotypes. The CD45+ve and CD45-ve subsets expressed similar levels of IL-6R, IGF-1R, CD49d and CD95, however, the CD45+ve population expressed increased CXCR4 and ICAM-1 (both p<0.001) compared to the CD45-ve population with both enhanced migration toward (1.55-fold, p=0.0037) and adhesion to (1.39-fold, p<0.0001) HS5 stromal cells, consistent with CD45 negativity correlating with greater metastatic potential. This phenotypic variation was recapitulated in a patient presenting with plasma cell leukaemia whereby the bone marrow MM cells were CD45HI, CXCR4HI, whereas the peripheral blood MM cells were CD45LOW, CXCR4LOW. Finally, the CD45-ve U266 cells exhibited increased resistance to bortezomib induced cell death when compared to their CD45+ve counterparts.
Utilising the U266 HMCL as a model of variable CD45 expression we have demonstrated that CD45 negativity was associated with an active EMT transcriptional programme and a range of adverse biological characteristics. Our data supports the hypothesis that in some instances MM disease progression may be driven by the irreversible activation of the EMT pathway leading to a more malignant and metastatic phenotype. Whether the loss of CD45 expression in vivo is likewise correlated with EMT-like characteristics or plays a causative role in their development remains to be determined.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.