Patent Publication Number: US-2022211669-A1

Title: Methods, Agents, and Compositions for the Treatment of Acute Myeloid Leukemia

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Application Ser. No. 62/490,184, filed Apr. 26, 2017, which application is hereby incorporated by reference in its entirety. 
    
    
     GOVERNMENT RIGHTS 
     This invention was made with government support under R01 CA-211614, R01 CA-178454, and R01 CA-182528 awarded by the National Institutes of Health. The Government has certain rights in the invention. 
    
    
     TECHNICAL FIELD 
     This disclosure relates to the field of cancer therapy. More specifically, this disclosure relates to benzodioxole compounds and their compositions and methods of use in the treatment of cancers associated with over-expression of Ten-eleven translocation 1 (TET1), particularly acute myeloid leukemia (AML). 
     BACKGROUND 
     Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies. Despite the improved risk stratifications and treatment-adapted strategies, with standard chemotherapies, still only 35%-40% of younger (aged &lt;60) and 5%-15% of older (aged ≥60) patients with AML can survive over 5 years. Many AML subtypes, such as the MLL-rearranged AMLs, are often associated with unfavorable outcome. Further, current treatment frequently involves intensive post-remission treatment with multiple cycles of high-dose cytarabine (Ara-C), which impairs the quality of life of the patients. While the incidence of AML is continually rising due to aging, most elderly 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. TET1, the founding member of the TET family, was first identified as a fusion partner of the MLL gene associated with t(10;11)(q22;q23) in AML. In contrast to the down-regulation and potential tumor-suppressor roles of all three TET genes reported in various types of solid tumors as well as the repression and tumor-suppressor role of TET2 observed in hematopoietic malignancies, the present inventors recently showed that TET1 was significantly up-regulated in MLL-rearranged AML and played an essential oncogenic role in the development of MLL-fusion-induced leukemia. An independent study by Zhao et al. confirmed the essential oncogenic role of Tet1 in the development of myeloid malignancies (Zhao, et al.,  Combined Loss of Tet 1  and Tet 2  Promotes B Cell, but Not Myeloid Malignancies, in Mice, Cell Rep  13: 1692-1704 (2015)). Thus, given the fact that knockout of Tet1 expression shows only very minor effects on normal development including hematopoiesis, TET1 is an attractive therapeutic target for AML. A need remains for effective therapies for acute myeloid leukemia (AML). 
     SUMMARY 
     Accordingly, through a series of in vitro drug screening and in vivo preclinical animal model studies, a class of benzodioxole chemical compounds has been identified as potent inhibitors that significantly and selectively suppress the viability of AML cells with high level of TET1 expression (i.e., TET1-high AML cells) and dramatically repress the progression of TET1-high AML in mice. These compounds directly bind STAT3/5 as STAT inhibitors and thereby suppress TET1 transcription and TET1 signaling, leading to potent anti-leukemic effects. 
     In one embodiment, a method of treating acute myeloid leukemia is provided, the method comprising administering to a subject in need thereof an effective amount of a compound, or a pharmaceutically acceptable salt thereof, having the formula: 
     
       
         
         
             
             
         
       
     
     wherein: 
     R 1 , R 2 , R 3 , R 4 , and R 5  are each independently selected from the group consisting of H, hydroxyl, alkyl, alkoxy, amine, halo, and trifluoromethyl, and wherein any two adjacent moieties of R 1 , R 2 , R 3 , R 4 , and R 5  may come together to form a heterocyclic ring; 
     R 6  is H or hydroxyl; and 
     R 7  is selected from H, 
     
       
         
         
             
             
         
       
     
