[Show abstract][Hide abstract] ABSTRACT: ABL tyrosine kinase inhibitors (TKI) like Imatinib, Dasatinib and Nilotinib are the gold standard in conventional treatment of CML. However, the emergence of resistance remains a major problem. Alternative therapeutic strategies of ABL TKI-resistant CML are urgently needed. We asked whether dual inhibition of BCR-ABL and Aurora kinases A-C could overcome resistance mediated by ABL kinase mutations. We therefore tested the dual ABL and Aurora kinase inhibitors PHA-739358 and R763/AS703569 in Ba/F3- cells ectopically expressing wild type (wt) or TKI-resistant BCR-ABL mutants. We show that both compounds exhibited strong anti-proliferative and pro-apoptotic activity in ABL TKI resistant cell lines including cells expressing the strongly resistant T315I mutation. Cell cycle analysis indicated polyploidisation, a consequence of continued cell cycle progression in the absence of cell division by Aurora kinase inhibition. Experiments using drug resistant variants of Aurora B indicated that PHA-739358 acts on both, BCR-ABL and Aurora Kinase B, whereas Aurora kinase B inhibition might be sufficient for the anti-proliferative activity observed with R763/AS703569. Taken together, our data demonstrate that dual ABL and Aurora kinase inhibition might be used to overcome ABL TKI resistant CML.
PLoS ONE 11/2014; 9(11):e112318. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) is a plasma cell neoplasm that results from clonal expansion of an Ig-secreting terminally differentiated B cell. Advanced MM is characterized by tissue damage that involves bone, kidney, and other organs and is typically associated with recurrent genetic abnormalities. IL-6 signaling via the IL-6 signal transducer GP130 has been implicated as an important driver of MM pathogenesis. Here, we demonstrated that ectopic expression of constitutively active GP130 (L-GP130) in a murine retroviral transduction-transplantation model induces rapid MM development of high penetrance. L-GP130-expressing mice recapitulated all of the characteristics of human disease, including monoclonal gammopathy, BM infiltration with lytic bone lesions, and protein deposition in the kidney. Moreover, the disease was easily transplantable and allowed different therapeutic options to be evaluated in vitro and in vivo. Using this model, we determined that GP130 signaling collaborated with MYC to induce MM and was responsible and sufficient for directing the plasma cell phenotype. Accordingly, we identified Myc aberrations in the L-GP130 MM model. Evaluation of human MM samples revealed recurrent activation of STAT3, a downstream target of GP130 signaling. Together, our results indicate that deregulated GP130 activity contributes to MM pathogenesis and that pathways downstream of GP130 activity have potential as therapeutic targets in MM.
Journal of Clinical Investigation 11/2014; · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Patients with BRAFV600E/K-driven melanoma respond to the BRAF inhibitor vemurafenib due to subsequent deactivation of the proliferative RAS/RAF/MEK/ERK pathway. In BRAF WT cells and those with mutations that activate or result in high levels of the BRAF activator RAS, BRAF inhibition can lead to ERK activation, resulting in tumorigenic transformation. We describe a patient with malignant melanoma who developed chronic lymphocytic leukemia (CLL) in the absence of RAS mutations during vemurafenib treatment. BRAF inhibition promoted patient CLL proliferation in culture and in murine xenografts and activated MEK/ERK in primary CLL cells from additional patients. BRAF inhibitor-driven ERK activity and CLL proliferation required B cell antigen receptor (BCR) activation, as inhibition of the BCR-proximal spleen tyrosine kinase (SYK) reversed ERK hyperactivation and proliferation of CLL cells from multiple patients, while inhibition of the BCR-distal Bruton tyrosine kinase had no effect. Additionally, the RAS-GTP/RAS ratio in primary CLL cells exposed to vemurafenib was reduced upon SYK inhibition. BRAF inhibition increased mortality and CLL expansion in mice harboring CLL xenografts; however, SYK or MEK inhibition prevented CLL proliferation and increased animal survival. Together, these results suggest that BRAF inhibitors promote B cell malignancies in the absence of obvious mutations in RAS or other receptor tyrosine kinases and provide a rationale for combined BRAF/MEK or BRAF/SYK inhibition.
