Frank Breitenbuecher

Insertion of FLT3 internal tandem duplication in the tyrosine kinase domain-1 is associated with resistance to chemotherapy and inferior outcome

To evaluate internal tandem duplication (ITD) insertion sites and length as well as their clinical impact in younger adult patients with FLT3-ITD-positive acute myeloid leukemia (AML), sequencing after DNA-based amplification was performed in diagnostic samples from 241 FLT3-ITD-mutated patients. All patients were treated on 3 German-Austrian AML Study Group protocols. Thirty-four of the 241 patients had more than 1 ITD, leading to a total of 282 ITDs; the median ITD length was 48 nucleotides (range, 15-180 nucleotides). ITD integration sites were categorized according to functional regions of the FLT3 receptor: juxtamembrane domain (JMD), n = 148; JMD hinge region, n = 48; beta1-sheet of the tyrosine kinase domain-1 (TKD1), n = 73; remaining TKD1 region, n = 13. ITD length was strongly correlated with functional regions (P < .001). In multivariable analyses, ITD integration site in the beta1-sheet was identified as an unfavorable prognostic factor for achievement of a complete remission (odds ratio, 0.22; P = .01), relapse-free survival (hazard ratio, 1.86; P < .001), and overall survival (hazard ratio, 1.59; P = .008). ITD insertion site in the beta1-sheet appears to be an important unfavorable prognostic factor in young adult patients with FLT3-ITD-positive AML. The clinical trials described herein have been registered as follows: AML HD93 (already published in 2003), AML HD98A (NCT00146120; http://www.ClinicalTrials.gov), and AMLSG 07-04 (NCT00151242; http://www.ClinicalTrials.gov).

Identification of a novel type of ITD mutations located in nonjuxtamembrane domains of the FLT3 tyrosine kinase receptor

In acute myeloid leukemia (AML), internal tandem duplications (ITDs) of the juxtamembrane (JM) of FLT3 have been shown to play a crucial role in driving proliferation and survival of the leukemic clone. Here, we report the identification of FLT3_ITD mutations located in non-JM domains of the FLT3-receptor. This novel type of FLT3_ITD mutation was found in 216 of 753 (28.7%) of unselected FLT3_ITD-positive AML cases. An FLT3 receptor harbouring a prototypic non-JM ITD (FLT3_ITD627E) mediated constitutive phosphorylation of FLT3 and of STAT5, suggesting that non-JM ITDs confer constitutive activation of the receptor. FLT3_ITD627E induced transformation of hematopoietic 32D cells and led to a lethal myeloproliferative disease in a syngeneic mouse model. Our results indicate that a significant proportion of activating FLT3_ITD mutations is not confined to the JM domain of FLT3. Further studies are warranted to define the biologic and clinical characteristics of non-JM ITDs.

A novel molecular mechanism of primary resistance to FLT3-kinase inhibitors in AML

Currently, FLT3 tyrosine kinase inhibitors (TKIs) are emerging as the most promising drug therapy to overcome the dismal prognosis of acute myelogenous leukemia (AML) patients harboring internal tandem duplications (ITDs) of FLT3. However, up-front drug resistance occurs in approximately 30% of patients, and molecular mechanisms of resistance are poorly understood. Here, we have uncovered a novel mechanism of primary resistance to FLT3 TKIs in AML: an FLT3 receptor harboring a nonjuxtamembrane ITD atypically integrating into the beta-2 sheet of the first kinase domain (FLT3_ITD627E) induces dramatic up-regulation of the anti-apoptotic myeloid cell leukemia 1 protein (MCL-1). Using RNA interference technology, deregulated MCL-1 protein expression was shown to play a major role in conferring the resistance phenotype of 32D_ITD627E cells. Enhanced and sustained binding of the adaptor protein GRB-2 to the FLT3_ITD627E receptor is involved in MCL-1 up-regulation and is independent from TKI (PKC412)-induced inhibition of the receptor kinase. Thus, we describe a new mechanism of primary resistance to TKIs, which operates by reprogramming local and distant signal transduction events of the FLT3 tyrosine kinase. The data presented suggest that particular ITDs of FLT3 may be associated with rewired signaling and differential responsiveness to TKIs.

Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway.

