ABSTRACT
Splicing factor (SF) gene mutations are frequent in myelodysplastic syndromes (MDS), and agents that modulate RNA splicing are hypothesized to provide clinical benefit. JNJ-64619178, a protein arginine methyltransferase 5 (PRMT5) inhibitor, was evaluated in patients with lower-risk (LR) MDS in a multi-part, Phase 1, multicenter study. The objectives were to determine a tolerable dose and to characterize safety, pharmacokinetics, pharmacodynamics, and preliminary clinical activity. JNJ-64619178 was administered on a 14 days on/7 days off schedule or every day on a 21-day cycle to patients with International Prognostic Scoring System (IPSS) Low or Intermediate-1 risk MDS who were red blood cell transfusion-dependent. Twenty-four patients were enrolled; 15 (62.5 %) patients had low IPSS risk score, while 18 (75.0 %) had an SF3B1 mutation. Median duration of treatment was 3.45 months (range: 0.03-6.93). No dose limiting toxicities were observed. The 0.5 mg once daily dose was considered better tolerated and chosen for dose expansion. Twenty-three (95.8 %) patients experienced treatment-emergent adverse events (TEAE). The most common TEAEs were neutropenia (15 [62.5 %]) and thrombocytopenia (14 [58.3 %]). JNJ-64619178 pharmacokinetics was dose-dependent. Target engagement as measured by plasma symmetric di-methylarginine was observed across all dose levels; however, variant allele frequency of clonal mutations in bone marrow or blood did not show sustained reductions from baseline. No patient achieved objective response or hematologic improvement per International Working Group 2006 criteria, or transfusion independence. A tolerable dose of JNJ-64619178 was identified in patients with LR MDS. However, no evidence of clinical benefit was observed.
Subject(s)
Anemia , Myelodysplastic Syndromes , Humans , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/therapeutic use , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/genetics , Anemia/drug therapy , Bone Marrow , Treatment OutcomeABSTRACT
PURPOSE: In this first-in-human, Phase 1, open-label, multicenter study, we evaluated JNJ-64619178, a selective and potent PRMT5 inhibitor, in patients with advanced malignant solid tumors or non-Hodgkin lymphomas (NHL). The primary objective was to evaluate the safety and to identify a recommended Phase 2 dose (RP2D) of JNJ-64619178. PATIENTS AND METHODS: Adult patients with treatment-refractory advanced solid tumors or NHL and measurable disease received escalating doses of JNJ-64619178 following two schedules (Schedule A: 14 days on/7 days off; Schedule B: every day on a 21-day cycle). Safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity were evaluated. RESULTS: Ninety patients received JNJ-64619178. Thrombocytopenia was identified as the only dose-limiting toxicity. JNJ-64619178 showed dose-proportional PK and robust target engagement, as measured by plasma symmetric dimethylarginine, across all dose levels. The objective response rate was 5.6% (5 of 90). Patients with adenoid cystic carcinoma (ACC) had an ORR of 11.5% (3 of 26) and a median progression-free survival of 19.1 months. CONCLUSIONS: JNJ-64619178 demonstrated manageable dose-dependent toxicity and preliminary evidence of antitumor activity in ACC and other tumor types. Plasma exposure was dose dependent, and target inhibition was maintained with intermittent and continuous dosing. On the basis of safety, clinical activity, PK, and PD findings, two provisional RP2Ds were selected: 1.5 mg intermittently and 1.0 mg once daily. Aside from ACC, clinical benefit was limited, and biomarkers to enrich for responsiveness to PRMT5 inhibition will be needed for further development.
