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Circulating tumor DNA (ctDNA) has emerged as a promising noninvasive biomarker to capture tumor genetics in patients with brain tumors. Research into its clinical utility, however, has not been standardized because the sensitivity and specificity of ctDNA remain undefined.To (1) review the primary literature about ctDNA in adults with glioma to compare the sensitivity and specificity of ctDNA in the cerebrospinal fluid vs the plasma and (2) to evaluate the effect of tumor grade on detection of ctDNA.PRISMA-guided systematic review and meta-analysis was performed using published studies that assessed ctDNA in either plasma or cerebrospinal fluid among adult patients with confirmed glioma. Summary receiver operating characteristic curves were generated using the Rücker-Schumacher method, and area under the curve (AUC) was calculated.Meta-analysis revealed improved biomarker performance for CSF (AUC = 0.947) vs plasma (AUC = 0.741) ctDNA, although this did not reach statistical significance ( P = .141). Qualitative analysis revealed greater sensitivities among single-allele PCR and small, targeted next-generation sequencing panels compared with broader panels. It additionally demonstrated higher sensitivity of ctDNA detection in high-grade vs low-grade gliomas, although these analyses were limited by a lack of specificity reporting in many studies.ctDNA seems to be a highly sensitive and specific noninvasive biomarker among adults with gliomas. To maximize its performance, CSF should be studied with targeted genetic analysis platforms, particularly in high-grade gliomas. Further studies on ctDNA are needed to define its clinical utility in diagnosis, prognostication, glioblastoma pseudoprogression, and other scenarios wherein neoadjuvant therapies may be considered.

Alterations detectable by Guardant360 gene panel

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Supplemental Figure 1: Case of advanced NSCLC with high level EGFR T790M mutation on plasma next-generation sequencing (NGS). Supplemental Figure 2: Distribution of variant AFs between 25% and 75% across 105 cases of plasma NGS. Supplemental Figure 3: Plasma NGS cases positive for EGFR T790M submitted for blinded validation.

Purpose: Plasma cell-free DNA (cfDNA) analysis is increasingly used clinically for cancer genotyping, but may lead to incidental identification of germline-risk alleles. We studied EGFR T790M mutations in non–small cell lung cancer (NSCLC) toward the aim of discriminating germline and cancer-derived variants within cfDNA.Experimental Design: Patients with EGFR-mutant NSCLC, some with known germline EGFR T790M, underwent plasma genotyping. Separately, deidentified genomic data and buffy coat specimens from a clinical plasma next-generation sequencing (NGS) laboratory were reviewed and tested.Results: In patients with germline T790M mutations, the T790M allelic fraction (AF) in cfDNA approximates 50%, higher than that of EGFR driver mutations. Review of plasma NGS results reveals three groups of variants: a low-AF tumor group, a heterozygous group (∼50% AF), and a homozygous group (∼100% AF). As the EGFR driver mutation AF increases, the distribution of the heterozygous group changes, suggesting increased copy number variation from increased tumor content. Excluding cases with high copy number variation, mutations can be differentiated into somatic variants and incidentally identified germline variants. We then developed a bioinformatic algorithm to distinguish germline and somatic mutations; blinded validation in 21 cases confirmed a 100% positive predictive value for predicting germline T790M. Querying a database of 31,414 patients with plasma NGS, we identified 48 with germline T790M, 43 with nonsquamous NSCLC (P < 0.0001).Conclusions: With appropriate bioinformatics, plasma genotyping can accurately predict the presence of incidentally detected germline risk alleles. This finding in patients indicates a need for genetic counseling and confirmatory germline testing. Clin Cancer Res; 23(23); 7351–9. ©2017 AACR.

