R-2HG Exhibits Anti-tumor Activity by Targeting FTO/m6A/MYC/CEBPA Signaling.

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling.

ETV6-NCOA2 fusion induces T/myeloid mixed-phenotype leukemia through transformation of nonthymic hematopoietic progenitor cells.

Mixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.

Rapid desensitization of humanized mice with anti-human FcεRIα monoclonal antibodies.

BACKGROUND: Anaphylaxis is classically mediated by allergen cross-linking of IgE bound to the α chain of FcεRI, the mast cell/basophil high affinity IgE receptor. Allergen cross-linking of the IgE/FcεRI complex activates these cells, inducing release of disease-causing mediators, cytokines, and enzymes. We previously demonstrated that IgE-mediated anaphylaxis could be safely prevented in wild-type BALB/c mice by rapid desensitization with anti-mouse FcεRIα mAb. OBJECTIVE: This study sought to use humanized mice to extend these results to humans. METHODS: We actively immunized huFcεRIα/F709 mice, which express human (hu) instead of mouse FcεRIα and a mutant IL-4 receptor that lacks inhibitory function. We passively immunized huFcεRIα mice, as well as human cord blood-reconstituted reNSGS mice, which are immune-deficient, produce mast cell-stimulating human cytokines, and develop numerous human mast cells. For desensitization, we used anti-huFcεRIα mAbs that bind FcεRIα regardless of its association with IgE (noncompeting mAbs), and/or mAbs that compete with IgE for huFcεRIα binding (competing mAbs). Anaphylaxis was induced by intravenous injection of antigen or anti-huIgE mAb. RESULTS: Anti-huFcεRIα mAb rapid desensitization was safer and more effective than allergen rapid desensitization and suppressed anaphylaxis more rapidly than omalizumab or ligelizumab. Rapid desensitization of naïve, IgE-sensitized huFcεRIα mice and huFcεRIα/F709 mice that were egg-allergic with anti-FcεRIα mAbs safely removed >98% of IgE from peritoneal mast cells and completely suppressed IgE-mediated anaphylaxis. Rapid desensitization of reNSGS mice with anti-FcεRIα mAbs also safely removed ∼98% of mast cell IgE and prevented IgE-mediated anaphylaxis. CONCLUSIONS: Rapid desensitization with anti-FcεRIα mAbs may be a safe, effective, and practical way to prevent IgE-mediated anaphylaxis.

The full transforming capacity of MLL-Af4 is interlinked with lymphoid lineage commitment.

Chromosome rearrangements involving the mixed-lineage leukemia gene (MLL) create MLL-fusion proteins, which could drive both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The lineage decision of MLL-fusion leukemia is influenced by the fusion partner and microenvironment. To investigate the interplay of fusion proteins and microenvironment in lineage choice, we transplanted human hematopoietic stem and progenitor cells (HSPCs) expressing MLL-AF9 or MLL-Af4 into immunodeficient NSGS mice, which strongly promote myeloid development. Cells expressing MLL-AF9 efficiently developed AML in NSGS mice. In contrast, MLL-Af4 cells, which were fully oncogenic under lymphoid conditions present in NSG mice, displayed compromised transformation capacity in a myeloid microenvironment. MLL-Af4 activated a self-renewal program in a lineage-dependent manner, showing the leukemogenic activity of MLL-Af4 was interlinked with lymphoid lineage commitment. The C-terminal homology domain (CHD) of Af4 was sufficient to confer this linkage. Although the MLL-CHD fusion protein failed to immortalize HSPCs in myeloid conditions in vitro, it could successfully induce ALL in NSG mice. Our data suggest that defective self-renewal ability and leukemogenesis of MLL-Af4 myeloid cells could contribute to the strong B-cell ALL association of MLL-AF4 leukemia observed in the clinic.

The cell polarity determinant CDC42 controls division symmetry to block leukemia cell differentiation.

