English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Plus monthly

Global: Zendy Tools annual

Global: Zendy Tools monthly

Global: Zendy Free Plan

Acute myeloid leukemia (AML) is characterized by high mortality, underlining the necessity for identifying tumor suppressors that counteract the leukemogenic potential of bona fide oncogenes such as the homeobox genes HOXA9 and CDX2. Homeobox genes are aberrantly expressed in the majority of patients with cytogenetically normal (CN)-AML and expression of CDX2 positively correlates with HOXA9 expression. Aberrant expression of Cdx2 in murine hematopoietic cells rapidly induced aggressive AML in mice. Recently, it was shown that a microRNA, miR-196, is encoded in the mammalian paralogous HOX gene cluster and that it has extensive evolutionarily conserved complementarity to sites in the 3' prime untranslated regions (3' UTR) of HOX genes (e.g. HOXA9, A7 and B8), directly regulating their expression in MLL-rearranged leukemic cells. To understand the role of miR-196b in AML more precisely, we first tried to identify transcripts generating mature miR-196b in human hematopoietic stem and progenitor cells (HSPC). We identified two novel non-coding transcripts encoding the miR-196b hairpin precursor sequence expressed from the HOXA9-10 locus in bone marrow HSPC (Online Supplementary Figure S1A, B) [NCBI accession number: MF139050, 486 basepairs (bp) and MF139051, 396 bp]. MF139051, a splice variant of the MF139050 transcript, showed 99.47% homology to MF139050. Both transcripts displayed high homology to the transcripts of other vertebrates and mammalian species. Retrovirally engineered expression of both transcripts resulted in significant overexpression of the mature miR-196b in HEK293T cells compared to the vector control (Figure 1A; Online Supplementary Figure S1C, D). Endogenous expression levels of both transcripts in the human CD34 bone marrow compartment were higher than those in mononuclear cells (Figure 1B). Second, we identified a highly conserved 802 bp long miR-196b promoter region, validated by luciferase reporter assay, located 201 bp upstream of the miR-196b stem loop precursor sequence on human chromosome 7 (Figure 1C; Online Supplementary Figure S1E, F). Transcription factor binding site prediction tools and published chromatin immunoprecipitation sequencing data showed, respectively, potential binding of transcription factors including SP1 and enrichment for proteins such as EZH2 on this promoter (Online Supplementary Figure S1G, H; Online Supplementary Table SW1). Previously, the miR-196b promoter region was described in the context of murine development, demonstrating in vivo enrichment for Cdx2 and Hoxd13. Next, we asked whether expression of miR-196b itself as well as the ratio between miR-196b expression and the expression of its direct target HOXA9 is perturbed in CN-AML patients: miRNA-sequencing analysis of healthy bone marrow-derived hematopoietic subpopulations revealed highest expression of miR-196b in the lymphoid-primed multi-potent progenitor subpopulation, following the same expression pattern as known for its target HOXA9, whose expression is also highest in immature cells (Figure 1D; Online Supplementary Table SE1). This resulted in a significant correlation between the expression levels of mature miR-196b or MF139051 and HOXA9 in HSPC (Online Supplementary Figure S2A; Online Supplementary Table SW2). In contrast, expression levels of miR-196b and of HOXA9 remained at similar levels across all three functionally validated leukemic bone marrow subpopulations from CN-AML patients (Figure 1D; Online Supplementary Table SE1). In contrast to the expression levels of the miR-196b transcript MF139051 and miR-196b, HOXA9 expression was dramatically increased in leukemic CD34 bone marrow compared to CD34 normal bone marrow cells. This difference could be confirmed in functionally validated leukemic stem cells compared to normal HSPC and in published data (Online Supplementary Figures S2B and S3; Online Supplementary Tables SW3-4). Based on this, the correlation between the expression levels of the mature miR-196b and its transcripts versus the expression of HOXA9 was lost in leukemic cells (Supplementary Figures S4). The difference in expression patterns resulted in a 111-fold higher ratio between the expression values of HOXA9 and mature miR-196b in CN-AML compared to normal CD34 bone marrow and a 57-fold higher ratio between NPM1c AML and normal CD34 bone marrow, which also held true for the short transcript MF139051 calculated for the same AML groups (Figure 1E; Online Supplementary Table SW4). This implies that compared to normal CD34 hematopoietic cells there are many fewer miR-196b transcripts and mature miRNA per HOXA9 transcript in CN-AML, including NPM1-mutated cases, previously shown to have particularly high HOX gene expression. Overexpression of miR-196b in normal murine HSPC did not affect clonogenicity and cell growth in vitro (Figure 2A; Online Supplementary Figure S5A, B). miR196b increased the proportion of circulating myeloid cells and three of 14 mice developed AML after a long latency, indicating that cooperating events may contribute to disease induction. (Figure 2B, F; Online Supplementary Table SW5). To test the impact of miR-196b expression in the context of Cdx2-induced AML, we retrovirally coexpressed Cdx2/miR-196b or Cdx2 alone in murine HSPC (Online Supplementary Figure S5C). Co-expression of miR-196b considerably reduced the Cdx2-induced proliferation, clonogenicity and spleen colony formation compared to those of Cdx2-transduced cells (Figure 2C, D; Online Supplementary Figure S5D-F). Cdx2-transplanted mice developed AML with a median latency of 193 days. In contrast, only 40% of the Cdx2/miR-196b-transplanted mice developed AML with a significantly longer latency (Figure 2E; Online Supplementary Table SW5). Moreover, miR-196b impaired growth and colony formation in the CDX2 and HOX gene-positive human AML cell lines OCI-AML3 and NB4 in vitro, in contrast to the CDX2 and HOX gene-negative human AML cell line Kasumi-1. Furthermore, miR-196b reduced engraftment of OCI-AML3 in NSG mice (Online Supplementary Figure S6A-K; Online Supplementary Table SW6). Overexpression of miR-196b did not induce apoptosis, cell cycle arrest or senescence in OCI-AML3 and NB4 cells (data not shown), suggesting that miR-196b preferentially targets selfrenewal by reducing HOX gene expression, thereby reducing clonogenicity and engraftment potential. Overexpression of miR-196b in HSPC alone resulted in 155 differentially expressed genes compared to the control. Among these 155 genes, 44% of the downregulated ones were significantly enriched for known miR-196b targets, whereas the upregulated ones did not show any miR-196b target enrichment (Online Supplementary Tables SE2-5; Online Supplementary Figure S7A-C). In Cdx2-transduced cells, overexpression of miR-196b induced 524 differentially expressed genes compared to Cdx2 alone (Online Supplementary Figure S7D; Online Supplementary Tables SE6 and SE7). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

The microRNA miR-196b acts as a tumor suppressor in Cdx2-driven acute myeloid leukemia