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Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies. Over 70% of patients with AML cannot survive over 5 years. Many AML subtypes, such as the MLL -rearranged AMLs, are often associated with unfavorable outcome. Current treatment frequently involves intensive chemotherapy, which impairs the quality of life of the patients. While the incidence of AML is continually rising due to aging, most elder patients cannot bear intensive chemotherapy and are associated with very poor survival. Thus, improved therapeutic strategies with less intensive treatment but a higher cure rate are urgently needed. The Ten-eleven translocation (TET) proteins (including TET1/2/3) are known to be able to convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), leading to DNA demethylation. In contrast to the repression and tumor-suppressor role of TET2 observed in hematopoietic malignancies, we recently showed that TET1, the founding member of the TET family, was significantly up-regulated in MLL -rearranged AML and played an essential oncogenic role. An independent study by Zhao et al. confirmed the essential oncogenic role of Tet1 in the development of myeloid malignancies. Thus, TET1 is an attractive therapeutic target for AML. In order to identify chemical compounds that may target TET1 signaling, we searched the drug-sensitivity/gene expression database of a total of 20,602 chemical compounds in the NCI-60 collection of cancer cell samples. The expression levels of endogenous TET1 showed a significant positive correlation with the responsiveness of cancer cells across the NCI-60 panel to 953 compounds (r > 0.2; P Further, secondary bone marrow transplantation followed by drug treatment was carried out to test the in vivo therapeutic effects of NSC-X1 and NSC-X2. Both candidate compounds significantly inhibited MLL-AF9 -induced AML, by prolonging the median survival from 49 days (control) to 94 (NSC-X1) or >200 (NSC-X2) days. Notably, 57% of the NSC-X2 treated mice were cured. In another AML model induced by AML-ETO9a (AE9a), NSC-X1 and NSC-X2 also exhibited remarkable therapeutic effects, with an elongated median survival from 46 days (control) to 95 (NSC-X1) and 122 (NSC-X2) days, respectively. To decipher the molecular mechanism by which NSC-X2 represses TET1 expression, we treated THP-1 AML cells with moderate to high concentration of NSC-X2 for over 100 days and then isolated a set of individual drug-resistant THP-1 single clones. Through RNA-seq of 6 of the NSC-X2-resistant clones, recurrent mutations were found in 14 genes, including JAK1 . Ingenuity pathway analysis (IPA) was used to analyze biological relationships amongst the 14 mutated genes. The top one network identified by IPA involving all of the 14 genes is closely associated with the JAK/STAT5 pathway. Results of chromatin immunoprecipitation (ChIP) assays showed TET1 is one of the direct downstream gene targets of STAT3 and STAT5. Through NMR chemical shift perturbation (CSP) and electrophoretic mobility shift assays (EMSAs), we showed a strong direct association between STAT3/5 DNA binding domain and the TET1 promoter, which could be severely interrupted by NSC-X2. Compared to currently available JAK/STAT inhibitors (e.g., Pacritinib, KW-2449, Stattic, and sc-355979), our compounds (NSC-370284 and UC-514321) exhibit a much higher selectivity and also a higher efficacy in targeting TET1 -high AML. Taken together, we identified chemical compounds NSC-X1 and especially, NSC-X2, as potent inhibitors that significantly and selectively suppress the viability of AML cells with high level of TET1 expression, and dramatically repress the progression of TET1 -high AML in mice. NSC-X2 directly binds STAT3/5 as STAT inhibitors and thereby suppress TET1 transcription and TET1 signaling, leading to potent anti-leukemic effects. Our results highlight the therapeutic potential of targeting the STAT/TET1 axis by selective inhibitors in AML treatment. Disclosures No relevant conflicts of interest to declare.