Subject(s)
Carcinoma, Adenoid Cystic , Lymphoma, Non-Hodgkin , Neoplasms , Adult , Humans , Protein-Arginine N-Methyltransferases/genetics , Neoplasms/drug therapy , Neoplasms/pathology , Pyrimidines , PyrrolesABSTRACT
Previous studies have demonstrated that the small molecule thrombopoietin (TPO) mimetic, eltrombopag (E), induces apoptosis in acute myeloid leukemia (AML) cells. Here, we sought to define the mechanism of the anti-leukemic effect of eltrombopag. Our studies demonstrate that, at a concentration of 5 µM E in 2% serum, E induces apoptosis in leukemia cells by triggering PARP cleavage and activation of caspase cascades within 2-6 hours. The induction of apoptotic enzymes is critically dependent on drug concentration and the concentration of serum. This effect is not associated with an alteration in mitochondrial potential but is associated with a rapid decrease in a reactive oxygen species (ROS) in particular hydrogen peroxide (H2O2). Interestingly, E also decreases mitochondrial maximal and spare respiratory capacities suggesting an induced mitochondrial dysfunction that may not be readily apparent under basal conditions but becomes manifest only under stress. Co-treatment of MOLM14 AML cells with E plus Tempol or H2O2 provides a partial rescue of cell toxicity. Ferric ammonioum citrate (FAC) also antagonized the E induced toxicity, by inducing notable increase in ROS level. Overall, we propose that E dramatically decreases ROS levels leading to a disruption of AML intracellular metabolism and rapid cell death.
Subject(s)
Antineoplastic Agents/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Reactive Oxygen Species/antagonists & inhibitors , Thyroxine/therapeutic use , Adenosine Triphosphate/metabolism , Adult , Aged , Apoptosis/drug effects , Blotting, Western , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Humans , Hydrogen Peroxide/antagonists & inhibitors , Leukemia, Myeloid, Acute/metabolism , Male , Middle Aged , Oxygen Consumption/drug effects , Tumor Cells, Cultured , Young AdultABSTRACT
T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor ß (FRß) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRß (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRß as well as endogenous FRß(+) AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRß(+) THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRß expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRß expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34(+) HSCs in vitro. Our results indicate that FRß is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA.
Subject(s)
Folate Receptor 2/antagonists & inhibitors , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/therapy , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Animals , Cells, Cultured , Female , Folate Receptor 2/genetics , Genetic Therapy/methods , HEK293 Cells , Humans , Immunotherapy, Adoptive/methods , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Molecular Targeted Therapy , Mutant Chimeric Proteins/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/pathologyABSTRACT
PURPOSE: Recent results have shown that myeloproliferative neoplasms (MPN) are strongly associated with constitutive activation of the Janus-activated kinase (JAK)2 tyrosine kinase. However, JAK2 inhibitors currently approved or under development for treating myeloproliferative neoplasms do not selectively deplete the malignant clone, and the inhibition of activity of the drug target (JAK2) has not been rigorously evaluated in the clinical studies. Therefore, in this study we developed an in vitro assay to gain insight into how effectively JAK2 activity is inhibited in the samples of patients. EXPERIMENTAL DESIGN: We treated primary cells from normal donors and patients with MPN with JAK2 inhibitors and measured phosphorylation of downstream targets STAT5 and STAT3 by flow cytometry. Obtained results were next correlated with JAK2 V617F allele burden and plasma cytokine level. RESULTS: We observed a dose-dependent decrease in pSTAT5 and pSTAT3 in ex vivo treated granulocytes. However, phosphorylation of STAT3 and STAT5 in cells from patients with myelofibrosis was significantly less inhibited when compared with cells from patients with polycythemia vera, essential thrombocythemia, and normal donors. Sensitivity to inhibition did not correlate with JAK2 V617F clonal burden. Mixing studies using plasma from patients with myelofibrosis did not transfer resistance to sensitive cells. Likewise, no single cytokine measured seemed to account for the observed pattern of resistance. CONCLUSIONS: Taken together, these observations suggest that there are cell intrinsic mechanisms that define a priori resistance to JAK2 inhibition in myelofibrosis, and the lesion is localized upstream of STAT3 and STAT5.