Effects of JQ1 and/or miR-150 nanoparticles on mouse blood cell differentiation four weeks after the last administration of drugs or control (PBS)

Acute myeloid leukemia (AML) is a common and fatal form of hematopoietic malignancy. Overexpression and/or mutations of FLT3 have been shown to occur in the majority of cases of AML. Our analysis of a large-scale AML patient cohort (N = 562) indicates that FLT3 is particularly highly expressed in some subtypes of AML, such as AML with t(11q23)/MLL-rearrangements or FLT3-ITD. Such AML subtypes are known to be associated with unfavorable prognosis. To treat FLT3-overexpressing AML, we developed a novel targeted nanoparticle system: FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) nanosized dendriplex encapsulating miR-150, a pivotal tumor suppressor and negative regulator of FLT3. We show that the FLT3L-guided miR-150 nanoparticles selectively and efficiently target FLT3-overexpressing AML cells and significantly inhibit viability/growth and promote apoptosis of the AML cells. Our proof-of-concept animal model studies demonstrate that the FLT3L-guided miR-150 nanoparticles tend to concentrate in bone marrow, and significantly inhibit progression of FLT3-overexpressing AML in vivo, while exhibiting no obvious side effects on normal hematopoiesis. Collectively, we have developed a novel targeted therapeutic strategy, using FLT3L-guided miR-150–based nanoparticles, to treat FLT3-overexpressing AML with high efficacy and minimal side effects. Cancer Res; 76(15); 4470–80. ©2016 AACR.

Clinical and molecular characteristics of the AML patients in GSE37642 cohort

Legends for supplemental figures.

Effects of the nanoparticles on mouse blood cell differentiation two (A) or four (B) weeks after the last administration of nanoparticles or control (PBS)

Supplemental Figure 1. Construction of G7-FLT3L dendrimers and their uptake ratio in AML cells. Supplemental Figure 2. G7 PAMAM dendrimers efficiently complex small, single stranded oligonucleotides. Supplemental Figure 3. G7-FLT3L-miR-150 nanoparticles repressed expression of direct or indirect target genes of miR-150 in vitro. Supplemental Figure 4. The specific targeting and inhibitory effect of G7-Flt3L-miR-150 nanoparticles to FLT3-overexpressing AML cells in vitro. Supplemental Figure 5. In vivo uptake and function of G7-Flt3L-miR-150 nanoparticles. Supplemental Figure 6. Analysis of the potential influence of the nanoparticles on hematopoietic system in normal mice.

Hydro-Jex® is a new enhanced method for heap leach treatment with significantly higher mineral recovery. In this case study at the Los Filos Mine in Mexico, the Hydro-Jex operation is evaluated for heap leach stability. This is pursued by numerical modeling of the heap leach pad stability and well monitoring of the phreatic surface before and after the Hydro-Jex operation. Experimental and numerical results both indicate an improvement in mineral processing under the influence of Hydro-Jex. Numerical results show an insignificant decrease and a significant increase in stability during and after Hydro-Jex injection application, respectively. The integrity of the liner is maintained in both cases. The zonal injection pressure, operationally below the overburden pressure, is found to be negligible compared with the dimensionality of the heap, and stability of the structure is not exposed to any significant risk, even up to 25% above overburden pressure. Furthermore, heap leach factor of safety increases after Hydro-Jex application. This is attributed to breakdown of the water solution build-up and thus a decrease in the phreatic surface depth below the top of the pad. It is suggested that injection wells be drilled, based on thorough geophysical data, in locations where overcompaction of heap material results in water solution build-up and pore pressure enhancement. Compared with traditional heap leaching, the Hydro-Jex technique not only expedites mineral processing by increasing chemical kinetics extraction but also increases the stability of the heap by unclogging drains.

