Waldenstrom's Macroglobulinemia (WM) is the clinical manifestation of lymphoproliferative disorders characterized by a clonal lymphoplasmacytic proliferation, excessive IgM secretion and elevated serum viscosity.
Most common B-cell non-Hodgkin lymphomas associated with WM are Lymphoplasmacytic Lymphomas (LPL) and Marginal Zone Lymphomas (MZL), especially those displaying plasmacytic differentiation (MZL-P). In order to elucidate common pathogenetic mechanisms that result in WM across lymphoma types, we profiled the methylome of 6 LPLs, 10 MZL-Ps, as well as normal B-cell subsets purified from human tonsillar tissue –germinal center B-cell controls (GCB, n=3) and plasma cell (PC, n=3) controls, using HELP assay and high-density oligonucleotide microarray from RocheNImblegen that queries DNA methylation level of 50,000 cytosine residues distributed among 14,000 gene promoters.
Unsupervised hierarchical clustering approach using Ward's method and Eucledian distances separated normal GCB and PC from the lymphoma cases and identified bi-directional changes in gene methylation: with aberrant gain or loss of methylation at specific genomic locations. We further utilized a t-test and identified 208 probesets that were differentially methylated between controls and lymphomas at p<0.01 and mean log-ratio difference between 2 groups >1.5 corresponding to 30% methylation difference. Ingenuity pathway analysis revealed CDKN1A and TGFb networks as the most aberrantly methylated in lymphomas.
Remarkably, we observed that Interleukin 10 Receptor a (IL10RA), was aberrantly hypomethylated in both lymphoma subtypes, resulting in its aberrant overexpression. IL10RA is a subunit of the IL10 cytokine receptor that is known to be a key factor in terminating the inflammatory responses via signaling through the JAK/STAT pathway resulting in STAT3 activation, which also plays an integral part in GCB differentiation and commitment to the plasma cell lineage. We thus hypothesized that aberrant epigenetic upregulation of IL10RA might promote survival and expansion of lymphoma cells. We predicted that stimulating IL10RA with IL10 ligand may lead to increased cell growth, while the blockade of IL10RA would inhibit cell growth. We selected 2 anti-IL10RA antibodies, which have previously been reported to have receptor blocking properties in vivo in mice. We tested 1ug/ml, 5ug/ml and 10ug/ml of each antibody and 10 ng/ml of the stimulatory IL10 ligand and observed that ligand provided stimulatory effect on the growth rate of a panel of B-cell lymphoma, including LPL cell line, while both anti-IL10RA antibodies had marked growth inhibitory effects. We further determined that growth inhibition resulted from dramatic induction of apoptosis in cells treated with the blocking anti-IL10RA antibodies. Further studies revealed that induction of apoptosis followed specific inhibition of signaling through JAK1/2 and phosphorylation of STAT3Y705 immediately after treatment and inhibition of signaling through MAPK and phosphorylation of STAT3S727 at later treatment time points.
In conclusion, we determined that IL10RA is aberrantly methylated and overexpressed in subtypes of low grade lymphomas exhibiting plasmacytic differentiation and manifesting as WM. Hence, strategies targeting this pathway should be explored as potential therapy for WM (irrespective of the underlying type of lymphoma).
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.