INIGEM   23989
INSTITUTO DE INMUNOLOGIA, GENETICA Y METABOLISMO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Analyzing the mutational landscape of two CLL mouse models overexpressing AID identifies proliferative fraction-associated tumor genes involved in leukemic development and progression
Autor/es:
YAN XIAO-YEN; CRISPO MARTINA; MICHEL C. NUSSENZWEIG; DI NOIA, JAVIER M.; OPPEZZO PABLO; MORANDE, PABLO E; SEIJA, NOÉ; DAVIDE ROBBIANI; PALACIOS FLORENCIA; CHIORAZZI NICHOLAS; JULIETA SPULVEDA; NATALIA REGO; SHIH-SHIH CHEN; NAVARRETE MARCELO
Lugar:
Edimburgo
Reunión:
Workshop; XVIII International Workshop on Chronic Lymphocytic Leukaemia; 2019
Institución organizadora:
iwCLL Consortium
Resumen:
Activation-induced cytidine deaminase (AID) induces physiological mutations in Ig genes initiatingsomatic hypermutation and class switch recombination process in B lymphocytes. However, it canalso act on other genomic regions resulting in off-target aberrations that have been linked tolymphomagenesis. In CLL, aberrant AID expression was described in patients with poor clinicaloutcome and specifically in a small subset of tumor cells known as ?proliferative fractions? (PF)(Palacios et al., Blood 2010; Patten et al., Blood, 2012). Although these studies suggest a linkbetween AID expression and CLL progression, direct evidence linking AID activity with specificmutations and CLL progression is missing. To better understand the role of AID during CLLevolution, we used a murine model that mimics a progressive CLL disease, the transgenic Eu-TCL1mouse, and crossed these with two different transgenic strains overexpressing AID; in one caseunder the control of the actin promoter (Okazaky et al., J. Exp. Med., 2003) to generate doubletransgenic -DT- actin-AID/Eu-TCL1 DT mice and in the other under the control of the Igκ promoter(Robbiani et al., Molecular Cell, 2009) to generate the DT Igκ-AID/Eu-TCL1 DT mice. Wepreviously reported by P. Morande et. al., YIM iwCLL2015, and by X-J Yan et al. iwCLL2015 thatboth DT strains are born healthy but experience accelerated leukemogenesis and development amore aggressive CLL with shortened life spans as compared to the monotransgenic Em-TCL1 mice.We now studied AID-induced changes in the genomic landscape of the leukemic clones from eachset of animals, and in its quiescent and proliferating subpopulations. AID mRNA and proteinexpression levels were visualized by qPCR and immunohistochemestry techniques, respectively.Lymphoid tissues of DT mice showed loss of normal B-cell follicles and higher AID proteinexpression as compared to their TCL-1 counterpart. Interestingly, while the DT model showed anincreased percentage of tumor cells expressing the Ki-67 proliferation marker, expression levels ofanti-apoptotic Bcl-2 remained unchanged. IgM+CD5+Ki-67+ (PF) and IgM+CD5+Ki-67- quiescentfraction (QF) were obtained from spleens of each DT-TCL1/AID strains and from Eμ-TCL1 modelby cell-sorting and finally genomic DNA extracted for all three genotypes (total = 15 mice, n=5 foreach genotype). Whole Exome Sequencing (WES) was performed and somatic variants identifiedusing VarScan2. Variants were classified according to their presence in AID hotspots and mutationalsignatures were extracted using the 96-trinucleotide context model.WES of the whole clone showed that the genomic mutational pattern of both DT-TCL1/AID modelswas dominated by C>T transitions at the RCY motif, indicating that observed variants preferentiallyoccurred at canonical AID hotspots (c-AID). A set of 40 genes associated with tumor developmentand/or progression were recurrently mutated at c-AID hotspots. Interestingly, a high percentage ofthese genes (>10%) were tumor gene drivers in human neoplasms based on previous reports. Toassess the role of AID activity in tumor proliferation, we next analyzed mutations occurring in thepurified PF and QF from each DT-TCL1/AID strain and from their control counterpart (Eu/TCL-1).This identify a set of 11 AID off-target genes with specific c-AID mutations that were shared byeither subset (PF and QF) or that were found specifically in the PF. Among the latter genes, weunderline 3 groups: i) oncogenes (Pim-1, Mcl-1, Myc), ii) tumor suppressors (Pax-5, Dusp2,Gata3, Klf2, Zfp36l2) and iii) Genome chromatin/histones (Hist1h1d, Hist1h1e, Hist2h2aa).Interestingly, we found that the site of somatic mutations in 4 of these genes (Pim-1, Mcl-1,Hist1h1d and Hist1h1e) were identical to those in human tumor cells. Crystallographic analyses ofthese mutations on human protein structures identified amino acid changes with putative functionsin these tumor driver genes suggesting a link between AID activity, point mutations, and tumor cellproliferation.In summary, we present two new double transgenic mouse models overexpressing the mutagenicenzyme AID in a CLL context. These DT mice develop a more aggressive leukemia, loss of normalB cell follicles, higher AID expression and increased tumor cell proliferation. WES analysis detectsthe development of a number of previously described AID off-target genes, identifies novel c-AIDmutations in other genes, and links these mutations with previously reported mutations in humantumor driver genes. Finally, these results suggest a direct link between aberrant AID expression,specific AID function and tumor clone aggressiveness in CLL.