Journal of Clinical Investigation 10/2014; · 13.77 Impact Factor
[Show abstract][Hide abstract] 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 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 lead 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 antiproliferative 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.Leukemia accepted article preview online, 24 September 2014. doi:10.1038/leu.2014.283.
[Show abstract][Hide abstract] ABSTRACT: Acute graft-versus-host disease (GvHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT), therefore a better understanding of its biology may improve therapeutic options. We observed miR-146a upregulation in T cells of mice developing acute GvHD compared to untreated mice. Transplanting miR-146a(-/-) T cells caused increased GvHD severity, elevated TNF serum levels and reduced survival. TNF receptor-associated factor 6(TRAF6), a verified target of miR-146a, was upregulated in miR-146a(-/-) T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased NF-κB activity and TNF production in miR-146a(-/-) T cells. Conversely, the detrimental effect of miR-146a deficiency in T cells was antagonized by TNF blockade, while phytochemical induction of miR-146a or its overexpression using a miR-146a mimic reduced GvHD severity. In humans, the minor genotype of the SNP rs2910164 in HCT donors, which reduces expression of miR-146a, was associated with severe acute GvHD (grade III/IV). We show that miR-146a functions as a negative regulator of donor T cells in GvHD by targeting TRAF6, leading to reduced TNF transcription. Since miR-146a expression can be exogenously enhanced, our results provide a novel, targeted molecular approach to mitigate GvHD.
[Show abstract][Hide abstract] 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.
Blood Cancer Journal 08/2014; 4:e240. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: FLT3 is the most frequently mutated kinase in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) in the juxta-membrane region constitute the majority of activating FLT3 mutations. Several FLT3 kinase inhibitors were developed and tested in the clinic with significant success. However, recent studies have reported the development of secondary drug resistance in patients treated with FLT3 inhibitors. Since FLT3-ITD is an HSP90 client kinase, we here explored if targeting the stability of drug-resistant FLT3 mutant protein could be a potential therapeutic option. We observed that HSP90 inhibitor treatment resulted in the degradation of inhibitor-resistant FLT3-ITD mutants and selectively induced toxicity in cells expressing FLT3-ITD mutants. Thus, HSP90 inhibitors provide a potential therapeutic choice to overcome secondary drug resistance following TKI treatment in FLT3-ITD positive AML.
PLoS ONE 05/2014; 9(5):e97116. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.
[Show abstract][Hide abstract] ABSTRACT: Graft-versus-host-disease (GvHD) is a severe complication of allogeneic hematopoietic cell transplantation (allo-HCT) characterized by the production of high levels of pro-inflammatory cytokines. Activated Janus kinases (JAKs) are required for T effector cell responses in different inflammatory diseases and their blockade could potently reduce acute GvHD. We observed that inhibition of JAK1/2 signaling resulted in reduced proliferation of effector T cells and suppression of pro-inflammatory cytokine production in response to alloantigen in mice. In vivo JAK 1/2 inhibition improved survival of mice developing acute GvHD and reduced histopathologic GvHD grading, serum levels of pro-inflammatory cytokines and expansion of alloreactive luc-transgenic T cells. Mechanistically, we could show that ruxolitinib impaired differentiation of CD4(+) T cells into IFN-γ and IL17A-producing cells, both T cell phenotypes are linked to GvHD. Conversely, ruxolitinib treatment in allo-HCT recipients increased FoxP3(+) regulatory T cells, which are linked to immunological tolerance. Based on these results, we treated 6 patients suffering from steroid-refractory GvHD with ruxolitinib. All patients responded with respect to clinical GvHD symptoms and serum levels of pro-inflammatory cytokines. In summary, ruxolitinib represents a novel targeted approach in GvHD by suppression of pro-inflammatory signaling that mediates tissue damage and by promotion of tolerogenic Treg cells.