     wherein R 8  is C or O. 
     In another embodiment, a pharmaceutical composition is provided, comprising (a) an effective amount of a Formula I compound, or a pharmaceutically acceptable salt thereof, and (b) at least one pharmaceutically acceptable carrier. 
     These and other objects, features, embodiments, and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . NSC-311068 and NSC-370284 suppress the viability of AML cells with high TET1 level. (a-b) TET1-high AML cell lines including MONOMAC-6, THP-1, KOCL-48, and KASUMI-1, along with a TET1-low control cell line (i.e., NB4), were treated with NSC-311068 (a) or NSC-370284 (b) at indicated doses (0, 50, 200, 500 nM). Cell viability was analyzed by MTS 48 hours post-treatment. (c) Repression of TET1 expression by NSC-311068 and NSC-370284 in AML cell lines. Cells were treated with DMSO, or 300 nM NSC-311068 or NSC-370284. TET1 expression levels were detected by qPCR 48 hrs post-treatment. (d) NSC-311068 and NSC-370284 (both at 300 nM) repressed global 5hmC level in THP-1 (left panels) and MONOMAC-6 (right panels) cells. (e,f) MONOMAC-6, THP-1 and KOCL-48 cells were treated with DMSO, or 25 nM NSC-311068 or NSC-370284. TET1 expression levels (e) and cell viability (f) were detected 24 hrs post-treatment. *, P&lt;0.05, two-tailed t-test. MM6, MONOMAC-6. Error bar indicates SD of triplicate experiments. 
         FIG. 2 . Therapeutic effects of NSC-311068 and NSC-370284 in AML in vivo. (a) NSC-311068 or NSC-370284 administration inhibits MLL-AF9-AML. The secondary BMT recipient mice were transplanted with leukemic BM blast cells collected from primary MLL-AF9 AML mice. Upon the onset of leukemia, the recipient mice were treated with DMSO (control; n=5), 2.5 mg/kg NSC-311068 (n=5) or NSC-370284 (n=7), i.p., once per day, for 10 days. Kaplan-Meier curves are shown. The P values were determined by log-rank test. (b) Wright-Giemsa staining of mouse peripheral blood (PB) and BM, or hematoxylin and eosin (H&amp;E) staining of mouse spleen and liver of the treated or control leukemic mice. (c,d) Tet1/2/3 gene expression levels (c) or Tet1 protein level (d) in BM blast cells of the treated or control leukemic mice. *, P&lt;0.05, two-tailed t-test. Error bar indicates SD of triplicate experiments. (e) Effects of NSC-311068 and NSC-370284 on the AE9a-AML mouse model. The secondary BMT recipient mice were transplanted with leukemic BM blast cells collected from the primary AE9a-AML mice. Upon the onset of leukemia, the recipient mice were treated with DMSO (control; n=5), 2.5 mg/kg NSC-311068 (n=6) or NSC-370284 (n=6), i.p., once per day, for 10 days. Kaplan-Meier curves are shown. The P values were determined by log-rank test. 
         FIG. 3 . STAT3 and STAT5 are potential direct targets of NSC-370284 in AML. (a) A top network identified with IPA involving all the 14 genes carrying recurrent mutations in THP-1 NSC-370284-resistant clones. Genes with mutations are labeled in bold font. (b) Knockdown of STAT3 and/or STAT5 reduces TET1 level. MONOMAC-6, THP-1 and NB4 cells were electroporated with control siRNA (siNC), siSTAT3, siSTAT5 or a combination of siSTAT3 and siSTAT5. TET1 expression level was detected by qPCR 48 hrs post-transfection. (c) Four genomic sites designed for ChIP-qPCR analysis to identify potential binding sites of STAT3 and STAT5 on TET1 promoter and other regions. (d-h) MONOMAC-6 cells (d-g) were treated with DMSO control or 500 nM NSC-370284. ChIP-qPCR assay was carried out 48 hrs after drug treatment. Enrichment of STAT3, STAT5, or IgG at the TET1 promoter region and other regions are shown. NB4 cells (h) were applied as a negative control. (i) Predicted binding of DNA (upper panel) or NSC-370284 (lower panel) with the DNA-binding domain (DBD) that is conserved between STAT3 and STAT5 proteins, from docking study on PDB ID 1bg1 using MolSoft ICM. (j) The association between STAT3 and NSC-370284 as determined with NMR chemical shift perturbation (CSP). Complex formation with compound 370284 induced extensive CSPs at the Be residues of STAT3 at 1:2 of protein:ligand molar ratio (red peaks: free STAT3; green peaks: STAT3-NSC-370284 complex). The CSP occurs at residues adjacent to the DNA-binding site (I464), and residues at or near the DBD. (k) NSC-370284 suppresses the binding between STAT3 and TET1 CpG island, as determined through EMSA. *, P&lt;0.05; **, P&lt;0.01, two-tailed t-test. Error bar indicates SD of triplicate experiments. 
         FIG. 4 . Effects of UC-514321, a structural analog of NSC-370284 in treating AMLs. (a) Structures of NSC-370284 and UC-514321. (b-e) Effects of NSC-370284, UC-514321 and other JAK/STAT pathway inhibitors, i.e., Pacritinib, KW-2449, Stattic and sc-355979, on the viability of AML cell lines MONOMAC-6 (b), THP-1 (c), KASUMI1 (d) and NB4 (e). Cells were treated with drugs at indicated doses. Cell viability was detected by MTS 48 hrs post-treatment. Error bar indicates SD of triplicate experiments. (f,g) Enhanced therapeutic effect of UC-514321, relative to NSC-370284, in treating TET1-high AMLs in vivo. Secondary BMT recipient mice were transplanted with primary leukemic BM cells with MLL-AF9 (f) or AAML-ETO9a (g). Upon the onset of leukemia, the recipient mice were treated with DMSO (control) (n=5), 2.5 mg/kg NSC-370284 (n=6) or UC-514321 (n=6), i.p., once per day, for 10 days. Kaplan-Meier curves are shown. The P values were determined by log-rank test. (h,i) Therapeutic effect of NSC-370284 and UC-514321 in treating MLL-AF10 AML (h) and FLT3-ITD/NPM1 mut  AML (i). Secondary BMT recipient mice were transplanted with primary leukemic BM cells with MLL-AF10 or FLT3-ITD/NPM1 mut . Upon the onset of leukemia, the recipient mice were treated with DMSO (control) (n=6 for each model), 2.5 mg/kg NSC-370284 (n=5) or UC-514321 (n=5), i.p., once per day, for 10 days. Kaplan-Meier curves are shown. The P values were determined by log-rank test. (j) Wright-Giemsa staining of mouse PB and BM, or H&amp;E staining of mouse spleen and liver of MLL-AF9 AML secondary BMT recipients with or without drug treatment. 
         FIG. 5 . NSC-370284 and UC-514321 function as TET1-transcription inhibitors in TET1-high AMLs and their anti-leukemic effects are TET1-dependent. (a) NSC-370284 and UC-514321 inhibit the transcription of TET1, but not TET2 or TET3. MONOMAC-6 cells were treated with DMSO control, 500 nM NSC-370284 or UC-514321 for 48 hrs. Gene expression levels are shown. (b) Effects of NSC-370284 and UC-514321 in downstream gene targets of TET1. (c) NSC-370284 and UC-514321 repressed global 5hmC level in MONOMAC-6 cells. (d) The association between STAT3 and UC-514321 as determined with NMR CSPs. (e-h) MONOMAC-6 cells were treated with DMSO control, 500 nM NSC-370284 or UC-514321. ChIP-qPCR assay was carried out 48 hrs after drug treatment. Enrichment of STAT3, STAT5, or IgG at the TET1 promoter region and other regions are shown. (i-1) Functions of NSC-370284 and UC-514321 depend on Tet1 expression. BM progenitor cells of wild-type or Tet1 −/−  mice were retrovirally transduced with MLL-AF9. Infected cells were treated with NSC-370284, UC-514321 and other JAK/STAT pathway inhibitors, i.e., Pacritinib, KW-2449, Stattic and sc-355979, at indicated doses (i,j) or at a particular dose (i.e., 500 nM; k,l) for 48 hrs. Relative cell viabilities are shown. (m) Cre-Tet1 fl/fl  mouse BM progenitor cells were retrovirally transduced with MLL-AF9. Transduced cells were induced with polyLC for 7 days, and then treated with 500 nM NSC-370284, UC-514321, or DMSO control for 48 hrs. Relative cell viability is shown. (n-o) THP-1 (n) and MONOMAC-6 (o) cells were infected with lentivirus of pLenti-puro vector-based Tet1 construct. Cells with or without doxycyclin inducing were treated with 250 nM NSC-370284, UC-514321 or DMSO control for 24 hrs. Relative cell viability is shown. *, P&lt;0.05; **, P&lt;0.01, two-tailed t-test. Error bar indicates SD of triplicate experiments. 
         FIG. 6 . Synergistic effect of NSC-370284 or UC-514321 with daunorubicin (DNR) and Ara-C in treating TET1-high AMLs in vitro and in vivo. (a) NSC-370284-resistant AML cells are sensitive to DNR. Three of the THP-1 NSC-370284-resistant clones (DRC-1-3) and the parental control were treated with DNR at indicated doses. Cell viability was tested 48 hours after the treatments. (b,c) THP-1 (b) and Kasumi-1 (c) cells were treated with DMSO (Ctrl), 25 nM NSC-370284 (upper panels) or UC-514321 (lower panels), and/or 100 nM DNR. Relative cell viability at 48 hours post drug treatment is shown. Error bar indicates SD of triplicate experiments. (d) Synergistic therapy of NSC-370284 or UC-514321 in combination with standard chemotherapy. Secondary BMT recipient mice were transplanted with primary MLL-AF9 leukemic BM cells. Upon the onset of leukemia, the mice were treated with DMSO (control), “5+3” regimen alone (i.e., a daily dose of 50 mg/kg Ara-C for five days along with a daily dose of 3 mg/kg DNR during the first three days of Ara-C treatment), or in combination with 10 days&#39; NSC-370284 or UC-514321 treatment, 2.5 mg/kg, i.p., once per day. 5-9 mice were included in each group. Kaplan-Meier curves are shown. The P values were determined by log-rank test. (e) Wright-Giemsa staining of mouse PB and BM, or H&amp;E staining of mouse spleen and liver of MLL-AF9 secondary BMT recipients treated with “5+3” alone, or combinational therapy. 
         FIG. 7 . Expression pattern of TET1 in TCGA database. TET1 gene expression levels were analyzed through cBioPortal website. 
         FIG. 8 . Cell viability screening of the top 20 chemical compounds that showed positive correlation between drug response and TET1 level in NCI-60 panel collection. MONOMAC-6 (MMC6), THP-1, KOCL-48 and KASUMI-1 cells were treated with the 20 top candidate chemical compounds individually at indicated doses for 48 hrs. Heatmap (a) and dose curves (b-e) are shown. 
         FIG. 9 . High expression and oncogenic role of TET1 in t(8;21) AML. (a-c) Comparison of TET1 expression between t(8;21) AML and other subtypes of AML or normal control (NC) samples. (a) In an in-house 109-sample dataset, including 30 t(8;21), 12 MLL-rearranged (MLL), 27 inv(16), and 31 t(15;17) AML, along with 9 normal control (NC) samples. (b) In the Netherlands 518-sample dataset (GSE14468), including 38 t(8;21), 19MLL-rearranged, 42 inv(16), 25 t(15;17), 214 normal karyotype (NK) and 180 other AML samples. (c) In the TCGA 179-sample dataset, including 7 t(8;21), 11 MLL-rearranged, 11 inv(16), 16 t(15;17), 75 normal karyotype (NK) and 59 other AML samples. Two-tailed t-test was used to calculate the P values. Bars show the median values. (d) Tet1 knockout inhibits t(8;21) fusion-induced cell transformation. Colony-forming/replating assays of wild-type or Tet1 −/−  mouse BM progenitor cells transduced with MSCV-PIG-AE (AE), or MSCV-PIG-AE9a (AE9a). Wild-type cells transduced with MSCV-PIG serve as a negative control. **, P&lt;0.01, two-tailed t-test. 
         FIG. 10 . NSC-370284 regulates TET1 expression through targeting STAT3/5. (a) THP-1 parental cells or representative drug-resistant clones (i.e., DRC-1, 2, 3) were treated with NSC-370284 at indicated doses for 48 hrs. Relative cell viabilities are shown. (b) THP-1 parental cells or drug resistant clones were treated with DMSO control or 500 nM NSC-370284 for 48 hrs. Levels of TET1 transcripts are shown. (c) Relative viability of MLL-AF9-AML cells transduced with MSCV-PIG-JAK1 A839G  mutant (Mut) or MSCV-PIG vector (ctrl) and then treated with DMSO or NSC-370284 (50 nM) for 48 hours. (d) Relative viability of THP-1 cells transduced with pLJM1-MSH3 V600I  or pLJM1-EGFP control and then treated with DMSO or NSC-370284 (50 nM) for 48 hours. (e-h) MONOMAC-6, THP-1 and NB4 cells were transfected with control siRNA (siNC), siSTAT3, or siSTAT5. Expression levels of STAT5 (e), STAT3 (f), TET2 (g) and TET3 (h) were detected by qPCR 48 hrs post-transfection. (i) THP-1 cells were transfected with control siRNA (siNC), siTET1, siJAK1 or siSTAT5. Gene expression levels were detected by qPCR 48 hrs post-transfection. MONOMAC-6 cells were treated with DMSO control or 500 nM NSC-370284. (j) ChIP-qPCR assay was carried out 48 hrs after drug treatment. Enrichment of TET1 or IgG at the JAK1 promoter region and other regions are shown. (k) MONOMAC-6 cells were treated with 250 nM NSC-370284 for 0 min, 15 min, 30 min, 2 hrs, and 24 hrs. Protein levels were detected thereafter. (l,m) THP-1 cells were treated with DMSO or 25 nM NSC-370284 for 24 hrs. Expression levels of TET1, HIF2a, IL2RA and FRA2 (1) were detected through qPCR. The enrichment of STAT5 on the promoters of TET1 (Site 2; CpG), HIF2α, IL2RA and FRA2 (m) was determined through ChIP-qPCR assays. *, P&lt;0.05; **, P&lt;0.01, two-tailed t-test. Error bar indicates SD of triplicate experiments. 
         FIG. 11 . Effects of 30 analogues of NSC-370284 on the viability of MONOMAC-6 cells. MONOMAC-6 cells were treated with 30 candidate analogues individually at indicated doses for 48 hrs. Heatmap of relative cell viability (as compared with DMSO treated control group) is shown. 
         FIG. 12 . Comparison of 5hmC enrichment between NSC-370284 or UC-514321-treated AML cells and control (DMSO-treated) cells. (a) Comparison of 5hmC peak numbers across the whole genome in ML-2 AML cells treated with NSC-370284 or UC-514321, or DMSO (control). 5hmC peak counts at different IDR cutoffs were shown. (b) Read density across 5hmC enriched regions. A unified catalog of 5hmC enriched regions was generated. Shown are heatmaps rank-ordered by reads per kilobase of exon model per million mapped reads (RPKM) of 5hmC-Seal samples minus that of the input samples. (c) Read density across all RefSeq genes. Shown are heatmaps of 5hmC read density changes across all RefSeq gene loci and 5 kb flanking regions (minus input values). 
         FIG. 13 . Unlike those of NSC-370284 and UC-514321, the effects of a set of JAK/STAT inhibitors are less TET1-dependent. (a-d) BM progenitor cells of wild-type or Tet1 −/−  mice were retrovirally transduced with MLL-AF9. Infected cells were treated with 200 nM JAK/STAT inhibitors individually, including Pacritinib (a), KW-2449 (b), Stattic (c) or sc-355979 (d), for 48 hrs. Relative cell viabilities are shown. (e) Cre-Tet1 fl/fl  mouse BM progenitor cells were retrovirally transduced with MLL-AF9. Transduced cells were induced with polyLC for 7 days, and then treated with 500 nM NSC-370284, UC-514321, or DMSO control for 48 hrs. Cell viabilities (normalized to non-induced Cre-Tet1 fl/fl  treated with DMSO control) are shown. ′K, P&lt;0.05; ′K′K, P&lt;0.01, two-tailed t-test 
         FIG. 14 . Comparison of effects of NSC-370284 and UC-514321 between AML cells and normal hematopoietic stem/progenitor cells (HSPCs; herein, c-Kit +  BM progenitor cells). BM cells of leukemic mice with MLL-AF9 fusion (MA9-AML) or FLT3-ITD/NPM1 mut , and c-Kit +  HSPCs from a pool of 6 healthy donor mice were cultured in liquid medium. Cells were treated with 50 nM NSC-370284, UC-514321, or DMSO as control. MTS (a), apoptosis assays (b) and qPCR (c) were conducted 3 days after cell seeding or drug treatments. Error bar indicates SD of triplicate experiments. 
         FIG. 15 . Acute toxicity profiling of NSC-370284 and UC-514321. Wild-type mice (n=5 for each group) were injected with DMSO (control), 2.5 mg/kg NSC-370284 or UC-514321, i.p., once per day, for 10 days. 24 hrs after the last administration, body weights (a), weights of spleens (b), livers (c), total PB WBC counts (d), BM Mac1 + Gr1 +  (e), Mac1 + Gr1 −  (f), Mac1 − Gr1 +  (g), and c-Kit +  (h) populations were analyzed. 
         FIG. 16 . Long-term toxicity profiling of NSC-370284 and UC-514321. Wild-type C57BL/6 mice (n=5 for each group) were injected with DMSO (control), 2.5 mg/kg NSC-370284 or UC-514321, i.p., once per day, for 10 days. 200 days after the last administration, body weights (a), weights of spleens (b), livers (c), total PB WBC counts (d), BM Mac1 + Gr1 +  (e), Mac1 + Gr1 −  (f), Mac1 − Gr1 +  (g), and c-Kit +  (h) populations were analyzed. 
         FIG. 17 . Blood concentration of UC-514321 after intraperitoneal injection. Error bar indicates SD of triplicate experiments. 
         FIG. 18 . Effects of standard chemotherapy reagents on the cell viability of THP-1 NSC-370284-resistant clones. Three representative THP-1 NSC-370284-resistant clones (DRC-1-3) and the parental control were treated with cytarabine (AraC) (a), all-trans retinoic acid (ATRA) (b), azacytidine (AZA) (c) and decitabine (DAC) (d) at indicated doses. Cell viability was tested 48 hours after the treatments. Error bar indicates SD of triplicate experiments. 
     
    
    