MicroRNAs (miRNAs) and N6-methyladenosine (m6A) are known to serve as key regulators of acute myeloid leukemia (AML). Our previous microarray analysis indicated miR-550-1 was significantly downregulated in AML. The specific biological roles of miR-550-1 and its indirect interactions and regulation of m6A in AML, however, remain poorly understood. At the present study, we found that miR-550-1 was significantly down-regulated in primary AML samples from human patients, likely owing to hypermethylation of the associated CpG islands. When miR-550-1 expression was induced, it impaired AML cell proliferation both in vitro and in vivo, thus suppressing tumor development. When ectopically expressed, miR-550-1 drove the G0/1 cell cycle phase arrest, differentiation, and apoptotic death of affected cells. We confirmed mechanistically that WW-domain containing transcription regulator-1 (WWTR1) gene was a downstream target of miR-550-1. Moreover, we also identified Wilms tumor 1-associated protein (WTAP), a vital component of the m6A methyltransferase complex, as a target of miR-550-1. These data indicated that miR-550-1 might mediate a decrease in m6A levels via targeting WTAP, which led to a further reduction in WWTR1 stability. Using gain- and loss-of-function approaches, we were able to determine that miR-550-1 disrupted the proliferation and tumorigenesis of AML cells at least in part via the direct targeting of WWTR1. Taken together, our results provide direct evidence that miR-550-1 acts as a tumor suppressor in the context of AML pathogenesis, suggesting that efforts to bolster miR-550-1 expression in AML patients may thus be a viable clinical strategy to improve patient outcomes.

It is becoming increasingly common in the mining industry for either crushed ore or filtered mine waste to be stacked to a significant height (>100 m) in a moist state with little compaction, resulting in deposits that can be potentially loose and unsaturated. This paper presents a case history describing the characterization of stacked filtered tailings at a mine site in South America. Cone penetration tests with pore pressure and seismic velocity measurements (SCPTu) were carried out along with selected drilling, sampling, and laboratory testing. Compression wave velocity (Vp) and shear wave velocity (Vs) profiles were obtained and compared with laboratory values on reconstituted saturated and unsaturated samples. Results indicate that shear wave velocity is sensitive to suction hardening effects and appears to capture the correct unsaturated in situ behavior. The cone resistance, which is a large strain measurement, can destroy the beneficial effects of suction hardening and appears to be insensitive to the unsaturated in situ behavior, but may capture the correct behavior after the beneficial effects of suction are removed if the soil becomes saturated.

Effective therapy of acute myeloid leukemia (AML) remains an unmet need. DNA methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Through a series of data analysis and drug screening, we identified two compounds (i.e., NSC-311068 and NSC-370284) that selectively suppress TET1 transcription and 5-hydroxymethylcytosine (5hmC) modification, and effectively inhibit cell viability in AML with high expression of TET1 (i.e., TET1-high AML), including AML carrying t(11q23)/MLL-rearrangements and t(8;21) AML. NSC-311068 and especially NSC-370284 significantly repressed TET1-high AML progression in vivo. UC-514321, a structural analog of NSC-370284, exhibited a more potent therapeutic effect and prolonged the median survival of TET1-high AML mice over three fold. NSC-370284 and UC-514321 both directly target STAT3/5, transcriptional activators of TET1, and thus repress TET1 expression. They also exhibit strong synergistic effects with standard chemotherapy. Our results highlight the therapeutic potential of targeting the STAT/TET1 axis by selective inhibitors in AML treatment.

Acute myeloid leukemia (AML) is a common and fatal form of hematopoietic malignancy. Overexpression and/or mutations of FLT3 have been shown to occur in the majority of cases of AML. Our analysis of a large-scale AML patient cohort (N = 562) indicates that FLT3 is particularly highly expressed in some subtypes of AML, such as AML with t(11q23)/MLL-rearrangements or FLT3-ITD. Such AML subtypes are known to be associated with unfavorable prognosis. To treat FLT3-overexpressing AML, we developed a novel targeted nanoparticle system: FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) nanosized dendriplex encapsulating miR-150, a pivotal tumor suppressor and negative regulator of FLT3. We show that the FLT3L-guided miR-150 nanoparticles selectively and efficiently target FLT3-overexpressing AML cells and significantly inhibit viability/growth and promote apoptosis of the AML cells. Our proof-of-concept animal model studies demonstrate that the FLT3L-guided miR-150 nanoparticles tend to concentrate in bone marrow, and significantly inhibit progression of FLT3-overexpressing AML in vivo, while exhibiting no obvious side effects on normal hematopoiesis. Collectively, we have developed a novel targeted therapeutic strategy, using FLT3L-guided miR-150–based nanoparticles, to treat FLT3-overexpressing AML with high efficacy and minimal side effects. Cancer Res; 76(15); 4470–80. ©2016 AACR.

