Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leukemia in Down syndrome

Abstract

Children with Down syndrome (DS) are prone to development of high-risk B-cell precursor ALL (DS-ALL), which differs genetically from most sporadic pediatric ALLs. Increased expression of cytokine receptor-like factor 2 (CRLF2), the receptor to thymic stromal lymphopoietin (TSLP), characterizes about half of DS-ALLs and also a subgroup of sporadic "Philadelphia-like" ALLs. To understand the pathogenesis of relapsed DS-ALL, we performed integrative genomic analysis of 25 matched diagnosis-remission and -relapse DS-ALLs. We found that the CRLF2 rearrangements are early events during DS-ALL evolution and generally stable between diagnoses and relapse. Secondary activating signaling events in the JAK-STAT/RAS pathway were ubiquitous but highly redundant between diagnosis and relapse, suggesting that signaling is essential but that no specific mutations are "relapse driving." We further found that activated JAK2 may be naturally suppressed in 25% of CRLF2^pos DS-ALLs by loss-of-function aberrations in USP9X, a deubiquitinase previously shown to stabilize the activated phosphorylated JAK2. Interrogation of large ALL genomic databases extended our findings up to 25% of CRLF2^pos, Philadelphia-like ALLs. Pharmacological or genetic inhibition of USP9X, as well as treatment with low-dose ruxolitinib, enhanced the survival of pre-B ALL cells overexpressing mutated JAK2. Thus, somehow counterintuitive, we found that suppression of JAK-STAT "hypersignaling" may be beneficial to leukemic B-cell precursors. This finding and the reduction of JAK mutated clones at relapse suggest that the therapeutic effect of JAK specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL.

Keywords: CRLF2; Down syndrome; JAK-STAT signaling; USP9X; acute lymphoblastic leukemia.

Fig. 1.

General distribution of somatic events in 31 patients with DS-ALL. The main panel lists recurrent SNV/indels and copy number variants (CNVs, represented by arrows). The rows correspond to the genes; the columns correspond to leukemia samples. Only SNVs/indels with VAF ≥ 5% are shown. Notice paired samples (i.e., diagnosis and relapse) are adjacent. The bars to the right indicate the number of patients harboring each alteration. The bars at the bottom indicate the total number of exonic somatic SNVs/indels in each sample (log₂ scale).

Fig. S1.

Analysis of SNV reveal increased C→G transversions in relapse. (A) The proportion of each of the 96 possible SNVs in their trinucleotide context. The proportion is calculated out of the total number of SNVs at each of the time points (total number of SNPs, DX (n = 492), R1 (n = 602), and R2 (n = 168). The three hypermutated samples (DSALLG11_R2, DSALLG1_R1, DSALLB1_R1), were excluded from the analysis. Asterisks represent significant difference between DX and R1, q-values < 0.05 (FDR-corrected Fisher’s exact test). (B) Similar analysis as in A, performed on an independent dataset of paired diagnosis-relapse ALLs from Oshima et al. (25).

Fig. S2.

Allelic frequency of P2RY8-CRLF2 rearrangements at diagnosis and relapse. Scatter plot comparing the inferred VAF of PAR1 deletion in all samples with the P2RY8–CRLF2 rearrangements, and the corresponding inferred tumor purity.

Fig. 2.

Temporal association of somatic mutations between diagnosis and relapse. (A) The bar plot reports the total number of paired patients (n = 25) with somatic variation in each of the loci listed. Variations were classified as shared regardless whether the identical or different genomic alterations in the same gene were present in both time points. (B) The genomic distribution of protein-changing mutations identified in our cohort in MSH6, MLH1, TP53, SETD2, and CREBBP. (C) Boxplot showing the mutational burden of each sample grouped by MLH1/MSH6 mutated (n = 4) or wild-type (n = 61).

Fig. S3.

HIST1 cluster deletions in DS-ALL. Exome coverage ratio (log2R values) in the HIST1 cluster region on chromosome 6 for all samples with putative deletions in the region.

Fig. S4.

IgH locus-MIR100HG translocation discovered by RNA sequencing. Raw coverage data and junction track of paired-end RNA-seq of patient DSALLB3, demonstrating noncanonic transcription from the MIR100HG locus and beginning upstream to MIR125B1. Chimeric transcripts mapping to IGH and MIR100HG were abundantly expressed in the two samples shown.

Fig. 3.

The dynamics of signaling mutations in DS-ALL. (A) VAF of JAK1, JAK2, NRAS, and KRAS mutated alleles at each time point. All 17 patients with mutations in at least one of the genes are shown. Patients are sorted by CRLF2 status at diagnosis. DX, diagnosis; R1, relapse 1; R2, relapse 2. (B) Graphical representation of somatic alterations in signaling genes along the different time points of relapsed DS-ALL. The vertices represent the mutated genes; edges between vertices indicate a patient with a mutation in both genes. Horizontal edges indicate that the connected genes were mutated in the same sample, and vertical edges connect genes mutated in the same patient at different time point. The thicker edges indicate events observed in more than one patient. The size of the vertices illustrates the number of patients with mutations in the gene. Only mutations with VAF ≥ 5% are included.

Fig. 4.

