A Role for Interferon Regulatory Factor 4 in the Development of CLL

Abstract 714

Chronic Lymphocytic Leukemia (CLL) representing 30% of all the adult leukemia is a heterogeneous B cell malignancy with complex clinical behavior and pathophysiology. Even though an increasing body of literature has helped in understanding the biology of CLL the precise molecular events that underlie the pathogenesis of this disease are still not fully understood. Recent Whole Genome Sequencing and Genome Wide Association Studies have linked different genetic loci to the origin and evolution of the disease. A Genome Wide Association Study (GWAS) by Di Bernardo et al identified Single Nucleotide Polymorphisms in the 3'UTR of Interferon Regulatory Factor 4 (IRF4) as a familial risk allele for CLL. These SNPs are associated with lower mRNA levels of IRF4 in individuals carrying the risk allele. Another study by Havelange V. et al identified heterozygous mutations in the DNA binding domain of IRF4 in patients with CLL. These studies have linked lower levels of IRF4 to the pathogenesis of CLL. Even though studies have linked low levels of IRF4 to the pathogenesis of CLL, whether low levels of IRF4 indeed contribute to CLL development remains to be determined.

In rodent, CLL cells are presumably derived from B1 cells. Like CLL cells, B1 cells are B220⁺CD5⁺ and possess self-reactive B cell receptors (BCR). B1 cells normally reside in peritoneal and pleural cavities and constitute only about 5% of total B lymphocytes in mice. However, B1 cell population is dramatically elevated in the V_H11 knock-in mice. V_H11 family of variable IgH chain is expressed exclusively on B1 cells where it pairs with V_k9 to form BCR that recognizes phosphatidylcholine on senescent RBCs. We did not observe an overt increase of CLL development in IRF4 deficient mice. However, when we crossed the V_H11 knock-in mice to an IRF4 deficient mice it resulted in a CLL like lymphoproliferative disorder with marked expansion of CD5⁺ B220^low/int population in blood, spleen and other lymphoid organs. To monitor pathogenesis of CLL, we collected blood from saphenous vein of IRF4^−/−V_H11 and IRF4^+/+V_H11 littermate control mice monthly. A total of ten mice from each group were used for this study. The appearance of a monoclonal B220^lowCD5⁺ population and its percentage among peripheral blood mononuclear cells (PBMC) were used for the initial CLL diagnosis. The diagnostic criterion for CLL in V_H11 mice is the appearance of monoclonal B220^lowCD5⁺ cells constituting over 20% of PBMCs. If the percentage of the B220^lowCD5⁺ clone is under 20% of the PBMC, the diagnosis would be monoclonal B cell lymphocytosis (MBL), a disease which can progress into CLL. Interestingly, 6 out of 10 IRF4^−/−VH11 mice developed CLL after just 5 months while the four remaining IRF4^−/−VH11 mice developed MBL. In contrast, none of the IRF4^+/+VH11 control mice developed CLL or MBL. Two of the mice developed aggressive CLL where leukemic cells infiltrated liver and other non-lymphoid organs. BrdU incorporation assay identified spleen as the primary site of CLL cells expansion although proliferating CLL cells were also identified in lymph nodes and bone marrow. We further isolated CLL cells from IRF4^−/−V_H11 mice and transplanted them into immunodeficient mice (Rag2^−/−gamma^−/−). Our result shows that the disease was transplantable as all the transplanted mice succumbed to CLL within 1 month post-transplantation. Treatment of IRF4^−/−V_H11 CLL cells with specific Syk and Btk inhibitors targeting BCR signaling showed that like human CLL, the leukemic cells were depended on active BCR signaling for their survival and proliferation.

In summary, we provide evidence for a causal relationship between low levels of IRF4 and development of CLL. Further studies are needed to provide mechanistic insights into how IRF4 is involved in the development and progression of this disease. The fine dissection of this relationship will allow for development of new therapeutic strategies and identification of new prognostic markers for diagnosis and treatment of CLL.