As a central regulator of cell polarity, the activity of CDC42 GTPase is tightly controlled in maintaining normal hematopoietic stem and progenitor cell (HSC/P) functions. We found that transformation of HSC/P to acute myeloid leukemia (AML) is associated with increased CDC42 expression and activity in leukemia cells. In a mouse model of AML, the loss of Cdc42 abrogates MLL-AF9-induced AML development. Furthermore, genetic ablation of CDC42 in both murine and human MLL-AF9 (MA9) cells decreased survival and induced differentiation of the clonogenic leukemia-initiating cells. We show that MLL-AF9 leukemia cells maintain cell polarity in the context of elevated Cdc42-guanosine triphosphate activity, similar to nonmalignant, young HSC/Ps. The loss of Cdc42 resulted in a shift to depolarized AML cells that is associated with a decrease in the frequency of symmetric and asymmetric cell divisions producing daughter cells capable of self-renewal. Importantly, we demonstrate that inducible CDC42 suppression in primary human AML cells blocks leukemia progression in a xenograft model. Thus, CDC42 loss suppresses AML cell polarity and division asymmetry, and CDC42 constitutes a useful target to alter leukemia-initiating cell fate for differentiation therapy.

A FOXO1-induced oncogenic network defines the AML1-ETO preleukemic program.

Understanding and blocking the self-renewal pathway of preleukemia stem cells could prevent acute myeloid leukemia (AML) relapse. In this study, we show that increased FOXO1 represents a critical mechanism driving aberrant self-renewal in preleukemic cells expressing the t(8;21)-associated oncogene AML1-ETO (AE). Although generally considered as a tumor suppressor, FOXO1 is consistently upregulated in t(8;21) AML. Expression of FOXO1 in human CD34+ cells promotes a preleukemic state with enhanced self-renewal and dysregulated differentiation. The DNA binding domain of FOXO1 is essential for these functions. FOXO1 activates a stem cell molecular signature that is also present in AE preleukemia cells and preserved in t(8;21) patient samples. Genome-wide binding studies show that AE and FOXO1 share the majority of their binding sites, whereby FOXO1 binds to multiple crucial self-renewal genes and is required for their activation. In agreement with this observation, genetic and pharmacological ablation of FOXO1 inhibited the long-term proliferation and clonogenicity of AE cells and t(8;21) AML cell lines. Targeting of FOXO1 therefore provides a potential therapeutic strategy for elimination of stem cells at both preleukemic and leukemic stages.

Supraphysiologic levels of the AML1-ETO isoform AE9a are essential for transformation.

Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34(+) cord blood cells in long-term culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.

The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation.

Antisense RNAs regulate the transcription and translation of the corresponding sense genes. Here, we report that an antisense RNA, AS-RBM15, is transcribed in the opposite direction within exon 1 of RBM15 RBM15 is a regulator of megakaryocyte (MK) differentiation and is also involved in a chromosome translocation t(1;22) in acute megakaryocytic leukemia. MK terminal differentiation is enhanced by up-regulation of AS-RBM15 expression and attenuated by AS-RBM15 knockdown. At the molecular level, AS-RBM15 enhances RBM15 protein translation in a CAP-dependent manner. The region of the antisense AS-RBM15 RNA, which overlaps with the 5'UTR of RBM15, is sufficient for the up-regulation of RBM15 protein translation. In addition, we find that transcription of both RBM15 and AS-RBM15 is activated by the transcription factor RUNX1 and repressed by RUNX1-ETO, a leukemic fusion protein. Therefore, AS-RBM15 is a regulator of megakaryocyte differentiation and may play a regulatory role in leukemogenesis.

CD44 variant isoform 9 emerges in response to injury and contributes to the regeneration of the gastric epithelium.

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a funcitonal role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-deficient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14-20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile anlayzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastic epithelium CD44v9 emerges within a regenerative cell lineage that coincides with macrophage inflitration within the injured mucosa. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Antibodies targeting human IL1RAP (IL1R3) show therapeutic effects in xenograft models of acute myeloid leukemia.

Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell-mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.

Xenograft models for normal and malignant stem cells.

