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3.
Bone Marrow Transplant ; 56(4): 841-852, 2021 04.
Article in English | MEDLINE | ID: mdl-33130821

ABSTRACT

Allogeneic stem cell transplantation (allo-SCT) offers a curative option in adult patients with acute lymphoblastic leukemia (ALL). Prognostic factors for survival after allo-SCT have not been sufficiently defined: pheno-/genotype, patients´ age, conditioning regimens and remission at allo-SCT are under discussion. We analyzed the outcome of 180 consecutive adult ALL-patients undergoing allo-SCT at our center between 1995 and 2018 to identify specific prognostic factors. In our cohort 19% were older than 55 years, 28% had Philadelphia-positive B-ALL, 24% T-ALL. 54% were transplanted in first complete remission (CR1), 13% in CR2 after salvage therapy, 31% reached no remission (8% within first-line, 23% within salvage therapy). In 66% conditioning contained total body irradiation (TBI). With a median follow-up of 10 years, we observed an overall survival of 33% at 10 years, and a progression free survival of 31%. The cumulative incidence of relapse was 41% at 10 years, the cumulative incidence of non-relapse mortality 28%. Acute graft-versus-host disease (GvHD) II°-IV° occurred in 31%, moderate/severe chronic GvHD in 27%. Survival was better in patients reaching CR before allo-SCT and in those receiving TBI. No difference between patients younger/older than 55 years and between different phenotypes was observed. Survival after allo-SCT improved considerably over the last decades.


Subject(s)
Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Adult , Disease-Free Survival , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Prognosis , Retrospective Studies , Transplantation Conditioning , Transplantation, Homologous
4.
J Stomatol Oral Maxillofac Surg ; 121(5): 599-603, 2020 11.
Article in English | MEDLINE | ID: mdl-31904529

ABSTRACT

Salivary duct carcinoma (SDC) is a rare and highly aggressive neoplasm of the salivary glands associated with high rates of local and distant recurrence and poor overall survival. We present a patient with SDC, who relapsed despite extensive multimodal therapy including surgery, postoperative radiochemotherapy, and heavy ion therapy. In the recurrent setting, immunohistochemical analysis confirmed androgen receptor positivity, prompting initiation of combined androgen deprivation therapy (ADT), which resulted in a fast and durable remission of the local tumor now lasting for 26 months. Analyzing the histopathologic specimens of all SDC patients treated at our department since 2009, we found significant AR expression in all patients. This is in line with other reports found in current literature and indicates AR positivity as a consistent feature of SDC, supporting ADT as a viable therapeutic option for SDC.


Subject(s)
Androgen Antagonists , Prostatic Neoplasms , Androgen Antagonists/therapeutic use , Humans , Male , Neoplasm Recurrence, Local/diagnosis , Neoplasm Recurrence, Local/drug therapy , Receptors, Androgen/genetics , Salivary Ducts
5.
Leukemia ; 31(12): 2732-2741, 2017 12.
Article in English | MEDLINE | ID: mdl-28484267

ABSTRACT

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a-/- dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2-STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a-/- DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK-STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.


Subject(s)
Dendritic Cells/metabolism , Gene Expression , Genes, MHC Class II , Janus Kinases/metabolism , MicroRNAs/genetics , STAT Transcription Factors/metabolism , Signal Transduction , Animals , Case-Control Studies , Dendritic Cells/immunology , Graft vs Host Disease/diagnosis , Graft vs Host Disease/etiology , Mice , Mice, Knockout , Polymorphism, Single Nucleotide , Severity of Illness Index , Stem Cell Transplantation/adverse effects
6.
Leukemia ; 30(8): 1725-33, 2016 08.
Article in English | MEDLINE | ID: mdl-27046463