     DETAILED DESCRIPTION 
     The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided herein. 
     While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently-disclosed subject matter belongs. 
     Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter. 
     As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method. 
     It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. 
     An “effective amount,” as used herein, refers to an amount of a substance (e.g., a therapeutic compound and/or composition) that elicits a desired biological response. In some embodiments, an effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay and/or alleviate one or more symptoms of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of; reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. Furthermore, an effective amount may be administered via a single dose or via multiple doses within a treatment regimen. In some embodiments, individual doses or compositions are considered to contain an effective amount when they contain an amount effective as a dose in the context of a treatment regimen. Those of ordinary skill in the art will appreciate that a dose or amount may be considered to be effective if it is or has been demonstrated to show statistically significant effectiveness when administered to a population of patients; a particular result need not be achieved in a particular individual patient in order for an amount to be considered to be effective as described herein. 
     As used herein, the term “alkyl” refers to a straight or branched saturated aliphatic hydrocarbon group having a single radical and 1-12 carbon atoms (i.e., C1-C12 alkyl). Non-limiting examples of alkyl groups include methyl, propyl, isopropyl, butyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like. A branched alkyl means that one or more alkyl groups such as methyl, ethyl, or propyl replace one or both hydrogens in a —CH2— group of a linear alkyl chain. In certain embodiments, alkyl is a C1-C6 alkyl or a C1-C4 alkyl. In other embodiments, alkyl is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. 
     Alkyl groups can optionally be unsubstituted or substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term “alkyl group substituent” includes but is not limited to alkyl, substituted alkyl, halo, hydroxyl, carboxyl, oxo, and the like. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen or alkyl. 
     “Alkoxy” refers to an alkyl-O— group wherein alkyl is as previously described. The term “alkoxy” as used herein can refer to, for example, one or more of methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, pentoxy, and the like. In certain embodiments, alkoxy is a C1-C12 alkoxy, a C1-C6 alkoxy, or a C1-C4 alkoxy. 
     The term “amine,” as used herein, includes primary, secondary, and tertiary amines. 
     The terms “halo,” as used herein, refers to fluoro, chloro, bromo, and iodo groups. 
     “Heterocyclic” means cyclic carbon rings having one or more heteroatoms (atoms other than carbon) in the ring. The ring may be saturated, partially saturated, and unsaturated, and the heteroatoms may be selected from the group consisting of nitrogen, sulfur and oxygen. Examples of saturated heterocyclic rings include saturated 3 to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, such as pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl; saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms, such as tetrahydrofuran, tetrahydropyranyl, dioxolane, and dioxane; saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as morpholinyl; saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, such as thiazolidinyl. Examples of partially saturated heterocyclic rings include furanyl, pyridinyl, imidazolyl, thiophenyl, pyrrolyl, pyrimidinyl, azole, dioxole, and the like. 
     When the term “independently selected” is used, the substituents being referred to (e.g., R groups, such as groups R 1  and R 2 ), can be identical or different. For example, both R 1  and R 2  can be the same substituent, or R 1  and R 2  can each be different substituents selected from a specified group. 
     The term “pharmaceutically acceptable excipient,” as used herein, means any physiologically inert, pharmacologically inactive material known to one skilled in the art, which is compatible with the physical and chemical characteristics of the particular active agent selected for use. Pharmaceutically acceptable excipients include, but are not limited to, polymers, resins, plasticizers, fillers, lubricants, diluents, binders, disintegrants, solvents, co-solvents, buffer systems, surfactants, preservatives, sweetening agents, flavoring agents, pharmaceutical grade dyes or pigments, and viscosity agents. 
     Conditions characterized by over-expression of TET1 include conditions associated with higher levels of TET1 messenger RNA or TET1 protein. In some embodiments, higher TET1 expression is induced by oncogenic signaling, such as STAT3/5-associated signaling. In certain embodiments, conditions characterized by over-expression of TET1 include hematopoietic malignancies having higher TET1 expression compared to normal control cells. 
     The term “hematopoietic malignancy,” as used herein, refers to malignancies of the hematopoietic and lymphoid tissues. Examples include, but are not limited to, lymphomas, leukemias, myeloproliferative neoplasms, plasma cell dyscrasias, histiocytic tumors, and dendritic cell neoplasms. In certain embodiments, hematopoietic malignancies include leukemias such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), and the like. In other embodiments, hematopoietic malignancies include lymphomas such as Hodgkin&#39;s lymphomas, non-Hodgkin&#39;s lymphomas, and the like. In certain embodiments, hematopoietic malignancies include myelomas, such as multiple myeloma. In a specific embodiment, the hematopoietic malignancy is AML, including TET1-high AML. 
     The terms “over-express” or “over-expressed” as used herein refer to a gene product which is expressed at levels greater than normal endogenous expression for that gene product. 
     TET1-high acute myeloid leukemia (TET1-high AML) refers to AML wherein leukemic cells exhibit elevated or high level expression of TET1 relative to normal hematopoietic cells. 
     The terms “treat,” “treatment,” and “treating,” as used herein, refer to a method of alleviating or abrogating a disease, disorder, and/or symptoms thereof in a subject, including a mammal. In certain embodiments, the subject is a human subject. 
     An “anti-cancer agent,” as used herein, refers to a chemical compound or chemotherapeutic agent useful in the treatment of cancer. In certain embodiments, an anti-cancer agent is a first line chemotherapeutic agent, or an agent generally regarded as the standard therapy or first-administered therapy for a given type of cancer. In certain embodiments, the second anti-cancer agent is a chemotherapeutic agent for the treatment of AML selected from the group consisting of cytarabine, cladribine, fludarabine, topotecan, doxorubicin, daunorubicin, epirubicin, idarubicin, decitabine, azacitidine, all-trans-retinoic acid, arsenic trioxide, gemtuzumab ozogamicin, midostaurin, nelarabine, clofarabine, dasatinib, imatinib, ponatinib, JQ1, methotrexate, corticosteroids, histamine dihydrochloride, interleukin 2, and combinations thereof. 
     Compounds and Methods of Treatment 
     DNA methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Through a series of data analysis and drug screening, a class of compounds that selectively suppresses TET1 transcription and 5-hydroxymethylcytosine (5hmC) modification is disclosed herein. These compounds effectively inhibit cell viability in AML with high level expression of TET1 (i.e., TET1-high AML), including AML carrying t(11q23)/MLL-rearrangements and t(8;21) AML. 
     While knockout of Tet1 expression shows only very minor effects on normal development including hematopoiesis, recent studies have demonstrated that TET1 plays a critical oncogenic role in AML through promoting expression of oncogenic targets (e.g. HOXA9, MEIS1 and PBX3, etc.) and repressing expression of tumor-suppressor targets (e.g., miR-22). Thus, targeting TET1 signaling is a promising therapeutic strategy to treat TET1-high AMLs. In order to target critical oncogenic proteins with catalytic activity, one of the most popular approaches is to interfere the catalytic activity of oncogenic proteins, such as FLT3 inhibitor Quizartinib that represses the kinase activity of FLT3, and STAT inhibitor Stattic that blocks the dimerization of STAT3, etc. However, as shown in a number of clinical reports, treatments of catalytic activity inhibitors often result in aberrant up-regulation of the target oncoproteins or trigger gene mutations, which eventually leads to drug resistance. The discovery of the bromodomain and extra-terminal (BET) inhibitor JQ1 as an effective strategy to target c-Myc signaling suggested an alternative strategy of repressing the TET1 signaling instead of directly targeting the enzymatic activity of TET1. Any drugs that efficiently target the expression, i.e., transcription, translation, or degradation, of the oncogenes or oncogenic proteins could largely avoid drug resistance caused by target oncogene up-regulation or constitutively activated gene mutations. Moreover, it was reported previously that TET1 can recruit polycomb proteins to the promoter region of the mir-22 gene and suppress the primary transcription of this critical tumor-suppressor microRNA, and such transcriptional suppression is independent from TET1&#39;s enzymatic activity. Therefore, instead of seeking inhibitors targeting TET1 enzymatic activity directly that are unable to fully repress the function of TET1, inhibitors that suppress TET1 expression were selected in this study for treating AML. 
     Through correlation analysis of cell response to 20,602 chemical compounds and TET1 levels of in the NCI-60 collection of cancer cell samples, followed by MTS assays of top drug candidates in AML cells, two candidate chemical compounds (NSC-311068 and NSC-370284) were identified that each suppress AML cell viability and TET1 expression: 
     
       
         
         
             
             
         
       
     
     Importantly, both NSC-311068 and especially NSC-370284 showed remarkable therapeutic effects in curing AML in vivo. UC-514321, a structural analog of NSC-370284, exhibited a more potent anti-leukemic activity in vitro and in vivo than NSC-370284: 
     
       
         
         
             
             
         
       
     
     Mechanistically, the disclosed TET1-signaling inhibitors directly target STAT3/5, which are direct upstream regulators of TET1 transcription. Remarkably, compared to currently available JAK/STAT inhibitors (e.g., Pacritinib, KW-2449, Stattic, and sc-355979), the compounds (NSC-370284 and UC-514321) exhibit a much higher selectivity and also a higher efficacy in targeting TET1-high AML, which likely due to the unique property of our TET1-signaling inhibitor as they directly bind to the DNA-binding domain (DBD) of STAT3/5, interfere with the binding of STAT3/5 to TET1 promoter region, and thereby repress the transcription of TET1. Moreover, both NSC-370284 and UC-514321 exhibit a synergistic effect with daunorubicin in treating TET1-high AML cells in vitro and in vivo. Notably, NSC-370284-resistant THP-1 AML cells are even more sensitive to daunorubicin than parental THP-1 cells. Taken together, these findings highlight the therapeutic potential of targeting TET1, a key oncogenic epigenetic regulator related to DNA demethylation, in AML. The data also reveal that STAT3 and STAT5 are direct upstream regulators of TET1 and are suitable targets to suppress TET1 signaling. The data suggest that application of small-molecule compounds that selectively and effectively target the STAT/TET1 signaling, particularly in combination with standard chemotherapy agents, represents an effective novel therapeutic strategy for the treatment of TET1-high AML (including MLL-rearranged AML and t(8;21) AML), which accounts for approximately 30% of total AML cases. Moreover, these effective inhibitors can also be employed as tool compounds in both basic and translational research to selectively target the STAT/TET1 signaling axis and suppress 5hmC globally. 
     Structural analysis suggested a potential direct binding of NSC-370284 to the conserved DBD of STAT3 or STAT5. Such binding and the binding sites were identified by use of NMR chemical shift perturbation (CSP). Complex formation with compound NSC-370284 induced extensive CSPs at the isoleucine (Ile) residues of STAT3 at 1:2 of protein:ligand molar ratio. 
     Based on the structure of compound NSC-370284, structural analogs were explored using BioVia Pipeline Pilot (Version 8.5.0.200) against the University of Cincinnati Compound Library, a collection of approximately 360,000 compounds. The 30 most structurally similar compounds were selected to explore the Structure Activity Relationships (SAR) (Tables A and B). The Table A compounds disclosed herein share the core aryl amine benzodioxole scaffold with NSC-370284, varying primarily in the amine substituents and in the aryl substituents. 
     Accordingly, provided herein are methods of treating a subject suffering from a condition characterized by over-expression of TET1, which comprise administering to the subject an effective amount of a compound, or pharmaceutically acceptable salt thereof, having the Formula: 
     
       
         
         
             
             
         
       
     
     wherein: 
     R 1 , R 2 , R 3 , R 4 , and R 5  are each independently selected from the group consisting of H, hydroxyl, alkyl, alkoxy, amine, halo, and trifluoromethyl, and wherein any two adjacent moieties of R 1 , R 2 , R 3 , R 4 , and R 5  may come together to form a heterocyclic ring; 
     R 6  is H or hydroxyl; and 
     R 7  is selected from H, 
     
       
         
         
             
             
         
       
     
     wherein R is C or O. 
     In certain embodiments, the condition characterized by over-expression of TET1 is a hematopoietic malignancy. In a specific embodiment, the condition is acute myeloid leukemia (AML). In a very specific embodiment, the condition is TET1-high AML. 
     In some embodiments, alkyl comprises straight or branched chain unsubstituted or substituted C1-C12 alkyl groups. In other embodiments, alkyl comprises straight or branched chain unsubstituted or substituted C1-C6 alkyl groups. In other embodiments, alkyl comprises straight or branched chain unsubstituted or substituted C1-C4 alkyl groups. In specific embodiments, alkyl is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. 
     In some embodiments, alkoxy comprises straight or branched chain unsubstituted or substituted C1-C12 alkoxy groups. In other embodiments, alkoxy comprises straight or branched chain unsubstituted or substituted C1-C6 alkoxy groups. In other embodiments, alkyl comprises straight or branched chain unsubstituted or substituted C1-C4 alkoxy groups. In specific embodiments, alkoxy is selected from the group consisting of methoxy, ethoxy, propoxy, butoxy, and pentyloxy. In a very specific embodiment, alkoxy comprises methoxy and ethoxy. 
     In certain embodiments, R 1 , R 2 , R 3 , R 4 , and R 5  are each independently selected from H, hydroxyl, methyl, ethyl, methoxy, ethoxy, amine, halo, and trifluoromethyl. In certain embodiments, any two adjacent R 1 , R 2 , R 3 , R 4 , and R 5  may come together to form a heterocyclic ring. In such embodiments, the heterocyclic ring may be a 5- or 6-membered ring. In specific embodiments, the heterocyclic ring formed by any two adjacent R 1 , R 2 , R 3 , R 4 , and R 5  is selected from the group consisting of azole, dioxole, and dioxolane. 
     Suitable Formula I compounds for use in the methods and compositions disclosed herein include any of the analogs set forth in Table A, alone or in combination. In specific embodiments, the Formula I compound is 6-{[4-Hydroxy-3,5-bis(2-methyl-2-propanyl)phenyl](4-morpholinyl)methyl}-1,3-benzodioxol-5-ol (UC-514321) or 6-(1-Pyrrolidinyl(3,4,5-trimethoxyphenyl)methyl)-1,3-benzodioxol-5-ol (NSC-370284). 
     
