Wenhuo Hu

Notch pathway activation targets AML-initiating cell homeostasis and differentiation

Notch behaves as a tumor suppressor in AML, and Notch activation induces cell cycle arrest, differentiation, and apoptosis of AML-initiating cells.

High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias

c-Kitlo HSCs exhibit enhanced self-renewal and long-term reconstitution potential and give rise to c-Kithi HSCs that have a megakaryocytic bias.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Hematopoietic Stem Cell Origin of BRAFV600E Mutations in Hairy Cell Leukemia

The cell of origin for the chronic lymphoproliferative disorder hairy cell leukemia is a long-term hematopoietic stem cell, as shown through human genetic data and murine genetic models. Finding the Origin Story for a Leukemia The cells that give rise to a cancer called hairy cell leukemia are hematopoietic stem cells, the precursors for all the types of normal blood cells, according to a new study by Chung et al. Although hairy cell leukemia is usually thought to be derived from mature B cells, it has not previously been matched with any specific stage of normal B cell development. Now, the authors performed detailed genetic analysis of human leukemia samples and also modeled this cancer in mice with different types of mutations, thus revealing the origin for hairy cell leukemia. Understanding the causes of this leukemia should help guide the design of effective treatments and may improve our understanding of similar cancers. Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder characterized by somatic BRAFV600E mutations. The malignant cell in HCL has immunophenotypic features of a mature B cell, but no normal counterpart along the continuum of developing B lymphocytes has been delineated as the cell of origin. We find that the BRAFV600E mutation is present in hematopoietic stem cells (HSCs) in HCL patients, and that these patients exhibit marked alterations in hematopoietic stem/progenitor cell (HSPC) frequencies. Quantitative sequencing analysis revealed a mean BRAFV600E-mutant allele frequency of 4.97% in HSCs from HCL patients. Moreover, transplantation of BRAFV600E-mutant HSCs from an HCL patient into immunodeficient mice resulted in stable engraftment of BRAFV600E-mutant human hematopoietic cells, revealing the functional self-renewal capacity of HCL HSCs. Consistent with the human genetic data, expression of BRafV600E in murine HSPCs resulted in a lethal hematopoietic disorder characterized by splenomegaly, anemia, thrombocytopenia, increased circulating soluble CD25, and increased clonogenic capacity of B lineage cells—all classic features of human HCL. In contrast, restricting expression of BRafV600E to the mature B cell compartment did not result in disease. Treatment of HCL patients with vemurafenib, an inhibitor of mutated BRAF, resulted in normalization of HSPC frequencies and increased myeloid and erythroid output from HSPCs. These findings link the pathogenesis of HCL to somatic mutations that arise in HSPCs and further suggest that chronic lymphoid malignancies may be initiated by aberrant HSCs.

MiR-29a maintains mouse hematopoietic stem cell self-renewal by regulating Dnmt3a

Hematopoietic stem cells (HSCs) possess the ability to generate all hematopoietic cell types and to self-renew over long periods, but the mechanisms that regulate their unique properties are incompletely understood. Herein, we show that homozygous deletion of the miR-29a/b-1 bicistron results in decreased numbers of hematopoietic stem and progenitor cells (HSPCs), decreased HSC self-renewal, and increased HSC cell cycling and apoptosis. The HSPC phenotype is specifically due to loss of miR-29a, because miR-29b expression is unaltered in miR-29a/b-1-null HSCs, and only ectopic expression of miR-29a restores HSPC function both in vitro and in vivo. HSCs lacking miR-29a/b-1 exhibit widespread transcriptional dysregulation and adopt gene expression patterns similar to normal committed progenitors. A number of predicted miR-29 target genes, including Dnmt3a, are significantly upregulated in miR-29a/b-1-null HSCs. The loss of negative regulation of Dnmt3a by miR-29a is a major contributor to the miR-29a/b-1-null HSPC phenotype, as both in vitro Dnmt3a short hairpin RNA knockdown assays and a genetic haploinsufficiency model of Dnmt3a restored the frequency and long-term reconstitution capacity of HSCs from miR-29a/b-1-deficient mice. Overall, these data demonstrate that miR-29a is critical for maintaining HSC function through its negative regulation of Dnmt3a.

