Deregulated KLF4 Expression in Myeloid Leukemias Alters Cell Proliferation and Differentiation through MicroRNA and Gene Targets.

Acute myeloid leukemia (AML) is characterized by increased proliferation and blocked differentiation of hematopoietic progenitors mediated, in part, by altered myeloid transcription factor expression. Decreased Krüppel-like factor 4 (KLF4) expression has been observed in AML, but how decreased KLF4 contributes to AML pathogenesis is largely unknown. We demonstrate decreased KLF4 expression in AML patient samples with various cytogenetic aberrations, confirm that KLF4 overexpression promotes myeloid differentiation and inhibits cell proliferation in AML cell lines, and identify new targets of KLF4. We have demonstrated that microRNA 150 (miR-150) expression is decreased in AML and that reintroducing miR-150 expression induces myeloid differentiation and inhibits proliferation of AML cells. We show that KLF family DNA binding sites are necessary for miR-150 promoter activity and that KLF2 or KLF4 overexpression induces miR-150 expression. miR-150 silencing, alone or in combination with silencing of CDKN1A, a well-described KLF4 target, did not fully reverse KLF4-mediated effects. Gene expression profiling and validation identified putative KLF4-regulated genes, including decreased MYC and downstream MYC-regulated gene expression in KLF4-overexpressing cells. Our findings indicate that decreased KLF4 expression mediates antileukemic effects through regulation of gene and microRNA networks, containing miR-150, CDKN1A, and MYC, and provide mechanistic support for therapeutic strategies increasing KLF4 expression.

MicroRNA-150 expression induces myeloid differentiation of human acute leukemia cells and normal hematopoietic progenitors.

In acute myeloid leukemia (AML) and blast crisis (BC) chronic myeloid leukemia (CML) normal differentiation is impaired. Differentiation of immature stem/progenitor cells is critical for normal blood cell function. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that interfere with gene expression by degrading messenger RNAs (mRNAs) or blocking protein translation. Aberrant miRNA expression is a feature of leukemia and miRNAs also play a significant role in normal hematopoiesis and differentiation. We have identified miRNAs differentially expressed in AML and BC CML and identified a new role for miR-150 in myeloid differentiation. Expression of miR-150 is low or absent in BC CML and AML patient samples and cell lines. We have found that expression of miR-150 in AML cell lines, CD34+ progenitor cells from healthy individuals, and primary BC CML and AML patient samples at levels similar to miR-150 expression in normal bone marrow promotes myeloid differentiation of these cells. MYB is a direct target of miR-150, and we have identified that the observed phenotype is partially mediated by MYB. In AML cell lines, differentiation of miR-150 expressing cells occurs independently of retinoic acid receptor α (RARA) signaling. High-throughput gene expression profiling (GEP) studies of the AML cell lines HL60, PL21, and THP-1 suggest that activation of CEPBA, CEBPE, and cytokines associated with myeloid differentiation in miR-150 expressing cells as compared to control cells contributes to myeloid differentiation. These data suggest that miR-150 promotes myeloid differentiation, a previously uncharacterized role for this miRNA, and that absent or low miR-150 expression contributes to blocked myeloid differentiation in acute leukemia cells.

Ets-1 is required for the activation of VEGFR3 during latent Kaposi's sarcoma-associated herpesvirus infection of endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma (KS), is present in the predominant tumor cells of KS, the spindle cells. Spindle cells express markers of lymphatic endothelium and, interestingly, KSHV infection of blood endothelial cells reprograms them to a lymphatic endothelial cell phenotype. KSHV-induced reprogramming requires the activation of STAT3 and phosphatidylinositol 3 (PI3)/AKT through the activation of cellular receptor gp130. Importantly, KSHV-induced reprogramming is specific to endothelial cells, indicating that there are additional host genes that are differentially regulated during KSHV infection of endothelial cells that contribute to lymphatic reprogramming. We found that the transcription factor Ets-1 is highly expressed in KS spindle cells and is upregulated during KSHV infection of endothelial cells in culture. The KSHV latent vFLIP gene is sufficient to induce Ets-1 expression in an NF-κB-dependent fashion. Ets-1 is required for KSHV-induced expression of VEGFR3, a lymphatic endothelial-cell-specific receptor important for lymphangiogenesis, and Ets-1 activates the promoter of VEGFR3. Ets-1 knockdown does not alter the expression of another lymphatic-specific gene, the podoplanin gene, but does inhibit the expression of VEGFR3 in uninfected lymphatic endothelium, indicating that Ets-1 is a novel cellular regulator of VEGFR3 expression. Knockdown of Ets-1 affects the ability of KSHV-infected cells to display angiogenic phenotypes, indicating that Ets-1 plays a role in KSHV activation of endothelial cells during latent KSHV infection. Thus, Ets-1 is a novel regulator of VEGFR3 and is involved in the induction of angiogenic phenotypes by KSHV.

The KSHV viral IL-6 homolog is sufficient to induce blood to lymphatic endothelial cell differentiation.

The predominant tumor cell of Kaposi's Sarcoma (KS) is the spindle cell, a cell of endothelial origin that expresses markers of lymphatic endothelium. In culture, Kaposi's Sarcoma-associated herpesvirus (KSHV) infection of blood endothelial cells drives expression of lymphatic endothelial cell specific markers, in a process that requires activation of the gp130 receptor and the JAK2/STAT3 and PI3K/AKT signaling pathways. While expression of each of the KSHV major latent genes in endothelial cells failed to increase expression of lymphatic markers, the viral homolog of human IL-6 (vIL-6) was sufficient for induction and requires the JAK2/STAT3 and PI3K/AKT pathways. Therefore, activation of gp130 and downstream signaling by vIL-6 is sufficient to drive blood to lymphatic endothelial cell differentiation. While sufficient, vIL-6 is not necessary for lymphatic reprogramming in the context of viral infection. This indicates that multiple viral genes are involved and suggests a central importance of this pathway to KSHV pathogenesis.

Activation of Akt through gp130 receptor signaling is required for Kaposi's sarcoma-associated herpesvirus-induced lymphatic reprogramming of endothelial cells.

Kaposi's sarcoma (KS) is the most common tumor of AIDS patients worldwide. KS-associated herpesvirus (KSHV) is the infectious cause of this highly vascularized skin tumor. The main cell type found within a KS lesion, the spindle cell, is latently infected with KSHV and has markers of both blood and lymphatic endothelial cells. During development, lymphatic endothelial cells differentiate from preexisting blood endothelial cells. Interestingly, KSHV infection of blood endothelial cells induces lymphatic endothelial cell differentiation. Here, we show that KSHV gene expression is necessary to maintain the expression of the lymphatic markers vascular endothelial growth factor receptor 3 (VEGFR-3) and podoplanin. KSHV infection activates many cell signaling pathways in endothelial cells and persistently activates STAT3 through the gp130 receptor, the common receptor of the interleukin 6 family of cytokines. We find that KSHV infection also activates the phosphatidylinositol 3-OH-kinase (PI3K)/Akt cell signaling pathway in latently infected endothelial cells and that gp130 receptor signaling is necessary for Akt activation. Using both pharmacological inhibitors and small interfering RNA knockdown, we show that the gp130 receptor-mediated activation of both the JAK2/STAT3 and PI3K/Akt cell signaling pathways is necessary for KSHV-induced lymphatic reprogramming of endothelial cells. The induction of the lymphatic endothelial cell-specific transcription factor Prox1 is also involved in KSHV-induced lymphatic reprogramming. The activation of gp130 receptor signaling is a novel mechanism for the differentiation of blood endothelial cells into lymphatic endothelial cells and may be relevant to the developmental or pathological differentiation of lymphatic endothelial cells as well as to KSHV pathogenesis.