New Insights in the Pathogenesis of MGUS, Multiple Myeloma (MM) and Waldenström`s Macroglobulinemia (WM): Chronic Antigenic Stimulation by Autoantigenic Targets of Paraproteins Is Mediated by CD4⁺ t-Cells

Abstract 472

Hyperphosphorylated paratarg-7 (pP-7) is the target of paraproteins in 15% of Caucasian, 4.5% of Asian and 28% of Afro-American patients with MGUS/MM. Carriers of pP-7 have an 8 to 13 times increased risk to develop IgA/IgG-MGUS and multiple myeloma (Grass et al., Lancet Oncology 2009; 10:950–956) and a 6.5 times increased risk to develop IgM-MGUS and Waldenstrom`s macroglobulinemia (Grass et al., Blood. 2011 Mar 10;117(10):2918–23.). Analysis of affected families showed that pP-7 is inherited in a dominant fashion, explaining cases of familial MGUS/MM/WM. Since paratarg-7 is hyperphosphorylated and inherited in a dominant fashion, we extended our studies to investigate the prevalence of further antigenic targets of paraproteins and their phosphorylation state. All (8/8) autoantigenic paraprotein target (paratargs no. 2, 5, 6, 7, 8, 9, 10, and 11) moleculary defined to date were shown to be hyperphosphorylated in the respective patients compared to normal controls, and the hyperphosphorylation state of the respective paraproteins targets was shown to be inherited in a dominant fashion for all autoantigenic paraprotein targets for which enough family members were available for such an analysis. The consistent finding of hyperphosphorylation of autoantigenic paraprotein targets suggests a common underlying mechanism associated with the pathogenesis of MGUS/MM, and implies a significant role of the hyperphosphorylation state in the pathogenesis of these neoplasms. Co-immunoprecipitation identified PKCzeta as the kinase responsible for the phosphorylation of most and PP2A as the phosphatase responsible for the dephosphorylation of all hyperphosphorylated autoantigenic targets of paraproteins. SNPs or mutations of PKCzeta and PP2A as the reason for the abnormal phosphorylation of the paraproteins targets were excluded. However, PP2A was shown to be inactivated by phosphorylation of its catalytic subunit at Y307. To determine the SNP or mutation which is associated with the inactivation of PP2A we carried out a genome-wide linkage analysis. We found strong evidence for a parametric linkage (full penetrant autosomal dominant model; disease marker allele frequency=0.02) with a LOD score of 5.9 on chromosome 4q35.

For pP-7 hyperphosphorylation we demonstrated that the hyperphosphrylation is due to an single additional phosphorylation at serine 17, which is located in the paraprotein-binding epitope of pP-7. Analysis of the T-cell response against normo- and hyperphosphorylated paratarg-7 derived peptides in affected and non-affected family members demonstrated that pP-7, but not wild-type p-7 induces a strong and specific CD4⁺ T-cell response in the patients with pP-7, which was shown to be HLA-DQ and HLA-DR restricted. This suggests that pP-7 confers a chronic antigenic stimulation which is mediated by CD4⁺ T-cells and that these pP-7 specific CD4⁺ T-cells stimulate a B-cell clone with the same specificity, eventually resulting in the clonal evolution of this B-cell clone. In summary, our results support the hypothesis of a role of chronic autoantigenic stimulation in the pathogenesis of MGUS/MM/WM. Indviduals carrying pP-7 are at particular risk, if their MHC-II haplotype allows the recognition of pP-7 derived antigenic peptides by their CD4⁺ T cells. We suggest that the different prevalence of pP-7 carriers and defined MHC-II haplotypes explain the different incidence rates of MGUS/MM/WM in different ethnic groups.