MicroRNAs are subject to precise regulation and have key roles in tumorigenesis. In contrast to the oncogenic role of miR-22 reported in myelodysplastic syndrome (MDS) and breast cancer, here we show that miR-22 is an essential anti-tumour gatekeeper in de novo acute myeloid leukaemia (AML) where it is significantly downregulated. Forced expression of miR-22 significantly suppresses leukaemic cell viability and growth in vitro, and substantially inhibits leukaemia development and maintenance in vivo. Mechanistically, miR-22 targets multiple oncogenes, including CRTC1, FLT3 and MYCBP, and thus represses the CREB and MYC pathways. The downregulation of miR-22 in AML is caused by TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. Furthermore, nanoparticles carrying miR-22 oligos significantly inhibit leukaemia progression in vivo. Together, our study uncovers a TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC signalling circuit and thereby provides insights into epigenetic/genetic mechanisms underlying the pathogenesis of AML, and also highlights the clinical potential of miR-22-based AML therapy., Mir-22 has been shown to be an oncogenic microRNA in breast cancer and myelodysplastic syndrome. Here, the authors show that mir-22 functions as a tumour suppressor in de novo acute myeloid leukaemia by inhibiting the expression of several oncogenes and that restoring mir-22 expression suppresses AML progression.

Effective therapy of acute myeloid leukemia (AML) remains an unmet need. DNA methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Through a series of data analysis and drug screening, we identified two compounds (i.e., NSC-311068 and NSC-370284) that selectively suppress TET1 transcription and 5-hydroxymethylcytosine (5hmC) modification, and effectively inhibit cell viability in AML with high expression of TET1 (i.e., TET1-high AML), including AML carrying t(11q23)/MLL-rearrangements and t(8;21) AML. NSC-311068 and especially NSC-370284 significantly repressed TET1-high AML progression in vivo. UC-514321, a structural analog of NSC-370284, exhibited a more potent therapeutic effect and prolonged the median survival of TET1-high AML mice over three fold. NSC-370284 and UC-514321 both directly target STAT3/5, transcriptional activators of TET1, and thus repress TET1 expression. They also exhibit strong synergistic effects with standard chemotherapy. Our results highlight the therapeutic potential of targeting the STAT/TET1 axis by selective inhibitors in AML treatment., Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Here, the authors identify 2 compounds that target the binding of STAT3/5 specifically to the TET1 promoter, inhibiting its expression and AML cell viability.

The following article introduces a model of the communication process as it occurs in a hostage negotiation. Unlike previous negotiation models, this model focuses on the communication of the negotiator rather than focusing on the communication or psychological state of the hostage taker. The model provides a framework of successful negotiation by analyzing and specifying the structure of previously successful negotiations. The resulting model consists of four phases, which are called Establishing Initial Dialogue, Building Rapport, Influencing, and Surrender. Theoretical basis and comparison to other existing negotiation models are discussed.

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

The use of filtering technology has improved considerably during the past few years, and tailings disposal facilities using filtered tailings are becoming more and more commonplace. Where once this method was only deemed suitable for relatively small-scale operations, technological advances are now being realised for large-scale operators. Often this application appears to be, and in fact is, relatively simple and straightforward. As with other tailings preparation methods, the use of filtered tailings provides no inherent panacea for tailings placement. Designers are advised to adopt designs thoughtfully, using site-specific data, information and tailings properties to ensure the method is employed rationally and that the resulting facility designs are flexible enough to accommodate changes to presumed operational design criteria. Practical experiences with facilities where less than optimal performance was achieved are presented.

