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1.
Sci Transl Med ; 16(748): eadl2720, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38776391

ABSTRACT

We present the preclinical pharmacology of BNT142, a lipid nanoparticle (LNP)-formulated RNA (RNA-LNP) encoding a T cell-engaging bispecific antibody that monovalently binds the T cell marker CD3 and bivalently binds claudin 6 (CLDN6), an oncofetal antigen that is absent from normal adult tissue but expressed on various solid tumors. Upon BNT142 RNA-LNP delivery in cell culture, mice, and cynomolgus monkeys, RNA is translated, followed by self-assembly into and secretion of the functional bispecific antibody RiboMab02.1. In vitro, RiboMab02.1 mediated CLDN6 target cell-specific activation and proliferation of T cells, and potent target cell killing. In mice and cynomolgus monkeys, intravenously administered BNT142 RNA-LNP maintained therapeutic serum concentrations of the encoded antibody. Concentrations of RNA-encoded RiboMab02.1 were maintained longer in circulation in mice than concentrations of directly injected, sequence-identical protein. Weekly injections of mice with BNT142 RNA-LNP in the 0.1- to 1-µg dose range were sufficient to eliminate CLDN6-positive subcutaneous human xenograft tumors and increase survival over controls. Tumor regression was associated with an influx of T cells and depletion of CLDN6-positive cells. BNT142 induced only transient and low cytokine production in CLDN6-positive tumor-bearing mice humanized with peripheral blood mononuclear cells (PBMCs). No signs of adverse effects from BNT142 RNA-LNP administration were observed in mice or cynomolgus monkeys. On the basis of these and other findings, a phase 1/2 first-in-human clinical trial has been initiated to assess the safety and preliminary efficacy of BNT142 RNA-LNP in patients with CLDN6-positive advanced solid tumors (NCT05262530).


Subject(s)
Antibodies, Bispecific , Claudins , Macaca fascicularis , T-Lymphocytes , Animals , Humans , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/pharmacokinetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Claudins/metabolism , Mice , RNA/metabolism , Female , Cell Line, Tumor , Xenograft Model Antitumor Assays , Liposomes , Nanoparticles
2.
Oncoimmunology ; 12(1): 2255041, 2023.
Article in English | MEDLINE | ID: mdl-37860278

ABSTRACT

IMAB362/Zolbetuximab, a first-in-class IgG1 antibody directed against the cancer-associated gastric-lineage marker CLDN18.2, has recently been reported to have met its primary endpoint in two phase 3 trials as a first-line treatment in combination with standard of care chemotherapy in CLDN18.2-positive Her2 negative advanced gastric cancer. Here we characterize the preclinical pharmacology of BNT141, a nucleoside-modified RNA therapeutic encoding the sequence of IMAB362/Zolbetuximab, formulated in lipid nanoparticles (LNP) for liver uptake. We show that the mRNA-encoded antibody displays a stable pharmacokinetic profile in preclinical animal models, mediates CLDN18.2-restricted cytotoxicity comparable to IMAB362 recombinant protein and inhibits human tumor xenograft growth in immunocompromised mice. BNT141 administration did not perpetrate mortality, clinical signs of toxicity, or gastric pathology in animal studies. A phase 1/2 clinical trial with BNT141 mRNA-LNP has been initiated in advanced CLDN18.2-expressing solid cancers (NCT04683939).


Subject(s)
Stomach Neoplasms , Animals , Humans , Mice , Cell Adhesion Molecules , Claudins/immunology , RNA, Messenger/genetics , Stomach Neoplasms/drug therapy , Stomach Neoplasms/genetics , Stomach Neoplasms/immunology , Antibodies/genetics , Antibodies/immunology
3.
Leukemia ; 36(3): 675-686, 2022 03.
Article in English | MEDLINE | ID: mdl-34732858

