Over-Expression of Activation-Induced Cytidine Deaminase in TCL1 Mice Leads to the Development of IGHV-Mutated CLL Clones That Associate with More Aggressive Disease

YAN XIAO-YEN; MORANDE PABLO; MEI QI; EZRA B KOLITZ; CRYSTAL D GRANT; GAUTAM NAYYAR; OPPEZZO PABLO; CHIORAZZI NICHOLAS

Atlanta, Georgia

Congreso; ASH Meeting 2017; 2017

ASH

Introduction. Activation-induced cytidine deaminase (AID) mediates somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes (IG s), both of which are necessary for an effective immune response in normal B cells. An unwanted consequence of AID´s role is its contribution to cellular transformation and tumor progression by aberrantly mutagenizing genes outside the IG locus (off-target mutations). In chronic lymphocytic leukemia (CLL), higher levels of IGHV clonal mutations in IGHV-mutated CLL (M-CLL) correlate strongly with better clinical outcomes, while, IGHV -unmutated (U-CLL) cases are associated with worse patient outcome. Paradoxically, U-CLL cells express significantly higher levels of AID than M- CLL cells.Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However, because SHM and CSR occur rarely in the leukemic clones of these animals, TCL1 Tg mice mimic only U-CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address this issue, we developed a TCL1 strain by interbreeding mice over-expressing AID in B lymphocytes (Eμ-TCL1xVκ-AID).Methods. B-cell clonal expansions were identified in spleen cells from 14 TCL1 and 23 TCL1xAID (DT) mice by amplifying cDNAs by PCR using consensus IGHV FR and isotype-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. Ig sequences were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Six DT and 2 TCL1 tumors were transferred to SCID mice (5 mice/donor)Results.Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 21 of 23 DT mice; each animal bore an IgMκ+ clone and 5 also exhibited a concomitant IgGκ+ clone. Approximately 50% used VH1-55, VH11-2, or VH12-3.SHM: The IgM+ TCL1 clones had a mutation frequency of 0.05% for IGHV and 0.09% for IGKV . In contrast, the IGHV and IGKV mutation frequencies were considerably higher in DT mice: IGHV 0.47% in IgM+ and 3.0% in IgG+ clones, and IGKV 0.9% for IgM+ and IgG+ combined. These mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1.However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%) than the TCL1 level (0.05%), it was considerably less than that found in the DT clones using other IGHV s (0.80%). In addition, no mutations were detected in VH1-55 clones from DT mice.IGHV gene use and SHM in clones that underwent CSR: Notably, in 2 of 5 instances the same IGHV-D-J rearrangement was found in IgMκ+ and IgGκ+ clones from the same mice. For the remaining 3, only the IgG+ version was detected. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%).Stereotyped IGHV-D-J and IGkV-Jk rearrangements: Among 32 IgM clones from DT mice, 4 expressed VH11-2 and Vκ14-126 and 8 expressed VH12-3 and Vκ4-91; these rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones.DT mice develop more aggressive disease: we observed shorter median survival in DT mice comparing to TCL1 mice (279 days vs 356 days, P =0.0034)Transfer tumor development: Spleen cells from 6 CLL-bearing mice were transferred into SCID recipients, leading to a selected outgrowth of VH12-3/Vκ4-91 and VH11-2/Vκ14-126 clones. Survival in these mice inversely correlated with IGHV mutations (Figure).Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes: certain IGHV s and IGK Vs are less susceptible to having major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell´s ability to synthesize AID (e.g., many IGHV1-69+ clones in U-CLL patients). AID overexpression also led to IgG+ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, transfer of DT clones to SCID mice led to more IGHV and IGKV mutations and shorter survival opposite to that in M-CLL patients. Possibly this adverse effect might be a consequence of AID´s aberrant action outside the IG loci which is facilitated in DT mice.