ABSTRACT

Mutations that activate FMS-like tyrosine kinase 3 (FLT3) are frequent occurrences in acute myeloid leukemia. Two distinct types of mutations have been described: internal duplication of the juxtamembranous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). Although both mutations lead to constitutive FLT3 signaling, only FLT3-ITD strongly activates signal transducer and activator of transcription 5 (STAT5). In a murine transplantation model, FLT3-ITD induces a myeloproliferative neoplasm, whereas FLT3-TKD leads to a lymphoid malignancy with significantly longer latency. Here we report that the presence of STAT5 is critical for the development of a myeloproliferative disease by FLT3-ITD in mice. Deletion of Stat5 in FLT3-ITD-induced leukemogenesis leads not only to a significantly longer survival (82 vs 27 days) of the diseased mice, but also to an immunophenotype switch with expansion of the lymphoid cell compartment. Interestingly, we were able to show differential STAT5 activation in FLT3-ITD(+) myeloid and lymphoid murine progenitors. STAT5 target genes such as Oncostatin M were highly expressed in FLT3-ITD(+) myeloid but not in FLT3-ITD(+) lymphoid progenitor cells. Strikingly, FLT3-TKD expression in combination with Oncostatin M is sufficient to reverse the phenotype to a myeloproliferative disease in FLT3-TKD mice. Thus, lineage-specific STAT5 activation in hematopoietic progenitor cells predicts the FLT3(+)-mediated leukemic phenotype in mice.


Subject(s)
Cell Lineage/genetics , Hematopoietic Stem Cells/metabolism , Leukemia, Myeloid, Acute/pathology , STAT5 Transcription Factor/genetics , Transcriptional Activation/genetics , fms-Like Tyrosine Kinase 3/genetics , Animals , Carcinogenesis/genetics , Leukemia, Myeloid, Acute/genetics , Lymphoid Progenitor Cells/metabolism , Mice , Mutation , Myeloid Cells/metabolism , Oncostatin M , STAT5 Transcription Factor/metabolism
7.
Bone Marrow Transplant ; 51(1): 127-31, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26437058

ABSTRACT

Central nervous system (CNS) complications have been described in patients undergoing allogeneic hematopoietic cell transplantation (alloHCT). Cerebrospinal fluid (CSF) analysis is included in the diagnostic workup in patients with neurological symptoms after alloHCT. CSF donor-recipient chimerism analysis usually is not used to evaluate patients with neurological complications after alloHCT. To assess the potential contribution of CSF donor-recipient chimerism in patients with neurological complications, we analyzed 85 CSF samples from 50 patients with neurological complications after alloHCT. After alloHCT, 21 patients showed the presence of recipient-derived DNA. In 13 of these patients, recurrence of the underlying disease was detected in CSF. There was a moderate correlation between the recipient DNA percentage as detected by short tandem repeat (STR) amplification and the cell concentration in CSF (Spearmann r: 0.66 P=0.004). The percentage of cells with immunophenotypic abnormalities from patients relapsing in the CNS detected by flow cytometry showed a strong correlation with the percentage of recipient-derived DNA in CSF assessed by STR analysis (Spearmann r: 0.83 P=0.0008). Donor-recipient chimerism analysis in CSF in patients with neurological symptoms after alloHCT is a practical, feasible and useful complementary method to the already established methodologies included in the diagnostic workup.


Subject(s)
Central Nervous System Diseases , Hematologic Neoplasms , Hematopoietic Stem Cell Transplantation , Transplantation Chimera/metabolism , Adult , Aged , Allografts , Central Nervous System Diseases/cerebrospinal fluid , Central Nervous System Diseases/etiology , Central Nervous System Diseases/pathology , Female , Hematologic Neoplasms/cerebrospinal fluid , Hematologic Neoplasms/pathology , Hematologic Neoplasms/therapy , Humans , Male , Middle Aged
8.
Ann Hematol ; 94(9): 1577-84, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26027858

ABSTRACT

Patients often experience bone marrow examinations (BMEs) as frightening and painful. Varying operators and uncertainty about who will perform the BME worsen their anxiety. In our study, clinical nurse specialists (CNSs) were trained to perform BMEs to ensure continuity and to test the feasibility, patient satisfaction, and biopsy quality. This exploratory evaluation assessed 574 BMEs at our tertiary center between January 2012 and February 2013, 398 BMEs performed by CNS and 176 by physicians. Our aims were to determine whether BMEs by CNS yield results similar to those of physicians, analyzing (1) patient satisfaction with the BME (a) consent and (b) performance, (2) induced pain, and (3) quality of aspirates and length of trephine biopsies. When performed by CNS, 100 % of the patients were satisfied with the consent procedure and 99 % with the BME performance (physicians 99 and 91 %, respectively). The median pain score was low when both CNS and physicians performed the BME, with no or only mild pain in 92 and 76 % of patients, respectively. Bone marrow (BM) aspirates by CNS and physicians were assessed as technically evaluable in ~70 %; moreover, the median length of trephine biopsies was similar when performed by CNS or physicians with 12 and 13 mm, respectively. In conclusion, BMEs conducted by motivated CNS and within a structured training program are feasible and yield equal outcomes compared to physicians. The use of adequate pain management during BMEs by trained and experienced operators results in an extremely rare use of sedatives, low pain scores, and high patient satisfaction.


