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

Abstract 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 2nd 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-L161RHet), homozygous mutation (Ikzf3-L161RHomo) or wild-type Ikzf3(Ikzf3WT). 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 Ikzf3WTor Ikzf3-L161RHet. In the spleen, however, the average proportion of marginal zone B cells (B220+CD23+CD21high) 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-L161RHetmice (p=0.01). In addition, we noted a higher proliferation rate of B cells from Ikzf3-L161RHetmice 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-L161RHomomice 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-L161RHetmice (9 mice/group; p 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-L161RHomoB cells (p<0.05) but not in Ikzf3-L161RHetB 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. Disclosures Wu: Neon Therapeutics: Equity Ownership

Activation of Notch and Myc signaling via B cell-restricted depletion of Dnmt3a generates a consistent murine model of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc-expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. SIGNIFICANCE: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition

A Murine Model of Chronic Lymphocytic Leukemia Based on B Cell-Restricted Expression of Sf3b1 Mutation and Atm Deletion

SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment