Abstract
Abstract 115
Extra copies of chromosome 21 (polysomy 21) is the most common somatic aneuploidy in B-cell acute lymphoblastic leukemia (B-ALL), including >90% of cases with high hyperdiploidy. In addition, children with Down syndrome (DS) have a 20-fold increased risk of developing B-ALL, of which ∼60% harbor CRLF2 rearrangements. To examine these associations within genetically defined models, we investigated B-lineage phenotypes in Ts1Rhr mice, which harbor triplication of 31 genes syntenic with the DS critical region (DSCR) on human chr.21. Murine pro-B cell (B220+CD43+) development proceeds sequentially through “Hardy fractions” defined by cell surface phenotype: A (CD24−BP-1−), B (CD24+BP-1−) and then C (CD24+BP-1+). Compared with otherwise isogenic wild-type littermates, Ts1Rhr bone marrow harbored decreased percentages of Hardy fraction B and C cells, indicating that DSCR triplication is sufficient to disrupt the Hardy A-to-B transition. Of note, the same phenotype was reported in human DS fetal liver B-cells, which have a block between the pre-pro- and pro-B cell stages (analogous to Hardy A-to-B).
To determine whether DSCR triplication affects B-cell proliferation in vitro, we analyzed colony formation and serial replating in methylcellulose cultures. Ts1Rhr bone marrow (B6/FVB background) formed 2–3-fold more B-cell colonies in early passages compared to bone marrow from wild-type littermates. While wild-type B-cells could not serially replate beyond 4 passages, Ts1Rhr B-cells displayed indefinite serial replating (>10 passages).
Ts1Rhr mice do not spontaneously develop leukemia, so we utilized two mouse models to determine whether DSCR triplication cooperates with leukemogenic oncogenes in vivo. First, we generated Eμ-CRLF2 F232C mice, which express the constitutively active CRLF2 mutant solely within B-cells. Like Ts1Rhr B-cells, (but not CRLF2 F232C B-cells) Ts1Rhr/CRLF2 F232C cells had indefinite serial replating potential. In contrast with Ts1Rhr B-cells, Ts1Rhr/CRLF2 F232C B-cells also engrafted into NOD.Scid.IL2Rγ−/− mice and caused fatal and serially transplantable B-ALL. Second, we retrovirally transduced BCR-ABL1 into unselected bone marrow from wild-type and Ts1Rhr mice and transplanted into irradiated wild-type recipients. Transplantation of transduced Ts1Rhr cells (106, 105, or 104) caused fatal B-ALL in recipient mice with shorter latency and increased penetrance compared to recipients of the same number of transduced wild-type cells. By Poisson calculation, the number of B-ALL initiating cells in transduced Ts1Rhr bone marrow was ∼4-fold higher than in wild-type animals (1:60 vs 1:244, P=0.0107). Strikingly, transplantation of individual Hardy A, B, and C fractions after sorting and BCR-ABL1 transduction demonstrated that the increased leukemia-initiating capacity almost completely resides in the Ts1Rhr Hardy B fraction; i.e., the same subset suppressed during Ts1Rhr B-cell differentiation.
To define transcriptional determinants of these phenotypes, we performed RNAseq of Ts1Rhr and wild-type B cells in methylcellulose culture (n=3 biologic replicates per genotype). As expected, Ts1Rhr colonies had ∼1.5-fold higher RNA abundance of expressed DSCR genes. We defined a Ts1Rhr signature of the top 200 genes (false discovery rate (FDR) <0.25) differentially expressed compared with wild-type cells. Importantly, this Ts1Rhr signature was significantly enriched (P=0.02) in a published gene expression dataset of DS-ALL compared with non-DS-ALL (Hertzberg et al., Blood 2009). Query of >2,300 signatures in the Molecular Signatures Database (MSigDB) C2 Chemical and Genetic Perturbations with the Ts1Rhr signature identified enrichment in multiple gene sets of polycomb repressor complex (PRC2) targets and H3K27 trimethylation. Most notably, SUZ12 targets within human embryonic stem cells were more highly expressed in Ts1Rhr cells (P=1.2×10−6, FDR=0.003) and the same SUZ12 signature was enriched in patients with DS-ALL compared to non-DS-ALL (P=0.007).
In summary, DSCR triplication directly suppresses precursor B-cell differentiation and promotes B-cell transformation both in vitro and by cooperating with proliferative alterations such as CRLF2 activation and BCR-ABL1 in vivo. Pharmacologic modulation of H3K27me3 effectors may overcome the pro-leukemogenic effects of polysomy 21.
Disclosures:
No relevant conflicts of interest to declare.
MdmX Promotes Bipolar Mitosis To Suppress Transformation and Tumorigenesis in p53-Deficient Cells and Mice