[Show abstract][Hide abstract] 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 β-catenin expression, a known activator of HES1 and important lung cancer oncogene. Characteristically for β-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.Oncogene advance online publication, 10 February 2014; doi:10.1038/onc.2013.592.
[Show abstract][Hide abstract] ABSTRACT: The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome-mediated IL-1β production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1β or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro-IL-1β cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1β originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1β were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication.
Journal of Experimental Medicine 08/2013; · 13.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSEThe prognosis of elderly patients with acute myeloid leukemia (AML) is still dismal even with intensive chemotherapy. In this trial, we compared the antileukemic activity of standard induction and consolidation therapy with or without the addition of the kinase inhibitor sorafenib in elderly patients with AML. PATIENTS AND METHODS
All patients received standard cytarabine and daunorubicin induction (7+3 regimen) and up to two cycles of intermediate-dose cytarabine consolidation. Two hundred one patients were equally randomly assigned to receive either sorafenib or placebo between the chemotherapy cycles and subsequently for up to 1 year after the beginning of therapy. The primary objective was to test for an improvement in event-free survival (EFS). Overall survival (OS), complete remission (CR) rate, tolerability, and several predefined subgroup analyses were among the secondary objectives.ResultsAge, sex, CR and early death (ED) probability, and prognostic factors were balanced between both study arms. Treatment in the sorafenib arm did not result in significant improvement in EFS or OS. This was also true for subgroup analyses, including the subgroup positive for FLT3 internal tandem duplications. Results of induction therapy were worse in the sorafenib arm, with higher treatment-related mortality and lower CR rates. More adverse effects occurred during induction therapy in the sorafenib arm, and patients in this arm received less consolidation chemotherapy as a result of higher induction toxicity.DiscussionIn conclusion, combination of standard induction and consolidation therapy with sorafenib in the schedule investigated in our trial is not beneficial for elderly patients with AML.
Journal of Clinical Oncology 07/2013; · 17.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In gastrointestinal stromal tumor (GIST), there is no biomarker available that indicates success or failure of therapy. We hypothesized that tumor specific CKIT or PDGFRA mutant DNA fragments can be detected and quantified in plasma samples of GIST patients.
We prospectively collected 291 plasma samples from 38 subjects with GIST harbouring activating mutations of CKIT or PDGFRA detected in tumor tissue, irrespective of current disease status or treatment. We used allele-specific Ligation PCR to detect mutant free circulating (fc)DNA.
We were able to detect fcDNA harbouring the tumor mutation in 15 out of 38 patients. Patients with active disease displayed significantly higher amounts of mutant fcDNA compared to patients in CR. The amount of mutant fcDNA correlated with disease course. We observed repeated positive test results or an increase of mutant fcDNA in five patients with progressive disease or relapse. A decline of tumor fcDNA or conversion from positive to negative was seen in five patients responding to treatment. A negative to positive conversion was seen in two patients with relapse and one patient with progression. In two cases, we aimed to identify additional mutations, and found four additional exchanges, including mutations not known from sequentially performed tumor biopsies.
Our results indicate that free circulating DNA harbouring tumor specific mutations in the plasma of patients with GIST can be used as tumor-specific biomarker. The detection of resistance mutations in plasma samples might allow earlier treatment changes and obviates the need for repeated tumor biopsies.
Clinical Cancer Research 07/2013; · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of HSP90 in stabilization of oncogenic tyrosine kinases made it an attractive therapeutic target for treating cancer but the molecular basis underlying the interaction between the HSP90 chaperone and client kinases is not elucidated yet. Using kinase inhibitors we show that the inactive conformation of ERBB2 does not interact with HSP90 chaperone and is thus not amenable to degradation upon HSP90 inhibitor treatment, while active ERBB2 kinase conformation promotes interaction with the HSP90 machinery and thus is degraded upon HSP90 inhibitor treatment. Interestingly, the kinase-chaperone interaction is disrupted in case of BCR-ABL and FLT3-ITD when bound to inhibitors irrespective of whether they block the kinase in an active or inactive conformation and thus our results indicate that the stability of the active kinase conformation varies between different kinases.
