MicroRNAs (miRNAs) are small noncoding RNAs, 19-24 nucleotides in length, that regulate gene expression and are expressed aberrantly in most types of cancer. MiRNAs also have been detected in the blood of cancer patients and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor-secreted miR-21 and miR-29a also can function by another mechanism, by binding as ligands to receptors of the Toll-like receptor (TLR) family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately may lead to tumor growth and metastasis. Thus, by acting as paracrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in tumor-immune system communication and is important in tumor growth and spread, thus representing a possible target for cancer treatment.icroRNAs (miRNAs) are small, noncoding RNAs, 19-24 nt in length, with gene-expression regulatory functions (1, 2) and are expressed aberrantly in most types of cancer (3, 4). MiRNAs also have been detected in the blood of cancer patients (5, 6) and can serve as circulating biomarkers (7). It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells (8) by canonical binding to their target messenger RNAs (8, 9). More recently, it has been demonstrated that, in addition to their role as gene-expression regulators, miRNAs also directly interact with proteins (10).Members of the Toll-like receptor (TLR) family (namely, murine TLR7 and human TLR8) can recognize and bind viral single-stranded RNA (ssRNA) sequences on dendritic cells and B lymphocytes, leading to cell activation and cytokine production (11,12). TLRs are a family of receptors through which the mammalian innate immune system recognizes the presence of invading pathogens (13,14). Both murine TLR7 and human TLR8 bind to and are activated by 20-nt-long ssRNAs, which represent physiological ligands for these two receptors (12), located in intracellular endosomes. Circulating mature miRNAs are 19-24 nt in length and could represent tumor-released ligands of TLR7 and TLR8 involved in intercellular communication in the tumor microenvironment. Results and Discussion Identification of Specific miRNAs Released in Cancer Cell-DerivedExosomes. To identify which miRNAs are present in tumor-secreted exosomes, we isolated exosomes from the supernatant of A-549 and SK-MES lung cancer cell lines. First, we assessed the purified supernatant exosome fraction for enrichment in CD9 and CD63, two known exosome markers (SI Appendix, Fig. S1A) (8,15). By performing NanoString analysis, we observed that nine miRNAs (miR-16, -21, -27b, -29a, -133a, -193a-3p, -544, -563, and -1283) were present in exosomes derived from ...

Aberrant DNA hypermethylation contributes to myeloid leukemogenesis by silencing structurally normal genes involved in hematopoiesis. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by targeting protein-coding mRNAs. Recently, miRNAs have been shown to play a role as both targets and effectors in gene hypermethylation and silencing in malignant cells. In the current study, we showed that enforced expression of miR-29b in acute myeloid leukemia cells resulted in marked reduction of the expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B at both RNA and protein levels. This in turn led to decrease in global DNA methylation and reexpression of p15 INK4b and ESR1 via promoter DNA hypomethylation. Although down-regulation of DNMT3A and DNMT3B was the result of a direct interaction of miR-29b with the 3 untranslated regions of these genes, no predicted miR-29b interaction sites were found in the DNMT1 IntroductionDNA methylation consists of an enzymatic addition of a methyl group at the carbon 5 position of cytosine in the context of the sequence 5Ј-cytosine-guanosine (CpG) and is mediated by DNA methyltransferases (DNMTs). 1 The promoter regions of approximately 50% of human genes contain regions of DNA with a cytosine and guanine content greater than expected (so-called CpG islands) that, once hypermethylated, mediate gene transcriptional silencing. 2 Distinct roles in genomic methylation have been reported for DNMT isoforms. Whereas DNMT1 preferentially replicates already existing methylation patterns, DNMT3A and 3B are responsible for establishing de novo methylation. 2 Silencing of structurally normal tumor suppressor genes by aberrant DNA hypermethylation has been reported in hematologic malignancies, including subsets of acute myeloid leukemia (AML). 3,4 Although the mechanisms leading to aberrant DNA hypermethylation remain to be fully elucidated, increased levels of DNMT1 and DNMT3A and 3B have been observed in malignant myeloid blasts compared with normal bone marrow (BM) mononuclear cells (MNCs), suggesting that DNMT overexpression contributes to gene promoter hypermethylation and in turn to leukemogenesis. 4 Growing evidence supports a role for microRNAs (miRNAs) as both targets and effectors in aberrant mechanisms of DNA hypermethylation. 5,6 miRNAs are noncoding RNAs of 19 to 25 nucleotides in length that regulate gene expression by inducing translational inhibition or cleavage of their target mRNAs through base pairing at partially or fully complementary sites. 7 Several groups have shown that miRNAs are altered in human malignancies and can function as tumor suppressor genes or oncogenes through expression regulation of their target genes. 7 Similar to tumor suppressor genes, miRNAs with tumor suppressor activity are often located in deleted genomic areas or are silenced by mutations or promoter hypermethylation in malignant cells. 5,[8][9][10] Saito et al recently demonstrated that miR-127 is silenced by promoter DNA hypermethylation and down-regulated in human bladder ...

The oncogenic BCR/ABL kinase activity induces and maintains chronic myelogenous leukemia (CML). We show here that, in BCR/ABL-transformed cells and CML blast crisis (CML-BC) progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the BCR/ABL-induced expression of the PP2A inhibitor SET. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and CML-BC progenitors, molecular and/or pharmacological activation of PP2A promotes dephosphorylation of key regulators of cell proliferation and survival, suppresses BCR/ABL activity, and induces BCR/ABL degradation. Furthermore, PP2A activation results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cells. Thus, functional inactivation of PP2A is essential for BCR/ABL leukemogenesis and, perhaps, required for blastic transformation.

The BCR/ABL oncogenic tyrosine kinase activates phosphatidylinositol 3-kinase (PI-3k) by a mechanism that requires binding of BCR/ABL to p85, the regulatory subunit of PI-3k, and an intact BCR/ABL SH2 domain. SH2 domain BCR/ABL mutants deficient in PI-3k activation failed to stimulate Akt kinase, a recently identified PI-3k downstream effector with oncogenic potential, but did activate p21 RAS and p70 S6 kinase. The PI-3k/Akt pathway is essential for BCR/ABL leukemogenesis as indicated by experiments demonstrating that wortmannin, a PI-3k specific inhibitor at low concentrations, suppressed BCR/ABL-dependent colony formation of murine marrow cells, and that a kinase-deficient Akt mutant with dominant-negative activity inhibited BCR/ABL-dependent transformation of murine bone marrow cells in vitro and suppressed leukemia development in SCID mice. In complementation assays using mouse marrow progenitor cells, the ability of transformation-defective SH2 domain BCR/ABL mutants to induce growth factor-independent colony formation and leukemia in SCID mice was markedly enhanced by expression of constitutively active Akt. In retrovirally infected mouse marrow cells, the BCR/ABL mutant lacking the SH2 domain was unable to upregulate the expression of c-Myc and Bcl-2; in contrast, expression of a constitutively active Akt mutant induced Bcl-2 and c-Myc expression, and stimulated the transcription activation function of c-Myc. Together, these data demonstrate the requirement for the BCR/ABL SH2 domain in PI-3k activation and document the essential role of the PI-3k/Akt pathway in BCR/ABL leukemogenesis.

We report for the first time that mutKIT, and particularly mutKIT17, confer higher relapse risk, and both mutKIT17 and mutKIT8 appear to adversely affect OS in AML with inv(16). We also confirm the adverse impact of mutKIT on relapse risk in t(8;21) AML. We suggest that patients with core-binding factor AML should be screened for mutKIT at diagnosis for both prognostic and therapeutic purposes, given that activated KIT potentially can be targeted with novel tyrosine kinase inhibitors.

Chronic myelogenous leukemia (CML) evolves from a chronic phase characterized by the Philadelphia chromosome as the sole genetic abnormality into blast crisis, which is often associated with additional chromosomal and molecular secondary changes. Although the pathogenic effects of most CML blast crisis secondary changes are still poorly understood, ample evidence suggests that the phenotype of CML blast crisis cells (enhanced proliferation and survival, differentiation arrest) depends on cooperation of BCR/ABL with genes dysregulated during disease progression. Most genetic abnormalities of CML blast crisis have a direct or indirect effect on p53 or Rb (or both) gene activity, which are primarily required for cell proliferation and survival, but not differentiation. Thus, the differentiation arrest of CML blast crisis cells is a secondary consequence of these abnormalities or is caused by dysregulation of differentiation-regulatory genes (ie, C IntroductionBlast crisis is the terminal phase of chronic myelogenous leukemia (CML), a clonal myeloproliferative disorder of the hematopoietic stem cell that typically evolves in 3 distinct clinical stages: chronic and accelerated phases and blast crisis. 1,2 The chronic phase lasts several years and is characterized by accumulation of myeloid precursors and mature cells in bone marrow, peripheral blood, and extramedullary sites. 1,2 The accelerated phase lasts 4 to 6 months and is characterized by an increase in disease burden and in the frequency of progenitor/precursor cells rather than terminally differentiated cells. The blast crisis lasts only a few months and is characterized by the rapid expansion of a population of myeloid or lymphoid differentiation-arrested blast cells. 3 CML is consistently associated with an acquired genetic abnormality, the Philadelphia chromosome (Ph 1 ), a shortened chromosome 22 resulting from a reciprocal translocation of the long arms of chromosomes 9 and 22. 4,5 This translocation generates the BCR/ABL fusion gene, which is translated in the p210 BCR/ABL oncoprotein 6,7 of almost all patients with CML.Two other BCR/ABL proteins, p190 and p230, generated by variant fusion genes, are only occasionally detected in classic CML. 8 Expression of p210 BCR/ABL is necessary and sufficient for malignant transformation, as demonstrated in in vitro assays and leukemogenesis in mice. [9][10][11][12][13] Transition to blast crisis is the unavoidable outcome of CML except in a cohort of patients receiving allogeneic bone marrow transplants early in the chronic phase. 14 The development of the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (Gleevec; formerly STI571; Novartis, Basel, Switzerland) as the treatment of choice for chronic-phase CML and its remarkable therapeutic effects suggest that blast crisis transition will be postponed for several years in most patients with CML. 15,16 However, the persistence of BCR/ABL transcripts in a cohort of patients with complete cytogenetic response raises the possibility that treatment with imatini...

Protein phosphatase 2A (PP2A), one of the major serine-threonine phosphatases in mammalian cells, maintains cell homeostasis by counteracting most of the kinase-driven intracellular signaling pathways. Unrestrained activation of oncogenic kinases together with inhibition of tumor suppressors is frequently required for the development of cancer. Because it has been found genetically altered or functionally inactivated in many solid cancers and leukemias, PP2A is indeed a bona fide tumor suppressor. For example, the phosphatase activity of PP2A is suppressed in chronic myelogenous leukemia and other malignancies characterized by the aberrant activity of oncogenic kinases. Notably, preclinical studies indicate that pharmacologic restoration of PP2A tumor suppressor activity by PP2A activating drugs (PADs, e.g. FTY720) effectively antagonizes cancer development and progression. Herein, we systematically discuss the importance of PP2A as a druggable tumor suppressor in light of the possible introduction of PADs into anti-cancer therapeutic protocols.

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