Background: Acute Myeloid leukemia (AML) is one of the most common and fatal form of hematologic malignancies. Recurring chromosomal aberrations and gene mutations have been shown to contribute to AML pathogenesis and clinical outcomes. However, no effective therapy is available to selectively target the cytogenetic and molecular abnormalities, except for PML-RARA in acute promyelocytic leukemia (APL) which can be targeted by all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). As a result, the majority of AML patients are suffering from unsatisfied treatment of standard chemotherapy, associated with high rate of relapse and inferior survival. Thus, better understanding of the molecular mechanisms underlying the pathogenesis and drug resistance of AML, and more effective treatments based on such understanding, are urgently needed. MiRNAs are a class of non-coding RNAs which post-transcriptionally regulate targeted gene expression. They usually consist of 20~24 nucleotides. MiRNAs are closely involved in almost all physiological and pathological processes. The widespread dysregulation of miRNA expressions have been shown to be correlated with various types of malignancies, including AML. In our recent publication, we show through Exiqon miRNA microarray analysis that several miRNAs are significantly down-regulated in most subtypes of de novo AML; miR-550 is one of them (Jiang et al., Cancer Cell, 2012). However, its role and regulatory mechanism in AML have been poorly elucidated. The present study is to investigate the biological functions and molecular mechanisms of miR-550 in AML. Methods: The expression levels of miR-550 were analyzed in multiple AML cell lines, AML patients' bone marrow (BM) mononuclear cells (MNC) and normal MNC control samples by using Taqman miRNA assay qPCR kit. Cell viability and proliferation assays, i.e., MTT assays, were performed in human AML cell lines with stable ectopic expression of miR-550 or control plasmids induced by retrovirus. Cell apoptosis and cell cycle were assessed via flow cytometry analysis. To determine the influence of miR-550 on the transformation capacity of mouse BM progenitor cells transduced with leukemic fusion genes, e.g. MLL-AF9 and AML-ETO9a (AE9a), colony-forming/replating assay (CFA) was carried out. To evaluate the effect of restoration of miR-550 expression/function in AML progression in vivo, we retrovirally infected leukemic blast cells carrying MLL-AF9 with miR-550 or empty vector, and performed secondary BM transplantation by i.v. injecting recipient mice with these donor cells. To identify potential target genes of miR-550, two independent AML patient datasets were analyzed and the correlation patterns between miR-550 and the candidate targets were shown. Results: Consistent with the results of our previous miRNA array, the expression level of miR-550 was significantly down-regulated in most AML patient samples and AML cell lines as compared with normal controls. In AML cell lines, retrovirus induced enforced expression of miR-550 resulted in G1-phase arrest, increased apoptosis, and inhibited cell growth and viability. In mouse BM progenitor cells, forced expression of miR-550 dramatically attenuated colony-forming capacity driven by MLL-AF9 or AE9a. Overexpression of miR-550 significantly inhibited progression of AML induced by MLL-AF9 (MLL-AF9+miR-550, with medium survival of 33 days; MLL-AF9, with medium survival of 27 days; P=0.01) in vivo. We further analyzed in two independent AML patient datasets the expressional correlation between miR-550 and its potential gene targets predicted by miRanda, miRWalk, PITA and Targetscan, etc. 77 candidate target genes inversely correlated with miR-550 in expression. Amongst these genes, FOXE1, IGFBP5 and KSR2 etc. have been shown to be oncogenes and are closely related with AML pathology. Therefore, these genes are candidate oncogenic targets that we will focus on in the future. Conclusions: The above results suggest that miR-550 is an important tumor suppressor in AML. Through targeting a series of oncogenes, miR-550 represses the viability and proliferation of leukemic cells, promotes apoptosis and differentiation, and inhibits cell transformation. Our study indicates that down-regulation of miR-550 likely plays a critical role in AML pathogenesis and restoration of miR-550 might hold great potential in treating AML in the future. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies. With standard chemotherapies, only 30-50% of younger (aged MicroRNAs (miRNA) are a class of small, non-coding RNAs that play important roles in post-transcriptional gene regulation. We recently reported that miR-150 functions as a pivotal tumor-suppressor gatekeeper in MLL-rearranged and other subtypes of AML, through targeting FLT3 and MYB directly, and the MYC/LIN28/HOXA9/MEIS1 pathway indirectly. Our data showed that MLL-fusion proteins up-regulate FLT3 level through inhibiting the maturation of miR-150. Therefore, our findings strongly suggest a significant clinical potential of restoration of miR-150 expression/function in treating FLT3 -overexpressing AML. In the present study, we first analyzed FLT3 expression patterns and prognostic impact in a large cohort of AML patients (n=562). We found that FLT3 is aberrantly highly expressed in FAB M1/M2/M5 AML or AML with t(11q23)/MLL -rearrangements, FLT3 -ITD or NPM1 mutations, and that increased expression of FLT3 is an independent predictor of poor prognosis in patients with FLT3 -overexpressing AML. To treat FLT3 -overexpressing AML, we developed a novel targeted nanoparticle system consisting of FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) dendriplexes encapsulating miR-150 