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-γ1 (PLC-γ1)-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-γ1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-γ1 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-γ1 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-γ1 and Bcr-Abl inhibition. The pivotal role of PLC-γ1 was further confirmed in a mouse leukemogenesis model.

Stabilization of Physical RAF/14-3-3 Interaction by Cotylenin A as Treatment Strategy for RAS Mutant Cancers

One-third of all human cancers harbor somatic RAS mutations. This leads to aberrant activation of downstream signaling pathways involving the RAF kinases. Current ATP-competitive RAF inhibitors are active in cancers with somatic RAF mutations, such as BRAF(V600) mutant melanomas. However, they paradoxically promote the growth of RAS mutant tumors, partly due to the complex interplay between different homo- and heterodimers of A-RAF, B-RAF, and C-RAF. Based on pathway analysis and structure-guided compound identification, we describe the natural product cotylenin-A (CN-A) as stabilizer of the physical interaction of C-RAF with 14-3-3 proteins. CN-A binds to inhibitory 14-3-3 interaction sites of C-RAF, pSer233, and pSer259, but not to the activating interaction site, pSer621. While CN-A alone is inactive in RAS mutant cancer models, combined treatment with CN-A and an anti-EGFR antibody synergistically suppresses tumor growth in vitro and in vivo. This defines a novel pharmacologic strategy for treatment of RAS mutant cancers.

Targeting MCL-1 sensitizes FLT3-ITD-positive leukemias to cytotoxic therapies

Patients suffering from acute myeloid leukemias (AML) bearing FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) have poor outcomes following cytarabine- and anthracyclin-based induction therapy. To a major part this is attributed to drug resistance of FLT3-ITD-positive leukemic cells. Against this background, we have devised an antibody array approach to identify proteins, which are differentially expressed by hematopoietic cells in relation to activated FLT3 signaling. Selective upregulation of antiapoptotic myeloid cell leukemia-1 (MCL-1) was found in FLT3-ITD-positive cell lines and primary mononuclear cells from AML patients as compared with FLT3-wild-type controls. Upregulation of MCL-1 was dependent on FLT3 signaling as confirmed by its reversion upon pharmacological inhibition of FLT3 activity by the kinase inhibitor PKC412 as well as siRNA-mediated suppression of FLT3. Heterologously expressed MCL-1 substituted for FLT3 signaling by conferring resistance of hematopoietic cells to antileukemia drugs such as cytarabine and daunorubicin, and to the proapoptotic BH3 mimetic ABT-737. Conversely, suppression of endogenous MCL-1 by siRNA or by flavopiridol treatment sensitized FLT3-ITD-expressing hematopoietic cells to cytotoxic and targeted therapeutics. In conclusion, MCL-1 is an essential effector of FLT3-ITD-mediated drug resistance. Therapeutic targeting of MCL-1 is a promising strategy to overcome drug resistance in FLT3-ITD-positive AML.

DOG1 regulates growth and IGFBP5 in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT or platelet-derived growth factor receptor α(PDGFRA), which can be therapeutically targeted by tyrosine kinase inhibitors (TKI) such as imatinib. Despite long-lasting responses, most patients eventually progress after TKI therapy. The calcium-dependent chloride channel DOG1 (ANO1/TMEM16A), which is strongly and specifically expressed in GIST, is used as a diagnostic marker to differentiate GIST from other sarcomas. Here, we report that loss of DOG1 expression occurs together with loss of KIT expression in a subset of GIST resistant to KIT inhibitors, and we illustrate the functional role of DOG1 in tumor growth, KIT expression, and imatinib response. Although DOG1 is a crucial regulator of chloride balance in GIST cells, we found that RNAi-mediated silencing or pharmacologic inhibition of DOG1 did not alter cell growth or KIT signaling in vitro. In contrast, DOG1 silencing delayed the growth of GIST xenografts in vivo. Expression profiling of explanted tumors after DOG1 blockade revealed a strong upregulation in the expression of insulin-like growth factor-binding protein 5 (IGFBP5), a potent antiangiogenic factor implicated in tumor suppression. Similar results were obtained after selection of imatinib-resistant DOG1- and KIT-negative cells derived from parental DOG1 and KIT-positive GIST cells, where a 5,000-fold increase in IGFBP5 mRNA transcripts were documented. In summary, our findings establish the oncogenic activity of DOG1 in GIST involving modulation of IGF/IGF receptor signaling in the tumor microenvironment through the antiangiogenic factor IGFBP5.

