ALBERT

Description: Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo–expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with β-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.

Description: t(8;21) is one of the most common chromosomal abnormalities associated with acute myeloid leukemia (AML). The resulting AML1-ETO fusion protein is directly involved in the pathogenesis of AML but is not able to cause leukemia on its own. We have shown previously that a C-terminal splice variant of t(8;21), AML1-ETO9a (AE9a), is sufficient to promote AML in mice. Here we show that the leukemia-initiating cells reside in the Lin-/Sca1-/cKit+ population. To identify molecular targets directly modulated by AE9a, we compared the gene expression profile to the promoter occupancy (ChIP-on-chip) profile of this population. We identified 1485 genes deregulated by AE9a, among which 544 genes are also ChIP-on-chip targets. CD45, a negative regulator of cytokine/growth factor receptor and JAK/STAT signaling, was greatly down-regulated in AE9a leukemia cells. We further show that t(8;21) AML-M2 patient samples have lower CD45 levels compared to non-t(8;21) AML-M2 samples. Interestingly, JAK1 and JAK2 were upregulated by AE9a and both CD45 and JAK1 are ChIP-on-chip targets. In addition we show that JAK/STAT signaling was enhanced in the leukemia cells. Consequently, the leukemia cells are more susceptible to JAK2 inhibitors than wild type cells. Our results indicate that AE9a enhances JAK/STAT signaling by directly modulating regulators of this signaling pathway, which provides a potential novel approach to treating t(8;21) AML.

Description: The t(8;21)(q22;q22) is common in adult acute myeloid leukemia (AML). The RUNX1-ETO fusion protein that is expressed by this translocation is poorly leukemogenic and requires additional mutations for transformation. Loss of sex chromosome (LOS) is frequently observed in t(8;21) AML. In the present study, to evaluate whether LOS cooperates with t(8;21) in leukemogenesis, we first used a retroviral transduction/transplantation model to express RUNX1-ETO in hematopoietic cells from XO mice. The low frequency of leukemia in these mice suggests that the potentially critical gene for suppression of t(8;21) leukemia in humans is not conserved on mouse sex chromosomes. The gene encoding the GM-CSF receptor α subunit (CSF2RA) is located on X and Y chromosomes in humans but on chromosome 19 in mice. GM-CSF promotes myeloid cell survival, proliferation, and differentiation. To determine whether GM-CSF signaling affects RUNX1-ETO leukemogenesis, hematopoietic stem/progenitor cells that lack GM-CSF signaling were used to express RUNX1-ETO and transplanted into lethally irradiated mice, and a high penetrance of AML was observed in recipients. Furthermore, GM-CSF reduced the replating ability of RUNX1-ETO–expressing cells. These results suggest a possible tumor-suppressor role of GM-CSF in RUNX1-ETO leukemia. Loss of the CSF2RA gene may be a critical mutation explaining the high incidence of LOS associated with the t(8;21)(q22;q22) translocation.

Description: Nonrandom and somatically acquired chromosomal translocations can be identified in nearly 50% of human acute myeloid leukemias. One common chromosomal translocation in this disease is the 8q22;21q22 translocation. It involves the AML1 (RUNX1) gene on chromosome 21 and the ETO (MTG8, RUNX1T1) gene on chromosome 8 generating the AML1-ETO fusion proteins. In this review, we survey recent advances made involving secondary mutational events and alternative t(8;21) transcripts in relation to understanding AML1-ETO leukemogenesis.

Description: Key Points Human RUNX1a orthologs are only found in primates. Alternative splicing of Runx1 involving exon 6 affects the pool size of hematopoietic stem cells.

Description: The t(8;21) chromosomal translocation is among the most frequent recurring cytogenetic abnormalities associated with acute myeloid leukemia (AML), found in 8-12% of de novo AML patients. The t(8;21) results in the stable fusion of the RUNX1 and RUNX1T1 genes, and formation of the oncofusion protein RUNX1-ETO (AML1-ETO). RUNX1-ETO is composed of the N-terminal DNA-binding domain of RUNX1 and nearly the entire ETO protein. RUNX1-ETO promotes leukemia development via the recruitment of transcription factor/transcriptional repression complexes (including NCOR, HDACs, p300, etc.) to regulatory regions of RUNX1 target genes known to be critical for myeloid differentiation and function, such as CEBPA, SPI1 (PU.1), NFE2, and CSF1R. Despite this knowledge, additional RUNX1-ETO target genes remain poorly characterized, and the complete molecular mechanism through which RUNX1-ETO leads to leukemic transformation remains to be elucidated. We propose that a better understanding of additional RUNX1-ETO target genes will lead to the potential for development of novel therapeutics to treat these patients. One such gene that we initially identified as markedly downregulated in RUNX1-ETO leukemia cells using a mouse model of t(8;21) AML is RASSF2 (Lo et al, Blood, 2012). Assessment of publicly available gene expression data revealed that RASSF2 is specifically downregulated in the bone marrow of t(8;21) AML patients compared to patients of different cytogenetic subtypes or to non-t(8;21) FAB subtype M2 AML patients. Additionally, RT-qPCR analysis confirmed that RASSF2 transcript is downregulated 10-100-fold in the t(8;21) AML cell lines, Kasumi-1 and SKNO-1, compared to non-t(8;21) AML cell lines and normal CD34+ hematopoietic cells. Expression of RUNX1-ETO in a non-t(8;21) AML cell line led to a reduction in RASSF2 mRNA expression, while knockdown of RUNX1-ETO in Kasumi-1 cells resulted in a ~5-fold increase in RASSF2 expression. Assessment of published ChIP-seq data showed that RUNX1-ETO directly binds at two regulatory regions within the RASSF2 genomic locus in t(8;21) AML cell lines and patient samples. Re-expression of RASSF2 at physiological levels in t(8;21) AML cell lines resulted in a modest negative growth phenotype, and greatly sensitized these cells to apoptosis following stimulation with various pro-apoptotic agents. Re-expression of RASSF2 in RUNX1-ETO-transduced primary mouse bone marrow caused these cells to lose their long-term self-renewal ability after 3 weeks in a serial replating/colony formation assay. This loss of self-renewal ability in co-transduced cells was accompanied by a marked increase in apoptosis during each of the first three weeks of replating. Mechanistically, re-expression of full-length RASSF2, but not of a deletion mutant lacking the SARAH heterodimerization domain (RASSF2ΔSARAH), in t(8;21) AML cell lines resulted in increased protein amount of the pro-apoptotic kinase, MST1. This suggests that RASSF2 may be a critical regulator of MST1 protein stability in AML cells. Importantly, modest (2-3-fold) overexpression of MST1 in t(8;21) AML cell lines resulted in a significant increase in apoptosis and caused growth arrest. The effects of RASSF2 or MST1 expression in non-t(8;21) AML cell lines were greatly reduced, suggesting that the cellular context of RUNX-ETO-driven leukemias makes them highly susceptible to MST1-dependent apoptosis. Overall, we have identified the importance of a MST1-driven pro-apoptotic signaling axis in t(8;21) leukemia. RUNX1-ETO-dependent transcriptional repression of RASSF2 may be essential for evasion of this apoptosis signaling during leukemic transformation via reduction of MST1 protein stability. MST1, perhaps better known as the mammalian orthologue of the drosophila Hippo kinase, is a critical tumor suppressor in many solid tumor types; and we believe our studies warrant the continued investigation of this pathway in hematological malignancy. Disclosures No relevant conflicts of interest to declare.