oligos (see Figure 1A). In FLT3 -overexpressing cell lines, the uptake ratios of the G7-FLT3L dendrimers were much higher (50.3~97.1%) than the G7-histone 2B (H2B) control nanoparticles (4.3~33.2%). And the uptake only took minutes. By integrating the miR-150 oligo with G7-FLT3L dendrimers, we constructed the G7-FLT3L-miR-150 dendriplexes, which significantly reduced the viability and increased the apoptosis of MONOMAC-6 cells carrying t(9;11) in a dose-dependent manner. To increase the stability of miR-150 oligos, we incorporated a 2'-o -methyl (2'-O Me) modification into the miRNA oligos. Indeed, the G7-FLT3L nanoparticles carrying 2'-O Me modified miR-150 exhibited a more sustained inhibition on cell growth. In order to further investigate the in vivo therapeutic effects of the miR-150 nanoparticles, we used a MLL -rearranged leukemia model. We transplanted wild-type recipient mice with primary mouse leukemic cells bearing the MLL-AF9 fusion. After the onset of leukemia, the mice were treated with G7-Flt3L or G7-NH2 control nanoparticles complexed with 2'-O Me-modified miR-150 oligos. In these treated animals, G7-Flt3L-miR-150 nanoparticles tended to be enriched in the bone marrow. The G7-Flt3L-miR-150 nanoparticles showed the best therapeutic effect (with median survival of 86 days), as compared with the control group (Ctrl; PBS treated; with median survival of 54 days) or the G7-NH2-miR-150 treated group (with median survival of 63 days). Nanoparticles carrying miR-150 mutant oligos showed no anti-leukemia effect at all. Notably, the G7-Flt3L-miR-150 treatment almost completely blocked MLL-AF9 -induced leukemia in 20% of the mice (Fig. 1B). Furthermore, the G7-Flt3L-miR-150 nanoparticles showed a synergistic effect with JQ1, a small-molecule inhibitor of the MYC pathway, in treating AML in vivo (Fig. 1C). Collectively, we have developed a novel targeted therapeutic strategy to treat FLT3-overexpressing AML, such as MLL-rearranged leukemias, which are resistant to currently available therapies, with both high specificity and efficacy. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with diverse chromosomal and molecular abnormalities. The majority of AML patients do not survive more than 5 years. Advanced genomic studies reveal that both genetic and epigenetic abnormalities frequently occur in de novo AML. However, it remains a challenge to understand the complicated genetic/epigenetic regulatory networks and identify the functionally important nodes in these networks. There is an urgent need to develop effective therapeutic strategies based on these new insights. The ten-eleven translocation (Tet) proteins are important epigenetic regulators, which can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and lead to DNA demethylation. Among the three TET family members (TET1/2/3), TET2 was identified as a tumor suppressor in myeloid malignancies. Our lab recently reported that TET1 is highly expressed in MLL/KMT2A (Mixed Lineage Leukemia)-rearranged AML, a subtype of AML with poor prognosis. It is a direct target activated by MLL-fusions, and functions as an essential oncogene (Huang et al., PNAS, 2013). However, the function and regulatory pathway(s) of TET1 in AML remain poorly understood. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play important roles in posttranscriptional gene regulation. Dysregulation of miRNAs is frequently observed in AML. Results of our profiling assays show that miR-22 is widely down-regulated in all major subtypes of de novo AML (Jiang et al., Cancer Cell, 2012), implying a tumor suppressor function. However, an oncogenic role for miR-22 was recently reported in myelodysplastic syndromes (MDS) and breast cancer, in which TET2 was repressed by miR-22 as its direct target gene. Here we show that, amongst a group of miRNAs (e.g. miR-495 and miR-150, etc.) whose expression levels are repressed in AML, miR-22 exhibits the most potent and consistent inhibition on MLL-AF9-induced transformation of mouse bone marrow (BM) progenitor cells. Moreover, forced expression of miR-22 dramatically inhibits cell transformation and leukemogenesis induced by multiple fusion genes, such as MLL-fusions and RUNX1/AML1-ETO9a. Furthermore, the maintenance of various subtypes of AML (e.g., those induced by MLL-fusion, AML1-ETO9a or FLT3-ITD/NPM1c+) is also dependent on the repression of miR-22. Thus, our data demonstrate a potent tumor-suppressor role of miR-22 in AML. Surprisingly, our analysis of three (in-house and outside) large-scale AML datasets revealed that TET2 (and likely also TET3) expression levels exhibited a significant positive correlation, whereas only TET1 exhibited a significant negative correlation (r Further, through a series of data analyses followed by experimental validations and functional studies, we show that a set of critical oncogenes, including CRTC1, FLT3 and MYCBP, are functionally important direct target genes of miR-22 in AML and thus, miR-22 negatively regulates the CREB and MYC signaling pathways. Our proof-of-concept study shows that miR-22 RNA oligos formulated with dendritic nanoparticles significantly inhibit leukemia progression and extend the overall median survival of MLL-AF9-induced leukemic mice from 29 days to 54 days (n=10 per group, p Taken together, our results demonstrate a potent tumor-suppressor role of miR-22 in AML, and suggest the potential clinical application of miR-22-nanoparticles in treating AML. We also identified a TET1⊣miR-22⊣CREB/MYC regulatory pathway, which is critical in AML pathogenesis (see Fig. 1). Our findings also highlight potential distinct genetic/epigenetic mechanisms underlying de novo AML and MDS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.