Subject(s)
Bone Marrow/pathology , Education, Nursing, Continuing , Nurse Clinicians/education , Pain/prevention & control , Adolescent , Adult , Aged , Aged, 80 and over , Biopsy , Female , Humans , Male , Middle Aged , Prospective Studies
9.
Leukemia ; 29(8): 1763-70, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25761934

ABSTRACT

FIP1L1-PDGFRA is a constitutively activated kinase described in chronic eosinophilic leukemia (CEL) and hypereosinophilic syndrome (HES). Imatinib is clinically active in FIP1L1-PDGFRA-positive diseases. Using in vitro screening to identify imatinib-resistant mutations, we frequently detected a Phe to Ser exchange at position 604 (F604S) of FIP1L1-PDGFRA alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S did not increase the biochemical or cellular IC50 value of imatinib when compared with unmutated FIP1L1-PDGFRA. However, FIP1L1-PDGFRA/F604S more efficiently induced growth factor independence in cell lines and primary mouse bone marrow cells. Pulse chase analysis revealed that the F604S exchange strongly stabilized FIP1L1-PDGFRA/F604S. The F604S mutation creates a binding site for the phosphatase domain of SHP-2, leading to lower autophosphorylation of FIP1L1-PDGFRA/F604S. This is associated with a reduced activation of SRC and CBL by FIP1L1-PDGFRA/F604S compared with the unmutated oncogene. As SRC inhibition and knockdown resulted in FIP1L1-PDGFRA stabilization, this explains the extended half-life of FIP1L1-PDGFRA/F604S. Interestingly, FIP1L1-PDGFRA/L629P, a recently identified mutation in an imatinib-resistant CEL patient, also showed protein stabilization similar to that observed with FIP1L1-PDGFRA/F604S. Therefore, resistance mutations in FIP1L1-PDGFRA that do not interfere with drug binding but rather increase target protein stability seem to be one of the drug-resistance mechanisms in FIP1L1-PDGFRA-positive disease.


Subject(s)
Mutation/genetics , Oncogene Protein pp60(v-src)/metabolism , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/genetics , Protein Kinase Inhibitors/pharmacology , Protein Stability/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Receptor, Platelet-Derived Growth Factor alpha/chemistry , Receptor, Platelet-Derived Growth Factor alpha/genetics , mRNA Cleavage and Polyadenylation Factors/chemistry , mRNA Cleavage and Polyadenylation Factors/genetics , Animals , Apoptosis , Blotting, Western , Cells, Cultured , HEK293 Cells , Humans , Hypereosinophilic Syndrome , Mice , NIH 3T3 Cells , Oncogene Protein pp60(v-src)/genetics , Precursor Cells, B-Lymphoid , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
10.
Leukemia ; 29(3): 535-47, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25092144

ABSTRACT

Almost 30% of all acute myeloid leukemias (AML) are associated with an internal tandem duplication (ITD) in the juxtamembrane domain of FMS-like tyrosine kinase 3 receptor (FLT3). Patients with FLT3-ITD mutations tend to have a poor prognosis. MicroRNAs (miRNAs) have a pivotal role in myeloid differentiation and leukemia. MiRNA-155 (MiR-155) was found to be upregulated in FLT3-ITD-associated AMLs. In this study, we discovered that FLT3-ITD signaling induces the oncogenic miR-155. We show in vitro and in vivo that miR-155 expression is regulated by FLT3-ITD downstream targets nuclear factor-κB (p65) and signal transducer and activator of transcription 5 (STAT5). Further, we demonstrate that miR-155 targets the myeloid transcription factor PU.1. Knockdown of miR-155 or overexpression of PU.1 blocks proliferation and induces apoptosis of FLT3-ITD-associated leukemic cells. Our data demonstrate a novel network in which FLT3-ITD signaling induces oncogenic miR-155 by p65 and STAT5 in AML, thereby targeting transcription factor PU.1.


