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1.
FASEB J ; 33(6): 7647-7666, 2019 06.
Article in English | MEDLINE | ID: mdl-30917007

ABSTRACT

The su(var)3-9, enhancer of zeste, trithorax (SET)/inhibitor 2 of protein phosphatase 2A (PP2A) oncoprotein binds and inhibits PP2A, composed of various isoforms of scaffolding, regulatory, and catalytic subunits. Targeting SET with a sphingolipid analog drug fingolimod (FTY720) or ceramide leads to the reactivation of tumor suppressor PP2A. However, molecular details of the SET-FTY720 or SET-ceramide, and mechanism of FTY720-dependent PP2A activation, remain unknown. Here, we report the first in solution examination of the SET-FTY720 or SET-ceramide complexes by NMR spectroscopy. FTY720-ceramide binding resulted in chemical shifts of residues residing at the N terminus of SET, preventing its dimerization or oligomerization. This then released SET from PP2ACα, resulting in PP2A activation, while monomeric SET remained associated with the B56γ. Our data also suggest that the PP2A holoenzyme, composed of PP2A-Aß, PP2A-B56γ, and PP2ACα subunits, is selectively activated in response to the formation of the SET-FTY720 complex in A549 cells. Various PP2A-associated downstream effector proteins in the presence or absence of FTY720 were then identified by stable isotope labeling with amino cells in cell culture, including tumor suppressor nonmuscle myosin IIA. Attenuation of FTY720-SET association by point mutations of residues that are involved in FTY720 binding or dephosphorylation of SET at Serine 171, enhanced SET oligomerization and the formation of the SET-PP2A inhibitory complex, leading to resistance to FTY720-dependent PP2A activation.-De Palma, R. M., Parnham, S. R., Li, Y., Oaks, J. J., Peterson, Y. K., Szulc, Z. M., Roth, B. M., Xing, Y., Ogretmen, B. The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction.


Subject(s)
Fingolimod Hydrochloride/pharmacology , Histone-Lysine N-Methyltransferase/metabolism , Magnetic Resonance Spectroscopy/methods , Protein Phosphatase 2/metabolism , Sphingosine 1 Phosphate Receptor Modulators/pharmacology , A549 Cells , Dimerization , Humans , Protein Binding
2.
Cureus ; 10(9): e3388, 2018 Sep 29.
Article in English | MEDLINE | ID: mdl-30524916

ABSTRACT

A case of a 51-year-old female with history of hypertension and a significant family history of premature coronary artery disease presented to the hospital after cardiac arrest. She successfully completed a targeted temperature management therapy with full neurologic recovery. Her hospital course was complicated by several bouts of ventricular fibrillation (VF) arrest which was rescued by timely defibrillation, high quality cardiorespiratory resuscitation, and administration of antiarrhythmic medications and inotropic agents. An automatic implantable cardioverter defibrillator (AICD) was inserted for secondary prevention of sudden cardiac death (SCD). A targeted genetic testing for idiopathic ventricular fibrillation revealed a mutation in the desmoglein-2 (DSG2) gene involved in arrhythmogenic right ventricular cardiomyopathy (ARVC). Eventually, a ventricular fibrillation radiofrequency ablation prevented recurrence of fatal arrhythmia and its associated symptoms.

3.
Cureus ; 10(6): e2877, 2018 Jun 25.
Article in English | MEDLINE | ID: mdl-30155379

ABSTRACT

We present a case of hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome complicated by acute pancreatitis that responded favorably to conservative measures. The microvascular abnormalities and heightened inflammatory state present in HELLP syndrome and severe preeclampsia might be responsible for pancreatic ischemia or cytokine-induced pancreatic damage, which could result in acute pancreatitis.

