B-Cell-Specific Transcription Factor BACH2 Involved in the Clinical Behavior Heterogeneity of Waldenström Macroglobulinemia

Approximately 30% of the patients who fulfil the criteria of Waldenström macroglobulinemia (WM) are diagnosed while asymptomatic, and will not require immediate therapy; these cases are called indolent WM (IWM). However, patients with a disease-related event will be considered for therapy, these cases are called symptomatic or aggressive WM (AWM). The physiopathology of these 2 groups remains unclear, and the mechanisms of progression have not been fully understood so far. We hypothesized that a gene signature that differentiates these two categories could be identified to better understand the underlying mechanisms of progression of WM.

Seventeen patients diagnosed with WM (8 IWM and 9 AWM) were included in this study. We selected tumour cells from the bone marrow (BM) using mononuclear cell isolation, then B cell enrichment (B cell isolation kit, Myltenyi-Biotec, USA). The purity was confirmed by flow cytometry. Total RNA was extracted using the Trizol method. Gene expression profiling was performed using U133A arrays (Affymetrix, USA). Gene expression was normalized using the RMA algorithm. We ranked genes by fold-change of expression levels on a first series of 11 patients (5 IWM and 6 AWM) calculated with the ‘limma’ package in R. Next, we used a supervised classification to establish a gene expression profile to distinguish IWM from AWM. Therewith, we validated this profile on an independent set of 6 patients (3 IWM and 3 AWM). We then performed a pathway analysis using Ingenuity^® analysis software. We confirmed gene expression deregulation with qRT-PCR on 3 candidate genes in the first series of patients. Genome-wide detection of copy number alteration and loss of heterozygosity were performed on 13 of the 17 WM cases, using the Genome-Wide Human SNP Array 6.0 (Affymetrix, USA). Finally, we investigated the functional consequences of the deregulation of these candidate genes in BCWM1 and MWCL1, both B cell lines originated from WM. Survival was studied using a colorimetric method with MTS (Promega, USA). Proliferation was analyzed using incorporation of a nucleoside analog (EdU) into DNA during active DNA synthesis (Invitrogen, USA).

The differential analysis has identified 82 probes, corresponding to 48 genes, significantly deregulated and capable of differentiating samples from IWM and AWM in an unsupervised classification. Moreover, with a supervised classification, this gene expression profile accurately classified 94% of the 17 WM samples, including the 6 WM of the independent validation set. The two molecular networks that appeared to play a major role in the physiopathology of IWM versus AWM were the plasma cell differentiation pathway and the AKT pathway. We have then identified 3 key genes in those 2 pathways, BACH2 and CIITA on the one hand and PTEN, respectively. We have then confirmed the deregulation of these gene expression levels by qRT-PCR in 3 IWM and 4 AWM; these 3 genes were over-expressed in IMW relatively to AMW. BACH2 is a B-cell-specific transcription factor known to be a tumour suppressor gene. It was shown that BACH2 reduces proliferation and induces cell death when over-expressed in B lymphoma tumour cells. We have thus pharmacologically over-expressed BACH2 in BCWM1 and MWCL1 and significantly reduced the proliferation and the survival of the two cell-lines. Further studies using BACH2 specific overexpression with lentiviral infection are underway, in vitro. The data will be presented at ASH. In order to further study the mechanisms of deregulation of BACH2 in IWM and AWM, we have conducted a genome wide SNP array study of 13 patients. Among those, 7 patients (4 IWM and 3 AWM) demonstrate a deletion of long arm of chromosome 6 (del6q), the most frequent chromosomal abnormality in WM. BACH2 gene is located on the 6q15 locus. Interestingly, we found that 3 out of the 3 AWM had a del6q that took in the 6q15 region, whereas 3 out of 4 of the IWM had a del6q preserving the 6q15 region. Therefore, haploinsufficiency could participate in the under-expression of BACH2 in aggressive WM; this hypothesis will be verified by using DNA qRT-PCR of BACH2.

To the best of our knowledge, we have identified for the first time a specific gene expression signature that differentiates IWM and AWM. We have exposed several genes from this dataset, including BACH2, which is a candidate to better understand the underlying mechanisms of progression of WM.

No relevant conflicts of interest to declare.