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Chronic myelogenous leukemia (CML) stem cells (LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells persist in the bone marrow (BM) despite effective inhibition of BCR-ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here, we show that although miR-126 supports the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels are lower in CML LSCs as compared to normal long-term hematopoietic stem cells (LT-HSCs). Down-regulation of miR-126 levels in CML LSCs is due to phosphorylation of SPRED1 by BCR-ABL, leading to inhibition of the RAN/EXP-5/RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using CML mouse models with conditional miR-126 knock-out (KO) in ECs and/or LSCs. Inhibition of BCR-ABL by TKI treatment causes an undesired increase in endogenous miR-126 levels, thereby enhancing LSC quiescence and persistence. miR-126 KO in LSCs and/or ECs, or treatment with a CpG-miR-126 inhibitor targeting miR-126 in both LSCs and ECs, enhances the in vivo anti-leukemic effects of TKI treatment and strongly diminishes LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in CML.

Chronic myeloid leukemia in chronic phase (CML-CP) is induced by BCR-ABL1 oncogenic tyrosine kinase. Tyrosine kinase inhibitors eliminate the bulk of CML-CP cells, but fail to eradicate leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) displaying innate and acquired resistance, respectively. These cells may accumulate genomic instability, leading to disease relapse and/or malignant progression to a fatal blast phase. In the present study, we show that Rac2 GTPase alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III (MRC-cIII), thereby generating high levels of reactive oxygen species (ROS) in CML-CP LSCs and primitive LPCs. MRC-cIII–generated ROS promote oxidative DNA damage to trigger genomic instability, resulting in an accumulation of chromosomal aberrations and tyrosine kinase inhibitor–resistant BCR-ABL1 mutants. JAK2(V617F) and FLT3(ITD)–positive polycythemia vera cells and acute myeloid leukemia cells also produce ROS via MRC-cIII. In the present study, inhibition of Rac2 by genetic deletion or a small-molecule inhibitor and down-regulation of mitochondrial ROS by disruption of MRC-cIII, expression of mitochondria-targeted catalase, or addition of ROS-scavenging mitochondria-targeted peptide aptamer reduced genomic instability. We postulate that the Rac2-MRC-cIII pathway triggers ROS-mediated genomic instability in LSCs and primitive LPCs, which could be targeted to prevent the relapse and malignant progression of CML.

We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%-7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. Mll PTD/WT mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. Mll PTD/WT mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.

IntroductionCytogenetic abnormalities involving chromosome band 11q23, such as translocations, deletions, and duplications, are seen in approximately 15% of patients with acute myeloid leukemia (AML) and most often result in gene fusions between the 5Ј-end of the MLL (ALL-1) gene and the 3Ј-end of a partner gene. 1 One type of MLL rearrangement not detectable by classic cytogenetics is the partial tandem duplication of MLL (MLL PTD). This rearrangement most commonly results from a duplication of a genomic region encompassing either MLL exons 5 through 11 or MLL exons 5 through 12 that is inserted into intron 4 of a full-length MLL gene, thus fusing introns 11 or 12 with intron 4 (exon designations used throughout are consistent with GenBank NT_033899.6). At the level of transcription this results in a unique in-frame fusion of exons 11 or 12 upstream of exon 5. 2,3 The presence of an MLL PTD at the DNA and RNA level has been demonstrated most often in adult de novo AML with normal cytogenetics or trisomy 11 (ϩ11), but it has also been observed in childhood leukemias, adult acute lymphoblastic leukemia (ALL), secondary leukemia, and a solid tumor cell line. [4][5][6] In adult de novo AML with a normal karyotype, the presence of the MLL PTD has been associated with a worse prognosis (ie, shorter duration of remission) when compared with normal karyotype AML without the MLL PTD. [7][8][9] The MLL PTD self-fusion has a duplicated N-terminal region that contains the AT hook DNA-binding motifs, a domain that preferentially binds an unmethylated cytosine in cytidine phosphate guanosine (CpG) dinucleotides on DNA, and a transcriptional repression domain. 3,10 In the absence of a fusion gene partner that would replace the 3Ј-end of the transcript, the mechanism by which this PTD functions in leukemogenesis, drug resistance, and leukemia relapse is currently unknown. We previously used Southern analysis to demonstrate that the MLL PTD defect is present on only one chromosome 11 and the other has a wild-type (WT) MLL allele in patients with AML with a normal karyotype. Likewise, in AML with ϩ11 and the MLL PTD, 2 chromosomes 11 contain MLL WT alleles, while the third copy contains the MLL PTD. 2 To examine the relationship between the MLL PTD and the MLL WT and to better understand the importance of the MLL PTD in leukemogenesis, we quantified each of these 2 gene products in primary AML blasts containing this molecular defect. Our results show the MLL WT transcript is not expressed in primary AML blasts that harbor the MLL PTD, in contrast to AML with either MLL WT genes only or the t(9;11)(p22;q23). Induction of MLL WT in response to a DNA methyltransferase (DNMT) inhibitor and/or a histone deacetylase (HDAC) inhibitor in these cases was selectively associated with enhanced sensitivity to cell death. As the absence of MLL WT protein is predicted to contribute to the leukemic phenotype, these data appear to identify a new molecular target for DNMT or HDAC inhibitors or both in patients with AML with the MLL PTD/WT genotyp...

