Rearrangement of CRLF2 in B-Progenitor and Down Syndrome Associated Acute Lymphoblastic Leukemia.

Abstract 182

Chromosomal alterations are a hallmark of acute lymphoblastic leukemia (ALL), but many cases lack a recurring cytogenetic abnormality. To identify novel alterations contributing to leukemogenesis, we previously performed genome-wide profiling of genetic alterations in pediatric ALL using single nucleotide polymorphism (SNP) microarrays. This identified a novel focal deletion involving the pseudoautosomal region (PAR1) of Xp/Yp in 15 B-progenitor ALL cases lacking sentinel chromosomal abnormalities, including six of eight cases of ALL associated with Down syndrome (DS-ALL). The deletion involved hematopoietic cytokine receptor genes, including IL3RA and CSF2RA, but due to poor array coverage, it was not possible to define the limits of deletion using SNP array data alone. To characterize this abnormality, we examined an expanded cohort of 329 B-ALL cases, including 22 B-progenitor DS-ALL cases. Strikingly, 12 (55%) DS-ALL cases harbored the PAR1 deletion. Mapping using high density CGH arrays showed the deletion to be identical in each case, and involved a 320kb region extending from intron 1 of the purinergic receptor gene P2RY8 to the promoter of CRLF2 (encoding cytokine receptor like factor 2, or thymic stromal lymphopoietin receptor). The deletion resulted in a novel fusion of the first, non-coding exon of P2RY8 to the entire coding region of CRLF2 in each case. The P2RY8-CRLF2 fusion resulted in elevated expression of CRLF2 detectable by quantitative RT-PCR, and flow cytometric analysis of leukemic cells. One DS-ALL case with elevated CRLF2 expression lacked the PAR1 deletion, but had an IGH@-CRLF2 translocation detected by fluorescence in situ hybridization (FISH). CRLF2 alteration was associated with gain of chromosome X (which was shown by FISH to result in duplication of the PAR1 deletion), deletion of 9p, and the presence of Janus kinase (JAK1 and JAK2) mutations. Ten (53%) of patients with CRLF2 alteration had JAK mutations, compared with two patients lacking CRLF2 abnormalities (P<0.0001). To validate these findings, we examined an additional 53 B-progenitor DS-ALL cases, and identified PAR1 deletion and P2RY8-CRLF2 fusion in 28 (52%) of cases. The deletion was also associated with elevated CRLF2 expression and JAK mutation in this cohort. CRLF2 forms a heterodimeric receptor with interleukin 7 receptor alpha, and binds thymic stromal lymphopoietin. CRLF2 signaling is important for T and dendritic cell development, but is not required for normal B cell development. The association of CRLF2 dysregulation and JAK mutations suggests a cooperative role in leukemogenesis, and to test this hypothesis, we examined the transforming effects of P2RY8-CRLF2 and JAK mutations in cytokine dependent murine Ba/F3 pro-B cells engineered to express IL-7 receptor alpha (Ba/F3-IL7R cells). Co-expression of both P2RY8-CRLF2 and JAK mutations (either the pseudokinase mutation R683G or the kinase domain mutation P933R) resulted in cytokine independent growth and constitutive Jak-Stat activation, but not expression of either P2RY8-CRLF2 or these JAK mutations alone. This transformation was attenuated by pharmacologic JAK inhibitors and shRNA mediated knockdown of CRLF2. Furthermore, co-immunoprecipitation demonstrated direct interaction of CRLF2 and phoshorylated JAK2. These findings identify CRLF2 alteration as a novel submicroscopic abnormality in B-progenitor ALL that is especially frequent in DS-ALL, and suggest that abnormal CRLF2 signaling, mediated by elevated CRLF2 expression and JAK mutation, is an important event in leukemogenesis. Furthermore, the association between CRLF2 alteration and JAK mutation may in part explain the lineage specificity of different JAK mutations in hematologic malignancies. Finally, these findings suggest that therapeutic inhibition of JAK-STAT signaling will be an important therapeutic approach in B-progenitor ALL with CRLF2 and JAK alterations.