ABSTRACT

With an incidence of ~50%, the absence or reduced protein level of p53 is much more common than TP53 mutations in acute myeloid leukemia (AML). AML with FLT3-ITD (internal tandem duplication) mutations has an unfavorable prognosis and is highly associated with wt-p53 dysfunction. While TP53 mutation in the presence of FLT3-ITD does not induce AML in mice, it is not clear whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. Here, we generated FLT3-ITD knock-in; p53 knockout (heterozygous and homozygous) double-transgenic mice and found that both alterations strongly cooperated in the induction of cytogenetically normal AML without increasing the self-renewal potential. At the molecular level, we found the strong upregulation of Htra3 and the downregulation of Lin28a, leading to enhanced proliferation and the inhibition of apoptosis and differentiation. The co-occurrence of Htra3 overexpression and Lin28a knockdown, in the presence of FLT3-ITD, induced AML with similar morphology as leukemic cells from double-transgenic mice. These leukemic cells were highly sensitive to the proteasome inhibitor carfilzomib. Carfilzomib strongly enhanced the activity of targeting AXL (upstream of FLT3) against murine and human leukemic cells. Our results unravel a unique role of p53 haploinsufficiency or loss in the development of FLT3-ITD + AML.


Subject(s)
Gene Expression Regulation, Leukemic , Haploinsufficiency , Leukemia, Myeloid, Acute/genetics , Tumor Suppressor Protein p53/genetics , fms-Like Tyrosine Kinase 3/genetics , Animals , Gene Duplication , Gene Knock-In Techniques , Mice , Mice, Inbred C57BL , Mutation
4.
J Med Chem ; 65(2): 1171-1205, 2022 01 27.
Article in English | MEDLINE | ID: mdl-34726055

ABSTRACT

The allosteric regulation of pyruvate kinase M2 (PKM2) affects the switching of the PKM2 protein between the high-activity and low-activity states that allow ATP and lactate production, respectively. PKM2, in its low catalytic state (dimeric form), is chiefly active in metabolically energetic cells, including cancer cells. More recently, PKM2 has emerged as an attractive target due to its role in metabolic dysfunction and other interrelated conditions. PKM2 (dimer) activity can be inhibited by modulating PKM2 dimer-tetramer dynamics using either PKM2 inhibitors that bind at the ATP binding active site of PKM2 (dimer) or PKM2 activators that bind at the allosteric site of PKM2, thus activating PKM2 from the dimer formation to the tetrameric formation. The present perspective focuses on medicinal chemistry approaches to design and discover PKM2 inhibitors and activators and further provides a scope for the future design of compounds targeting PKM2 with better efficacy and selectivity.


Subject(s)
Carrier Proteins/antagonists & inhibitors , Chemistry, Pharmaceutical , Glycolysis , Membrane Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Allosteric Regulation , Allosteric Site , Humans , Thyroid Hormones , Thyroid Hormone-Binding Proteins
5.
Curr Top Med Chem ; 21(25): 2258-2271, 2021.
Article in English | MEDLINE | ID: mdl-34348626

ABSTRACT

INTRODUCTION: Pyruvate kinase isozyme M2 (PKM2) was observed to be overexpressed and play a key role in cell growth and cancer cells' metabolism. During the past years, phytochemicals have been developed as new treatment options for chemoprevention and cancer therapy. Natural resources, like shikonin (naphthoquinone) and its derivatives, have emerged to be high potential therapeutics in cancer treatment. METHODS: Our study aimed to design novel anti-tumour agents (PKM2 inhibitors) focusing on the shikonin scaffold with a better activity using computational methods. We applied a three-dimensional quantitative structure-activity relationship (3D-QSAR) approach using Field-based QSAR. RESULTS: The Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) were performed on a series of forty shikonin derivatives, including shikonin, to develop robust models and rationalize the PKM2 inhibitory activity profile by building a correlation between structural features and activity. CONCLUSION: These predictive computational models will further help the design and synthesis of potent PKM2 inhibitors and their fast biological assessment at a low cost.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Naphthoquinones/chemistry , Pyruvate Kinase/antagonists & inhibitors , Quantitative Structure-Activity Relationship , Isoenzymes/antagonists & inhibitors
6.
Blood Adv ; 5(9): 2294-2304, 2021 05 11.
Article in English | MEDLINE | ID: mdl-33929500

