B Cell Restricted Expression of Mutated IKZF3 modulates BCR Signaling and Homing Pathways in a Mouse Model of CLL

Abstract

Mutation in IKZF3 (AIOLOS), a lymphoid transcription factor with a key role in B cell development and function, has been identified as a putative driver of chronic lymphocytic leukemia (CLL) through large-scale WES studies of CLL patients. Prevalent in ~3% CLLs and associated with fludarabine resistance, mutated IKZF3 has been detected uniquely as a hotspot mutation (L162R), localized within the DNA binding domain. The functional effects exerted by this mutation on lymphomagenesis remain unexplored, and were the subject of the current study. We generated a B-cell restricted knock-in mouse line by crossing mice with the Ikzf3 mutant floxed allele with Cd19-Cre animals (i.e. Cre recombinase expression under the Cd19 promoter), and established cohorts carrying either heterozygous mutant-Ikzf3 (Ikzf3Het), homozygous mutant-Ikzf3 (Ikzf3Homo) or wild-type Ikzf3 (Ikzf3WT). We thereafter monitored the animals for leukemia development by serial flow cytometry analyses of peripheral blood. Notably, we detected appearance of ~10-50% clonal B220+CD5+Igk+in 8 of 30 (27%) of the Ikzf3Het mice by 20 months of age, but not in age-matched Ikzf3WTmice (n=30). The cohort of Ikzf3Homo mice has been monitored thus far for 18 months, with identification already of one of 30 (3%) with leukemia with a ~15 months onset. Across animals with CLL-like disease, the pattern of disease localization was consistently observed as detected in the spleen but not in the bone marrow, suggesting preferential homing of Ikzf3-mutant cells to the splenic microenvironment. To dissect the mechanisms underlying Ikzf3-mediated leukemogenesis, we asked whether presence of mutated-Ikzf3 was associated with changes in B cell transcriptional programs in young mice without disease. We performed RNA-sequencing using splenic B cells from Ikzf3Het, Ikzf3Homo or Ikzf3wt animals (3 mice/group, 3-month old). The most striking changes were identified between Ikzf3Homo to Ikzf3wt mice, with ~1400 differentially expressed genes in Ikzf3Homocells compared to Ikzf3wt (>2 fold, adjusted p<0.05). Within this Ikzf3Homo-specific signature, we identified genes regulating B cell positioning within the light zone of germinal centers (GCs, p<0.0001) and BCR signaling pathway members (p<0.05), by gene set enrichment analysis (GSEA). The enhanced activation of the BCR pathway was further confirmed by western blot analysis of splenic B cells stimulated with anti-IgM, with Ikzf3Homo and Ikzf3het mice showing upregulation of phosphorylation in SYK, AKT and ERK after BCR engagement, as compared to Ikzf3WT. Allo-immunization with the T-cell dependent antigen sheep red blood cells (SRBC) for 10 days elicited a higher rate of germinal centers (GC) formation in Ikzf3Homo mice than Ikzf3WT (p<0.01, ANOVA with Tukey's correction), further indicating an enhanced response to BCR stimulation in presence of the mutation. Overall, phenotypic changes of Ikzf3het appeared less pronounced than Ikzf3Homo, in young mice. To further define the functional effects of mutant-Ikzf3 in transcriptional regulation, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using anti-AIOLOS antibody in Ikzf3WT and Ikzf3Homo B cells. While we observed no significant changes in the conserved DNA binding motif or the genomic localization of AIOLOS binding sites, stronger AIOLOS binding at the transcription start sites (TSS) was detected in Ikzf3Homo versus Ikzf3WTcells (p=1.3747x10-5, Student's t-test). These results indicate an enhanced binding of mutated AIOLOS to DNA which may in turn dysregulate gene expression. As an example, upregulation of the chemokine receptor Cxcr4 in Ikzf3Homo B cells was associated with an enrichment of mutant AIOLOS at its promoter region. Functionally, we confirmed that such upregulation was associated with increased migration of Ikzf3Homo cells to SDF-1 in vitro, in a transwell-based chemotaxis assay (p<0.01, ANOVA with Tukey's correction). Overall, this novel model provides evidence of an oncogenic role of IKZF3 mutation in CLL. Ikzf3 mutation induces broad transcriptional and functional changes associated with dysregulation of BCR signaling and homing mechanisms, which may favor disease initiation and progression in vivo. Comparative analyses between Ikzf3-mutant murine CLLs and IKZF3-mutant primary samples are ongoing, to further refine the relevance of Ikzf3 in B cell function and lymphomagenesis. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.