Description: RUNX1-ETO (also known as AML1-ETO and AML1-MTG8) is a fusion gene generated from t(8;21), which is a common chromosome translocation in acute myeloid leukemia (AML). It has been shown that t(8;21) requires additional aberrations to induce leukemia. Interestingly, 32-59% of t(8;21) patients also display loss of a sex chromosome (LOS) in their leukemia cells. Therefore, loss of the genes located on the sex chromosomes, especially in the pseudoautosomal regions (PARs) that are shared between the X and Y chromosomes, may contribute to RUNX1-ETO leukemia development. One gene of interest in the PARs is CSF2RA, which encodes the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. When the GM-CSF receptor is bound to its ligand, downstream signaling events promote various functional outcomes including proliferation, differentiation, self-renewal, and survival of myeloid cells. Thus, GM-CSF signaling has the potential to regulate both normal and malignant hematopoiesis. We previously reported that mice expressing RUNX1-ETO in GM-CSF deficient hematopoietic cells displayed higher incidence of leukemia (Matsuura S et al. 2012 Blood 119:3155). This result suggests that GM-CSF signaling is inhibitory to RUNX1-ETO dependent leukemogenesis. Furthermore, GM-CSF treatment reduces the self-renewal potential of RUNX1-ETO expressing cells and promotes myeloid differentiation in replating assays. We therefore hypothesize that the negative effect of GM-CSF on RUNX1-ETO induced leukemia development is due to the activation of selected GM-CSF downstream signaling pathway(s) that diminish self-renewal capacity and promote myeloid differentiation. To understand the molecular mechanism of the negative effect of GM-CSF on t(8;21) leukemogenesis, in the current report, we conducted a gene expression profiling assay to examine the effect of GM-CSF on RUNX1-ETO cells. MigR1 vector control or MigR1-RUNX1-ETO retrovirus transduced lineage negative/c-Kit positive (Lin-/c-Kit+) murine hematopoietic stem/progenitor cells (HSPCs) were cultured with or without GM-CSF for 24 hours. Then, Lin-/c-Kit+/GFP+ HSPCs were isolated for the profiling study. We observed little response to GM-CSF in control HSPCs, with only 4 genes being differentially expressed after a 2-fold cutoff. Conversely, 122 genes were differentially expressed in RUNX1-ETO cells treated with GM-CSF. These results clearly indicate that RUNX1-ETO specifically enhances GM-CSF responsiveness in HSPCs. Gene Set Enrichment Analysis (GSEA) of the differentially expressed genes in RUNX1-ETO cells reveals that this response resembles that of GM-CSF-induced myeloid differentiation. Furthermore, pathway analysis of these differentially expressed genes predicts MEK1/2 and ERK1/2 to be activated after GM-CSF treatment in RE cells. We previously reported that ERK1/2, downstream targets of MEK1/2, are hyper-phosphorylated after GM-CSF treatment of RUNX1-ETO cells, and MEK-ERK activation has been shown to regulate cell proliferation and myelopoiesis. Other GM-CSF induced genes are predicted targets of MYD88. MYD88 is upregulated during myeloid differentiation. Its in vivo knockout has been reported to result in an increase of hematopoietic stem cells (HSCs) and reduction of mature granulocytes. Most interestingly, a subset of genes upregulated in GM-CSF treated RUNX1-ETO cells are predicted to be activated by CEBPβ. CEBPβ can heterodimerize with CEBPα and is induced during myelopoiesis, critical for macrophage differentiation, capable of promoting granulopoiesis, and involved in regulating granulopoiesis in vivo. In conclusion, our data suggest that RUNX1-ETO expression results in hyper-responsiveness to GM-CSF. Such enhanced GM-CSF signaling activates the expression of a specific group of genes and results in the reduced self-renewal capacity and increased myeloid differentiation of HSPCs. These GM-CSF effects are likely involved in reducing the leukemogenic potential of RUNX1-ETO and may be considered for specific therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.