Type: Oral
Session: 602. Myeloid Oncogenesis: Basic: Therapeutic Targeting of Myeloid Malignancies
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Biological Processes, molecular biology
To determine whether TIF1β promoted the development of BCR-ABL-induced myeloid leukemia in vivo, we generated BCR-ABL;Tif1βf/f;Cre-ERT2 compound mice by crossing Rosa26 locus BCR-ABL conditional knock-in mice and Tif1βf/f;Cre-ERT2 conditional KO mice. We transplanted BM cells isolated from Cre-ERT2 (WT), Tif1βf/f;Cre-ERT2 (Tif1β KO), BCR-ABL;Cre-ERT2 (BCR-ABL), and BCR-ABL;Tif1βf/f;Cre-ERT2 (BCR-ABL;Tif1β KO) mice into lethally irradiated CD45.1+ WT recipient mice, and injected tamoxifen 4 weeks after BM transplantation. While BCR-ABL KI mice showed rapid expansion of leukemic blast cells and died in a short period of time, the deletion of Tif1β gene resulted in a significantly longer survival with reduced tumor burden in BM, liver and spleen in BCR-ABL KI mice. BCR-ABL KI mice mostly developed myeloid leukemia, in contrast, BCR-ABL;Tif1β KO mice dominantly showed B-cell leukemia or bone marrow failure diseases. A subset of BCR-ABL;Tif1β KO mice survived without development of lethal leukemia in the primary and the secondary transplantation settings, indicating that TIF1β maintained the self-renewal capacity of leukemic stem cells (LSCs) but also controlled the myeloid/lymphoid lineage commitment.
To elucidate the mechanisms underlying the impaired development of leukemia in BCR-ABL;Tif1β KO mice, we performed RNA sequencing analyses of HSCs isolated from WT, Tif1β KO, BCR-ABL, BCR-ABL;Tif1β KO mice. Gene set enrichment analysis revealed that the loss of Tif1β repressed BCR-ABL-induced activation of proliferative stem cell signatures, but increased expression of myeloid differentiation genes including PU.1 gene in HSCs. We found that BCR-ABL;Tif1β KO HSCs reduced expression levels of Myc and Myc-target genes including Fra1 transcription factor (TF), which was known to promote tumor cell proliferation. Notably, human CML-CP and CML-BC CD34+ cells markedly increased expression levels of MYC and FRA1 genes.
To clarify how the Tif1β protein controlled self-renewal of LSCs, we performed ATAC-sequencing in murine HSCs and TIF1β-ChIP-sequencing using BCR-ABL-expressing human leukemia cells. ATAC-seq revealed that BCR-ABL KI HSCs showed enrichments of binding motif of Fra1 in open chromatin and that of PU.1 in closed chromatin, compared to WT HSCs, while BCR-ABL;Tif1β KO HSCs cancelled those enrichments of binding motifs of TFs. TIF1β-ChIP-seq revealed that the TIF1β protein directly bound to a promoter region of the FRA1 gene. TIF1β binding sites were enriched with canonical PU.1- and JUN/FRA1-binding motifs. We also found that treatment with imatinib decreased enrichment of FRA1-binding motif among TIFβ-binding regions in BCR-ABL+ cells, indicating that TIFβ opened chromatin accessibility to activate expression of and transcriptional function of the Fra1 gene in a BCR-ABL dependent manner, but repressed the accessibility of myeloid differentiation regulator TFs. Furthermore, knockdown of Fra1 significantly impaired the proliferation of BCR-ABL stem cells in an in vitro condition.
In conclusion, we demonstrate that the Tif1β gene was required for the propagation of BCR-ABL-induced myeloid leukemia stem cell in mice. Tif1β enhanced chromatin accessibility of Fra1 TF to promote transcription of LSC signature genes, but also reduced chromatin accessibility at the PU.1-binding motif, resulting in the enhanced proliferation and limited differentiation of LSCs.
Disclosures: No relevant conflicts of interest to declare.
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