LS104, a non-ATP-competitive small-molecule inhibitor of JAK2, is potently inducing apoptosis in JAK2V617F-positive cells

The activating JAK2V617F mutation has been described in the majority of patients with BCR-ABL-negative myeloproliferative disorders (MPD). In this report, we characterize the small-molecule LS104 as a novel non-ATP-competitive JAK2 inhibitor: Treatment of JAK2V617F-positive cells with LS104 resulted in dose-dependent induction of apoptosis and inhibition of JAK2 autophosphorylation and of downstream targets. Activation of these targets by JAK2 was confirmed in experiments using small interfering RNA. LS104 inhibited JAK2 kinase activity in vitro. This effect was not reversible using elevated ATP concentrations, whereas variation of the kinase substrate peptide led to modulation of the IC50 value for LS104. In line with these data, combination treatment using LS104 plus an ATP-competitive JAK2 inhibitor (JAK inhibitor I) led to synergistically increased apoptosis in JAK2V617F-positive cells. Furthermore, LS104 strongly inhibited cytokine-independent growth of endogenous erythroid colonies isolated from patients with JAK2V617F-positive MPD in vitro, whereas there was no significant effect on growth of myeloid colonies obtained from normal controls. Based on these data, we have recently started a phase I clinical trial of LS104 for patients with JAK2V617F-positive MPDs. To the best of our knowledge, this is the first report on a non-ATP-competitive kinase inhibitor being tested in a clinical trial. [Mol Cancer Ther 2008;7(5):1176–84]

Inactivation of Capicua drives cancer metastasis

Metastasis is the leading cause of death in people with lung cancer, yet the molecular effectors underlying tumor dissemination remain poorly defined. Through the development of an in vivo spontaneous lung cancer metastasis model, we show that the developmentally regulated transcriptional repressor Capicua (CIC) suppresses invasion and metastasis. Inactivation of CIC relieves repression of its effector ETV4, driving ETV4-mediated upregulation of MMP24, which is necessary and sufficient for metastasis. Loss of CIC, or an increase in levels of its effectors ETV4 and MMP24, is a biomarker of tumor progression and worse outcomes in people with lung and/or gastric cancer. Our findings reveal CIC as a conserved metastasis suppressor, highlighting new anti-metastatic strategies that could potentially improve patient outcomes.

Development of a highly sensitive and specific method for detection of circulating tumor cells harboring somatic mutations in non-small-cell lung cancer patients.

Background Oncogenic mutations are powerful predictive biomarkers for molecularly targeted cancer therapies. For mutation detection patients have to undergo invasive tumor biopsies. Alternatively, archival samples are used which may no longer reflect the actual tumor status. Circulating tumor cells (CTC) could serve as an alternative platform to detect somatic mutations in cancer patients. We sought to develop a sensitive and specific assay to detect mutations in the EGFR gene in CTC from lung cancer patients. Methods We developed a novel assay based on real-time polymerase chain reaction (PCR) and melting curve analysis to detect activating EGFR mutations in blood cell fractions enriched in CTC. Non-small-cell lung cancer (NSCLC) was chosen as disease model with reportedly very low CTC counts. The assay was prospectively validated in samples from patients with EGFR-mutant and EGFR-wild type NSCLC treated within a randomized clinical trial. Sequential analyses were conducted to monitor CTC signals during therapy and correlate mutation detection in CTC with treatment outcome. Results Assay sensitivity was optimized to enable detection of a single EGFR-mutant CTC/mL peripheral blood. CTC were detected in pretreatment blood samples from all 8 EGFR-mutant lung cancer patients studied. Loss of EGFR-mutant CTC signals correlated with treatment response, and its reoccurrence preceded relapse. Conclusions Despite low abundance of CTC in NSCLC oncogenic mutations can be reproducibly detected by applying an unbiased CTC enrichment strategy and highly sensitive PCR and melting curve analysis. This strategy may enable non-invasive, specific biomarker diagnostics and monitoring in patients undergoing targeted cancer therapies.