Sandra Braun

Strong negative feedback from Erk to Raf confers robustness to MAPK signalling

Protein levels within signal transduction pathways vary strongly from cell to cell. Here, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. We systematically perturbed the protein levels of Erk, the terminal kinase in the MAPK signalling pathway in a panel of human cell lines. We found that the steady‐state phosphorylation of Erk is very robust against perturbations of Erk protein level. Although a multitude of mechanisms exist that may provide robustness against fluctuating protein levels, we found that one single feedback from Erk to Raf‐1 accounts for the observed robustness. Surprisingly, robustness is provided through a fast post‐translational mechanism although variation of Erk levels occurs on a timescale of days.

Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling

The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild‐type B‐Raf (B‐Raf wt) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B‐Raf wt and several of its gain‐of‐function (g‐o‐f) mutants. In contrast, the B‐Raf V600E, B‐Raf insT and B‐Raf G469A oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B‐Raf wt, B‐Raf V600E displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B‐Raf wt and Raf‐1wt mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf‐1 by kinase‐inactivated B‐Raf require an intact DIF. Surprisingly, the B‐Raf DIF is not required for dimerisation between Raf‐1 and B‐Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two‐step mechanism consisting of dimerisation and DIF‐dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras‐driven Raf‐mediated (paradoxical) ERK activation.

Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors

Grb2-associated binder 2 (Gab2) serves as a critical amplifier in the signaling network of Bcr-Abl, the driver of chronic myeloid leukemia (CML). Despite the success of tyrosine kinase inhibitors (TKIs) in CML treatment, TKI resistance, caused by mutations in Bcr-Abl or aberrant activity of its network partners, remains a clinical problem. Using inducible expression and knockdown systems, we analyzed the role of Gab2 in Bcr-Abl signaling in human CML cells, especially with respect to TKI sensitivity. We show for the first time that Gab2 signaling protects CML cells from various Bcr-Abl inhibitors (imatinib, nilotinib, dasatinib and GNF-2), whereas Gab2 knockdown or haploinsufficiency leads to increased TKI sensitivity. We dissected the underlying molecular mechanism using various Gab2 mutants and kinase inhibitors and identified the Shp2/Ras/ERK and the PI3K/AKT/mTOR axes as the two critical signaling pathways. Gab2-mediated TKI resistance was associated with persistent phosphorylation of Gab2 Y452, a PI3K recruitment site, and consistent with this finding, the protective effect of Gab2 was completely abolished by the combination of dasatinib with the dual PI3K/mTOR inhibitor NVP-BEZ235. The identification of Gab2 as a novel modulator of TKI sensitivity in CML suggests that Gab2 could be exploited as a biomarker and therapeutic target in TKI-resistant disease.

Distinct signal transduction processes by IL-4 and IL-13 and influences from the Q551R variant of the human IL-4 receptor alpha chain

BackgroundAlthough IL-4 and IL-13 share the IL-13 receptor, IL-13 exhibits unique functions. To elicit the cellular basis of these differences, signal transduction processes have been compared. Additionally, the role of the IL-4 receptor alpha (IL-4Rα) variant Q551R was investigated.MethodsPeripheral blood mononuclear cells from donors were stimulated with IL-4 and IL-13. The phosphorylation status of effector substrates was detected by immunostaining. Binding of SHP-2 to IL-4Rα was investigated by using synthetic peptides.ResultsSHP-2 bound IL-4Rα synthetic peptide; this binding was reduced in the presence of the R551 variant. Stimulation with IL-4 increased SHP-1 phosphorylation, however, stimulation with IL-13 increased SHP-2 phosphorylation. PI3-kinase phosphorylation was elevated following stimulation with IL-13 in all individuals and with IL-4 only in R551 individuals. Jak1, Tyk2 and IRS-2 signals were reduced after IL-13 stimulation in Q551 individuals. STAT3 phosphorylation was markedly increased in R551 individuals, following stimulation with both IL-4 and IL-13. However, STAT3 was only detected immediately in nuclear extracts from variant individuals after stimulation with IL-13; in wildtype individuals STAT3 was only detected after IL-4 treatment.ConclusionIL-4 and IL-13 appear to promote distinct signal transduction cascades. SHP-1 seems to be predominately activated by IL-4 and to influence the PI3-kinase, in contrast, SHP-2 seems to be predominately activated by IL-13 and to influence Jak1, Tyk2 and IRS-2. Both phosphatases control STAT3. In the presence of the variant R551, SHP-1/2 activation is reduced and signal transduction is altered. STAT3 signaling appears be further regulated on the level of nuclear translocation.

