Consequences of myd88 mutations for b cell tolerance

Abstract

MYD88 mutations, found in 39% of activated B cell type diffuse large B cell lymphoma (ABC-DLBCL) and almost 100% of Waldenstrom macroglobulinemia (WM), have emerged as one of the most frequently recurring mutations in mature B cell malignancies. Strikingly, a single MYD88 L265P mutation predominates in both ABC-DLBCL and WM, and frequently overlaps with mutations in CD79B and TNFAIP3. However, little is known about the consequences of acquiring these mutations for the behaviour of normal mature B cells. In this thesis, I analysed the consequence of these mutations using retroviral gene transfer strategy on normal activated mature mouse B cells. I found that single MYD88 L265P mutation dysregulates TLR tolerance, resulting in self-TLR ligand driven B cell proliferation in vitro. However this break of TLR tolerance is transient, and is curtailed by the induction of TNFAIP3-dependent negative feedback on NFkappaB activation, and by Bim-dependent and Bcl2-inhibited apoptosis. These data revealed the effects of MYD88 mutations on normal B cells and the several checkpoints that are in place to prevent single MYD88 mutation from triggering constitutive dysregulated B cell proliferation. Since the oncogenic activity of MYD88 L265P relies on the activation of nucleic acid sensing TLRs, inhibitors targeting this pathway have recently emerged as potential novel therapeutic agents for treating diseases with MYD88 L265P. Paradoxically, I found that the inhibition of endosomal TLRs through Unc93b1 (3d) mutation or Tlr9 deficiency does not suppress MYD88 L265P B cell growth in vivo, and instead, triggering enhanced plasmablast differentiation, revealing an unexpected and powerful inhibitory effect of TLR9 on MYD88 L265P. This raises the hypothesis that multiple ligands can activate the MYD88 L265P mutation for aberrant B cell growth. One of these ligands is the cytokine BAFF, as the additional of BAFF in vitro drives MYD88 L265P B cell proliferation and differentiation when TLR9 activity is absent. These results open the potential for targeting multiple pathways in treating MYD88 L265P diseases. In the second part of this thesis, I analysed the effects of MYD88 L265P on B cell tolerance in the context of chronic exposure to self-antigen, exploring the possibility of the cross-activation of self-sensing BCR and TLRs. MYD88 L265P mutation fails to protect B cells from self-antigen induced cell death unless coupled with Bcl2 overexpression or CD79B overexpression/mutation, with the latter also inducing plasmablast differentiation and autoantibody production. Here, I demonstrated that the accumulating number of self-BCR-reactive MYD88 L265P B cells correlates with increasing surface IgM expression, which is conferred by the cooperation between MYD88 L265P and CD79B ITAM mutations. Moreover, these experiments revealed an additional layer of checkpoint for B cell tolerance, in which MYD88 L265P attenuates BCR-dependent pro-survival signal from the BCR by repressing the surface expression of CD79B and IgM. Overall, this thesis demonstrates the consequences of the recurring MYD88 L265P mutation for B cell tolerance to self TLR and BCR ligands, proposes that constitutive self-ligand stimulation of these receptors may contribute to B cell malignancies, and provides insights into potential novel therapeutic targets for MYD88 mutated lymphomas.