ABSTRACT

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) is widely applicable and prognostic prior to allogeneic hematopoietic cell transplantation (alloHCT). We evaluated the prognostic role of clonal hematopoiesis-associated DNMT3A, TET2, and ASXL1 (DTA) and non-DTA mutations for MRD monitoring post-alloHCT to refine MRD marker selection. Of 154 patients with AML, 138 (90%) had at least one mutation at diagnosis, which were retrospectively monitored by amplicon-based error-corrected NGS on day 90 and/or day 180 post-alloHCT. MRD was detected in 34 patients on day 90 and/or day 180 (25%). The rate of MRD positivity was similar when DTA and non-DTA mutations were considered separately (17.6% vs 19.8%). DTA mutations had no prognostic impact on cumulative incidence of relapse, relapse-free survival, or overall survival in our study and were removed from further analysis. In the remaining 131 patients with at least 1 non-DTA mutation, clinical and transplantation-associated characteristics were similarly distributed between MRD-positive and MRD-negative patients. In multivariate analysis, MRD positivity was an independent adverse predictor of cumulative incidence of relapse, relapse-free survival, and overall survival but not of nonrelapse mortality. The prognostic effect was independent of different cutoffs (above limit of detection, 0.1% and 1% variant allele frequency). MRD log-reduction between diagnosis and post-alloHCT assessment had no prognostic value. MRD status post-alloHCT had the strongest impact in patients who were MRD positive prior to alloHCT. In conclusion, non-DTA mutations are prognostic NGS-MRD markers post-alloHCT, whereas the prognostic role of DTA mutations in the posttransplant setting remains open.


Subject(s)
Leukemia, Myeloid, Acute , High-Throughput Nucleotide Sequencing , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/therapy , Mutation , Neoplasm, Residual , Retrospective Studies
7.
Cancers (Basel) ; 13(8)2021 Apr 07.
Article in English | MEDLINE | ID: mdl-33916994

ABSTRACT

In recent years, onco-metabolites like D-2-hydroxyglutarate, which is produced in isocitrate dehydrogenase-mutated tumors, have gained increasing interest. Here, we report a metabolite in human specimens that is closely related to 2-hydroxyglutarate: the intramolecular ester of 2-hydroxyglutarate, 2-hydroxyglutarate-γ-lactone. Using 13C5-L-glutamine tracer analysis, we showed that 2-hydroxyglutarate is the endogenous precursor of 2-hydroxyglutarate-lactone and that there is a high exchange between these two metabolites. Lactone formation does not depend on mutated isocitrate dehydrogenase, but its formation is most probably linked to transport processes across the cell membrane and favored at low environmental pH. Furthermore, human macrophages showed not only striking differences in uptake of 2-hydroxyglutarate and its lactone but also in the enantiospecific hydrolysis of the latter. Consequently, 2-hydroxyglutarate-lactone may play a critical role in the modulation of the tumor microenvironment.