Subject(s)
Gene Expression Regulation, Leukemic , Leukemia, Myeloid, Acute/genetics , MicroRNAs/genetics , Proto-Oncogene Proteins/genetics , STAT5 Transcription Factor/genetics , Trans-Activators/genetics , Transcription Factor RelA/genetics , fms-Like Tyrosine Kinase 3/genetics , Adolescent , Adult , Aged , Animals , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Male , Mice , Mice, Transgenic , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , Middle Aged , Mutation , Myeloid Cells/metabolism , Myeloid Cells/pathology , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction , Trans-Activators/metabolism , Transcription Factor RelA/metabolism , fms-Like Tyrosine Kinase 3/metabolism
11.
Leukemia ; 29(4): 858-68, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25249015

ABSTRACT

The SH2-containing adaptor protein Grb10 was first identified in a yeast screen as a new binding partner for BCR-ABL and associates with BCR-ABL in a tyrosine-dependent manner. However, its function in BCR-ABL-mediated leukemogenesis in vivo is still unknown. Here we describe an important role of Grb10 in BCR-ABL-induced leukemia by using a versatile system for efficient oncogene expression and simultaneous Grb10 knockdown from a single vector. Primary bone marrow (BM) cells coexpressing Grb10-miR/BCR-ABL showed a significant decrease in colony formation and cell cycle progression. Transplantation of Grb10miR/BCR-ABL- or control-miR/BCR-ABL- transduced BM leads to a CML/B-ALL-like phenotype with significantly delayed disease onset and progression resulting in prolonged overall survival in Grb10-miR-transplanted mice. Methylcellulose experiments exhibit additive effects of imatinib treatment and Grb10 knockdown. Cell cycle analysis suggests an anti-proliferative effect of Grb10 knockdown in BCR-ABL(+) primary BM cells. However, Grb10 abrogation was not capable of completely abolishing the BCR-ABL-induced disease. Our findings were confirmed in the human BCR-ABL(+) cell line K562, where we demonstrate reduced viability, cell cycle progression and induction of apoptosis by stable Grb10 microRNA expression. Taken together, our results suggest that Grb10 knockdown in vivo leads to impaired proliferation, longer survival and reduced colony formation, suggesting an important role of Grb10 in BCR-ABL-mediated leukemogenesis.


Subject(s)
Bone Marrow Cells/pathology , Fusion Proteins, bcr-abl/genetics , GRB10 Adaptor Protein/genetics , Gene Expression Regulation, Leukemic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzamides/pharmacology , Bone Marrow Cells/metabolism , Cell Cycle/drug effects , Cell Proliferation/drug effects , Disease Models, Animal , Female , Fusion Proteins, bcr-abl/metabolism , GRB10 Adaptor Protein/antagonists & inhibitors , GRB10 Adaptor Protein/metabolism , Humans , Imatinib Mesylate , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mice, Inbred BALB C , MicroRNAs/genetics , MicroRNAs/metabolism , Piperazines/pharmacology , Primary Cell Culture , Pyrimidines/pharmacology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction
12.
Oncogene ; 34(5): 578-88, 2015 Jan 29.
Article in English | MEDLINE | ID: mdl-24509876