Subject(s)
Drug Resistance , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Primary Myelofibrosis/metabolism , Protein Kinase Inhibitors/pharmacology , Adult , Aged , Aged, 80 and over , Alleles , Carbazoles/pharmacology , Cytokines/pharmacology , Female , Furans , Gene Frequency , Humans , Janus Kinase 2/genetics , Male , Middle Aged , Mutation , Neutrophils/drug effects , Neutrophils/metabolism , Phosphorylation , Primary Myelofibrosis/genetics , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/metabolismABSTRACT
Targeting chromatin regulators for the treatment of malignancies has shown great promise, but also revealed significant challenges. By employing an elegant shRNA screen and a selective pharmacological inhibitor, a recent study published in Nature establishes the bromodomain protein Brd4 as novel target in acute myeloid leukemia (AML).
ABSTRACT
6-Phenylpyrrolocytosine (PhpC) is a cytosine mimic with excellent base-pairing fidelity, thermal stability, and high fluorescence. In this work, PhpC-containing small interfering RNAs (siRNAs) are shown to possess thermal stability and gene silencing activity that is virtually identical to that of natural siRNA. The emissive properties of PhpC allow the cellular trafficking of PhpC-containing siRNAs to be monitored by fluorescence microscopy. Accumulation in the cytoplasm of HeLa cells was observed using real time imaging. These findings demonstrate that PhpC-modified siRNAs retain the properties of natural siRNAs while allowing for fluorescence-based detection and monitoring, providing an ideal system for probing siRNA uptake and trafficking.
Subject(s)
Cytosine/analogs & derivatives , Cytosine/metabolism , RNA Transport , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , Cytosine/chemistry , Fluorescence , Gene Silencing , HeLa Cells , Humans , Molecular Structure , RNA, Small Interfering/analysis , RNA, Small Interfering/genetics , Stereoisomerism , TemperatureABSTRACT
We report that combining a DNA analog (2'F-ANA) with rigid RNA analogs [2'F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1-1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides. These heavily or fully modified duplexes are active against a range of mRNA targets. Effective patterns of modification were chosen based on screens using two sequences targeting firefly luciferase. We then applied the most effective duplex designs to the knockdown of the eIF4E binding proteins 4E-BP1 and 4E-BP2. We identified modified duplexes with potency comparable to native siRNA. Modified duplexes showed dramatically enhanced stability to serum nucleases, and were characterized by circular dichroism and thermal denaturation studies. Chemical modification significantly reduced the immunostimulatory properties of these siRNAs in human peripheral blood mononuclear cells.
Subject(s)
Arabinonucleotides/chemistry , Oligonucleotides/chemistry , RNA Interference , RNA, Small Interfering/chemistry , RNA/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins , Circular Dichroism , DNA/chemistry , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Humans , Interferons/biosynthesis , Luciferases, Firefly/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA Stability , RNA, Messenger/metabolism , RNA, Small Interfering/metabolismABSTRACT
Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transferase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contributes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.
Subject(s)
Leukemia/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Proto-Oncogene Proteins/metabolism , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Histone-Lysine N-Methyltransferase , Histones/metabolism , Homeodomain Proteins/genetics , Humans , Leukemia/genetics , Leukemia/pathology , Mice , Myeloid-Lymphoid Leukemia Protein/genetics , Protein Binding , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myb/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Biologic characterization of SB-559457 (SB), a nonpeptidyl hydrazone class of thrombopoietin receptor (Mpl) agonist, revealed toxicity toward human leukemia cells. Antiproliferative effects followed by significant, nonapoptotic, cell death within 72 hours occurred in 24 of 26 acute myeloid leukemia, 0 of 6 acute lymphoblastic leukemia, and 3 of 6 chronic myeloid leukemia patient samples exposed to SB, but not recombinant human thrombopoietin (rhTpo), in liquid suspension culture. Further investigation revealed increased phosphorylation of p70S6/S6 kinases in SB-, but not in rhTpo-, treated cells. Expression profiling of cells exposed to SB versus rhTpo revealed statistically significant, more than 2-fold changes in GAPDH and REDD1 gene expression, confirmed by quantitative reverse-transcribed polymerase chain reaction. These genes, induced in energy or hypoxia stressed cells, have been implicated in cell death pathways, and may provide important clues to the mechanism of SB-induced, leukemic cell death. These results suggest that nonpeptidyl, hydrazone class Mpl agonists may be clinically useful antileukemic agents by virtue of their combined thrombopoietic and antileukemic effects.