Many heap leach operations are very large, covering many hectares and containing millions of tonnes of low-grade ore. Operators estimate recovery from laboratory column tests on ore samples that typically produce metal values somewhat lower than the column leach tests with the resultant metal inventory remaining in the heaps. After the ore material is placed and ripped, the only operational technique to improve recovery revolves around solution and reagent management. The key to reducing metal values in the heap inventory is to promote the contact of the leaching lixiviant to the valuable mineral and then to rinse the dissolved metal from the ore. This is normally accomplished by applying additional solution to the surface of the leach pads in a two dimensional fashion via emitters and sprinklers. Hydro Jex, a proprietary metallurgical technique, was developed to take heap leaching into three dimensions, a new concept for the metals extractive industry. This technology has been used by Newmont Mining Corporation on gold heap leach pads on the Carlin Trend in Nevada for the last three years to recover the inventory gold remaining in heaps after traditional solution application. It is the nature of Hydro-Jex to deliver significant amounts of solution, in a controlled manner, to discrete areas within a heap. As a consequence, the resulting pore pressures an elevated phreatic surface could have a considerable and adverse influence on the slope stability of a heap. It is therefore vital that the possible effects of the Hydro- Jex operation on slope stability be thoughtfully investigated prior to its implementation. Such investigation and analytical processes are presented herein and preliminary advice pertaining to slope stability is provided.

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

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

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

MicroRNAs (miRNAs), are small non-coding RNA molecules known to be important regulators of cancer biology. Notably, we and others have shown that miRNAs play important roles in Acute Myeloid Leukemia (AML), a heterogeneous malignancies with multiple chromosomal and molecular abnormalities. Patients with chromosomal rearrangements involving mixed lineage leukemia (MLL), the mammalian homology of trithorax gene, are associated with poor survival. Previously, we have found that MLL-rearranged AML drives aberrant expression of several miRNAs, most notably microRNA-9 (miR-9). Expression of miR-9 with MLL-AF9, a common MLL-translocation, was sufficient to promote transformation normal hematopoietic progenitor cells in vitro and leukemogenesis in vivo. We previously found that miR-9 reduces expression of several genes but we did not know which genes were critical tumor suppressors. We found that the polycomb group member RING1- and YY1-Bindin Protein (RYBP) was consistently inhibited upon miR-9 expression. To assess the regulation of RYBP we used publically available data from the Cancer Genome Atlas (TCGA) and looked at genome-wide Illumina 450K methylation data. We did not find a strong correlation with methylation and RYBP expression, suggesting that expression of RYBP is likely not regulated by the DNA methylation machinery in patients. Upon looking at copy number alterations we found that a small population of AML patients contained either homozygous or heterozygous loss of RYBP, suggesting a potential role of RYBP in leukemia pathogenesis. To assess the role of RYBP we did a series of in vitro experiments. We found that expression of RYBP was sufficient to attenuate colony-forming growth driven by MLL- AF9. Furthermore, RYBP expression was able to reduce proliferation, increase apoptosis, and significantly reduce immature cell population. To determine the role of RYBP expression in vivo, we transplanted lethally irradiated mice with progenitors retrovirally transduced with MLL-AF9 compared to MLL-AF9 and RYBP. We found that expression of RYBP was sufficient to reduce leukemia burden in vivo as well as induce differentiation as shown by flow cytometry and histological analysis. Thus, this demonstrates that RYBP is a functional tumor suppressor in MLL-rearranged AML. In conclusion, we have demonstrated that chromosomal rearrangements involving MLL, the mammalian homology of trithorax, downregulates a member of the polycomb complex through upregulation of miR-9. Disclosures No relevant conflicts of interest to declare.