Current treatments for acute myeloid leukemia (AML) are designed to target rapidly dividing blast populations with limited success in eradicating the functionally distinct leukemia stem cell (LSC) population, which is postulated to be responsible for disease resistance and relapse. We have previously reported high miR-126 expression levels to be associated with a LSC-gene expression profile. Therefore, we hypothesized that miR-126 contributes to “stemness” and is a viable target for eliminating the LSC in AML. Here we first validate the clinical relevance of miR-126 expression in AML by showing that higher expression of this microRNA (miR) is associated with worse outcome in a large cohort of older (≥60 years) cytogenetically normal AML patients treated with conventional chemotherapy. We then show that miR-126 overexpression characterizes AML LSC-enriched cell subpopulations and contributes to LSC long-term maintenance and self-renewal. Finally, we demonstrate the feasibility of therapeutic targeting of miR-126 in LSCs with novel targeting nanoparticles (NP) containing antagomiR-126 resulting in in vivo reduction of LSCs likely by depletion of the quiescent cell subpopulation. Our findings suggest that by targeting a single miR, i.e., miR-126, it is possible to interfere with LSC activity, thereby opening potentially novel therapeutic approaches to treat AML patients.

Purpose Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore we asked whether adding already established effective drugs such as Topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML. Experimental Design The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML. Results Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared to each single therapy. Conclusions Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of AML patients that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression, and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML.

The MLL-partial tandem duplication (PTD) associates with high-risk cytogenetically normal acute myeloid leukemia (AML). Concurrent presence of FLT3-internal tandem duplication (ITD) is observed in 25% of patients with MLL-PTD AML. However, mice expressing either Mll-PTD or Flt3-ITD do not develop AML, suggesting that 2 mutations are necessary for the AML phenotype. Thus, we generated a mouse expressing both Mll-PTD and IntroductionAcute myeloid leukemia (AML) is a genetically heterogeneous disease. Recurrent cytogenetic and molecular gene aberrations have been used to classify AML patients into distinct subsets that differ in biologic, clinical, and prognostic characteristics. The MLL gene, located at chromosome band 11q23, encodes for a protein involved in epigenetic regulation of gene expression. 1 In AML, this gene is frequently involved in chromosome translocations at 11q23 and, at the molecular level, is fused with one of more than 50 different partners. 2 We first reported an internal duplication of MLL, an in-frame repeat producing an elongated protein retaining all functional domains, in cytogenetically normal (CN)-AML. 3 Approximately 5% to 7% of CN-AML patients have a MLL-partial tandem duplication (PTD) mutation, which is associated with unfavorable prognosis, 3-5 but if and how it contributes to myeloid leukemogenesis remain to be elucidated. Approximately 25% of CN-AML patients with the MLL-PTD had constitutive activation of FLT3, a tyrosine kinase receptor regulating proliferation and survival of hematopoietic cells, via an internal tandem duplication (FLT3-ITD), and have a very poor prognosis. 6 This suggests that both MLL-PTD and FLT3-ITD mutations are necessary for an AML phenotype, as supported by the broadly accepted 2-hit model. 7 Indeed, a Mll-PTD mouse, created by knocking in exons 5 to 11 in-frame and driven off of the endogenous Mll promoter, did not develop AML. 8 8,9 This model, which develops AML, is the first that requires the MLL-PTD. In this model, the 2 mutated genes are regulated by their respective endogenous promoters to recapitulate the Mll PTD/WT :Flt3 ITD/WT AML found in humans. This differs from some other doublemutant mouse models of human AML that carry one mutation driven by the endogenous promoter and the other mutation driven by transgenes, 10 or introduced via viral transduction, 11 or 2 mutations driven off 2 endogenous promoters but requiring BM transplantation. 12 Methods Mouse strainsThe Mll PTD/WT , Flt3 ITD/WT , and Flt3 ITD/ITD mice were generated and genotyped as previously described. 8,9 Male Flt3 ITD/WT Balb/c mice were backcrossed onto the C57Bl/6J strain to purity and then bred with Mll PTD/WT mice to generate Mll PTD/WT :Flt3 ITD/WT double knock-in offspring. Genotyping was performed as previously described. 8,9 All animals studied were compared within litters and/or age-and sex-matched. All experiments were conducted under an approved The Ohio State University Institutional Submitted March 2, 2012; accepted May 17, 2012. Prepublished online as Blood Firs...