KZF-L162R Mutation Affects Splenic Mature B Cell Development and Alters Expression of Aiolos Target Genes

Large-scale DNA sequencing efforts in chronic lymphocytic leukemia (CLL) have identified a broad array of putative cancer drivers arising from somatic mutations in this disease, but functional understanding of the impact of these genetic events on CLL onset and progression remains to be elucidated. One such example is mutation in the IKZF3 gene, encoding the zinc finger protein AIOLOS, mutated in ~2% of CLLs and associated with fludarabine-refractory disease. AIOLOS is a lymphoid-restricted transcription factor and a chromatin remodeler that plays an essential role in B cell development and maturation. In CLL, the IKZF3 mutation, also reported in few cases of diffuse large B cell lymphoma and mantle cell lymphoma,targets a highly conserved hotspot (L162R, homologous to murine L161R) that is localized in the 2^nd zinc finger of the DNA-binding domain, required for DNA sequence recognition. Given the localization of this hotspot mutation, we hypothesized that it impacts the function of AIOLOS to drive CLL.

To characterize the effects of the IKZF3-L162R mutation, we generated a knock-in mouse line that conditionally expresses the point mutation in a B cell lineage context through crossing with Cd19-cre mice, generating mouse lines carrying Ikzf3-L161R as either a heterozygous mutation (Ikzf3-L161R^Het), homozygous mutation (Ikzf3-L161R^Homo) or wild-type Ikzf3(Ikzf3^WT).

Given the established role of Aiolos in lymphoid differentiation, we first asked how the mutation impacts B cell development. By flow cytometry, using established markers to detect marrow pro-B, pre-B, transitional and mature B cell populations, or peritoneal B1a and B1b cell populations, no differences in the proportion of cells were observed between Ikzf3^WTor Ikzf3-L161R^Het. In the spleen, however, the average proportion of marginal zone B cells (B220+CD23+CD21^high) was markedly reduced in heterozygousmice compared to wild type mice (6 mice/group: 4.9% vs. 11.5%, p=0.006), while the average proportion of follicular B cells (B220+CD23+CD21-) was increased (76% vs. 63%; p=0.003). Immunohistochemical staining of spleen sections confirmed that the marginal zone area was significantly reduced in Ikzf3-L161R^Hetmice (p=0.01). In addition, we noted a higher proliferation rate of B cells from Ikzf3-L161R^Hetmice when stimulated with LPS and IL-4 for 3 days (p=0.01), suggesting that the mutation confers a survival advantage to B cells. Similar analyses in Ikzf3-L161R^Homomice are ongoing.

By immunofluorescence and immunoprecipitation, neither Aiolos binding with its partners CHD4, SIN3 or HDAC1, nor its cellular distribution were impacted by the mutation. Of note, the total protein level of Aiolos was increased in Ikzf3-L161R^Hetmice (9 mice/group; p<0.05). Since the mutation localizes to a DNA binding domain, we hypothesized that it modifies the ability of Aiolos to control expression of its target genes. We therefore performed CHIP-seq in Ikzf3^WTsplenic B cells, and identified Aiolos-associated high confidence peaks (fold change (FC) enrichment compared to input > 20) corresponding to DNA binding sites in the promoters of genes such as Rps19, Ogg1, Dusp2, Phf23 or Brfp1 and confident peaks (FC>10) in the anti-apoptotic gene Mcl1 and in genes involved in BCR signaling (i.e.Syk, Pi3kr1, Nfkbid), suggesting that their expression is under the control of Aiolos.

Comparison of the expression by qPCR of these 8 genes in splenic B cells from the 3 mouse lines revealed Dusp2, Mcl1, Syk, Nfkbid and Phf23 to be upregulated in Ikzf3-L161R^HomoB cells (p<0.05) but not in Ikzf3-L161R^HetB cells. These findings suggest that the mutation directly impacts the expression level of Aiolos target genes. The upregulation of Mcl1 expression is particularly relevant in the context of CLL as dysregulation of anti-apoptotic signaling is characteristic of the disease.

In conclusion, these data show that Aiolos mutation affects B cell subpopulation ontogeny, inducing a disproportionate abundance of follicular B cells endowed with high proliferative capacity. The mutation impacts Aiolos transcription capacity leading to upregulation of genes belonging to pathways cardinal to CLL development, including BCR signaling and apoptosis. Ongoing studies focus combining RNA-seq and CHIP-seq in mutant B cells, with the aim of identifying the breadth of differential expressed genes and dysregulated cellular pathways in mutant B cells in an unbiased manner.