miR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription-quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options.

Antileukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia.

In B-cell acute lymphoblastic leukemia (B-ALL), activation of Notch signaling leads to cell-cycle arrest and apoptosis. We aimed to harness knowledge acquired by understanding a mechanism of Notch-induced cell death to elucidate a therapeutically viable target in B-ALL. To this end, we identified that Notch activation suppresses Polo-like kinase 1 (PLK1) in a B-ALL-specific manner. We identified that PLK1 is expressed in all subsets of B-ALL and is highest in Philadelphia-like (Ph-like) ALL, a high-risk subtype of disease. We biochemically delineated a mechanism of Notch-induced PLK1 downregulation that elucidated stark regulation of p53 in this setting. Our findings identified a novel posttranslational cascade initiated by Notch in which CHFR was activated via PARP1-mediated PARylation, resulting in ubiquitination and degradation of PLK1. This led to hypophosphorylation of MDM2Ser260, culminating in p53 stabilization and upregulation of BAX. shRNA knockdown or pharmacologic inhibition of PLK1 using BI2536 or BI6727 (volasertib) in B-ALL cell lines and patient samples led to p53 stabilization and cell death. These effects were seen in primary human B-ALL samples in vitro and in patient-derived xenograft models in vivo These results highlight PLK1 as a viable therapeutic target in B-ALL. Efficacy of clinically relevant PLK1 inhibitors in B-ALL patient-derived xenograft mouse models suggests that use of these agents may be tailored as an additional therapeutic strategy in future clinical studies.

Anti-tumor immunity induced by ectopic expression of viral antigens is transient and limited by immune escape.

Immunotherapeutic treatments in head and neck cancer clinical trials include cancer vaccines targeting foreign viral antigens or mutational neoantigens derived from cancer-expressed proteins. Anti-tumor immune responses place cancer cells under selective pressure to lose or downregulate target antigens; therefore, vaccination against virus- or host- "driver" oncogenes are proposed as a strategy to overcome immune escape. Herein, we demonstrate the impact of immunogenic viral antigens on anti-tumor response and immune editing in MOC2-E6E7, a syngeneic murine oral cancer cell line expressing HPV-16 E6 and E7 oncoproteins. Using orthotopic syngeneic models, we observed in vivo tumor growth kinetics of MOC2-E6E7 is delayed in immunocompetent mice compared to parental MOC2 tumors. In contrast, tumor growth remained similar in Rag1-/- mice lacking adaptive immunity. MOC2-E6E7 tumors demonstrated an "inflamed" or immune-activated tumor microenvironment and greater infiltration of CD8+ T cells compared to MOC2. By real-time PCR, we detected downregulation of E6 and E7 genes in MOC2-E6E7 tumors only in immunocompetent mice, suggesting the loss of ectopic viral antigen expression due to immune editing. We then assessed the efficacy of a biomaterials-based mesoporous silica rod (MSR) cancer vaccine targeting HPV-16 E7 in our model. Vaccination induced robust infiltration of antigen-specific CD8+ T cells, which led to tumor growth delay and modestly prolonged survival in MOC2-E6E7 tumors. Increased efficacy was seen in a separate head and neck cancer tumor model, mEER, which obligately expresses E7 antigen. Collectively, our data highlight the need for both immunogenicity and 'driver' status of target antigens to be considered in cancer vaccine design.

MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199.

Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.

Selective BCL-2 inhibition by ABT-199 causes on-target cell death in acute myeloid leukemia.

B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here, we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nmol/L, and cell death occurred within 2 hours. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on-target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.

Notch activation inhibits AML growth and survival: a potential therapeutic approach.

Although aberrant Notch activation contributes to leukemogenesis in T cells, its role in acute myelogenous leukemia (AML) remains unclear. Here, we report that human AML samples have robust expression of Notch receptors; however, Notch receptor activation and expression of downstream Notch targets are remarkably low, suggesting that Notch is present but not constitutively activated in human AML. The functional role of these Notch receptors in AML is not known. Induced activation through any of the Notch receptors (Notch1-4), or through the Notch target Hairy/Enhancer of Split 1 (HES1), consistently leads to AML growth arrest and caspase-dependent apoptosis, which are associated with B cell lymphoma 2 (BCL2) loss and enhanced p53/p21 expression. These effects were dependent on the HES1 repressor domain and were rescued through reexpression of BCL2. Importantly, activated Notch1, Notch2, and HES1 all led to inhibited AML growth in vivo, and Notch inhibition via dnMAML enhanced proliferation in vivo, thus revealing the physiological inhibition of AML growth in vivo in response to Notch signaling. As a novel therapeutic approach, we used a Notch agonist peptide that led to significant apoptosis in AML patient samples. In conclusion, we report consistent Notch-mediated growth arrest and apoptosis in human AML, and propose the development of Notch agonists as a potential therapeutic approach in AML.

Lack of NF-kappaB1 (p105/p50) attenuates unloading-induced downregulation of PPARalpha and PPARalpha-regulated gene expression in rodent heart.