8.
Leukemia ; 35(5): 1301-1316, 2021 05.
Article in English | MEDLINE | ID: mdl-32948843

ABSTRACT

Clonal hematopoiesis of indeterminate potential (CHIP) is linked to leukemia gene mutations and associates with an increased risk for coronary artery disease and poor prognosis in ischemic cardiomyopathy. Two recurrently mutated genes in CHIP and adult acute myeloid leukemia (AML) encode for isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). Global expression of mutant IDH2 in transgenic mice-induced dilated cardiomyopathy and muscular dystrophy. In this retrospective observational study, we investigated whether mutant IDH1/2 predisposes to cardiovascular disease in AML patients. Among 363 AML patients, IDH1 and IDH2 mutations were detected in 26 (7.2%) and 39 patients (10.7%), respectively. Mutant IDH1 patients exhibited a significantly higher prevalence of coronary artery disease (26.1% vs. 6.4%, p = 0.002). Applying inverse probability-weighting analysis, patients with IDH1/2 mutations had a higher risk for a declining cardiac function during AML treatment compared to IDH1/2 wild type patients [left ventricular ejection fraction pretreatment compared to 10 months after diagnosis: 59.2% to 41.9% (p < 0.001) vs 58.5% to 55.4% (p = 0.27), respectively]. Mechanistically, RNA sequencing and immunostaining in hiPS-derived cardiomyocytes indicated that the oncometabolite R-2HG exacerbated doxorubicin mediated cardiotoxicity. Evaluation of IDH1/2 mutation status may therefore help identifying AML patients at risk for cardiovascular complications during cytotoxic treatment.


Subject(s)
Coronary Artery Disease/etiology , Coronary Artery Disease/genetics , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute/genetics , Mutation/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Coronary Artery Disease/pathology , Female , Genotype , Humans , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Prognosis , Propensity Score , Retrospective Studies , Stroke Volume , Ventricular Function, Left/genetics , Young Adult
9.
Haematologica ; 106(2): 565-573, 2021 02 01.
Article in English | MEDLINE | ID: mdl-32241846

ABSTRACT

Mutant IDH1 (mIDH1) inhibitors have shown single-agent activity in relapsed/refractory AML, though most patients eventually relapse. We evaluated the efficacy and molecular mechanism of the combination treatment with azacitidine, which is currently the standard of care in older AML patients, and mIDH1 inhibitor BAY1436032. Both compounds were evaluated in vivo as single agents and in combination with sequential (azacitidine, followed by BAY1436032) or simultaneous application in two human IDH1 mutated AML xenograft models. Combination treatment significantly prolonged survival compared to single agent or control treatment (P<.005). The sequential combination treatment depleted leukemia stem cells (LSC) by 470-fold. Interestingly, the simultaneous combination treatment depleted LSCs by 33,150-fold compared to control mice. This strong synergy is mediated through inhibition of MAPK/ERK and RB/E2F signaling. Our data strongly argues for the concurrent application of mIDH1 inhibitors and azacitidine and predicts improved outcome of this regimen in IDH1 mutated AML patients.


Subject(s)
Azacitidine , Leukemia, Myeloid, Acute , Aged , Aniline Compounds , Animals , Benzimidazoles , Humans , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Mice
11.
Leukemia ; 34(2): 416-426, 2020 02.
Article in English | MEDLINE | ID: mdl-31586149

ABSTRACT

Mutations in isocitrate dehydrogenase 1 (IDH1) are found in 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R-2HG), which induces histone- and DNA-hypermethylation through the inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. Here we report the biochemical characterization, in vivo antileukemic effects, structural binding, and molecular mechanism of the inhibitor HMS-101, which inhibits the enzymatic activity of mutant IDH1 (IDH1mut). Treatment of IDH1mut primary AML cells reduced 2-hydroxyglutarate levels (2HG) and induced myeloid differentiation in vitro. Co-crystallization of HMS-101 and mutant IDH1 revealed that HMS-101 binds to the active site of IDH1mut in close proximity to the regulatory segment of the enzyme in contrast to other IDH1 inhibitors. HMS-101 also suppressed 2HG production, induced cellular differentiation and prolonged survival in a syngeneic mutant IDH1 mouse model and a patient-derived human AML xenograft model in vivo. Cells treated with HMS-101 showed a marked upregulation of the differentiation-associated transcription factors CEBPA and PU.1, and a decrease in cell cycle regulator cyclin A2. In addition, the compound attenuated histone hypermethylation. Together, HMS-101 is a unique inhibitor that binds to the active site of IDH1mut directly and is active in IDH1mut preclinical models.