4.
Nat Commun ; 9(1): 2791, 2018 07 18.
Article in English | MEDLINE | ID: mdl-30022024

ABSTRACT

Common fragile sites (CFSs) are prone to chromosomal breakage and are hotspots for chromosomal rearrangements in cancer cells. We uncovered a novel function of Fanconi anemia (FA) protein FANCM in the protection of CFSs that is independent of the FA core complex and the FANCI-FANCD2 complex. FANCM, along with its binding partners FAAP24 and MHF1/2, is recruited to CFS-derived structure-prone AT-rich sequences, where it suppresses DNA double-strand break (DSB) formation and mitotic recombination in a manner dependent on FANCM translocase activity. Interestingly, we also identified an indispensable function of Rad52 in the repair of DSBs at CFS-derived AT-rich sequences, despite its nonessential function in general homologous recombination (HR) in mammalian cells. Suppression of Rad52 expression in combination with FANCM knockout drastically reduces cell and tumor growth, suggesting a synthetic lethality interaction between these two genes, which offers a potential targeted treatment strategy for FANCM-deficient tumors with Rad52 inhibition.


Subject(s)
Chromosome Fragile Sites , Colonic Neoplasms/genetics , DNA Helicases/genetics , Gene Expression Regulation, Neoplastic , Rad52 DNA Repair and Recombination Protein/genetics , Recombinational DNA Repair , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Colonic Neoplasms/therapy , DNA/genetics , DNA/metabolism , DNA Breaks, Double-Stranded , DNA Helicases/antagonists & inhibitors , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fanconi Anemia Complementation Group Proteins , Female , HCT116 Cells , HEK293 Cells , Humans , Injections, Subcutaneous , Mice , Mice, Nude , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rad52 DNA Repair and Recombination Protein/antagonists & inhibitors , Rad52 DNA Repair and Recombination Protein/metabolism , Signal Transduction , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor Assays
5.
Case Rep Vasc Med ; 2017: 9613863, 2017.
Article in English | MEDLINE | ID: mdl-29181220

ABSTRACT

We herein report a serious vascular complication of diagnostic cardiac catheterization due to an embolization of an Angio-Seal closure device causing acute lower limb ischemia. The Angio-Seal was deployed via the right femoral artery following the catheterization which embolized several hours later to the right popliteal artery. Fogarty embolectomy restored perfusion to the right lower limb; however, compartment syndrome subsequently developed which required evacuation of a hematoma and repair of right popliteal artery.

6.
Cureus ; 9(1): e974, 2017 Jan 12.
Article in English | MEDLINE | ID: mdl-28191378

ABSTRACT

We report a case of a 43-year-old Israeli male who presented with an intermittent fever associated with a gradual appearance of diffusely scattered erythematous non-pruritic maculopapular lesions, generalized body malaise, muscle aches, and distal extremity weakness. He works in the Israeli military and has been exposed to dogs that are used to search for people in tunnels and claimed that he had removed ticks from the dogs. In the hospital, he presented with fever, a diffuse maculopapular rash, and an isolated round black eschar. He was started on doxycycline based on suspected Mediterranean spotted fever (MSF) in which he improved significantly with resolution of his clinical complaints. His immunoglobulin G (IgG) MSF antibody came back positive.

7.
Sci Signal ; 8(381): ra58, 2015 Jun 16.
Article in English | MEDLINE | ID: mdl-26082434

ABSTRACT

During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. We found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C'3-OH) in S1P and Asp(684) in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with makorin ring finger protein 1 (MKRN1), an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell-derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth.


Subject(s)
Carcinoma, Lewis Lung/metabolism , Cell Nucleus/metabolism , Lysophospholipids/metabolism , Molecular Docking Simulation , Neoplasm Proteins/metabolism , Sphingosine/analogs & derivatives , Telomerase/metabolism , Allosteric Regulation/genetics , Animals , Carcinoma, Lewis Lung/genetics , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/pathology , Humans , Lysophospholipids/genetics , Mice , Mice, Knockout , Mice, SCID , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Phosphorylation/genetics , Protein Binding , Sphingosine/genetics , Sphingosine/metabolism , Telomerase/antagonists & inhibitors , Telomerase/genetics
8.
Front Oncol ; 4: 388, 2014.
Article in English | MEDLINE | ID: mdl-25642418