Subject(s)
Antineoplastic Agents/pharmacology , Hydrazones/pharmacology , Leukemia, Myeloid/pathology , Receptors, Thrombopoietin/agonists , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Cell Death/drug effects , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Gene Expression Regulation, Leukemic/drug effects , Humans , Hydrazones/chemistry , Hydrazones/toxicity , Leukemia, Myeloid/enzymology , Leukemia, Myeloid/genetics , Peptides/pharmacology , Phosphorylation/drug effects , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, CulturedABSTRACT
The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment, proliferation, and differentiation. Given its critical functions, c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay, we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic, functionality of binding was shown. The mimic decreased c-Myb expression, and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest, the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally, in studies using normal human CD34(+) cells, we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation, and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate, these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis.
Subject(s)
Blood Cells/metabolism , Feedback, Physiological/physiology , Gene Expression Regulation , Genes, myb/physiology , Hematopoiesis/physiology , MicroRNAs/metabolism , 3' Untranslated Regions/genetics , Antigens, CD34/metabolism , Base Sequence , Binding Sites/genetics , Blood Cells/cytology , Blotting, Western , Cell Cycle/genetics , Cell Differentiation/physiology , Flow Cytometry , Homeostasis/physiology , Humans , Immunoprecipitation , K562 Cells , Leukemia/genetics , Leukemia/metabolism , Molecular Sequence Data , Protein Binding , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myb/genetics , Proto-Oncogene Proteins c-myb/metabolism , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Children with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate. We used in vitro and mouse transplantation assays to study hematopoiesis in trisomy 21 fetal livers with normal GATA1 alleles. Remarkably, trisomy 21 progenitors exhibited enhanced production of erythroid and megakaryocytic cells that proliferated excessively. Our findings indicate that trisomy 21 itself is associated with cell-autonomous expansion of erythro-megakaryocytic progenitors. This may predispose to TMD and AMKL by increasing the pool of cells susceptible to malignant transformation through acquired mutations in GATA1 and other cooperating genes.
Subject(s)
Cell Differentiation/genetics , Down Syndrome/embryology , Erythrocytes/physiology , Megakaryocytes/physiology , Animals , Down Syndrome/pathology , Erythroid Precursor Cells/physiology , Female , Fetal Tissue Transplantation/physiology , Hematopoietic System/embryology , Humans , Liver/cytology , Liver/embryology , Liver/pathology , Liver Transplantation/physiology , Mice , Mice, SCID , Myeloid Progenitor Cells/physiology , PregnancyABSTRACT
The cytotoxic RNase, Onconase (ONC), isolated from amphibian oocytes, was used to study its effect on the radiation response in A549 human NSCLC in vitro and in vivo. In cell culture studies, we found that ONC increased the radiation response by ONC-induced inhibition of O2 consumption (QO2). The occurrence of apoptosis was increased by ONC and was dependent on dosages and time exposure (measured by a Tunnel in situ cell death detection assay). Moreover, ONC inhibited sublethal damage repair (SLDR), confirmed by a split dose experiment. In animal studies, ONC significantly increased the radiation-induced tumor growth delay of A549 tumors in vivo. Using a non-invasive DCE-MRI technology, ONC-induced changes of perfusion were observed in A549 tumors. We concluded that the ONC-induced enhancement in tumor oxygenation was mainly due to the reduction in QO2 rather than an increase in tumor blood flow. This investigation suggests important potential clinical uses of ONC for the treatment of NSCLC cancer patients.