Subject(s)
Binding Sites/drug effects , Enzyme Inhibitors/pharmacology , Isocitrate Dehydrogenase/antagonists & inhibitors , Mutation/drug effects , Animals , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Differentiation/drug effects , DNA Methylation/drug effects , Histones/metabolism , Humans , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Up-Regulation/drug effects
12.
Ann Hematol ; 98(8): 1905-1918, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31104089

ABSTRACT

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.


Subject(s)
Drug Delivery Systems/methods , Fusion Proteins, bcr-abl/antagonists & inhibitors , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/therapy , Nanoparticles/administration & dosage , RNA, Small Interfering/pharmacology , Animals , Bone Marrow/drug effects , Bone Marrow/metabolism , Bone Marrow/pathology , Cell Survival/drug effects , Disease Models, Animal , Female , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Expression , Gene Targeting/methods , Humans , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Lipids/administration & dosage , Lipids/chemistry , Mice , Mice, Nude , Nanoparticles/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA, Small Interfering/pharmacokinetics , Survival Analysis , Xenograft Model Antitumor Assays
13.
Blood ; 132(16): 1703-1713, 2018 10 18.
Article in English | MEDLINE | ID: mdl-30190321

ABSTRACT

Molecular measurable residual disease (MRD) assessment is not established in approximately 60% of acute myeloid leukemia (AML) patients because of the lack of suitable markers for quantitative real-time polymerase chain reaction. To overcome this limitation, we established an error-corrected next-generation sequencing (NGS) MRD approach that can be applied to any somatic gene mutation. The clinical significance of this approach was evaluated in 116 AML patients undergoing allogeneic hematopoietic cell transplantation (alloHCT) in complete morphologic remission (CR). Targeted resequencing at the time of diagnosis identified a suitable mutation in 93% of the patients, covering 24 different genes. MRD was measured in CR samples from peripheral blood or bone marrow before alloHCT and identified 12 patients with persistence of an ancestral clone (variant allele frequency [VAF] >5%). The remaining 96 patients formed the final cohort of which 45% were MRD+ (median VAF, 0.33%; range, 0.016%-4.91%). In competing risk analysis, cumulative incidence of relapse (CIR) was higher in MRD+ than in MRD- patients (hazard ratio [HR], 5.58; P < .001; 5-year CIR, 66% vs 17%), whereas nonrelapse mortality was not significantly different (HR, 0.60; P = .47). In multivariate analysis, MRD positivity was an independent negative predictor of CIR (HR, 5.68; P < .001), in addition to FLT3-ITD and NPM1 mutation status at the time of diagnosis, and of overall survival (HR, 3.0; P = .004), in addition to conditioning regimen and TP53 and KRAS mutation status. In conclusion, NGS-based MRD is widely applicable to AML patients, is highly predictive of relapse and survival, and may help refine transplantation and posttransplantation management in AML patients.


Subject(s)
Biomarkers, Tumor/genetics , Hematopoietic Stem Cell Transplantation/mortality , High-Throughput Nucleotide Sequencing/methods , Leukemia, Myeloid, Acute/diagnosis , Mutation , Neoplasm Recurrence, Local/diagnosis , Neoplasm, Residual/diagnosis , Adult , Aged , Cohort Studies , Female , Follow-Up Studies , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/therapy , Male , Middle Aged , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/therapy , Neoplasm, Residual/genetics , Neoplasm, Residual/mortality , Neoplasm, Residual/therapy , Nucleophosmin , Prognosis , Remission Induction , Survival Rate , Transplantation, Homologous , Young Adult
14.
Oncotarget ; 8(43): 73871-73883, 2017 Sep 26.
Article in English | MEDLINE | ID: mdl-29088753