CD99 is a therapeutic target on disease stem cells in myeloid malignancies

CD99 is a potential therapeutic target enriched on disease stem cells in myeloid malignancies. Stemming the tide of leukemia development Acute myeloid leukemia and myelodysplastic syndromes are maintained by specific populations of malignant stem cells, and successful treatment requires the eradication of these disease-causing cells. Chung et al. identified CD99 as a marker expressed on the surface of leukemic stem cells but not normal hematopoietic stem cells, suggesting its potential as a therapeutic target. A monoclonal antibody against CD99 had promising preclinical effectiveness in xenograft models and was selective for malignant stem cells, paving the way for further development of this approach. Acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) are initiated and sustained by self-renewing malignant stem cells; thus, eradication of AML and MDS stem cells is required for cure. We identified CD99 as a cell surface protein frequently overexpressed on AML and MDS stem cells. Expression of CD99 allows for prospective separation of leukemic stem cells (LSCs) from functionally normal hematopoietic stem cells in AML, and high CD99 expression on AML blasts enriches for functional LSCs as demonstrated by limiting dilution xenotransplant studies. Monoclonal antibodies (mAbs) targeting CD99 induce the death of AML and MDS cells in a SARC family kinase–dependent manner in the absence of immune effector cells or complement, and anti-CD99 mAbs exhibit antileukemic activity in AML xenografts. These data establish CD99 as a marker of AML and MDS stem cells, as well as a promising therapeutic target in these disorders.

The role of microRNAs in hematopoietic stem cell and leukemic stem cell function

Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and reconstitute all elements of the hematopoietic system. Acute myeloid leukemia (AML) is thought to arise from, and be maintained by, leukemic stem cells (LSCs), which exhibit similar features to HSCs, including the abilities to self-renew and differentiate into non-self-renewing cells. Acquisition of stem-cell-like characteristics by the LSCs is likely mediated in part by molecular mechanisms that normally regulate HSC function. Thus, understanding the shared and unique aspects of the molecular regulation of these cell populations will be important to understanding the relationship between normal hematopoiesis and leukemogenesis. MicroRNAs (miRNAs) are small noncoding RNAs that act at the posttranscriptional level to regulate protein expression. Unfortunately, most investigations of the role of miRNAs in normal hematopoiesis have been restricted to studies of their effects on lineage commitment in progenitors and mature effector cell function, but not on HSCs. Recent studies have identified miRNAs that enhance HSC function, and an abundance of profiling studies using primary AML samples have identified dysregulated miRNAs that may target genes implicated in self-renewal (HOX genes, P53, and PTEN), thus providing a potential link between normal and malignant stem cells. While these studies as well as recent in vivo models of miRNA-induced leukemogenesis (e.g. miR-29a, miR-125b) suggest a role for miRNAs in the development of AML, future studies using serial transplantation of primary AML blasts, from both mouse models and primary human AML specimens, will be necessary to assess the roles of miRNAs in LSC biology.

miR-99 regulates normal and malignant hematopoietic stem cell self-renewal

The microRNA-99 (miR-99) family comprises a group of broadly conserved microRNAs that are highly expressed in hematopoietic stem cells (HSCs) and acute myeloid leukemia stem cells (LSCs) compared with their differentiated progeny. Herein, we show that miR-99 regulates self-renewal in both HSCs and LSCs. miR-99 maintains HSC long-term reconstitution activity by inhibiting differentiation and cell cycle entry. Moreover, miR-99 inhibition induced LSC differentiation and depletion in an MLL-AF9–driven mouse model of AML, leading to reduction in leukemia-initiating activity and improved survival in secondary transplants. Confirming miR-99’s role in established AML, miR-99 inhibition induced primary AML patient blasts to undergo differentiation. A forward genetic shRNA library screen revealed Hoxa1 as a critical mediator of miR-99 function in HSC maintenance, and this observation was independently confirmed in both HSCs and LSCs. Together, these studies demonstrate the importance of noncoding RNAs in the regulation of HSC and LSC function and identify miR-99 as a critical regulator of stem cell self-renewal.

Measuring microRNA expression in mouse hematopoietic stem cells.

MicroRNAs (miRNAs) are important regulators of diverse biologic processes. In the hematopoietic system, miRNAs have been shown to regulate lineage fate decisions, mature immune effector cell function, apoptosis, and cell cycling, and a more limited number of miRNAs has been shown to regulate hematopoietic stem cell (HSC) self-renewal. Many of these miRNAs were initially identified as candidate regulators of HSC function by comparing miRNA expression in hematopoietic stem and progenitors cells (HSPCs) to their mature progeny. While the measurement of miRNA expression in rare cell populations such as HSCs poses practical challenges due to the low amount of RNA present, a number of techniques have been developed to measure miRNAs in small numbers of cells. Here, we describe our protocol for measuring miRNAs in purified mouse HSCs using a highly sensitive real-time quantitative PCR strategy that utilizes microfluidic array cards containing pre-spotted TaqMan probes that allows the detection of mature miRNAs in small reaction volumes. We also describe a simple data analysis method to evaluate miRNA expression profiling data using an open-source software package (HTqPCR) using mouse HSC miRNA profiling data generated in our lab.