USP9X genomic aberrations. (A) Log2R values (Log₂ ratio sample/normal) in the USP9X genomic region in the three DS-ALL patients with deletions in the region. Data shown are derived from a high-resolution SNP array (DSALLB7 and DSALLG14), or from exome coverage data (DSALLA2; indicated with “§”). (B) Coding mutations found in USP9X in independent published cohorts of B-ALLs (, –44). The mutation found in patient DSALLG11 is indicated in red. (C) Scatter plots of the VAF of mutations in diagnosis and in relapse in three patients with USP9X genomic lesions (Und, undetected). (D) CRLF2 and USP9X RNA-seq expression in Ph-like ALLs downloaded from the PeCan database (45). Samples classified as either carrying CRLF2 fusions (n = 28) or negative for the fusions (n = 96). Samples with the known USP9X–DDX3X fusion transcript are indicated with darker color.

Fig. S5.

USP9X genomic aberrations. (A) Log2R exome coverage data in the two leukemia samples of patient DSALLB7. Two deletion peaks are identified in the diagnosis sample (DSALLB7_DX), mapping to PAR1 region and to USP9X region, together with a gain of the whole X chromosome. Only USP9X deletion was also identified in the relapse sample (DSALLB7_R1). The X chromosome carrying the P2RY8–CRLF2 rearrangement was lost. (B) USP9X expression in 16 samples with RNA-seq data. (Samples from patient DSALLB7 are indicated with red). (C) Sanger sequencing of cDNA from sample DSALLG11_DX, showing the transcription from the mutated USP9X locus (c.3344insAGGGC – shaded area). (D) Genomic view of the insertion mutation in USP9X in sample DSALLG11_DX exome data, demonstrating the mutation validated in C. (E) VAF of USP9X and other mutations in signaling genes in the B-ALL cohort studied by Oshima et al. (25). (F) Density plot of the VAF of the mutations in sample DSALLA2_DX, demonstrating USP9X deletion correspond to the major clone.

Fig. 5.

Silencing of USP9X enhances leukemia cell survival by buffering JAK signaling. (A) The effect of increasing doses of the USP9X inhibitor BRD0476 on viability of 018z BCP-ALL cells transduced with CRLF2/JAK2^R683G. Shown is flow cytometry analysis of the fraction of viable transduced cells expressing surface CRLF2 (CRLF2+) 2 wk after treatment (*P< 0.05). (B) Phosphoflow cytometry analysis of STAT5 phosphorylation (pSTAT5) 30 min after treatment with BRD0476 (10 μM). (C) Immunoblot for USP9X in wild-type and CRISPR/Cas9-knockout 018z leukemia cells. (D) The effect of USP9X knockout on the relative growth of CRLF2/JAK2^R683G transduced 018z cells after 2 wk in culture. Shown is flow cytometry analysis of the fraction of viable transduced cells expressing surface CRLF2 (CRLF2+) (*P < 0.05). (E) The effect of the JAK inhibitor ruxolitinib on proliferation of 018z cells transduced with CRLF2/JAK2^R683G one week following treatment. (F) Phospho flow cytometry analysis of STAT5 phosphorylation (pSTAT5) after treatment with ruxolitinib (0.25 μM).

Fig. S6.

USP9X and JAK inhibition in CRLF2/JAK2^R683G transduced cells (related to Fig. 5). (A) CRLF2 expression by flow cytometry of 018z cells 3 d following transduction with CRLF2/JAK2^R683G. (B) No effect of increasing doses of the USP9X inhibitor BRD0476 on viability of 018z cells transduced with lentiviral backbone control vector carrying GFP (BB-GFP). y axis: percentage of viable BB-GFP transduced 018z cells (GFP⁺) after 2 wk of treatment. (C) Phospho flow cytometry analysis of phospho STAT3 (pSTAT3) after treatment with BRD0476 (10 μM) for 30 min demonstrating no effect. (D) cDNA sequence of wild-type 018z compared with cDNA after transduction with CRISPR/Cas9 lentiviral vector demonstrating disruption of USP9X. (E) No effect of USP9X knockout on the viability of BB-GFP transduced 018z cells. y axis: percentage of viable BB-GFP transduced 018z cells (GFP⁺) after 2 wk of culture. (F) Treatment with low doses of JAK inhibitor does not affect the proliferation of BB-GFP transduced 018z cells. y axis: percentage of viable BB-GFP transduced 018z cells (GFP⁺) after 1 wk of treatment with ruxolitinib (**P ≤ 0.01; ***P ≤ 0.001). (G) Phospho flow cytometry analysis of phospho STAT3 (pSTAT3) after treatment with ruxolitinib (0.25 μM) for 30 min.

Fig. S7.

Interaction between USP9X, IL7R, and JAK1. (A) Affinity pull-down of USP9X and associated proteins by biotinylated BRD0476 in INS-1E cells. (B) The effect of increasing doses of the USP9X inhibitor BRD0476 on viability of 018z BCP-ALL cells transduced with CRLF2/IL7R^INS. Shown is flow cytometry analysis of the fraction of viable transduced cells expressing surface CRLF2 (CRLF2+) 2 wk after treatment (*P < 0.05). (C) Phospho flow cytometry analysis of STAT5 phosphorylation (pSTAT5) 30 min after treatment with BRD0476 (10 μM) in CRLF2/IL7R^INS transduced cells. (D) The effect of USP9X knockout on the relative growth of CRLF2/IL7R^INS transduced 018z cells after 2 wk in culture. Shown is flow cytometry analysis of the fraction of viable transduced cells expressing surface CRLF2 (CRLF2+) (*P < 0.05). (E) The effect of the JAK inhibitor ruxolitinib on proliferation of 018z cells transduced with CRLF2/IL7R^INS 1 wk following treatment.