ABSTRACT

Lung cancer is the leading cause of cancer-related deaths worldwide. Recently, we have shown that Notch1 inhibition resulted in substantial cell death of non-small cell lung cancer (NSCLC) cells in vitro. New compounds targeting Notch signal transduction have been developed and are now being tested in clinical trials. However, the tumorigenic role of individual Notch receptors in vivo remains largely unclear. Using a Kras(G12D)-driven endogenous NSCLC mouse model, we analyzed the effect of conditional Notch1 and Notch2 receptor deletion on NSCLC tumorigenesis. Notch1 deficiency led to a reduced early tumor formation and lower activity of MAPK compared with the controls. Unexpectedly, Notch2 deletion resulted in a dramatically increased carcinogenesis and increased MAPK activity. These mice died significantly earlier due to rapidly growing tumor burden. We found that Notch1 regulates Ras/MAPK pathway via HES1-induced repression of the DUSP1 promoter encoding a phosphatase specifically suppressing pERK1/2. Interestingly, Notch1 but not Notch2 ablation leads to decreased HES1 and DUSP1 expression. However, Notch2-depleted tumors showed an appreciable increase in ß-catenin expression, a known activator of HES1 and important lung cancer oncogene. Characteristically for ß-catenin upregulation, we found that the majority of Notch2-deficient tumors revealed an undifferentiated phenotype as determined by their morphology, E-Cadherin and TTF1 expression levels. In addition, these carcinomas showed aggressive growth patterns with bronchus invasion and obstruction. Together, we show that Notch2 mediates differentiation and has tumor suppressor functions during lung carcinogenesis, whereas Notch1 promotes tumor initiation and progression. These data are further supported by immunohistochemical analysis of human NSCLC samples showing loss or downregulation of Notch2 compared with normal lung tissue. In conclusion, this is the first study characterizing the in vivo functions of Notch1 and Notch2 in Kras(G12D)-driven NSCLC tumorigenesis. These data highlight the clinical importance of a thorough understanding of Notch signaling especially with regard to Notch-targeted therapies.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Cell Transformation, Neoplastic/genetics , Receptor, Notch1/biosynthesis , Receptor, Notch2/biosynthesis , Animals , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/genetics , Disease Models, Animal , Dual Specificity Phosphatase 1/biosynthesis , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/biosynthesis , Humans , Mice , Proto-Oncogene Proteins p21(ras)/genetics , Receptor, Notch1/genetics , Receptor, Notch2/genetics , Signal Transduction/genetics , Transcription Factor HES-1 , beta Catenin/biosynthesis
13.
Blood Cancer J ; 4: e240, 2014 Aug 22.
Article in English | MEDLINE | ID: mdl-25148222

ABSTRACT

The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYK(wt), TEL-SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYK(wt) enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK-SYK caused lethal T-cell lymphomas and the cytoplasmic TEL-SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL-SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.


Subject(s)
Gene Deletion , Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Oncogene Proteins, Fusion , Primary Myelofibrosis , STAT5 Transcription Factor , Animals , Cell Line , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/prevention & control , Male , Mice , Mice, Inbred BALB C , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , Myelodysplastic Syndromes/prevention & control , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Primary Myelofibrosis/genetics , Primary Myelofibrosis/metabolism , Primary Myelofibrosis/pathology , Primary Myelofibrosis/prevention & control , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Syk Kinase , ETS Translocation Variant 6 Protein
16.
Leukemia ; 25(5): 848-55, 2011 May.
Article in English | MEDLINE | ID: mdl-21331071

ABSTRACT

Mature donor-derived T cells in allogeneic bone marrow (BM) transplants mediate the graft-versus-tumor (GVT) effect by recognizing alloantigens on leukemic cells. However, alloantigen reactivity towards non-malignant tissues also induces graft-versus-host disease (GVHD). Defining T-cell subpopulations that mediate the GVT effect in the absence of GVHD induction remains a major challenge in allogeneic BM transplantation. In this study, we show that in vitro-generated alloantigen-specific CD8(+) cytotoxic T cells (CTLs) established by weekly stimulation with alloantigen-expressing antigen-presenting cells did not induce GVHD in two major histocompatibility complex-mismatched BM transplantation models, where induction of lethal GVHD is dependent on the presence of either CD4(+) or CD8(+) T cells. Despite their strong alloantigen specificity, transplantation of CTLs did not induce the expression of GVHD-associated cytokines IFN-γ and TNF-α or clinical or histological signs of GVHD, and lead to a survival rate of above 90%. However, transplantation of unstimulated CD8(+) T cells, which were not primed by the alloantigen in vitro, induced GVHD in both the transplantation models. Although CTLs were impaired in GVHD induction, they efficiently eradicated Bcr-Abl-transformed B-cell leukemias or mastocytomas. Thus, in vitro-derived CTLs might be useful for optimizing anti-tumor therapy in the absence of GVHD induction.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Graft vs Host Disease/immunology , Graft vs Tumor Effect/immunology , Isoantigens/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Blotting, Western , CD8-Positive T-Lymphocytes/transplantation , Cytokines/blood , Female , Flow Cytometry , Graft vs Host Disease/mortality , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Spleen/cytology , Spleen/metabolism , Survival Rate
17.
Oncogene ; 30(8): 933-43, 2011 Feb 24.
Article in English | MEDLINE | ID: mdl-20972453