       
         
           
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                 Formula I Compounds 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Lab  
                   
               
               
                   
                   
                 Reference 
                   
               
               
                 Com- 
                   
                 Number  
                   
               
               
                 pound 
                   
                 (NSC or  
                   
               
               
                 Num- 
                   
                 UC  
                 MW 
               
               
                 ber 
                 Structure 
                 number) 
                 Formula 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 765626 
                 365.3463 
               
               
                   
               
               
                   
                 6-{1-Pyrrolidinyl[4-  
                   
                   
               
               
                   
                 (trifluoromethyl)phenyl]methyl}-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5-ol 
                   
                   
               
               
                   
               
               
                 2 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 517451 
                 403.4257 
               
               
                   
               
               
                   
                 6-[4-Morpholinyl(3,4,5- 
                   
                   
               
               
                   
                 trimethoxyphenyl)methyl]-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5-ol 
                   
                   
               
               
                   
               
               
                 3 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472619 
                 359.3731 
               
               
                   
               
               
                   
                 6-[(2-Hydroxy-3-methoxyphenyl)(4- 
                   
                   
               
               
                   
                 morpholinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 4 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472616 
                 387.4263 
               
               
                   
               
               
                   
                 6-[1-Pyrrolidinyl(2,4,6- 
                   
                   
               
               
                   
                 trimethoxyphenyl)methyl]-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5-ol 
                   
                   
               
               
                   
               
               
                 5 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 517463 
                 356.4156 
               
               
                   
               
               
                   
                 6-((4-(Dimethylamino)phenyl)(4- 
                   
                   
               
               
                   
                 morpholinyl)methyl)-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 6 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472585 
                 343.3737 
               
               
                   
               
               
                   
                 6-[(2-Methoxyphenyl)(4- 
                   
                   
               
               
                   
                 morpholinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 7 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 943568 
                 379.3729 
               
               
                   
               
               
                   
                 6-{piperidino[4- 
                   
                   
               
               
                   
                 (trifluoromethyl)phenyl]methyl}-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5-ol 
                   
                   
               
               
                   
               
               
                 8 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472591 
                 373.3997 
               
               
                   
               
               
                   
                 6-((2,4-Dimethoxyphenyl)-4- 
                   
                   
               
               
                   
                 morpholinylmethyl)-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 9 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 514329 
                 327.3743 
               
               
                   
               
               
                   
                 2-[1,3-Benzodioxol-5-yl(4- 
                   
                   
               
               
                   
                 morpholinyl)methyl]-4-methylphenol 
                   
                   
               
               
                   
               
               
                 10 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472587 
                 373.3997 
               
               
                   
               
               
                   
                 6-[(2,3-Dimethoxyphenyl)(4- 
                   
                   
               
               
                   
                 morpholinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 11 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 943336 
                 381.3457 
               
               
                   
               
               
                   
                 6-{morpholino[4- 
                   
                   
               
               
                   
                 (trifluoromethyl)phenyl]methyl}-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5-ol 
                   
                   
               
               
                   
               
               
                 12 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 514341 
                 355.3844 
               
               
                   
               
               
                   
                 6-[1,3-Benzodioxol-5-yl(4- 
                   
                   
               
               
                   
                 morpholinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 13 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 514321 
                 441.5598 
               
               
                   
               
               
                   
                 6-{[4-Hydroxy-3,5-bis(2-methyl-2- 
                   
                   
               
               
                   
                 propanyl)phenyl](4- 
                   
                   
               
               
                   
                 morpholinyl)methyl}-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 14 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472606 
                 403.4257 
               
               
                   
               
               
                   
                 6-[4-Morpholinyl(2,3,4- 
                   
                   
               
               
                   
                 trimethoxyphenyl)methyl]-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5-ol 
                   
                   
               
               
                   
               
               
                 15 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 943337 
                 331.3382 
               
               
                   
               
               
                   
                 6-[(4-Fluorophenyl)(4- 
                   
                   
               
               
                   
                 morpholinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 16 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 514318 
                 340.4162 
               
               
                   
               
               
                   
                 6-{[4-(Dimethylamino)phenyl](1- 
                   
                   
               
               
                   
                 pyrrolidinyl)methyl}-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 17 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 514328 
                 318.3212 
               
               
                   
               
               
                   
                 6-(3,4,5-Trimethoxybenzyl)-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5-ol 
                   
                   
               
               
                   
               
               
                 18 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 491129 
                 354.4427 
               
               
                   
               
               
                   
                 6-{[4-(Dimethylamino)phenyl](1- 
                   
                   
               
               
                   
                 piperidinyl)methyl}-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 19 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472589 
                 329.3472 
               
               
                   
               
               
                   
                 6-((2-Hydroxyphenyl)(4- 
                   
                   
               
               
                   
                 morpholinyl)methyl)-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 20 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472610 
                 357.4003 
               
               
                   
               
               
                   
                 6-[(2,3-Dimethoxyphenyl)(1- 
                   
                   
               
               
                   
                 pyrrolidinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 21 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 514363 
                 357.3573 
               
               
                   
               
               
                   
                 6-[1,3-Benzodioxol-5-yl(4- 
                   
                   
               
               
                   
                 morpholinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 22 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472614 
                 343.3737 
               
               
                   
               
               
                   
                 6-[(2-Hydroxy-3-methoxyphenyl)(1- 
                   
                   
               
               
                   
                 pyrrolidinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 23 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472607 
                 357.4003 
               
               
                   
               
               
                   
                 6-[(2,4-Dimethoxyphenyl)(1- 
                   
                   
               
               
                   
                 pyrrolidinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 24 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 517453 
                 341.3579 
               
               
                   
               
               
                   
                 6-[1,3-Benzodioxol-5-yl(1- 
                   
                   
               
               
                   
                 pyrrolidinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 25 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472617 
                 327.3743 
               
               
                   
               
               
                   
                 6-[(2-Methoxyphenyl)(1- 
                   
                   
               
               
                   
                 pyrrolidinyl)methyl]-1,3-benzodioxol- 
                   
                   
               
               
                   
                 5-ol 
                   
                   
               
               
                   
               
               
                 26 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 472595 
                 371.4269 
               
               
                   
               
               
                   
                 6-[(2,4-Dimethoxyphenyl)(1- 
                   
                   
               
               
                   
                 piperidinyl)methyl]-1,3-benzodioxol-5- 
                   
                   
               
               
                   
                 ol 
                   
                   
               
               
                   
               
               
                 27 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 370284 
                   
               
               
                   
               
               
                   
                 6-(1-Pyrrolidinyl(3,4,5- 
                   
                   
               
               
                   
                 trimethoxyphenyl)methyl)-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5-ol 
               
               
                   
               
            
           
         
       
     
     Also provided herein are methods of treating a subject suffering from a condition characterized by over-expression of TET1, the method comprising administering to the subject an effective amount of a compound, or a pharmaceutically acceptable salt thereof, as set forth in Table B. 
     
       
         
           
               
             
               
                 TABLE B 
               
             
            
               
                   
               
               
                 Additional analogs of NSC-370284 
               
            
           
           
               
               
               
               
            
               
                 Com- 
                   
                   
                   
               
               
                 pound 
                   
                 Lab  
                   
               
               
                 Num- 
                   
                 Reference 
                 MW  
               
               
                 ber 
                 Structure 
                 Number 
                 Formula 
               
               
                   
               
               
                 28 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 514315 
                 416.3781 
               
               
                   
               
               
                   
                 4-Hydroxy-3-[(6-hydroxy-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5-yl)(3,4,5- 
                   
                   
               
               
                   
                 trimethoxyphenyl)methyl]-2(5H)- 
                   
                   
               
               
                   
                 furanone 
                   
                   
               
               
                   
               
               
                 29 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 517461 
                 441.5167 
               
               
                   
               
               
                 30 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 378752 
                 263.3321 
               
               
                   
               
               
                   
                 1-[(7-Methoxy-1,3- 
                   
                   
               
               
                   
                 benzodioxol-5- 
                   
                   
               
               
                   
                 yl)methyl]azepane 
                   
                   
               
               
                   
               
               
                 31 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 517447 
                 419.4682 
               
               
                   
               
               
                   
                 4,5-Dimethoxy-2-[4- 
                   
                   
               
               
                   
                 morpholinyl(3,4,5- 
                   
                   
               
               
                   
                 trimethoxyphenyl)methyl]phenol 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, the Formula I or Table B compounds disclosed herein are administered with a second anti-cancer agent to provide a synergistic or enhanced efficacy or inhibition of cancer cell growth. For example, the Formula I or Table B compounds of the present disclosure can be administered in combination with a chemotherapeutic agent including, for example, cytarabine, cladribine, fludarabine, topotecan, doxorubicin, daunorubicin, epirubicin, idarubicin, decitabine, azacitidine, all-trans-retinoic acid, arsenic trioxide, gemtuzumab ozogamicin, midostaurin, nelarabine, clofarabine, dasatinib, imatinib, ponatinib, JQ1, methotrexate, corticosteroids, histamine dihydrochloride, interleukin 2, and combinations thereof. In some embodiments, the Formula I or Table B compound is co-administered with the second anti-cancer agent. In certain embodiments, “co-administered” means a Formula I or Table B compound is administered together with a second anti-cancer agent in the same unit dosage. In other embodiments, “co-administered” means a Formula I or Table B compound and a second anti-cancer agent are administered in separate dosage forms, concurrently or consecutively. 
     Pharmaceutical Compositions 
     Provided herein are compositions comprising an effective amount of a compound according to Formula I or Table B, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 
     It will be appreciated that the Formula I or Table B compounds disclosed herein can be administered to a patient or subject either alone or as part of a pharmaceutical composition. The Formula I or Table B compounds can be administered to patients either orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), intracistemally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments, or drops), or as a buccal or nasal spray. 
     Compositions comprising the Formula I or Table B compounds of the present disclosure suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. 
     These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. 
     Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. 
     Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like. 
     Solid dosage forms such as tablets, dragées, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients. 
     Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances, and the like. 
     Besides such inert diluents, the compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. 
     Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like. 
     Compositions for rectal or vaginal administrations are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component. 
     Dosage forms for topical administration of Formula I or Table B compounds of the present invention include ointments, powders, sprays, and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention. 
     In addition, the Formula I or Table B compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present disclosure. Enantiomers and racemates of the Formula I or Table B compounds are also suitable for use in the compositions and methods disclosed herein. 
     The Formula I or Table B compounds of the present invention can be administered to a patient at dosage levels in the range of about 1.5 mg to about 150 mg per day; it is also possible to administer larger amounts, such as from about 150 mg to 1 g per day. A unit dosage form of Formula I or Table B compounds is an amount which would be administered as a single dose. For a normal human adult having a body weight of about 70 kilograms, a dosage in the range of about 0.2 mg to about 2.0 mg per kilogram of body weight per day is suitable. The specific dosage used, however, can vary. For example, the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the compound being used. The determination of optimum dosages for a particular patient is well known to those skilled in the art. The Formula I or Table B compounds of the present invention can be given in single and/or multiple dosages. 
     The Formula I or Table B compounds disclosed herein are synthesized using standard organic synthetic techniques known to the ordinary skilled artisan. 
     EXAMPLES 
     The following detailed methodology and materials are set forth to support and illustrate particular aspects and embodiments of the invention, and should not be construed as limiting the scope thereof. 
     Example 1. Chemical Compounds NSC-311068 and NSC-370284 Effectively Inhibit the Viability of TET1-High AML Cells 
     The high expression and oncogenic role of TET1 in AML has been previously reported. In fact, high expression of TET1 was found not only in AML, but also in various tumors including uterine cancer, glioma, etc., and especially, in testicular germ cell malignancies ( FIG. 7 ). This indicates potential oncogenic role of TET1 in many cancers where TET1 expression level is relatively high. 
     In order to identify chemical compounds that may target TET1 signaling, the drug-sensitivity/gene expression database of a total of 20,602 chemical compounds in the NCI-60 collection of cancer cell samples was searched. 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&gt;0.2; P&lt;0.05). The top 120 with the highest r values were selected and their effects on cell viability of a TET1-high AML cell line, i.e., MONOMAC-6/t(9;11) were tested. Then, the top 20 showing the most significant inhibitory effects (Table 1) were further tested in three other TET1-high AML cell lines including THP-1/t(9;11), KOCL-48/t(4;11) and KASUMI-1/t(8;21) AML cells, along with MONOMAC-6 cells as a positive control ( FIG. 8 a - e    and Table 2). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Top 20 candidate chemical compounds that showed positive  
               
