CRLF2 overexpression results in reduced B-cell differentiation and upregulated E2F signaling in the Dp16 mouse model of Down syndrome

Abstract

Children with Down syndrome (DS) are 10-fold more likely to develop B-cell acute lymphoblastic leukemia (B-ALL), with a higher frequency of rearrangements resulting in overexpression of cytokine receptor-like factor 2 (CRLF2). Here, we investigated the impact of CRLF2 overexpression on B-cell progenitor proliferation, immunophenotype, and gene expression profile in the Dp(16)1Yey (Dp16) mouse model of DS compared with wild-type (WT) mice. CRLF2 overexpression enhanced immature B-lymphoid colony development and increased the proportion of less differentiated pre-pro-B cells, with a greater effect in Dp16 versus WT. In CRLF2-rearranged (CRLF2-R) B-ALL patient samples, cells with higher CRLF2 expression exhibited a less differentiated B-cell immunophenotype. CRLF2 overexpression resulted in a gene expression signature associated with E2F signaling both in Dp16 B-progenitors and in DS-ALL patient samples, and PI3K/mTOR and pan-CDK inhibitors, which reduce E2F-mediated signaling, exhibited cytotoxicity in CRLF2-R B-ALL cell lines and patient samples. CRLF2 overexpression alone in Dp16 stem and progenitor cells did not result in leukemic transformation in recipient mice. Thus, CRLF2 overexpression results in reduced B-cell differentiation and enhanced E2F signaling in Dp16 B-progenitor cells and DS-ALL patient samples. These findings suggest a functional basis for the high frequency of CRLF2-R in DS-ALL as well as a potential therapeutically targetable pathway.

Figure 1.. CRLF2 overexpression enhances B-lymphoid colony formation and reduces B cell maturation, with a greater fold change in the Dp16 background.

(A) CRLF2 overexpression in BM HSPCs from both WT and Dp16 mice increased B-lymphoid colony growth compared to GFP control. Histogram depicts colony counts from six samples (two technical replicates across three independent experiments, Student’s t-test, *p<0.01, **p<0.001). Error bars indicate standard deviation. (B) CRLF2 overexpression in WT and Dp16 HSPCs grown in B cell-differentiation conditions resulted in an increased percentage of the less-differentiated pre-pro-B cells. This effect was greater in the Dp16 versus WT genetic background. Histogram shows percentage of pre-pro-B cells in the GFP⁺ gate of each experimental group, with mean values from three independent experiments with two biological replicates each (Student’s t-test, *p<0.05, **p<0.001). Error bars indicate standard deviation.

Figure 2.. CRLF2 overexpression is associated with reduced B cell differentiation in B-ALL patient samples.

(A) In two CRLF2-overexpressing B-ALL patient samples (DS-ALL 839 and non-DS ALL 105127-R) demonstrating both pro-B and pre-B populations, the high-CRLF2 mean fluorescence intensity (MFI) quartile demonstrated a significantly higher mean proportion of pro-B cells (p=0.024). (B) Representative flow plots showing CRLF2 overexpression correlates with a less differentiated immunophenotype in B-ALL patient samples 839 (DS) and 105127-R (non-DS).

Figure 3.. Dp16 CRLF2 cells demonstrate enrichment for E2F targets.

Gene set enrichment analysis plots show upregulation of E2F targets in Dp16 CRLF2 cells compared to (A) Dp16 GFP cells and (B) WT CRLF2 cells. Normalized enrichment score (NES), false discovery rate (FDR) q-value, and familywise error rate (FWER) p-value are also displayed.