Rearrangement of CRLF2 in B-progenitor- and Down syndrome-associated acute lymphoblastic leukemia

Abstract

Aneuploidy and translocations are hallmarks of B-progenitor acute lymphoblastic leukemia (ALL), but many individuals with this cancer lack recurring chromosomal alterations. Here we report a recurring interstitial deletion of the pseudoautosomal region 1 of chromosomes X and Y in B-progenitor ALL that juxtaposes the first, noncoding exon of P2RY8 with the coding region of CRLF2. We identified the P2RY8-CRLF2 fusion in 7% of individuals with B-progenitor ALL and 53% of individuals with ALL associated with Down syndrome. CRLF2 alteration was associated with activating JAK mutations, and expression of human P2RY8-CRLF2 together with mutated mouse Jak2 resulted in constitutive Jak-Stat activation and cytokine-independent growth of Ba/F3 cells overexpressing interleukin-7 receptor alpha. Our findings indicate that these two genetic lesions together contribute to leukemogenesis in B-progenitor ALL.

Figure 1. Deletion involving pseudoautosomal region 1 (PAR) and P2RY8-CRLF2 fusion in B-progenitor ALL

a, representative log₂ratio SNP 6.0 microarray DNA copy number data of six cases with PAR1 deletion. White is normal, blue is deletion, and red is gain. Paired data is shown, with normal (N) on the left, and tumor (T) on the right for each case. DS, Down syndrome. b, mapping of the extent of the PAR1 deletion is shown for a representative case. The sparse SNP 6.0 probe coverage of the region, particularly the lack of coverage of the CRLF2/CSF2RA/IL3RA region is shown. Location of SNP 6.0 probes is shown by vertical black lines, and the coverage of the one million feature Agilent array shown as green lines, with the corresponding log₂ratio copy number data shown as red lines. The region of deletion defined by each platform is shown as a horizontal arrow. c, RT-PCR demonstrating P2RY8-CRLF2 fusion transcripts. NTC, no template control. d, the fusion junction is identical in each P2RY8-CRLF2 positive case, and involves the first non-coding exon of P2RY8 and the entire open reading frame of CRLF2.

Figure 2. Transforming effects of P2RY8-CRLF2 and JAK mutations

a, Ba/F3 cells expressing murine IL-7 receptor alpha chain (Ba/F3-IL7R, or B7 cells) were transduced with retroviral constructs expressing P2RY8-CRLF2 (P2C) and/or Jak2 alleles (wild type murine Jak2, or Jak2 683G and 933R). Cells were washed and cultured in the absence of cytokine. Co-expression of P2RY8-CRLF2 and Jak mutants resulted in cytokine-independent growth, but not expression of either P2RY8-CRLF2 or Jak mutants alone. b, western blotting demonstrating constitutive Jak-Stat activation in Ba/F3-IL7R cells expressing P2RY8-CRLF2 and Jak2 mutants. Ba/F3 cells grown in IL-3, and Ba/F3-IL7R cells grown in IL-7 (without starvation and stimulation) are included at the left of the figure as positive controls. A representative blot of three independent experiments is shown. c, pharmacological Jak inhibition (with Jak inhibitor I) inhibits the growth of Ba/F3-IL7R cells transduced with P2RY8-CRLF2 and Jak2 683G or 933R. Cells were washed three times, plated at 0.5×10⁶ cells/ml in triplicate, and counted after 48 hours. The ETV6-RUNX1 B-progenitor ALL cell line REH, which does not harbor P2RY8-CRLF2 or JAK mutations, was used as a control. d, Knockdown of CRLF2 expression by lentiviral shRNAs attenuates cytokine-independent growth of Ba/F3-IL7R cells expressing P2RY8-CRLF2 and Jak2 683G. Cells were transduced with non-target (scrambled) shRNA, each of three CRLF2 shRNAs (181, 286 and 757) and a pool of all three shRNAs. This resulted in substantial but incomplete attenuation of CRLF2 expression (see Supplementary Fig. 12 online) and reduced cytokine-independent growth. Cells with near-total down-regulation of CRLF2 expression following shRNA knockdown (isolated by flow sorting for CRLF2) showed marked abrogation of cytokine-independent growth. t-test (compared to cells transduced with non-target shRNA) P-value: *, 0.05<P<0.10; **P<0.01; ***P<0.001. All error bars show s.e.m.