               
                 correlation in drug response and TET1 level in 
               
               
                 NCI-60 collection 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Lab 
                   
               
               
                   
                 Compound 
                 Reference 
                   
               
               
                   
                 Number 
                 Number 
                 Correlation 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                   
                 652026 
                 0.59 
               
               
                   
                   
                 633268 
                 0.551 
               
               
                   
                   
                 600290 
                 0.538 
               
               
                   
                   
                 82339 
                 0.535 
               
               
                   
                   
                 534 
                 0.532 
               
               
                   
                   
                 624167 
                 0.525 
               
               
                   
                   
                 633272 
                 0.525 
               
               
                   
                   
                 622600 
                 0.523 
               
               
                   
                   
                 628949 
                 0.512 
               
               
                   
                   
                 49512 
                 0.51 
               
               
                   
                   
                 622589 
                 0.508 
               
               
                   
                   
                 122291 
                 0.506 
               
               
                   
                   
                 635297 
                 0.506 
               
               
                   
                   
                 34757 
                 0.505 
               
               
                   
                   
                 625882 
                 0.502 
               
               
                   
                   
                 650738 
                 0.501 
               
               
                   
                   
                 634781 
                 0.5 
               
               
                   
                 27 
                 370284 
                 0.499 
               
               
                   
                   
                 667730 
                 0.499 
               
               
                   
                   
                 311068 
                 0.498 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
             
            
               
                 IC50 of the top 20 candidate chemical compounds in MONOMAC-6, THP-1, KOCL-48 and KASUMI-1 cells. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 652026 
                 633268 
                 600290 
                 82339 
                 534 
                 624167 
                 633272 
                 622600 
                 628949 
                 49512 
               
               
                   
               
               
                 MMC6 
                 6.88E+04 
                 5.36E−02 
                 2.21E+04 
                 2.05E+01 
                 5.03E+00 
                 7.35E+04 
                 7.90E−01 
                 7.35E+04 
                 2.53E−04 
                 7.46E−05 
               
               
                 THP-1 
                 1.09E+00 
                 1.92E−06 
                 8.42E−01 
                 6.09E−01 
                 7.70E−01 
                 N/A 
                 1.31E+00 
                 9.49E−04 
                 N/A 
                 8.30E−03 
               
               
                 KOCL-48 
                 5.09E+00 
                 5.79E−06 
                 1.36E+00 
                 1.30E+00 
                 1.23E+00 
                 6.85E−01 
                 3.28E−01 
                 1.10E−02 
                 1.63E−05 
                 9.34E−01 
               
               
                 KASUMI1 
                 3.68E−04 
                 4.83E+06 
                 2.00E−06 
                 2.33E−04 
                 6.08E−01 
                 N/A 
                 N/A 
                 5.79E−06 
                 2.47E+00 
                 1.56E+00 
               
               
                 Average 
                 1.72E+04 
                 1.21E+06 
                 5.53E+03 
                 5.61E+00 
                 1.91E+00 
                 3.68E+04 
                 8.10E−01 
                 1.84E+04 
                 8.24E−01 
                 6.26E−01 
               
               
                   
               
               
                   
                 622589 
                 122291 
                 635297 
                 34757 
                 625882 
                 650738 
                 634781 
                 370284 
                 667730 
                 311068 
               
               
                   
               
               
                 MMC6 
                 1.22E+00 
                 8.93E−01 
                 7.59E+00 
                 8.09E+01 
                 8.28E−01 
                 9.58E+00 
                 N/A 
                 3.85E−04 
                 N/A 
                 6.74E−01 
               
               
                 THP-1 
                 N/A 
                 N/A 
                 1.07E−02 
                 1.16E−01 
                 2.10E−04 
                 1.30E+00 
                 2.29E+00 
                 3.56E−04 
                 7.10E+00 
                 2.35E−04 
               
               
                 KOCL-48 
                 5.62E+00 
                 4.76E+00 
                 2.20E+00 
                 8.56E+00 
                 1.46E−01 
                 2.02E+00 
                 N/A 
                 3.82E−02 
                 1.30E+00 
                 7.26E−01 
               
               
                 KASUMI1 
                 2.46E−05 
                 3.81E−04 
                 4.16E−04 
                 2.38E−04 
                 N/A 
                 7.27E−05 
                 N/A 
                 4.93E−06 
                 4.93E+00 
                 4.20E−06 
               
               
                 Average 
                 2.28E+00 
                 1.89E+00 
                 2.47E+00 
                 2.24E+01 
                 3.25E−01 
                 3.23E+00 
                 2.30E+00 
                 9.75E−03 
                 4.45E+00 
                 3.50E−01 
               
               
                   
               
            
           
         
       
     
     It was found that TET1 is highly expressed not only in MLL-rearranged AML as reported previously, but also in AML carrying t(8;21); moreover, depletion of Tet1 expression also significantly inhibited t(8;21) fusion gene-induced colony-forming/replating capacity of mouse bone marrow (BM) progenitor cells (see  FIG. 9 ). Results showed that NSC-311068 and NSC-370284 exhibited the most significant effects in inhibiting cell viability of all four TET1-high AML cell lines, whereas showing no significant inhibition on viability of NB4/t(15;17) AML cells, a control cell line with very low level of TET1 expression ( FIG. 1 a,b   ). In the NCI-60 collection, cell lines with relatively higher TET1 expression levels showed more obvious positive correlation between TET1 expression level and activity of both NSC-311068 and NSC-370284, compared to that across the entire NCI-60 panel, whereas cell lines with relatively lower TET1 expression levels exhibited no obvious positive correlation (NSC-311068) or even negative correlation (NSC-370284) ( FIG. 8 c - d   ). In TET1-high AML cells, NSC-311068 and 370284 significantly repressed the level of TET1 expression ( FIG. 1 c   ), as well as the global 5hmC level ( FIG. 1 d   ). In order to rule out the possibility of non-specific toxicity, the dose of NSC-311068 and NSC-370284 was reduced to 25 nM, and gene expression and cell viability 24 hours after treatment were tested. The low dose, short-term treatments again resulted in a significant down-regulation of TET1 transcription, accompanied with a very minor decrease in the viability of MONOMAC-6, THP-1 and KOCL-48 cells ( FIG. 1 e - f   ). Thus, it is unlikely that the inhibitory effects of NSC-311068 and NSC-370284 on TET1 expression were due to nonspecific toxicity. 
     Example 2. NSC-311068 and NSC-370284 Suppress AML Progression In Vivo 
     The potential in vivo therapeutic effects of NSC-311068 and 370284 were then tested with the MLL-AF9 AML model. NSC-311068 and especially 370284 treatments significantly inhibited MLL-AF9-induced AML in secondary BM transplantation (BMT) recipient mice, by prolonging the median survival from 49 days (control) to 94 (NSC-311068) or &gt;200 (NSC-370284) days ( FIG. 2 a   ). Notably, 57% (4 out of 7) of the NSC-370284 treated mice were cured, as the pathological morphologies in peripheral blood (PB), BM, spleen and liver tissues all turned to normal ( FIG. 2 b   ). The in vivo down-regulation of Tet1 expression by the compounds at both RNA and protein levels was validated by qPCR ( FIG. 2 c   ) and Western blotting ( FIG. 2 d   ), respectively. In another AML model induced by AML-ETO9a (AE9a) 28 , NSC-311068 and NSC-370284 also exhibited remarkable therapeutic effects, with an elongated median survival from 46 days (control) to 95 (NSC-311068) and 122 (NSC-370284) days, respectively ( FIG. 2 e   ). Interestingly, NSC-370284 has been reported previously as an analog of the natural product podophyllotoxin (PPT) that was associated with anti-leukemic activity. Given the better therapeutic effect of NSC-370284 in vivo ( FIG. 2   a,b,e ), NSC-370284 was selected for further studies. 
     Example 3. NSC-370284 Suppresses TET1 Expression Through Targeting STAT3/5 
     To decipher the molecular mechanism by which NSC-370284 represses TET1 expression, the strategy developed by Kapoor and colleagues ENREF 31 was adapted to identify direct target protein(s) of NSC-370284. Briefly, multiple-drug-resistant clones were established and transcriptome sequencing was conducted to find mutations in each clone; the assumption was that the critical components of the signaling of the drug target(s) would have a high chance to carry mutations in drug-resistant clones. To this end, THP-1 AML cells were treated with high to moderate concentration of NSC-370284 for over 100 days and then a set of individual drug-resistant THP-1 single clones were isolated (see the representatives in  FIG. 10 a   ). These drug-resistant cells showed no significant down-regulation of TET1 expression upon treatment of NSC-370284 ( FIG. 10 b   ). Through RNA-seq of 6 of the NSC-370284-resistant clones, recurrent mutations were found in 14 genes in at least two individual clones. Ingenuity pathway analysis (IPA) was used to analyze biological relationships amongst the 14 mutated genes (Table 3). The top five networks identified by IPA, based on Fisher&#39;s exact test, are associated with cancer, hematological disease, immunological disease, etc. (Table 4). The top one network identified by IPA involving all of the 14 genes is closely associated with the JAK/STAT5 pathway ( FIG. 3 a   ). A number of these genes have been reported to be associated with the JAK/STAT signaling. It is likely that mutations in such genes may overcome NSC-370284-mediated inhibitory effect on AML cell viability/growth, and thereby confer drug resistance to the AML clones. To test this, we chose JAK1 and MSH3 as two representatives and cloned constructs carrying the JAK1 A893G  mutant or the MSH3 V600I  mutant that was detected in our drug-resistant THP-1 cells (Table 3). As expected, forced expression of either mutant at least partially reversed the inhibitory effect of NSC-370284 on AML cell viability ( FIG. 10 c - d   ). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Genes with recurrent mutations in THP-1 NSC-370284-resistant clones. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Gene 
                 ExonicFunc 
                 AAChange 
                 Chr 
                 Pos 
                 Ref 
                 Obs 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MSH3 
                 nonsynonymous SNV 
                 NM_002439:c.G1798A:p.V600I 
                 chr5 
                 80057399 
                 G 
                 A 
               
               
                 COG3 
                 nonsynonymous SNV 
                 NM_031431:c.A1903G:p.I635V 
                 chr13 
                 46090371 
                 A 
                 G 
               
               
                 IPO5 
                 nonsynonymous SNV 
                 NM_002271:c.G2524A:p.V842I 
                 chr13 
                 98668012 
                 G 
                 A 
               
               
                 TMED5 
                 nonsynonymous SNV 
                 NM_001167830:c.T415C:p.W139R 
                 chr1 
                 93621913 
                 A 
                 G 
               
               
                 HSP90AB1 
                 nonsynonymous SNV 
                 NM_001271969:c.T593G:p.V198G 
                 chr6 
                 44217836 
                 T 
                 G 
               
               
                 CCDC132 
                 nonsynonymous SNV 
                 NM_017667:c.A1877T:p.N626I 
                 chr7 
                 92952944 
                 A 
                 T 
               
               
                 MIER2 
                 nonsynonymous SNV 
                 NM_017550:c.G1396A:p.A466T 
                 chr19 
                 307339 
                 C 
                 T 
               
               
                 SYF2 
                 nonsynonymous SNV 
                 NM_207170:c.G206C:p.W69S 
                 chr1 
                 25554653 
                 C 
                 G 
               
               
                 VDAC2 
                 nonsynonymous SNV 
                 NM_001184823:c.G826C:p.G276R 
                 chr10 
                 76990688 
                 G 
                 C 
               
               
                 SWAP70 
                 nonsynonymous SNV 
                 NM_0150555:c.A1527T:p.E509D 
                 chr11 
                 9769576 
                 A 
                 T 
               
               
                 NOTCH2 
                 frameshift deletion 
                 NM_001200001:c.17_18del:p.6_6del 
                 chr1 
                 120612003 
                 GG 
                 — 
               
               
                 MARCKS 
                 nonsynonymous SNV 
                 NM_002356:c.G130C:p.D44H 
                 chr6 
                 114180886 
                 G 
                 C 
               
               
                 SRRM2 
                 frameshift deletion 
                 NM_016333:c.5444_5445del:p.1815_1815del 
                 chr16 
                 2815973 
                 GG 
                 — 
               
               
                 JAK1 
                 nonsynonymous SNV 
                 NM_002227:c.A893G:p.E298G 
                 chr1 
                 65332646 
                 T 
                 C 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Top 5 signaling pathways and associated diseases of the genes with  
               
               
                 recurrent mutations in THP-1 NSC-370284-resistant clones. 
               