PLoS ONE 07/2013; 8(7):e68394. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Formation of asymmetric kinase dimers is required for wt-EGFR activation upon ligand stimulation. The role of receptor dimerization in oncogenic EGFRvIII mutant activation is not completely understood and the molecular details of EGFRvIII interactions within homo-dimers and hetero-dimers are not elucidated yet. FINDINGS: By employing mutations that disrupt the asymmetric kinase dimer interface in EGFRvIII, we demonstrate that the mechanism of oncogenic EGFRvIII mutant activation is similar to that of the full-length wild-type EGFR. Surprisingly, the monomeric EGFRvIII lacks autophosphorylation and the formation of asymmetric kinase dimers is indispensable for oncogenic kinase activation. In addition, we show that ERBB3 can act as an activator of EGFRvIII by forming asymmetric kinase dimer in a ligand-independent manner. Interestingly, we found that the formation of asymmetric kinase dimer is dispensable for ERBB3 phosphorylation by the activated EGFR kinase as well as the ERBB2 kinase thus revealing a novel model for receptor function. CONCLUSIONS: Lateral signaling is a novel mechanism of signal propagation via ERBB3 upon activation by EGFR/ERBB2 kinase even in the absence of their ability to form asymmetric kinase dimers.
Cell Communication and Signaling 06/2013; 11(1):39. · 4.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A phase I dose-escalation study of MSC1992371A, an oral aurora kinase inhibitor, was carried out in patients with hematologic malignancies. Patients received escalating doses either on days 1-3 and 8-10 (n=36) or on days 1-6 (n=39) of a 21-day cycle. The maximum tolerated doses were 37 and 28mg/m(2)/day, respectively. Dose-limiting toxicities included severe neutropenia with infection and sepsis, mucositis/stomatitis, and diarrhea. Complete responses occurred in 3 patients. Four disease-specific expansion cohorts then received the dose and schedule dictated by the escalation phase but the study was prematurely discontinued due to hematologic and gastrointestinal toxicity at clinically effective doses.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: Dasatinib and nilotinib are active in imatinib resistant CML and many patients undergo sequential treatment. We aimed at modelling sequential TKI resistance in vitro to compare the sequences imatinib-nilotinib-dasatinib and imatinib-dasatinib-nilotinib. EXPERIMENTAL DESIGN: We designed an in vitro-model for sequential TKI resistance in CML. Replicates of imatinib resistant cell lines were treated with dasatinib or nilotinib. Second line resistant replicates were exposed to 3rd line treatment. RESULTS: Growth of all replicates in all three lines of treatment was associated with T315I. However, T315I occurred with low abundance and did not increase during sequential treatment. Nilotinib 2nd line more often gave rise to sequential resistance compared to dasatinib due to pre-existing P-loop mutations, especially at suboptimal drug concentration. In contrast, mutations predisposing to dasatinib resistance such as F317C/V and V299L did not occur before dasatinib exposure. Nilotinib 3rd line did not overcome imatinib-dasatinib resistance due to pre-existing T315I or P-loop/V299L or P-loop/F317 exchanges. Dasatinib 3rd line suppressed imatinib-nilotinib resistant replicates with residual sensitivity. CONCLUSIONS: Sequential acquisition of BCR-ABL drug resistance mutations in CML might be underestimated. Resistance to sequential TKI monotherapy in vitro more often was associated with stepwise acquisition of drug-specific compound mutations compared to T315I. Pre-existing mutations strongly limited the activity of both 3rd line treatments, and the activity of nilotinib 2nd line in vitro critically depended on drug concentration.
Clinical Cancer Research 04/2013; · 8.19 Impact Factor