Subject(s)
Antineoplastic Agents , Lung Neoplasms , Ribonucleases/pharmacology , Transplantation, Heterologous , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor/drug effects , Cell Line, Tumor/radiation effects , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/radiotherapy , Mice , Mice, Nude , Neoplasm Transplantation , Ribonucleases/therapeutic useABSTRACT
The C-Myb transcription factor is essential for hematopoiesis, including in the T-cell lineage. The C-Myb locus is a common site of retroviral insertional mutagenesis, however no recurrent genomic involvement has been reported in human malignancies. Here, we identified 2 types of genomic alterations involving the C-MYB locus at 6q23 in human T-cell acute leukemia (T-ALL). First, we found a reciprocal translocation, t(6;7)(q23;q34), that juxtaposed the TCRB and C-MYB loci (n = 6 cases). Second, a genome-wide copy-number analysis by array-based comparative genomic hybridization (array-CGH) identified short somatic duplications that include C-MYB (MYB(dup), n = 13 cases of 84 T-ALL, 15%). Expression analysis, including allele-specific approaches, showed stronger C-MYB expression in the MYB-rearranged cases compared with other T-ALLs, and a dramatically skewed C-MYB allele expression in the TCRB-MYB cases, which suggests that a translocation-driven deregulated expression may overcome a cellular attempt to down-regulate C-MYB. Strikingly, profiling of the T-ALLs by clinical, genomic, and large-scale gene expression analyses shows that the TCRB-MYB translocation defines a new T-ALL subtype associated with a very young age for T-cell leukemia (median, 2.2 years) and with a proliferation/mitosis expression signature. By contrast, the MYB(dup) alteration was associated with the previously defined T-ALL subtypes.
Subject(s)
Chromosomes, Human, Pair 6/genetics , Chromosomes, Human, Pair 7/genetics , Leukemia-Lymphoma, Adult T-Cell/genetics , Proto-Oncogene Proteins c-myb/genetics , Translocation, Genetic , Adolescent , Adult , Age Distribution , Aged , Base Sequence , Child , Child, Preschool , Female , Gene Dosage , Gene Expression Profiling , Genome, Human , Humans , Infant , Male , Middle Aged , Molecular Sequence Data , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Sequence Homology, Nucleic AcidABSTRACT
BACKGROUND: The cytotoxic RNase, ranpirnase (ONCONASE, ONC), may have promising therapeutic implication as an alternative for cisplatin for the treatment of lung cancer, due to inhibition of protein synthesis by t-RNA cleavage. MATERIALS AND METHODS: A549 and NCI-H1975 human NSCLC cell lines were cultured in the presence and absence of ONC. Cytotoxicity was monitored using a clonogenic assay. Using an inverted phase and fluorescence microscope, we studied whether apoptosis was induced by ONC in gefitinib-induced apoptosis-resistant A549 tumor cells. The therapeutic effectiveness of ONC was studied via single and multiple administrations on A549 human non-small cell lung cancer (NSCLC), including tumors previously untreatable by cisplatin. ONC-induced changes in ATP levels were also monitored by non-localized phosphorus MR spectroscopy. RESULTS: ONC significantly inhibited the cell growth of A549 tumors. Apoptosis was significantly induced by ONC in a dose-dependent manner. In animal studies, multiple small doses of ONC were more effective than one large single dose for the inhibition of tumor growth with reduced side-effects, probably due to the normalization of leaky tumor vessels. ONC in combination with cisplatin significantly reduced tumor growth of A549 tumors. In large tumors, including those unsuccessfully treated with cisplatin, ONC showed inhibition of tumor growth, while a second treatment of cisplatin did not. During monitoring by non-localized phosphorus MR spectroscopy, ATP levels decreased, likely due to ONC-induced inhibition of oxygen consumption (QO2). CONCLUSION: ONC significantly inhibited tumor growth of A549 NSCLC cells in both in vitro and in vivo studies. This investigation suggests important potential clinical uses of ONC for the treatment of NSCLC cancer patients.
Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Ribonucleases/pharmacology , Adenosine Triphosphate/metabolism , Animals , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Growth Processes/drug effects , Cell Line, Tumor , Cisplatin/administration & dosage , Drug Administration Schedule , Female , Humans , Lung Neoplasms/metabolism , Mice , Mice, Nude , Ribonucleases/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
Myb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G(1)/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G(2)/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-myb expression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G(2)/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-myb functions in regulating human hematopoiesis.
Subject(s)
Cell Division , Cyclin B/metabolism , G2 Phase , Gene Expression Regulation , Hematopoietic System , Antigens, CD34/metabolism , Base Sequence , Cells, Cultured , Cyclin B/genetics , Cyclin B1 , Gene Expression Regulation/drug effects , Humans , Interleukin-2/pharmacology , Phytohemagglutinins/pharmacology , Proto-Oncogene Proteins c-myb/genetics , Proto-Oncogene Proteins c-myb/metabolism , RNA, Small Interfering/genetics , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
Many algorithms based on computational analysis and thermodynamic parameters have been developed to predict the secondary structure of RNA. Still, many antisense oligodeoxynucleotides (AS ODNs), or siRNA molecules designed according to these predictions fail to silence the intended target, whereas other, not fulfilling those criteria prove highly active. We have developed a reliable mapping strategy, which allows us to predict the sites accessible for hybridization within target mRNA in vitro and in vivo. Our mapping experiments employed self-quenching reporter molecules (SQRMs) and were first carried out in a cell free system, and later confirmed in vivo.
Subject(s)
DNA-Binding Proteins/genetics , Drug Design , Oligonucleotides, Antisense , RNA, Small Interfering , Blotting, Western/methods , Cell Line, Tumor , Genetic Therapy , Humans , Lymphoma/genetics , Lymphoma/therapy , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/genetics , Polymerase Chain Reaction/methods , Proto-Oncogene Proteins c-bcl-6 , RNA, Messenger/genetics , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Sequence AnalysisABSTRACT
Our work is motivated by the belief that RNA targeted gene silencing agents can be developed into effective drugs for treating hematologic malignancies. In many experimental systems, antisense nucleic acids of various composition, including antisense oligodeoxynucleotides (AS ODNs) and short interfering RNA (siRNA), have been shown to perturb gene expression in a sequence specific manner. Nevertheless, our clinical experience, and those of others, have led us to conclude that the antisense nucleic acids (ASNAs) we, and others, employ need to be optimized with regard to intracellular delivery, targeting, chemical composition, and efficiency of mRNA destruction. We have hypothesized that addressing these critical issues will lead to the development of practical and effective nucleic acid drugs. An overview of our recent work which seeks to addresses these core issues is contained within this review.
Subject(s)
Hematologic Neoplasms/drug therapy , Nucleic Acids/therapeutic use , Humans , Nucleic Acids/administration & dosage , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/therapeutic use , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/chemistry , RNA, Small Interfering/therapeutic useABSTRACT
We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20-30 base oligodeoxynucleotides with 5-6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem-loop of the SQRM suggests that SQRM be made to target natural stem-loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.
Subject(s)
Fluorescent Dyes/chemistry , Nucleic Acid Hybridization/methods , Oligodeoxyribonucleotides, Antisense/chemistry , RNA, Messenger/chemistry , Animals , Cell Line , Cricetinae , DNA-Binding Proteins/genetics , Humans , Protein Biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-6 , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-myb/genetics , RNA, Messenger/metabolism , Rabbits , Transcription Factors/genetics , Transcription, GeneticABSTRACT
We studied the effects of Lyn ablation on the survival of drug-resistant chronic myelogenous leukemia (CML) blast crisis cells using siRNA. Lyn siRNA reduced Lyn protein in both normal hematopoietic cells and BCR-ABL1-expressing (BCR-ABL1(+)) blasts by 80-95%. Within 48 h, siRNA-treated BCR-ABL1(+) blasts underwent apoptosis, whereas normal cells remained viable. This increased dependence on Lyn signaling for BCR-ABL1(+) blast survival provides the basis for rational treatment of drug-resistant CML blast crisis, particularly when lymphoid in nature.