OBJECTIVE: Unloading of the rodent heart activates the fetal gene program, decreases peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARalpha-regulated gene expression (MCAD), and induces cardiomyocyte atrophy. NF-kappaB regulates the fetal gene program and PPARalpha-regulated gene expression during cardiac hypertrophy and induces atrophy in skeletal muscle. Our objective was to test the hypothesis that NF-kappaB is the regulator for activation of the fetal gene program, for downregulation of PPARalpha and PPARalpha-regulated gene expression, and for cardiomyocyte atrophy in the heart subjected to mechanical unloading. METHODS: Activation of the inhibitory kappa B kinase beta (IKKbeta)/NF-kappaB pathways were measured in the heterotopically transplanted rat heart using Western blotting of total and phospho-IKKbeta and using transcription factor ELISA's for the five members of the NF-kappaB family (p65 (Rel A), p105/p50, c-Rel, RelB, and p100/p52). In loss of function experiments, we transplanted hearts of p105/p50 knockout mice into wildtype mice and compared changes in gene expression and cardiomyocyte size with wildtype hearts transplanted into wildtype mice. RESULTS: Total and phospho-IKKbeta levels significantly increased in the transplanted heart seven days after surgery. The activation of IKKbeta was paralleled by increased DNA binding activity of p65 and p105/p50. Mechanical unloading induced myosin heavy chain beta expression and decreased cardiomyocyte size in hearts of both wildtype and p105/p050 knockout animals. In contrast, the downregulation of PPARalpha and MCAD was significantly attenuated or prevented in the hearts of p105/p50 knockout mice. CONCLUSIONS: The IKKbeta/p65/p50 pathway is activated in the unloaded rodent heart and a likely regulator for the downregulation of PPARalpha and PPARalpha-regulated gene expression.

Activation of PPARgamma enhances myocardial glucose oxidation and improves contractile function in isolated working hearts of ZDF rats.

It is suggested that insulin resistance and metabolic maladaptation of the heart are causes of contractile dysfunction. We tested the hypothesis whether systemic PPARgamma activation, by changing the metabolic profile in a model of insulin resistance and type 2 diabetes (the ZDF rat) in vivo, improves contractile function of the heart in vitro. Male Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats, at 53-56 days of age, were treated with either GI-262570 (a nonthiazolidinedione PPARgamma agonist; A) or vehicle (V) for 1 wk. Agonist treatment resulted in correction of hyperglycemia and dyslipidemia, as well as in reduced hyperinsulinemia. The accumulation of triacylglycerols in the myocardium, characteristic of the ZDF rat, disappeared with treatment. Cardiac power and rates of glucose oxidation in the isolated working heart were significantly reduced in ZDF-V rats, but both parameters increased to nondiabetic levels with agonist treatment. In ZDF-V hearts, transcript levels of PPARalpha-regulated genes and of myosin heavy chain-beta were upregulated, whereas GLUT4 was downregulated compared with ZL. Agonist treatment of ZDF rats reduced PPARalpha-regulated genes and increased transcripts of GLUT4 and GLUT1. In conclusion, by changing the metabolic profile, reducing myocardial lipid accumulation, and promoting the downregulation of PPARalpha-regulated genes, PPARgamma activation leads to an increased capacity of the myocardium to oxidize glucose and to a tighter coupling of oxidative metabolism and contraction in the setting of insulin resistance and type 2 diabetes.

Intramyocardial lipid accumulation in the failing human heart resembles the lipotoxic rat heart.

In animal models of lipotoxicity, accumulation of triglycerides within cardiomyocytes is associated with contractile dysfunction. However, whether intramyocardial lipid deposition is a feature of human heart failure remains to be established. We hypothesized that intramyocardial lipid accumulation is a common feature of non-ischemic heart failure and is associated with changes in gene expression similar to those found in an animal model of lipotoxicity. Intramyocardial lipid staining with oil red O and gene expression analysis was performed on heart tissue from 27 patients (9 female) with non-ischemic heart failure. We determined intramyocardial lipid, gene expression, and contractile function in hearts from 6 Zucker diabetic fatty (ZDF) and 6 Zucker lean (ZL) rats. Intramyocardial lipid overload was present in 30% of non-ischemic failing hearts. The highest levels of lipid staining were observed in patients with diabetes and obesity (BMI>30). Intramyocardial lipid deposition was associated with an up-regulation of peroxisome proliferator-activated receptor alpha (PPARalpha) -regulated genes, myosin heavy chain beta (MHC-beta), and tumor necrosis factor alpha (TNF-alpha). Intramyocardial lipid overload in the hearts of ZDF rats was associated with contractile dysfunction and changes in gene expression similar to changes found in failing human hearts with lipid overload. Our findings identify a subgroup of patients with heart failure and severe metabolic dysregulation characterized by intramyocardial triglyceride overload and changes in gene expression that are associated with contractile dysfunction.

Linking gene expression to function: metabolic flexibility in the normal and diseased heart.

Metabolism transfers energy from substrates to ATP. As a "metabolic omnivore," the normal heart adapts to changes in the environment by switching from one substrate to another. We propose that this flexibility is lost in the maladapted, diseased heart. Both adaptation and maladaptation are the results of metabolic signals that regulate transcription of key cardiac regulatory genes. We propose that metabolic remodeling precedes, initiates, and sustains functional and structural remodeling. The process of metabolic remodeling then becomes a target for pharmacological intervention restoring metabolic flexibility and normal contractile function of the heart.

IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a.

Nuclear exclusion of the forkhead transcription factor FOXO3a by protein kinase Akt contributes to cell survival. We investigated the pathological relationship between phosphoylated-Akt (Akt-p) and FOXO3a in primary tumors. Surprisingly, FOXO3a was found to be excluded from the nuclei of some tumors lacking Akt-p, suggesting an Akt-independent mechanism of regulating FOXO3a localization. We provide evidence for such a mechanism by showing that IkappaB kinase (IKK) physically interacts with, phosphorylates, and inhibits FOXO3a independent of Akt and causes proteolysis of FOXO3a via the Ub-dependent proteasome pathway. Cytoplasmic FOXO3a correlates with expression of IKKbeta or Akt-p in many tumors and associates with poor survival in breast cancer. Further, constitutive expression of IKKbeta promotes cell proliferation and tumorigenesis that can be overridden by FOXO3a. These results suggest the negative regulation of FOXO factors by IKK as a key mechanism for promoting cell growth and tumorigenesis.