The model systems available for studying human hematopoiesis, malignant hematopoiesis, and hematopoietic stem cell (HSC) function in vivo have improved dramatically over the last decade, primarily due to improvements in xenograft mouse strains. Several recent reviews have focused on the historic development of immunodeficient mice over the last 2 decades, as well as their use in understanding human HSC and leukemia stem cell (LSC) biology and function in the context of a humanized mouse. However, in the intervening time since these reviews, a number of new mouse models, technical approaches, and scientific advances have been made. In this review, we update the reader on the newest and best models and approaches available for studying human malignant and normal HSCs in immunodeficient mice, including newly developed mice for use in chemotherapy testing and improved techniques for humanizing mice without laborious purification of HSC. We also review some relevant scientific findings from xenograft studies and highlight the continued limitations that confront researchers working with human HSC and LSC in vivo.

MEIS1 regulates an HLF-oxidative stress axis in MLL-fusion gene leukemia.

Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment.

RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia.

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

RUNX1-ETO Leukemia.

AML1-ETO leukemia is the most common cytogenetic subtype of acute myeloid leukemia, defined by the presence of t(8;21). Remarkable progress has been achieved in understanding the molecular pathogenesis of AML1-ETO leukemia. Proteomic surveies have shown that AML-ETO forms a stable complex with several transcription factors, including E proteins. Genome-wide transcriptome and ChIP-seq analyses have revealed the genes directly regulated by AML1-ETO, such as CEBPA. Several lines of evidence suggest that AML1-ETO suppresses endogenous DNA repair in cells to promote mutagenesis, which facilitates acquisition of cooperating secondary events. Furthermore, it has become increasingly apparent that a delicate balance of AML1-ETO and native AML1 is important to sustain the malignant cell phenotype. Translation of these findings into the clinical setting is just beginning.

OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues.

Immunodeficient mice are now readily engrafted with human hematopoietic cells. However, these mice are susceptible to graft-versus-host disease (GVHD) induced by the engraftment and rapid expansion of coinjected human T cells. Therefore, highly purified sample populations must be used, adding significant time, expense, and effort. Here, we have explored in vivo and in vitro methods utilizing anti-T-cell antibodies to circumvent this problem. Intraperitoneal injection of the antibody within 48 hours prevented GVHD. Alternatively, short-term in vitro incubation of cells with antibody immediately before transplant was equally effective. Although in vitro antithymocyte globulin treatment resulted in a dramatic loss of SCID-repopulating cells (SRCs), treatment with OKT3 or UCHT1 abrogated GVHD risk and preserved engraftment potential. Leukemia samples that presented with substantial human T-cell contamination were effectively rescued from GVHD. In addition, OKT3 treatment of unfractionated cord blood resulted in robust engraftment of primary and secondary mice that was indistinguishable from grafts obtained using purified CD34(+) cells. Limiting dilution analysis of unfractionated blood demonstrated a SRC frequency of 1 in 300 to 500 CD34(+) cells, similar to that of purified hematopoietic stem and progenitor cells. This protocol streamlines xenograft studies while significantly reducing the cost and time of the procedure.

Downregulation of RUNX1/CBFß by MLL fusion proteins enhances hematopoietic stem cell self-renewal.

RUNX1/CBFß (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFß recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFß activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFß protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFß. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfß levels accelerates MLL-AF9-mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFß by MLL fusion proteins and suggest that targeting RUNX1/CBFß levels may be a potential therapy for MLLs.

Functional Niche Competition Between Normal Hematopoietic Stem and Progenitor Cells and Myeloid Leukemia Cells.

Hematopoietic stem and progenitor cells (HSPCs) reside in a specialized niche that regulates their proliferative capacity and their fate. There is increasing evidence for similar roles of marrow niches on controlling the behavior of leukemic cells; however, whether normal hematopoietic stem cell (HSC) and leukemic cells reside in or functionally compete for the same marrow niche is unclear. We used the mixed lineage leukemia-AF9 (MLL-AF9) murine acute myeloid leukemia (AML) in a competitive repopulation model to investigate whether normal HSPC and leukemic cells functionally compete for the same marrow niches. Irradiated recipient mice were transplanted with fixed numbers of MLL-AF9 cells mixed with increasing doses of normal syngeneic whole bone marrow (WBM) or with purified HSPC (LSK). Survival was significantly increased and leukemic progression was delayed proportional to increasing doses of normal WBM or normal LSK cells in multiple independent experiments, with all doses of WBM or LSK cells studied above the threshold for rapid and complete hematopoietic reconstitution in the absence of leukemia. Confocal microscopy demonstrated nests of either leukemic cells or normal hematopoietic cells but not both in the marrow adjacent to endosteum. Early following transplantation, leukemic cells from animals receiving lower LSK doses were cycling more actively than in those receiving higher doses. These results suggest that normal HSPC and AML cells compete for the same functional niche. Manipulation of the niche could impact on response to antileukemic therapies, and the numbers of normal HSPC could impact on leukemia outcome, informing approaches to cell dose in the context of stem cell transplantation.