ABSTRACT

Myeloproliferation with prominent eosinophilia is associated with rearrangements of PDGFR-A or -B. The most common rearrangement is FIP1L1-PDGFRA (FP). The majority of patients with PDGFR-rearranged myeloproliferation respond to treatment with imatinib. In contrast to BCR-ABL-positive chronic myelogenous leukemia, only few cases of imatinib resistance and mutations of the FP kinase domain have been described so far. We hypothesized that the number of critical residues mediating imatinib resistance in FP in contrast to BCR-ABL might be limited. We performed an established systematic and comprehensive in vitro resistance screen to determine the pattern and frequency of possible TKI resistance mutations in FP. We identified 27 different FP kinase domain mutations including 25 novel variants, which attenuated response to imatinib, nilotinib or sorafenib. However, the majority of these exchanges did not confer complete inhibitor resistance. At clinically achievable drug concentrations, FP/T674I predominated with imatinib, whereas with nilotinib and sorafenib, FP/D842V and the compound mutation T674I+T874I became prevalent. Our results suggest that the PDGFR kinase domain contains a limited number of residues where exchanges critically interfere with binding of and inhibition by available PDGFR kinase inhibitors at achievable concentrations, which might explain the low frequency of imatinib resistance in this patient population. In addition, these findings would help to select the appropriate second-line drug in cases of imatinib-resistant disease and may be translated to other neoplasms driven by activated forms of PDGFR-A or -B.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/genetics , Myeloproliferative Disorders/genetics , Protein Kinase Inhibitors/pharmacology , Receptor, Platelet-Derived Growth Factor alpha/chemistry , Receptor, Platelet-Derived Growth Factor alpha/genetics , Amino Acid Sequence , Benzamides , Benzenesulfonates/pharmacology , Blotting, Western , Cell Line, Tumor , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Myeloproliferative Disorders/drug therapy , Niacinamide/analogs & derivatives , Phenylurea Compounds , Piperazines/pharmacology , Protein Structure, Tertiary , Pyridines/pharmacology , Pyrimidines/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Sorafenib , Structure-Activity Relationship
19.
Oncogene ; 29(5): 739-51, 2010 Feb 04.
Article in English | MEDLINE | ID: mdl-19881535

ABSTRACT

In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.


Subject(s)
Fusion Proteins, bcr-abl/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Phospholipase C gamma/metabolism , Protein Serine-Threonine Kinases/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction/physiology , Animals , Apoptosis/physiology , Benzamides , Blotting, Western , Enzyme Activation/drug effects , Enzyme Activation/physiology , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Mice , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/pharmacology , RNA, Small Interfering , Signal Transduction/drug effects , TOR Serine-Threonine Kinases
20.
Oncogene ; 27(31): 4380-4, 2008 Jul 17.
Article in English | MEDLINE | ID: mdl-18362889

ABSTRACT

Imatinib inhibits the kinase activity of Bcr-Abl and is currently the most effective drug for treatment of chronic myeloid leukemia (CML). Imatinib also blocks c-Abl, a physiological tyrosine kinase activated by a variety of stress signals including damaged DNA. We investigated the effect of pharmacological inhibition of c-Abl on the processing of irradiation-induced DNA damage in Bcr-Abl-negative cells. Cell lines and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were treated with imatinib or dasatinib before gamma-irradiation. Inhibition of c-Abl caused an enhanced irradiation-induced mutation frequency and slowdown of DNA repair, whereas imatinib was ineffective in cells expressing a T315I variant of c-Abl. Mutation frequency and repair kinetics were also studied in c-Abl-/- murine embryonic fibroblasts (MEFs) retransfected with wild-type c-Abl (wt-Abl) or a kinase-defect variant of Abl (KD-Abl). Enhanced mutation frequency as well as delayed DNA repair was observed in cells expressing KD-Abl. These data indicate that pharmacological inhibition of c-Abl compromises DNA-damage response.


Subject(s)
DNA Repair , Fusion Proteins, bcr-abl/biosynthesis , Gene Expression Regulation, Neoplastic , Proto-Oncogene Proteins c-abl/physiology , Animals , Antineoplastic Agents/pharmacology , Benzamides , Chromosome Aberrations , DNA Damage , Dasatinib , Fibroblasts/metabolism , Humans , Imatinib Mesylate , Kinetics , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/radiation effects , Mice , Piperazines/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Pyrimidines/pharmacology , Thiazoles/pharmacology
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