Modeling Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia in Transgenic Mice Using Human MYD88L256P Mutated Protein

Abstract Lymphoplasmacytic lymphoma (LPL)/Waldenstrom macroglobulinemia (WM) is a B-cell disorder resulting from the accumulation of cloncal lymphoplasmacytic cells in the bone marrow (BM) along with excessive secretion of immunoglobulin M paraprotein. Despite recent advances in treatment LPL/WM remains incurable. Generating a refined LPL/WM mouse model that would facilitate studying this disease as well as enable stringent in vivo validation of potential therapeutic approaches has been a long-sought goal for the filed. Interestingly, among the most common mutations identified by recent high-throughput studies there was a single gain of function point mutation in the MYD88 gene, with a predicted non-synonymous amino acid (L265P) change, found in over 91% of patients. Therefore, we hypothesized that generation of transgenic mice overexpressing MYD88L256Pcould recapitulate the human setting of LPL/WM. To overcome problems inherently related to embryonic lethality, we generated MYD88WT and MYD88L256P conditional transgenic mice expressing human proteins. To remove a transcriptional stop cassette and activate MYD88 expression in vivo, we mated the conditional mice with mice expressing Cre recombinase under the control of Activation-Induced Cytidine Deaminase (AID) gene promoter to generate AIDCre/-;MYD88WT/- and AIDCre/-;MYD88L256P/- double-mutant mice. Recombinase activity driven by the AID gene promoter caused removal of the stop cassette and expression of MYD88 in germinal center (GC) and post-GC B cells, the appropriate B-cell compartment from which LPL/WM clones are thought to originate in humans. Young (up to 30 weeks of age) AIDCre/-;MYD88WT/- and AIDCre/-;MYD88L256P/- mice where indistinguishable from AIDCre/- mice as assessed by external physical examination. Moreover, obvious histologic and IHC differences in spleens, lymph nodes, and bone marrows between the mutant genotypes and the AIDCre/- control mice were not found. No clonal bands for the IgH gene, nor M-spikes were detected in Southern blots and serum protein electrophoresis, respectively. However, after 30 weeks of age, and at variable times thereafter, some of the AIDCre/-; MYD88L256P/- aging cohort (n=27) mice developed skin rash and excoriation as well as loss of hair in the submandibular areas. A fraction of AIDCre/-; MYD88L256P/- animals that weren't sacrificed because of the severe dermatitis had enlarged lymph nodes and appeared sick. To assess whether MYD88 transgenic mice develop B-cell lymphomas or major pathologic alterations consistent with the diagnosis of LPL/WM, animals were euthanized at different ages and lymphoid organs were rigorously examined. In spleens and lymph nodes of 8 of the AIDCre/-; MYD88L256P/- animals, a nodular lymphoid infiltrate composed predominantly of small atypical cells varyingly infiltrated with CD138+ plasma cells was observed. Lymphoid cells infiltrated other organs including the BM. Enhanced expression of MYD88 and activation of NF-κB activity within atypical lymphoid nodules in diseased transgenic mice was documented by IHC staining for MYD88 and p65. However, no clonal bands of the IgH gene were detected in Southern blots, suggesting that overexpression of MYD88L256P/- mutant protein in activated B-cells gave rise to a polyclonal lymphoproliferative disorder. Consistently, serum protein electrophoresis analysis showed diffuse, polyclonal increase in γ-globulins. Moreover, stains of peripheral blood smears revealed rouleaux formation suggesting increased concentrations of plasma proteins. In addition, one of AIDCre/-; MYD88L256P/- animals developed a more aggressive monoclonal disease resembling human diffuse large B-cell lymphoma. However, large atypical lymphoid cells in this mouse lacked expression of MYD88 and nuclear p65. All AIDCre/- controls and AIDCre/-; MYD88WT/- mice lacked described phenotypes and were histologically unremarkable. Taken together, these results demonstrate that although MYD88L256P/- alone might not be sufficient to induce a malignant transformation in the mature B-cell compartment, it gave rise to a low-grade mature non-clonal B-cell lymphoproliferative disorder with some of the characteristic futures of LPL/WM. Therefore, the MYD88L256P/- mutation might be necessary but not sufficient for LPL/WM development and must be present with concomitant “second hit” genetic alterations for a neoplasm to develop. Disclosures Treon: Pharmacyclics: Consultancy, Research Funding.