               
                   
               
             
            
               
                 (a) Top 5 canonical pathways involving the genes with recurrent  
               
               
                 mutations in THP-1 NSC-370284-resistant clones. 
               
            
           
           
               
               
            
               
                 Name of Canonical Pathways 
                 P value 
               
               
                   
               
               
                 IL-22 Signaling 
                 1.17E-04 
               
               
                 Role of JAK family kinases in IL-6-type  
                 1.27E-04 
               
               
                 Cytokine Signaling 
                   
               
               
                 IL-9 Signaling 
                 2.37E-04 
               
               
                 Oncostatin M Signaling 
                 2.37E-04 
               
               
                 Role of JAK2 in Hormone-like Cytokine  
                 2.37E-04 
               
               
                 Signaling 
               
               
                   
               
            
           
           
               
            
               
                 (b) Top 5 diseases and disorders involving the genes with recurrent  
               
               
                 mutations in THP-1 NSC-370284-resistant clones. 
               
            
           
           
               
               
            
               
                 Diseases and Disorders 
                 P value range 
               
               
                   
               
               
                 Cancer 
                 4.84E-02 ~ 2.14E-05 
               
               
                 Hematological Disease 
                 4.26E-02 ~ 2.14E-05 
               
               
                 Immunological Disease 
                 4.26E-02 ~ 2.14E-05 
               
               
                 Organismal Injury and Abnormalities 
                 4.84E-02 ~ 2.14E-05 
               
               
                 Developmental Disorder 
                 4.13E-02 ~ 2.14E-05 
               
               
                   
               
            
           
         
       
     
     Consistent with this, it was shown that knockdown of STAT3 and/or STAT5 in MONOMAC-6 and THP-1 cells resulted in a down-regulation of TET1, but not TET2 or TET3 ( FIG. 3 b    and  FIG. 10 c - f   ). Through searching the UCSC Genome Browser (https://genome.ucsc.edu/index.html), it was found that STAT5 has a putative binding site ( ttccctgaa cagcttttaca tgtg (SEQ ID NO: 1); the consensus binding motif: ttcnnngaa (SEQ ID NO: 2);  FIG. 3 c   , Site 4) located within the promoter region of TET1 gene, suggesting TET1 might be a direct target of the STAT proteins. The direct binding of STAT3 and STAT5 on the TET1 loci was further validated in MONOMAC-6 cells through chromatin immunoprecipitation (ChIP)-qPCR assay, and such binding could be disturbed by NSC-370284 treatment ( FIG. 3 c - g   ). In NB4 cells, no significant binding of STAT3 or STAT5 on the TET1 loci was detected ( FIG. 3 h   ). JAK1 was known as the upstream activator of the STAT pathway. Surprisingly, knockdown of TET1 resulted in a reduction of JAK1 transcription in AML cells ( FIG. 10 i   ). ChIP-qPCR results showed direct binding of TET1 to the JAK1 promoter ( FIG. 10 j   ). The above findings suggest JAK/STAT pathway promotes TET1 transcription via direct binding of STAT3/5 to the TET1 promoter, and TET1 also binds to the JAK1 promoter and activates JAK1 transcription. This indicates a feedback loop between JAK1/STAT/TET1 in AML. 
     Structural analysis suggested a potential direct binding of NSC-370284 to the conserved DNA-binding domain (DBD) of STAT3 or STAT5 ( FIG. 3 i   ). Such binding and the binding sites were identified by use of NMR chemical shift perturbation (CSP). Complex formation with compound NSC-370284 induced extensive CSPs at the isoleucine (Ile) residues of STAT3 at 1:2 of protein:ligand molar ratio ( FIG. 3 j   ). The CSPs occurred at residues adjacent to the DNA-binding site (I464) and those at or near DBD ( FIG. 3 j   ), indicating that compound NSC-370284 binds to STAT3 at or near DBD. The association between STAT3 and the TET1 CpG island was further verified with electrophoretic mobility shift assay (EMSA); this association could almost be completely blocked by NSC-370284 ( FIG. 3 k   ). In order to test whether NSC-370284 also inhibits the phosphorylation of STAT3 and STAT5, MONOMAC-6 cells were treated with NSC-370284 for a series of time points. Western blotting showed no significant alterations of the levels of STAT3 and STAT5 phosphorylation ( FIG. 10 k   ). It is likely NSC-370284 mainly competes against DNA for STAT binding, but not does suppress STAT activation. 
     These results indicate that STAT3 and STAT5 are direct targets of NSC-370284, which can interfere with the binding of STAT3/5 to TET1 promoter region and thereby suppress the transcription of TET1. Similarly, a previous study also reported a compound C48, a structural analog of NSC-370284, can bind directly to the DNA binding domain of STAT3 protein and lead to apoptosis and inhibition of tumor cell growth. 
     Notably, the inhibitory effects of NSC-370284 on the expression of other STAT5 target genes, such as HIF2α, IL2RA and FRA2, were not as obvious as that on TET1 ( FIG. 10 l   ). The basal enrichment of STAT5 on the promoter of HIF2α, IL2RA or FRA2 was very low, as compared with that on the TET1 promoter; and the interruption by NSC-370284 on such association was much less obvious ( FIG. 10 m   ). The very weak, if any, basal affinity of STAT5 with most of its target genes&#39; promoters without cytokine stimulation (e.g., IL-2, IL-3 or EPO) has been reported before. Therefore, these results revealed a very strong enrichment of STAT3/5 on TET1 promoter ( FIG. 3 e - g , 3 k   ;  FIG. 10 l - m   ). Moreover, results indicate that different from typical STAT inhibitors that target STAT kinase activity, NSC-370284 may exert its function mainly through interfering with the binding of STAT protein to the DNA regions which have relatively higher basal affinity to STATs, like the TET1 promoter, in AML cells. 
     Enhanced Therapeutic Efficacy of NSC-370284 Analog UC-514321 
     Based on the structure of compound NSC-370284, structural analogs were explored using BioVia Pipeline Pilot (Version 8.5.0.200) against the University of Cincinnati Compound Library, a collection of approximately 360,000 compounds. The 30 most structurally similar compounds were selected to explore the Structure Activity Relationships (SAR) (Tables A and B). The Table A compounds share the core aryl amine benzodioxole scaffold with NSC-370284, varying primarily in the amine substituents and in the aryl substituents. Results of MTS assays showed that one of the analog compounds, UC-514321, most significantly repressed MONOMAC-6 cell viability ( FIG. 4 a    and  FIG. 11 ). UC-514321 showed an enhanced effect in repressing the viability of TET1-high AML (including MONOMAC-6, THP-1, and KASUMI-1) cells, as compared with NSC-370284 and other JAK/STAT inhibitors, e.g. Pacritinib, KW-2449, STAT3/5 inhibitor Stattic, or STAT5 inhibitor sc-355979 ( FIG. 4 b - d   ). Similar to NSC-370284, UC-514321 showed no inhibitory effect on the viability of TET1-low AML (i.e., NB4) cells ( FIG. 4 e   ). Thus, the anti-tumor effect of UC-514321 is also TET1-signaling dependent. Notably, the STAT3/5 specific inhibitors Stattic and sc-355979 did not show significant inhibitory effects on the viability of these AIL cells, probably because their IC 50  values are much higher than 500 nM, the maximum concentration we tested. Overall, compared to other JAK and/or STAT inhibitors, UC-514321 is more effective and selective in inhibiting the viability of TET1-high AML cells. 
     Moreover, compared to the parental compound (NSC-370284), UC-514321 also showed an improved therapeutic effect in AML mouse models in vivo. In MLL-AF9-AML mice, UC-514321 prolonged median survival from 49 days (control) to &gt;200 days, better than NSC-370284 ( FIG. 4 f   ). In the AE9a-AML model, the median survival of UC-514321 treated mice was 160 days, much longer than the control (46 days) and NSC-370284 (114 days) treated groups ( FIG. 4 g   ). Thus, in both AML animal models, UC-514321 prolonged the median survival over 3 folds ( FIG. 4 f - g   ). Moreover, the therapeutic effects of NSC-370284 and UC-514321 were tested in two other AML models, i.e., MLL-AF10 AML and FLT3-ITD/NPM1 mut  AML. NSC-370284 prolonged median survival of MLL-AF10 leukemic mice from 35 days to &gt;50 days, and that of FLT3-ITD/NPM1 mut  leukemic mice from 21 days to 34 days. UC-514321 showed an even better therapeutic effect, as it prolonged median survival of both MLL-AF10 and FLT3-ITD/NPM1 mut  leukemic mice to &gt;50 days ( FIG. 4 i,j   ). Notably, UC-514321 treatment cured 66.7% (4 out of 6) of the MLL-AF9 AML mice ( FIG. 4 f,h   ), and none of the UC-514321-treated FLT3-ITD/NPM1 mut  AML recipients developed full-blown AML within 50 days ( FIG. 4 j   ). 
     To determine whether the mechanism underlying UC-514321 function is similar to that of NSC-370284, a series of mechanistic studies were performed. As expected, similar to NSC-370284, UC-514321 significantly repressed expression of TET1, but not TET2 or TET3, along with the down-regulation of putative target genes of TET1, e.g., HOXA7, HOXA10, MEIS1, PBX3 and FLT3, etc., and the up-regulation of negative targets, e.g., miR-22, in MONOMAC-6 cells ( FIG. 5 a,b   ). Notably, compared to NSC-370284, UC-514321 exhibited an enhanced effect on the changes in gene expressions ( FIG. 5 a,b   ). Both NSC-370284 and UC-514321 significantly reduced global 5hmC levels ( FIG. 5 c   ), and their inhibitory effect was further confirmed with genome-wide 5hmC-seq in AML cells. We found both NSC-370284 and UC-514321 treatments resulted in around 75% reduction of 5hmC peak enrichment in the whole genome ( FIG. 12 a - c   ). Similar to NSC-370284 ( FIG. 3 j   ), the largest CSPs induced by UC-514321 treatment were also observed at the residues adjacent to DNA-binding surface (I431) and those near or in DBD ( FIG. 5 d   ). In contrast, Stattic and sc-355979 target the STAT SH2 domain. Therefore, both NSC-370284 and UC-514321 induce CSP at residues at or adjacent to the DNA-binding surface, and thus they likely interfere with STAT protein&#39;s binding to DNA. This was further verified with ChIP-qPCR assays. In MONOMAC-6 cells, UC-514321 treatment also resulted in a remarkable repression on the binding of STAT3/5 to TET1 promoter, even more significant than NSC-370284 at the CpG region ( FIG. 5 e - h   ). 
     In wild-type mouse BM progenitor cells transduced with MLL-AF9, treatment with NSC-370284 or UC-514321 resulted in a remarkable repression of cell viability, and no such inhibition was observed in Tet1 deficient counterpart cells ( FIG. 5 i - l   ). Other JAK/STAT inhibitors (i.e. Pacritinib, KW-2449, Stattic, and sc-355979) showed no significant differences in affecting cell viability between wild-type and Tet1 deficient cells ( FIG. 5 i,j    and  FIG. 13 a - d   ). Further, in MLL-AF9-transduced BM progenitor cells from conditional Tet1 knockout mice, NSC-370284 and UC-514321 also showed no significant effect in inhibiting the viability of the cells with induced Tet1 deficiency ( FIG. 5 m    and  FIG. 13 e   ). In addition, a pLenti-puro vectored Tet1 construct was created and transduced into THP-1 and MONOMAC-6 cells, which showed that ectopic expression of Tet1 sufficiently reversed the inhibitory effects of NSC-370284 and UC-514321 and restored viability of the AML cells ( FIG. 5 n - o   ). Taken together with the selective effect of NSC-370284 on TET1-high AML cell lines ( FIG. 1 b   ), the above results suggest that the anti-leukemic effects of NSC-370284 and UC-514321 are TET1-signaling dependent. 
     Example 5. NSC-370284 and UC-514321 Show No Obvious Toxicity in Healthy Mice 
     Cell viability and apoptosis assays were first carried out to assess the effects of NSC-370284 and UC-514321 on normal hematopoietic stem/progenitor cells (HSPCs; herein we used c-Kit+ BM cells) in vitro. Remarkably, NSC-370284 or UC-514321 treatment dramatically suppressed the viability of AML cells, but not that of normal HSPCs ( FIG. 14 a   ). In addition, the compounds significantly increased apoptosis in AML cells, but not in normal HSPCs ( FIG. 14 b   ). Thus, these two compounds showed no obvious toxicity on normal HSPCs. This is consistent with endogenous Tet1 expression pattern, as AML cells with MLL-AF10 or FLT3-ITD/NPM1 mut  have relatively higher Tet1 expression levels, as compared with normal HSPCs ( FIG. 14 c   ). 
     To assess potential toxicity of NSC-370284 and UC-514321 in normal tissues, especially the hematopoietic system, in vivo, NSC-370284 or UC-514321 were injected into normal C57BL/6 mice and assessed potential acute toxicity (24 hrs) or long-term (200 days) toxicity after 10 succeeding days&#39; administration of either NSC-370284 or UC-514321. Body weights, spleen and liver weights, white blood cell (WBC) counts, all peripheral blood lineages, as well as granulocytes (Mac1 + Gr1 + ), monocytes (Mac1 + Gr1 − ) and progenitor (c-Kit + ) lineages of BM cells were assessed, and no evidence of either acute or long-term toxicity was observed ( FIG. 15, 16  and Table 5). 
                     TABLE 5                  Acute or long-term effects of NSC-370284 and UC-514321 on mouse blood cell       differentiation after 10 consecutive days&#39; administration       (a) Acute effects (24 hrs post the last administration) of NSC-370284 and UC-514321 on mouse        blood cell differentiation.                                                     WBC   NE   LY   MO   EO   BA   RBC   PLT           (K/μl)   (K/μl)   (K/μl)   (K/μl)   (K/μl)   (K/μl)   (M/μl)   (K/μl)               DMSO   10.32 ±   1.97 ±   7.49 ±   0.27 ±   0.2 ±   0.07 ±   8.61 ±   552.32 ±           3.2   0.65   2.6   0.02   0.08   0.03   1.86   131.22       370284   8.35 ±   1.67 ±   6.74 ±   0.22 ±   0.15 ±   0.05 ±   8.55 ±   460.21 ±           2.34   0.21   2.55   0.14   0.01   0.05   3.34   42.42       514321   8.64 ±   1.94 ±   6.54 ±   0.27 ±   0.14 ±   0.07 ±   9.54 ±   559.43 ±           3.68   0.56   2.7   0.1   0.12   0.05   0.74   116.9       Normal range   1.8-10.7   0.1-2.4   0.9-9.3   0.0-0.4   0.0-0.2   0.0-0.2   6.36-9.42   592-2972                         (b) Long-term effects (200 days post the last administration) of NSC-370284 and UC-       514321 on mouse blood cell differentiation.                                                     WBC   NE   LY   MO   EO   BA   RBC   PLT           (K/μl)   (K/μl)   (K/μl)   (K/μl)   (K/μl)   (K/μl)   (M/μl)   (K/μl)               DMSO   11.97 ±   1.83 ±   9.79 ±   0.22±   0.06 ±   0.01 ±   8.5 ±   954.95 ±           3.26   0.51   2.85   0.09   0.04   0.01   1.59   99.25       370284   10.34 ±   2.26 ±   7.68 ±   0.31±   0.11 ±   0.03 ±   7.38 ±   929.62 ±           2.19   0.54   1.55   0.03   0.01   0.02   1.7   154.61       514321   11.89 ±   2.11 ±   8.55 ±   0.25 ±   0.1 ±   0.07 ±   7.07 ±   681.56 ±           1.64   0.44   1.18   0.05   0.01   0.04   0.42   402.64       Normal range   1.8-10.7   0.1-2.4   0.9-9.3   0.0-0.4   0.0-0.2   0.0-0.2   6.36-9.42   592-2972               Note:       Normal C57BL/6 mice were treated with PBS (control), 2.5 mg/kg NSC-370284 or UC-514321, i.p., once every day for 10 days. Shown are data collected at the indicated time points (i.e. 24 hrs (a) or 200 days (b)) post the last i.p. injection of the chemical compounds or DMSO control (DMSO). WBC = white blood cells; NE = neutrophils; LY = lymphocytes; MO = monocytes; EO = eosinophils; BA = basophils; RBC = red blood cells; PLT = platelets. Means ± Standard deviations are shown.            
The maximum tolerated dose of NSC-370284 and UC-514321 in mice was 65-85.6 mg/kg (Table 6). The LD50 was around 123 mg/kg (Table 6). Analysis of the pharmacokinetic properties of UC-514321 showed that the compound had a half-life of 11.02 hrs in mouse blood (Table 7;  FIG. 17 ).
 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Maximum tolerated dose (MTD) and median lethal dose (LD 50 ) of  
               