ABSTRACT

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is a primary regulator of cellular proliferation through targeting of proliferative kinases, cell cycle regulators, and apoptosis inhibitors. It is through the regulation of these regulatory elements that gives PP2A tumor suppressor functions. In addition to mutations on the regulatory subunits, the phosphatase/tumor suppressing activity of PP2A is also inhibited in several cancer types due to overexpression or modification of the endogenous PP2A inhibitors such as SET/I2PP2A. This review focuses on the current literature regarding the interactions between the lipid signaling molecules, selectively sphingolipids, and the PP2A inhibitor SET for the regulation of PP2A, and the therapeutic potential of sphingolipids as PP2A activators for tumor suppression via targeting SET oncoprotein.

9.
J Clin Invest ; 123(10): 4144-57, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23999433

ABSTRACT

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/ß-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and ß-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/ß-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.


Subject(s)
Antineoplastic Agents/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Neoplastic Stem Cells/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Phosphatase 2/metabolism , Animals , Apoptosis , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Resistance, Neoplasm , Enzyme Activators/pharmacology , Fingolimod Hydrochloride , Fusion Proteins, bcr-abl/metabolism , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/enzymology , Humans , Janus Kinase 2/metabolism , K562 Cells , Mice , Mice, Transgenic , Neoplastic Stem Cells/enzymology , Propylene Glycols/pharmacology , Sphingosine/analogs & derivatives , Sphingosine/pharmacology , Wnt Signaling Pathway , Xenograft Model Antitumor Assays , beta Catenin/metabolism
10.
Blood ; 122(17): 3034-44, 2013 Oct 24.
Article in English | MEDLINE | ID: mdl-23970380

ABSTRACT

As tyrosine kinase inhibitors (TKIs) fail to induce long-term response in blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL), novel therapies targeting leukemia-dysregulated pathways are necessary. Exportin-1 (XPO1), also known as chromosome maintenance protein 1, regulates cell growth and differentiation by controlling the nucleocytoplasmic trafficking of proteins and RNAs, some of which are aberrantly modulated in BCR-ABL1(+) leukemias. Using CD34(+) progenitors from CML, B-ALL, and healthy individuals, we found that XPO1 expression was markedly increased, mostly in a TKI-sensitive manner, in CML-BC and Ph(+) B-ALL. Notably, XPO1 was also elevated in Ph(-) B-ALL. Moreover, the clinically relevant XPO1 inhibitor KPT-330 strongly triggered apoptosis and impaired the clonogenic potential of leukemic, but not normal, CD34(+) progenitors, and increased survival of BCR-ABL1(+) mice, 50% of which remained alive and, mostly, became BCR-ABL1 negative. Moreover, KPT-330 compassionate use in a patient with TKI-resistant CML undergoing disease progression significantly reduced white blood cell count, blast cells, splenomegaly, lactate dehydrogenase levels, and bone pain. Mechanistically, KPT-330 altered the subcellular localization of leukemia-regulated factors including RNA-binding heterogeneous nuclear ribonucleoprotein A1 and the oncogene SET, thereby inducing reactivation of protein phosphatase 2A tumor suppressor and inhibition of BCR-ABL1 in CML-BC cells. Because XPO1 is important for leukemic cell survival, KPT-330 may represent an alternative therapy for TKI-refractory Ph(+) leukemias.


Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Hydrazines/pharmacology , Karyopherins/antagonists & inhibitors , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Triazoles/pharmacology , Adult , Animals , Antigens, CD34/genetics , Antigens, CD34/metabolism , Apoptosis/drug effects , Cell Proliferation/drug effects , Clinical Trials, Phase I as Topic , DNA-Binding Proteins , Drug Evaluation, Preclinical , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Histone Chaperones/antagonists & inhibitors , Histone Chaperones/genetics , Histone Chaperones/metabolism , Humans , Karyopherins/genetics , Karyopherins/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Mice , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Kinase Inhibitors/pharmacology , Protein Transport , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Ribonucleoproteins/antagonists & inhibitors , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , Exportin 1 Protein
11.
Blood ; 122(11): 1923-34, 2013 Sep 12.
Article in English | MEDLINE | ID: mdl-23926298

ABSTRACT

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.


Subject(s)
Janus Kinase 2/metabolism , Leukemia/drug therapy , Propylene Glycols/pharmacology , Protein Phosphatase 2/metabolism , Sphingosine/analogs & derivatives , Animals , Cell Line, Transformed , Cell Line, Tumor , Cells, Cultured , Class Ib Phosphatidylinositol 3-Kinase , DNA-Binding Proteins , Enzyme Activation/drug effects , Fingolimod Hydrochloride , Histone Chaperones , Humans , Immunoblotting , Immunosuppressive Agents/pharmacology , Janus Kinase 2/genetics , Kaplan-Meier Estimate , Leukemia/genetics , Leukemia/pathology , Mice , Mice, SCID , Mutation , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Phosphatase 2/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Sphingosine/pharmacology , Treatment Outcome
12.
J Hematol Oncol ; 6: 21, 2013 Mar 16.
Article in English | MEDLINE | ID: mdl-23497456

ABSTRACT

BACKGROUND: Activating mutations [internal tandem duplication (ITD)] or overexpression of the FMS-like tyrosine kinase receptor-3 (FLT3) gene are associated with poor outcome in acute myeloid leukemia (AML) patients, underscoring the need for novel therapeutic approaches. The natural product silvestrol has potent antitumor activity in several malignancies, but its therapeutic impact on distinct molecular high-risk AML subsets remains to be fully investigated. We examined here the preclinical activity of silvestrol in FLT3-ITD and FLT3 wild-type (wt) AML. METHODS: Silvestrol in vitro anti-leukemic activity was examined by colorimetric cell viability assay, colony-forming and flow cytometry assays assessing growth inhibition and apoptosis, respectively. Pharmacological activity of silvestrol on FLT3 mRNA translation, mRNA and protein expression was determined by RNA-immunoprecipitation, qRT-PCR and immunoblot analyses, respectively. Silvestrol in vivo efficacy was investigated using MV4-11 leukemia-engrafted mice. RESULTS: Silvestrol shows antileukemia activity at nanomolar concentrations both in FLT3-wt overexpressing (THP-1) and FLT3-ITD (MV4-11) expressing AML cell lines (IC50 = 3.8 and 2.7 nM, respectively) and patients' primary blasts [IC50 = ~12 nM (FLT3-wt) and ~5 nM (FLT3-ITD)]. Silvestrol increased apoptosis (~4fold, P = 0.0001), and inhibited colony-formation (100%, P < 0.0001) in primary blasts. Silvestrol efficiently inhibited FLT3 translation reducing FLT3 protein expression by 80-90% and decreased miR-155 levels (~60%), a frequently co-regulated onco-miR in FLT3-ITD-positive AML. The median survival of silvestrol-treated vs vehicle-treated mice was 63 vs 29 days post-engraftment, respectively (P < 0.0001). CONCLUSIONS: Silvestrol exhibits significant in vivo and in vitro antileukemic activities in AML through a novel mechanism resulting in inhibition of FLT3 and miR-155 expression. These encouraging results warrant a rapid translation of silvestrol for clinical testing in AML.


Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Interleukin Receptor Common gamma Subunit/physiology , Leukemia, Myeloid, Acute/drug therapy , MicroRNAs/antagonists & inhibitors , Triterpenes/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Animals , Apoptosis/drug effects , Blast Crisis/drug therapy , Blast Crisis/metabolism , Blast Crisis/pathology , Blotting, Western , Cell Proliferation/drug effects , Flow Cytometry , Humans , In Vitro Techniques , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , MicroRNAs/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolism
13.
EMBO Mol Med ; 5(1): 105-21, 2013 01.
Article in English | MEDLINE | ID: mdl-23180565

ABSTRACT

Mechanisms that alter protein phosphatase 2A (PP2A)-dependent lung tumour suppression via the I2PP2A/SET oncoprotein are unknown. We show here that the tumour suppressor ceramide binds I2PP2A/SET selectively in the nucleus and including its K209 and Y122 residues as determined by molecular modelling/simulations and site-directed mutagenesis. Because I2PP2A/SET was found overexpressed, whereas ceramide was downregulated in lung tumours, a sphingolipid analogue drug, FTY720, was identified to mimick ceramide for binding and targeting I2PP2A/SET, leading to PP2A reactivation, lung cancer cell death, and tumour suppression in vivo. Accordingly, while molecular targeting of I2PP2A/SET by stable knockdown prevented further tumour suppression by FTY720, reconstitution of WT-I2PP2A/SET expression restored this process. Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis. The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720. Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1(-/-) MEFs. Thus, these data suggest that targeting I2PP2A/SET by FTY720 suppresses lung tumour growth, at least in part, via PP2A activation and necroptosis mediated by the kinase domain of RIPK1.


Subject(s)
Antineoplastic Agents/pharmacology , Histone Chaperones/antagonists & inhibitors , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Propylene Glycols/pharmacology , Protein Phosphatase 2/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Sphingosine/analogs & derivatives , Transcription Factors/antagonists & inhibitors , Animals , Cell Line, Tumor , DNA-Binding Proteins , Fingolimod Hydrochloride , Gene Knockdown Techniques , Histone Chaperones/chemistry , Histone Chaperones/genetics , Histone Chaperones/metabolism , Humans , Lung Neoplasms/pathology , Mice , Mice, SCID , Models, Molecular , Necrosis , Phosphorylation , Propylene Glycols/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Sphingosine/metabolism , Sphingosine/pharmacology , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Xenograft Model Antitumor Assays
14.
Blood ; 117(22): 5941-52, 2011 Jun 02.
Article in English | MEDLINE | ID: mdl-21527515

ABSTRACT

The mechanisms by which sphingosine kinase-1 (SK-1)/sphingosine 1-phosphate (S1P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-1/S1P enhances Bcr-Abl1 protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl1 stability was enhanced by ectopic SK-1 expression. Conversely, siRNA-mediated SK-1 knockdown in K562/IMA-3 cells, or its genetic loss in SK-1(-/-) MEFs, significantly reduced Bcr-Abl1 stability. Regulation of Bcr-Abl1 by SK-1/S1P was dependent on S1P receptor 2 (S1P2) signaling, which prevented Bcr-Abl1 dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-1/S1P2 restored PP2A-dependent Bcr-Abl1 dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34(+) mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T315I) Bcr-Abl1 in situ. Accordingly, impaired SK-1/S1P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T315I-Bcr-Abl1-derived mouse allografts. Since SK-1/S1P/S1P2 signaling regulates Bcr-Abl1 stability via modulation of PP2A, inhibition of SK-1/S1P2 axis represents a novel approach to target wild-type- or mutant-Bcr-Abl1 thereby overcoming drug resistance.


Subject(s)
Drug Resistance, Neoplasm , Fusion Proteins, bcr-abl/chemistry , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Lysophospholipids/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Phosphatase 2/metabolism , Receptors, Lysosphingolipid/metabolism , Sphingosine/analogs & derivatives , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Benzamides , Cell Line, Tumor , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mice, SCID , Phosphorylation/drug effects , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Phosphotransferases (Alcohol Group Acceptor)/genetics , Piperazines/administration & dosage , Protein Phosphatase 2/genetics , Pyrimidines/administration & dosage , RNA, Small Interfering/genetics , Receptors, Lysosphingolipid/genetics , Signal Transduction , Sphingosine/metabolism , Ubiquitination
15.
Cell ; 140(5): 652-65, 2010 Mar 05.
Article in English | MEDLINE | ID: mdl-20211135

ABSTRACT

MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.


Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , MicroRNAs/metabolism , Animals , Blast Crisis , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Line, Tumor , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Mice , Proto-Oncogene Proteins c-pim-1/metabolism , RNA-Induced Silencing Complex/metabolism
16.
Blood ; 111(2): 816-28, 2008 Jan 15.
Article in English | MEDLINE | ID: mdl-17925491

ABSTRACT

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are aberrantly regulated at transcriptional or posttranslational levels by the constitutive kinase activity of the BCR/ABL oncoprotein. As a result, altered expression/function of RBPs leads to increased resistance to apoptotic stimuli, enhanced survival, growth advantage, and differentiation arrest of CD34+ progenitors from patients in CML blast crisis. Here, we identify the mRNAs bound to the hnRNP-A1, hnRNP-E2, hnRNP-K, and La/SSB RBPs in BCR/ABLtransformed myeloid cells. Interestingly, we found that the mRNA encoding the transcription factor E2F3 associates to hnRNP-A1 through a conserved binding site located in the E2F3 3' untranslated region (UTR). E2F3 levels were up-regulated in CML-BCCD34+ in a BCR/ABL kinase- and hnRNP-A1 shuttling-dependent manner. Moreover, by using shRNA-mediated E2F3 knock-down and BCR/ABL-transduced lineage-negative bone marrow cells from E2F3+/+ and E2F3-/- mice, we show that E2F3 expression is important for BCR/ABL clonogenic activity and in vivo leukemogenic potential. Thus, the complexity of the mRNA/RBP network, together with the discovery of E2F3 as an hnRNP-A1-regulated factor, outlines the relevant role played by RBPs in posttranscriptional regulation of CML development and progression.


Subject(s)
3' Untranslated Regions/metabolism , Blast Crisis/metabolism , Cell Transformation, Neoplastic/metabolism , E2F3 Transcription Factor/biosynthesis , E2F3 Transcription Factor/metabolism , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Leukemic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Protein-Tyrosine Kinases/metabolism , RNA, Neoplasm/metabolism , RNA-Binding Proteins/metabolism , 3' Untranslated Regions/genetics , Animals , Antigens, CD34/genetics , Antigens, CD34/metabolism , Apoptosis/genetics , Blast Crisis/genetics , Blast Crisis/pathology , Cell Survival/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , E2F3 Transcription Factor/genetics , Female , Fusion Proteins, bcr-abl/genetics , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Mice , Mice, Knockout , Protein-Tyrosine Kinases/genetics , RNA, Neoplasm/genetics , RNA-Binding Proteins/genetics
17.
J Clin Invest ; 117(9): 2408-21, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17717597

ABSTRACT

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190(BCR/ABL) myeloid and lymphoid cell lines and CML-BC(CD34+) and Ph1 ALL(CD34+/CD19+) progenitors but not of normal CD34+ and CD34+/CD19+ bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190(BCR/ABL)-driven [including p210/p190(BCR/ABL)(T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.


Subject(s)
Blast Crisis/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Propylene Glycols/therapeutic use , Sphingosine/analogs & derivatives , Animals , Benzamides , Blast Crisis/genetics , Blast Crisis/metabolism , Blast Crisis/pathology , Cell Survival/drug effects , Dasatinib , Drug Resistance, Neoplasm/drug effects , Fingolimod Hydrochloride , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Neoplastic , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Molecular Structure , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Piperazines/pharmacology , Propylene Glycols/chemistry , Protein Phosphatase 2 , Pyrimidines/pharmacology , Signal Transduction/drug effects , Sphingosine/chemistry , Sphingosine/therapeutic use , Thiazoles/pharmacology , Time Factors , Tumor Cells, Cultured
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