TET1 plays an essential oncogenic role in MLL-rearranged leukemia.

The ten-eleven translocation 1 (TET1) gene is the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine to 5-hydroxymethylcytosine. Although TET1 was first identified as a fusion partner of the mixed lineage leukemia (MLL) gene in acute myeloid leukemia carrying t(10,11), its definitive role in leukemia is unclear. In contrast to the frequent down-regulation (or loss-of-function mutations) and critical tumor-suppressor roles of the three TET genes observed in various types of cancers, here we show that TET1 is a direct target of MLL-fusion proteins and is significantly up-regulated in MLL-rearranged leukemia, leading to a global increase of 5-hydroxymethylcytosine level. Furthermore, our both in vitro and in vivo functional studies demonstrate that Tet1 plays an indispensable oncogenic role in the development of MLL-rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical cotargets, including homeobox A9 (Hoxa9)/myeloid ecotropic viral integration 1 (Meis1)/pre-B-cell leukemia homeobox 3 (Pbx3) genes. Collectively, our data delineate an MLL-fusion/Tet1/Hoxa9/Meis1/Pbx3 signaling axis in MLL-rearranged leukemia and highlight TET1 as a potential therapeutic target in treating this presently therapy-resistant disease.

miR-9 is an essential oncogenic microRNA specifically overexpressed in mixed lineage leukemia-rearranged leukemia.

MicroRNAs (miRNAs), small noncoding RNAs that regulate target gene mRNAs, are known to contribute to pathogenesis of cancers. Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies with various chromosomal and/or molecular abnormalities. AML with chromosomal translocations involving the mixed lineage leukemia (MLL) gene are usually associated with poor survival. In the present study, through a large-scale, genomewide miRNA expression assay, we show that microRNA-9 (miR-9) is the most specifically up-regulated miRNA in MLL-rearranged AML compared with both normal control and non-MLL-rearranged AML. We demonstrate that miR-9 is a direct target of MLL fusion proteins and can be significantly up-regulated in expression by the latter in human and mouse hematopoietic stem/progenitor cells. Depletion of endogenous miR-9 expression by an appropriate antagomiR can significantly inhibit cell growth/viability and promote apoptosis in human MLL-rearranged AML cells, and the opposite is true when expression of miR-9 is forced. Blocking endogenous miR-9 function by anti-miRNA sponge can significantly inhibit, whereas forced expression of miR-9 can significantly promote, MLL fusion-induced immortalization/transformation of normal mouse bone marrow progenitor cells in vitro. Furthermore, forced expression of miR-9 can significantly promote MLL fusion-mediated leukemogenesis in vivo. In addition, a group of putative target genes of miR-9 exhibited a significant inverse correlation of expression with miR-9 in a series of leukemia sample sets, suggesting that they are potential targets of miR-9 in MLL-rearranged AML. Collectively, our data demonstrate that miR-9 is a critical oncomiR in MLL-rearranged AML and can serve as a potential therapeutic target to treat this dismal disease.

A ROS-Activatable Agent Elicits Homologous Recombination DNA Repair and Synergizes with Pathway Compounds.

We designed ROS-activated cytotoxic agents (RACs) that are active against AML cancer cells. In this study, the mechanism of action and synergistic effects against cells coexpressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD were investigated. One RAC (RAC1) had an IC50 value of 1.8±0.3 µm, with ninefold greater selectivity for transformed cells compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway-targeting therapies in AML cells but less so in untransformed cells. Together, these results demonstrate that RAC1 can selectively target poor prognosis AML and that it does so by creating DNA double-strand breaks that require homologous recombination.