               
                 NSC-370284 and UC-514321. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Number 
                 Number of 
               
               
                 Animals 
                 Routes 
                 Dose 
                 of Death 
                 Exposure 
               
               
                   
               
            
           
           
               
            
               
                 NSC-370284 (LD 50  = 123.1 mg/kg) 
               
            
           
           
               
               
               
               
               
            
               
                 C57BL/6 
                 I.P. 
                   200 mg/kg 
                 10 
                 10 
               
               
                   
                 I.P. 
                   150 mg/kg 
                 6 
                 10 
               
               
                   
                 I.P. 
                 113.5 mg/kg 
                 3 
                 10 
               
               
                   
                 I.P. 
                  85.6 mg/kg 
                 1 
                 10 
               
               
                   
                 I.P. 
                   65 mg/kg 
                 0 
                 10 
               
            
           
           
               
            
               
                 UC-514321 (LD 50  = 123.7 mg/kg) 
               
            
           
           
               
               
               
               
               
            
               
                 C57BL/6 
                 I.P. 
                   200 mg/kg 
                 10 
                 10 
               
               
                   
                 I.P. 
                   150 mg/kg 
                 7 
                 10 
               
               
                   
                 I.P. 
                 113.5 mg/kg 
                 2 
                 10 
               
               
                   
                 I.P. 
                  85.6 mg/kg 
                 1 
                 10 
               
               
                   
                 I.P. 
                   65 mg/kg 
                 0 
                 10 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 Blood pharmacokinetic parameters of UC-514321 
               
               
                 (data provided as mean ± SD). 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Pharmacokinetic 
                 C57BL/6 Mice 
               
               
                   
                 Parameters 
                 I.P. 15 mg/kg 
               
               
                   
                 Half-life (h) 
                 11.02 ± 2.51 
               
               
                   
                 AUC  0-∞ (μg · h · mL −1 ) 
                 29.35 ± 2.75 
               
               
                   
                 Mean Residence Time (h) 
                 16.12 ± 1.79 
               
               
                   
                 Volume of Distribution 
                 21.66 ± 1.74 
               
               
                   
                 (L · kg −1 ) 
                   
               
               
                   
                 Clearance (L · kg −1  · h −1 ) 
                  1.36 ± 0.08 
               
               
                   
                   
               
            
           
         
       
