Notch2 Is Critical for CLL Development in the IRF4^-/-Vh11 Mice

Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the western world. Despite considerable progress in our current understanding of CLL, the molecular events underlying the complex pathogenesis and clinical behavior of CLL have not been fully elucidated. IRF4 belongs to the IRF superfamily of transcription factors and plays critical roles at multiple stages of B cell development. Genome Wide Association Studies had linked Single Nucleotide Polymorphism (SNP) mediated IRF4 down regulation to the pathogenesis of human CLL (Di Bernardo et al. Nature Genetics, 2008). Moreover, low levels of IRF4 were found to correlate with poorer disease outcomes in human CLL (Chang et al. Blood, 2002). More recently, the studies from our group have established a causal relationship between low levels of IRF4 and development of CLL. We used Vh11 knock-in (Vh11) mice that express a prearranged immunoglobulin variable heavy chain (Vh11) leading to preferential generation of B1 cells (CLL precursors). Interestingly, we have shown that mice expressing the Vh11 transgene in the IRF4 deficient background (IRF4^-/-Vh11) spontaneously develop CLL at complete penetrance (Shukla et al. Blood, 2013). Additionally, we have also shown that low levels of IRF4 dramatically accelerate CLL development in the New Zealand Black (NZB) mice (Ma et al. JBC, 2013). Although our studies have demonstrated a critical role for IRF4 in the pathogenesis of CLL, the molecular mechanism through which IRF4 controls CLL development remains unclear.

In an attempt to identify the molecular networks regulated by IRF4 in CLL cells we performed gene expression profiling of CLL cells isolated from IRF4^-/-Vh11 mice. Intriguingly, our analysis revealed that Notch signaling was hyperactive in the IRF4^-/-Vh11 CLL cells compared to the IRF4^+/+Vh11 B1 cells. This is particularly important because recent high depth sequencing studies have identified mutations in Notch1 protein leading to its stabilization and activation in ~10% of all CLL patients (Puente et al. Nature, 2011, Fabbri et al. JEM, 2011). Furthermore, the individuals carrying these mutations have an aggressive disease course and poor overall survival (Fabbri et al. JEM, 2011). Recently, studies by Simonetti et al have shown that the IRF4 deficient B cells express high levels of Notch receptors and display increased Notch activity (Simonetti et al. JEM, 2013). We also detected high levels of Notch2 but not Notch1 on the cell surface of IRF4^-/-Vh11 CLL cells. To examine the contribution of Notch signaling in the pathogenesis of CLL, we crossed IRF4^-/-Vh11 mice with Notch2 conditional mutant mice and with mice expressing cre recombinase in B cells (CD19cre). This approach allowed us to genetically delete Notch2 receptor specifically in B cells of IRF4^-/-Vh11 mice (CD19creNotch2^fl/flIRF4^-/-Vh11). Surprisingly, CLL cells could still emerge from the majority of CD19creNotch2^fl/flIRF4^-/-Vh11 mice. However, further analysis revealed that those CLL cells expressed high levels of Notch2 on their surface and were apparently derived from IRF4^-/-Vh11 B1 cells that escaped cre-mediated deletion. Importantly, our analysis also showed that the majority of B1 and B2 cells in the CD19creNotch2^fl/flIRF4^-/-Vh11 mice had lost the expression of Notch2, indicating that Notch2 is dispensable for their generation.

We also performed a series of analysis to determine how Notch signaling affects B1 and CLL cells in vitro. We used a co-culture system to engage Notch receptors to their ligand (Delta like 1 or DL1) expressed on S17 stromal cells (S17-DL1). Co-culture of wild type B1 cells with S17-DL1 stromal cells led to enhanced proliferation and survival of B1 cells when compared with cells co-cultured on control vector transfected S17 stromal cells (S17-R1). Importantly, Notch2 receptor deficient B1 cells failed to respond to DL1 expressing S17 stromal cells. Similarly, IRF4^-/-Vh11 CLL cells also showed enhanced survival on S17-DL1 stromal cells compared to S17-R1 stromal cells.

In summary, our studies show that Notch2 is absolutely required for CLL development in the IRF4^-/-Vh11 mice, thereby supporting a critical role for Notch signaling in the pathogenesis of CLL.