ABSTRACT

The neurotrophins (NTs) play a key role in neuronal survival and maintenance. The TRK (tropomyosin-related kinase) tyrosine kinase receptors (TRKA, TRKB, TRKC) are high affinity receptors for NTs. There is increasing data demonstrating an important role of the TRK family in cancer initiation and progression. NTs have been known for many years to promote chemotaxis, maturation, and survival of mast cells. However, the role of NT signaling in the pathogenesis of mastocytosis is not well understood. In this study, we demonstrate that activation of TRKA by its ligand nerve growth factor (NGF) is potent to trigger a disease in mice with striking similarities to human systemic mastocytosis (SM). Moreover, activation of TRKA by NGF strongly rescues KIT inhibition-induced cell death of mast cell lines and primary mast cells from patients with SM, and this rescue effect can be efficiently blocked by entrectinib (a new pan TRK specific inhibitor). HMC-1 mast cell leukemia cells that are resistant to KIT inhibition induced by TRKA activation show reactivation of MAPK/ERK (extracellular signal-regulated kinase) and strong upregulation of early growth response 3 (EGR3), suggesting an important role of MAPK-EGR3 axis in the development of resistance to KIT inhibition. Targeting both TRK and KIT significantly prolongs survival of mice xenotransplanted with HMC-1 cells compared with targeting KIT alone. Thus, these data strongly suggest that TRKA signaling can improve neoplastic mast cell fitness. This might explain at least in part why treatment with KIT inhibitors alone so far has been disappointing in most published clinical trials for mastocytosis. Our data suggest that targeting both KIT and TRKs might improve efficacy of molecular therapy in SM with KIT mutations.

15.
Biol Blood Marrow Transplant ; 23(7): 1095-1101, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28389256

ABSTRACT

Molecular genetics may influence outcome for patients with myelofibrosis. To determine the impact of molecular genetics on outcome after allogeneic stem cell transplantation, we screened 169 patients with primary myelofibrosis (n = 110), post-essential thrombocythemia/polycythemia vera myelofibrosis (n = 46), and myelofibrosis in transformation (n = 13) for mutations in 16 frequently mutated genes. The most frequent mutation was JAK2V617F (n = 101), followed by ASXL1 (n = 49), calreticulin (n = 34), SRSF2 (n = 16), TET2 (n = 10), U2AF1 (n = 11), EZH2 (n = 7), MPL (n = 6), IDH2 (n = 5), IDH1 (n = 4), and CBL (n = 1). The cumulative incidence of nonrelapse mortality (NRM) at 1 year was 21% and of relapse at 5 years 25%. The 5-year rates progression-free (PFS) and overall survival (OS) were and 56%, respectively. In a multivariate analysis CALR mutation was an independent factor for lower NRM (HR, .415; P = .05), improved PFS (HR, .393; P = .01), and OS (HR, .448; P = .03). ASXL1 and IDH2 mutations were independent risk factors for lower PFS (HR, 1.53 [P = .008], and HR, 5.451 [P = .002], respectively), whereas no impact was observed for "triple negative" patients. Molecular genetics, especially CALR, IDH2, and ASXL1 mutations, may thus be useful to predict outcome independently from known clinical risk factors after allogeneic stem cell transplantation for myelofibrosis.


Subject(s)
Hematopoietic Stem Cell Transplantation/methods , Molecular Biology/methods , Primary Myelofibrosis/genetics , Transplantation Conditioning/methods , Transplantation, Homologous/methods , Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult
16.
RNA ; 22(10): 1535-49, 2016 10.
Article in English | MEDLINE | ID: mdl-27492256

ABSTRACT

Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders with prevalent mutations in several splicing factors, but the splicing programs linked to specific mutations or MDS in general remain to be systematically defined. We applied RASL-seq, a sensitive and cost-effective platform, to interrogate 5502 annotated splicing events in 169 samples from MDS patients or healthy individuals. We found that splicing signatures associated with normal hematopoietic lineages are largely related to cell signaling and differentiation programs, whereas MDS-linked signatures are primarily involved in cell cycle control and DNA damage responses. Despite the shared roles of affected splicing factors in the 3' splice site definition, mutations in U2AF1, SRSF2, and SF3B1 affect divergent splicing programs, and interestingly, the affected genes fall into converging cancer-related pathways. A risk score derived from 11 splicing events appears to be independently associated with an MDS prognosis and AML transformation, suggesting potential clinical relevance of altered splicing patterns in MDS.