     
     Example 6. Synergistic Effect with Standard Chemotherapy 
     Long-term treatment with a drug may cause drug resistance in patients, and thus combinatory therapy is often required. To treat AML cells that have gained resistance to inhibitors, the effects of a set of first-line AML chemotherapy drugs including daunorubicin (DNR), cytarabine (AraC), all-trans retinoic acid (ATRA), azacytidine (AZA), and decitabine (DAC) on the viability of parental THP-1 cells and three NSC-370284-resistant clones were investigated ( FIG. 6 a    and  FIG. 18 a - d   ). Strikingly, all 3 drug-resistant clones appeared to be much more sensitive to DNR than the parental control ( FIG. 6 a   ). NSC-370284 or UC-514321 works synergistically with DNR on inhibiting the viability of THP-1 and KASUMI-1 cells ( FIG. 6 b,c   ). The synergistic effect between NSC-370284 or 514321 and the standard chemotherapy (i.e. the “5+3” regimen) was further validated in vivo. Even administrated at relative low doses, the combinatorial treatment of NSC-370284+DNR/AraC or UC-514321+DNR/AraC showed a better therapeutic effect in curing MLL-AF9 AML, as the combinatorial administrations cured 83.3% (5 out of 6) of the AML mice ( FIG. 6 d,e   ). 
     Through analysis of the RNA-seq data of the NSC-370284 resistant clones and parental cells, we showed that several gene clusters that are known to be associated with drug response, especially response to topoisomerase II inhibitors such as DNR, are enriched in NSC-370284 resistant cells. These gene clusters include JAK/STAT signaling, G2M checkpoint, MYC targets and E2F targets, etc. A potential DNR sensitizing mechanism might be through targeting G2M checkpoint. It was shown that overexpression of CDC25, a key phosphatase of G2M checkpoint control, could significantly sensitize tumor cells to doxorubicin treatment. RNA-seq data showed increased CDC25 levels in NSC-370284 resistant AML clones, relative to the parental cells. Also consistent is the enrichment of G2M checkpoint gene cluster in control samples relative to NSC-370284 treated samples. The activation of JAK/STAT pathway might, directly or indirectly, contribute to G2M checkpoint abnormality, as reported previously by others. Therefore, very likely the deregulation of G2M checkpoint in NSC-370284 resistant clones at least partially explains why the resistant clones, compared to the parental AML cells, are more sensitive to DNR treatment. Overall, the combination of NSC-370284 or UC-514321 with DNR represents a promising therapeutic strategy that will not only be effective in treating patients with TET1-high AMLs at relative low doses, but also avoid the occurrence of resistance to NSC-370284 or UC-514321. 
     Example 7. Materials and Methods 
     The Maintenance, Monitoring, and End-Point Treatment of Mice 
     C57BL/6 (CD45.2), B6.SJL (CD45.1) mice were purchased from the Jackson Lab (Bar Harbor, Me., USA) or Harlan Laboratories, Inc (Indianapolis, Ind., USA). Both male and female mice were used for the experiments. All laboratory mice were maintained in the animal facility at University of Cincinnati or University of Chicago. All experiments on mice in the research protocol were approved by Institutional Animal Care and Use Committee (IACUC) of University of Cincinnati or University of Chicago. All methods were performed in accordance with the relevant guidelines and regulations. The maintenance, monitoring, and end-point treatment of mice were conducted as described previously (Jiang, X., et al.,  miR -22  has a potent anti - tumour role with therapeutic potential in acute myeloid leukaemia, Nat Commun  7: 11452 (2016); Jiang, X., et al.,  Eradication of Acute Myeloid Leukemia with FLT 3  Ligand - Targeted miR -150  Nanoparticles, Cancer Res  76; 4470-80 (2016); Jiang, X., et al.  Blockade of miR -150  Maturation by MLL - Fusion/MYC/LIN -28  Is Required for MLL - Associated Leukemia, Cancer Cell  22; 524-35 (2012)). Randomization, allocation concealment and blind outcome assessment were conducted throughout all the experiments. 
     Mouse Bone Marrow Transplantation (BMT) Followed with Drug Treatment 
     Secondary mouse BMT was carried out as described previously. Upon the onset of leukemia (when mice had an engraftment (CD45.1) of over 20% and/or white blood cell counts higher than 4×10 9 /L, usually 10 days post transplantation), the recipient mice were injected with DMSO control, 2.5 mg/kg NSC-311068, NSC-370284 or UC-514321, i.p., once per day, for ten days. For the “5+3” and NSC-370284 or UC-514321 combination treatment experiment, after the onset of AML, the recipient mice were treated with PBS control, or NSC-370284 or UC-514321 alone, i.p., once per day, for ten days or together with the “5+3” treatment. For the “5+3” treatment, AraC (Cytarabine, Bedford Laboratories) and DNR (Daunorubicin, Sigma-Aldrich) were reconstituted with PBS, filtered, and stored in aliquots at −20° C. The “5+3” therapy regimen consists of five consecutive daily doses of 50 mg/kg AraC along with 3 mg/kg DNR daily during the first three days of treatment. Drugs were delivered by tail vein and intraperitoneal injection. Weights were taken daily during treatment and doses were recalculated to ensure the mice received a consistent dose of 50 mg/kg AraC and 3 mg/kg DNR every treatment. 
     Cell Culture and Drug Treatment 
     MONOMAC-6, THP-1, KOCL-48, KASUMI-1, ML-2 and NB4 cells were purchased from ATCC (Manassas, Va.), and cultured as described previously (Jiang, X., et al. Blockade of miR-150 Maturation by MLL-Fusion/MYC/LIN-28 Is Required for MLL-Associated Leukemia.  Cancer Cell  22, 524-535 (2012); Li, Z., et al. miR-196b directly targets both HOXA9/MEIS1 oncogenes and FAS tumour suppressor in MLL-rearranged leukaemia. (Li, Z., et al.,  miR -196 b directly targets both HOXA 9 /MEIS 1  oncogenes and FAS tumour suppressor in MLL - rearranged leukaemia, Nat Commun  2:688 (2012)). All cell lines were tested for mycoplasma contamination yearly using a PCR Mycoplasma Test Kit (PromoKine) and were proven to be mycoplasma negative. All cell lines were authenticated through STR profiling yearly. 
     Cell Transfection and Retrovirus Infection 
     siRNAs were transfected into MONOMAC-6 cells with Cell Line Nucleofector Kit V following program T-037, using the Amaxa Nucleofector Technology (Amaxa Biosystems, Berlin, Germany). Experiments were performed 48 h after transfection. Retrovirus infection of mouse BM progenitor cells were conducted as described previously with some modifications. Briefly, retrovirus vectors were co-transfected with pCL-Eco packaging vector (IMGENEX, San Diego, Calif.) into HEK293T cells using Effectene Transfection Reagent (Qiagen, Valencia, Calif.) to produce retrovirus. BM cells were harvested from a cohort of 4- to 6-week-old C57BL/6, Tet1 −/− , or Cre-Tet fl/fl  donor mice after five days of 5-fluorouracil (5-FU) treatment, and primitive hematopoietic progenitor cells were enriched with Mouse Lineage Cell Depletion Kit (Miltenyi Biotec Inc., Auburn, Calif.). An aliquot of enriched hematopoietic progenitor cells was added to retroviral supernatant together with polybrene in cell culture plates, which were centrifuged at 2,000 g for 2 hours at 32° C. (i.e., spinoculation) and then the medium was replaced with fresh media and incubated for 20 hours at 37° C. Next day, the same procedure was repeated once. Infected cells were grown in RPMI 1640 medium containing 10 ng/mL murine recombinant IL-3, 10 ng/mL IL-6, 100 ng/mL murine recombinant SCF (R&amp;D Systems, Minneapolis, Minn.), along with 1.0 mg/ml of G418. Experiments were performed 7 days after infection. 
     RNA Extraction and Quantitative RT-PCR 
     Total RNA was extracted with the miRNeasy extraction kit (Qiagen) and was used as template for quantitative RT-PCR (qPCR) analysis. 
     Cell Apoptosis, Viability and Proliferation Assays 
     These experiments were conducted as described previously (Jiang, X., et al.,  Blockade of miR -150  Maturation by MLL - Fusion/MYC/LIN -28  Is Required for MLL - Associated Leukemia, Cancer Cell  22:524-35 (2012); Li, Z., et al.,  miR -196 b directly targets both HOXA 91 MEIS 1  oncogenes and FAS tumour suppressor in MLL - rearranged leukaemia, Nat Commun  2:688 (2012)) with ApoLive-Glo Multiplex Assay Kit, or CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay Kit (Promega, Madison, Wis.). 
     NMR Chemical Shift Perturbation (CSP) 
     Specific Ile-methyl labeled STAT3 for NMR studies was prepared as described previously (Byrd, J. C., et al. Repetitive cycles of high-dose cytarabine benefit patients with acute myeloid leukemia and inv(16)(p13q22) or t(16;16)(p13;q22): results from CALGB 8461. (Namanja, A. T., et al.,  Allosteric Communication across STAT 3  Domains Associated with STAT 3  Function and Disease - Causing Mutation, J Mol Biol  428:579-89 (2016)). For each compound, STAT3 was expressed and purified fresh, and a reference spectrum was acquired. Then, the complexes of STAT3 with each of the compounds in the same buffer as the free STAT3 (reference) sample were prepared and two-dimensional (2D)  1 H- 13 C-HMQC spectra of STAT3 were acquired. The protein samples contained 20 μM STAT3. Both compounds were added to a final concentration of 40 μM, respectively. All 2D  1 H- 13 C HMQC spectra were collected with 2048×128 complex points at 35° C. on the Bruker Ascend 700 spectrometer equipped with a cryoprobe. The spectra were analyzed with the program Sparky (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco). 
     Chromatin Immunoprecipitation-qPCR (ChIP-qPCR) 
     ChIP assay was conducted as described previously (Barry, S. P., et al.,  STAT 3  modulates the DNA damage response pathway, Int J Exp Pathol  91:506-14 (2010)), with SABiosciences Corporation&#39;s ChampionChIP One-Day kit (Qiagen, Frederick, Md.) following the manufacturer&#39;s protocol. Chromatin from MONOMAC-6 cells were cross-linked, sonicated into an average size of ˜500 bp, and then immunoprecipitated with antibodies against STAT3 (Santa Cruz, Dallas, Tex.), STAT5 (BD Biosciences, San Jose, Calif.), or IgG (Abcam, Cambridge, Mass.). Purified DNA was amplified by real-time qPCR using primers targeting the promoter of TET1 as described before (Huang, H., et al.,  TET 1  plays an essential oncogenic role in MLL - rearranged leukemia, Proc Natl Acad Sci USA  110:11994-99 (2013)). Sequences of qPCR primers are: Site 1 forward: 5′-ACTTTGACCTCCCAAAGTGCTGGA-3′ (SEQ ID NO: 3), reverse: 5′-ACCTGAGTGATGCTGAGACTTCCT-3′ (SEQ ID NO: 4); Site 2 forward: 5′-TTTGGGAACCGACTCCTCACCT-3′ (SEQ ID NO: 5), reverse: 5′-TCGGGCAAACTTTCCAACTCGC-3′ (SEQ ID NO: 6); Site 3 forward: 5′-ACGCTGGGCATTTCTGATCCACTA-3′ (SEQ ID NO: 7), reverse: 5′-TATTGTGCAGCTCGTTTAGTGCCC-3′ (SEQ ID NO: 8); Site 4 forward: 5′-CCATCTCCCGACACACA-3′ (SEQ ID NO: 9); reverse: 5′-TTGGCAGTGACCTTGAGA-3′ (SEQ ID NO: 10). 
     Electrophoretic-Mobility Shift Assay (EMSA) 
     EMSA was conducted with EMSA Assay Kit (Signosis, Santa Clara, Calif.) according to the manufacturer&#39;s protocol with minor modifications. Briefly, purified STAT3 protein was incubated with Biotin-labeled TET1-CPG probe (hot probe) and/or cold probe, and then protein/DNA complexes were separated on a non-denaturing polyacrylamide gel. Bands were detected using Streptavidin-HRP conjugate and a chemiluminescent substrate. The sequences of the TET1-CPG probe are: Forward: 5′ Biotin-CCGGTAGGCGTCCTCCGCGACCCGC-3′ (SEQ ID NO: 11); Reverse: 5′ Biotin-GCGGGTCGCGGAGGACGCCTACCGG-3′ (SEQ ID NO: 12). 
     Western Blotting 
     Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and ruptured with RIPA buffer (Pierce, Rockford, Ill.) containing 5 mM EDTA, PMSF, cocktail inhibitor, and phosphatase inhibitor cocktail. Cell extracts were microcentrifuged for 20 min at 10,000 g and supernatants were collected. Cell lysates were resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked for 1 hour with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 and incubated overnight at 4° C. with anti-Tet1 antibody (GT1462, GeneTex, Irvine, Calif.) or anti-ACTIN antibody (8H10D10, Cell Signaling Technology Inc., Danvers, Mass.). Membranes were washed 30 min with Tris-buffered saline containing 0.1% Tween-20, incubated for 1 hour with appropriate secondary antibodies conjugated to horseradish peroxidase, and developed using chemiluminescent substrates. 
     5hmC Labeling Reaction and Dot-Blotting 
     The 5-hydroxymethylcytosine (5-hmC) labeling reactions and 5hmC dot blotting were performed as described previously (Huang, H., et al. TET1 plays an essential oncogenic role in MLL-rearranged leukemia.  Proc Natl Acad Sci USA  110, 11994-11999 (2013)). Briefly, 3 jig sonicated genomic DNA (100-500 bp) fragments were incubated in solution containing 50 mM Hepes buffer (pH 7.9), 25 mM MgCl2, 100 jiM UDP-6-N3-Glc, and 1 jiM beta-glucosyltransferase (f3-GT) for 1 hr at 37° C. The CLICK was performed with addition of 150 jiM dibenzocyclooctyne modified biotin into the purified DNA solution, and the reaction mixture was incubated for 2 hrs at 37° C. Six hundred nanograms of labeled genomic DNA samples were spotted on an Amersham Hybond-N+ membrane (GE Healthcare, Little Chalfont, UK). DNA was fixed to the membrane by Stratagene UV Stratalinker 2400 (auto-crosslink). The membrane was then blocked with 5% BSA and incubated with Avidin-HRP (1:40,000) (Bio-Rad, Hercules, Calif.), and then visualized by enhanced chemiluminescence. 
     5hmC-Seal Library Construction 
     100 ng genomic DNA extracted from ML-2 cell were fragmented in 50 jiL Tagmentation buffer at 55° C. Fragmented DNA was purified by Zymo DNA clean&amp;concentrator Kit (Zymo Research, Tustin, Calif.). Then, the selective 5hmC chemical labeling was performed in 25 jiL glucosylation buffer (50 mM HEPES buffer pH 8.0, 25 mM MgCl2) containing above fragmented DNA, 100 uM N3-UDP-Glc, 1 uM β-GT, and incubated at 37° C. for 2 hr. After purified in 45 μL ddH2O, 1.5 pL DBCO-PEG4-Biotin (Click Chemistry Tools, 4.5 mM stored in DMSO) was added and incubated at 37° C. for 2 hr. The biotin labeled DNA was pulled down by 5 pL C1 Streptavidin beads (Life Technologies, Carlsbad, Calif.) for 15 min at room temperature. Next, the captured DNA fragments were subjected to 13 cycles of PCR amplification using Nextera DNA sample preparation kit (Illumina, San Diego, Calif.). The resulting amplified product was purified by 1.0× AMPure XP beads. Input library was made by direct PCR from fragmented DNA without chemical labeling and capture. The libraries were quantified by a Qubit fluorometer (Life Technologies) and sequenced on an Illumina HiSEQ4000 sequencer with paired-end 50-bp reads. 
     Statistical Software and Statistical Analyses 
     The gene network was analyzed with Ingenuity Pathway Analysis (Qiagen). The modeling of protein-DNA/chemical compound binding was conducted with Molsoft ICM-Pro (Molsoft L.L.C., San Diego, Calif.). The t-test, Kaplan-Meier method, and log-rank test, etc. were performed with WinSTAT (R. Fitch Software), GraphPad Prism version 5.00 (GraphPad Software, San Diego, Calif.), and/or Partek Genomics Suite (Partek Inc). The P-values less than 0.05 were considered as statistically significant. For 5hmC sequencing analysis, illumina sequencing reads were mapped to UCSC hg19 human reference genome using bowtie program. Only uniquely mapped reads were retained for the following data analysis. PCR duplicates were removed using samtools. The identification of 5hmC peaks in each sample was performed using MACS, and an IDR cutoff of 0.01 was used to filter high confident peaks. Peaks from different samples were merged together into a unified catalog of 5hmC enriched regions using HOMER. To visualize sequencing signals in IGV, BigWig files were generated by deepTools with RPKM normalization method. All the data meet the assumptions of the tests, with acceptable variation within each group, and similar variance between groups. 
     Data Availability 
     Data referenced in this study are available in The Gene Expression Omnibus. The 5hmC sequencing data is available under GSE97407 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=etodaoikjtwnpgb&amp;acc=GSE97407).