Subject(s)
Mutation , Myelodysplastic Syndromes/genetics , Phosphoproteins/genetics , RNA Splice Sites , RNA Splicing Factors/genetics , Serine-Arginine Splicing Factors/genetics , Splicing Factor U2AF/genetics , Adult , Aged , Aged, 80 and over , Case-Control Studies , Female , Humans , Male , Middle Aged , Myelodysplastic Syndromes/pathology , RNA Splicing
17.
Curr Cancer Drug Targets ; 16(9): 818-828, 2016.
Article in English | MEDLINE | ID: mdl-27321378

ABSTRACT

Hematopoietic stem and progenitor cell differentiation are blocked in acute myeloid leukemia (AML) resulting in cytopenias and a high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation promoting compounds. High MN1 expression confers poor prognosis to AML patients and induces resistance to cytarabine and alltrans-retinoic acid (ATRA) induced differentiation. Using a high-throughput drug screening, we identified the dihydrofolate reductase (DHFR) antagonist pyrimethamine to be a potent inducer of apoptosis and differentiation in several murine and human leukemia cell lines. Oral pyrimethamine treatment was effective in two xenograft mouse models and specifically targeted leukemic cells in human AML cell lines and primary patient cells, while CD34+ cells from healthy donors were unaffected. The antileukemic effects of PMT could be partially rescued by excess folic acid, suggesting an oncogenic function of folate metabolism in AML. Thus, our study identifies pyrimethamine as a candidate drug that should be further evaluated in AML treatment.


Subject(s)
Antineoplastic Agents/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Pyrimethamine/pharmacology , Animals , Apoptosis/drug effects , Cell Line, Tumor , Folic Acid Antagonists/pharmacology , High-Throughput Screening Assays/methods , Humans , Mice , Xenograft Model Antitumor Assays
18.
Exp Hematol ; 43(8): 685-97, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26032956

ABSTRACT

The discovery of oncogenic mutations in isocitrate dehydrogenase (IDH) enzymes has highlighted the delicate interplay of metabolism, cellular signaling, and transcriptional regulation that was off-focus for some time in the genomic era. Although IDH inhibitors are being evaluated for clinical efficacy, an in-depth understanding of disease pathogenesis linked to IDH mutations is required to develop rational combination treatments and to be evaluated in the clinic. To gain such an understanding, several questions need to be addressed: Why do IDH mutations occur selectively in subsets of a disease entity although they are found to be present in a very heterogeneous set of unrelated tumors? Why are 2-hydroxyglutarate-producing tumors specifically selected for the R-enantiomer and not for the S-enantiomer? Are the changes in 2-hydroxyglutarate-induced DNA methylation primary or secondary alterations in tumorigenesis? What are the roles of hypoxia-inducible factor (HIF) and its prolyl 4-hydroxylases in IDH-mutant tumors? Here, we address these questions and discuss the consequences for basic and clinical research related to IDH-mutant tumors.


Subject(s)
Enzyme Inhibitors/therapeutic use , Hematologic Neoplasms , Isocitrate Dehydrogenase , Mutation , Animals , DNA Methylation/drug effects , DNA Methylation/genetics , Glutarates/metabolism , Hematologic Neoplasms/drug therapy , Hematologic Neoplasms/enzymology , Hematologic Neoplasms/genetics , Humans , Isocitrate Dehydrogenase/antagonists & inhibitors , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Stereoisomerism
20.
PLoS Genet ; 10(8): e1004558, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25122120

ABSTRACT

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.


Subject(s)
Cytokinesis/genetics , Microtubules/genetics , Septins/genetics , Stathmin/genetics , Animals , Cell Proliferation/genetics , Fibroblasts/drug effects , Fibroblasts/metabolism , Gastrula/growth & development , Humans , Mice , Phenylurea Compounds/pharmacology , Pyridines/pharmacology , Septins/biosynthesis , Sequence Deletion , Stathmin/biosynthesis
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