Studies on linkage and association of atopy with the chromosomal region 12q13–24

Several studies have shown linkage of bronchial asthma, allergic rhinitis and total serum IgE concentration to the chromosomal region 12q13–24 in ethnical diverse populations. This region harbours a number of candidate genes for asthma and atopy, including stem cell factor (SCF), leukotriene A4 hydrolase (LTA4H), thyroid receptor 2 (TR2), and signal transducer and activator of transcription 6 (STAT6). However, the same region was shown as well to be linked to other diseases with inflammatory character. So far no variants in any of these genes have been published which would allow association studies and confirm the pathogenicity of any of these genes.

Cooperation of BRAF F595L and mutant HRAS in histiocytic sarcoma provides new insights into oncogenic BRAF signaling

Activating BRAF mutations, in particular V600E/K, drive many cancers and are considered mutually exclusive with mutant RAS, whereas inactivating BRAF mutations in the D594F595G596 motif cooperate with RAS via paradoxical MEK/ERK activation. Due to the increasing use of comprehensive tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We investigated an atypical BRAF mutation, F595L, which was identified along with mutant HRAS in histiocytic sarcoma and also occurs in epithelial cancers, melanoma and neuroblastoma, and determined its interaction with mutant RAS. Unlike other DFG motif mutants, BRAFF595L is a gain-of-function variant with intermediate activity that does not act paradoxically, but nevertheless cooperates with mutant RAS to promote oncogenic signaling, which is efficiently blocked by pan-RAF and MEK inhibitors. Mutation data from patients and cell lines show that BRAFF595L, as well as other intermediate-activity BRAF mutations, frequently coincide with mutant RAS in various cancers. These data define a distinct class of activating BRAF mutations, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway and underscore the value of comprehensive genomic testing for uncovering the vulnerabilities of individual tumors.

Amino acid variants in Surfactant protein D are not associated with bronchial asthma

Surfactant protein D (SFTPD) belongs to the family of collectins and is part of the innate immune system. Thereby it plays an important role in the defense of various pathogens. Besides it is involved in the development of acute and chronic inflammation of the lung. Levels of SFTPD are elevated in serum and alveolar lavage of asthmatic patients. As SFTPD binds and neutralizes common allergens like house dust mites it is especially important in allergic asthma. Three common amino acid variants have been identified in SFTPD and association of the first variant has been described to severe infection with respiratory syncytial virus. Furthermore the functional impact of all three amino acid variants has been demonstrated. Due to its function SFTPD represents an ideal candidate gene for bronchial asthma and we were interested whether the polymorphisms were in association with asthma in children. The three polymorphisms leading to amino acid exchanges (Met11Thr, Ala160Thr, and Ser270 Thr) were typed by restriction fragment length polymorphisms in 322 asthmatic children and 270 controls. Association analyses were performed by Armitages trend test. In addition haplotypes were calculated by FASTEHPLUS and FAMHAP. None of the polymorphisms was in association with bronchial asthma. Haplotype analyses revealed four major haplotypes all of which were evenly distributed between the populations. We conclude from our data that functional amino acid variants in SFTPD do not play a major role in the genetic pre‐disposition to bronchial asthma in children.

Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B‐Raf signaling in vivo. Here, we generated a conditional knock‐in mouse allowing the expression of the B‐RafAVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase‐impaired protein, the BrafAVKA allele does not phenocopy the lethality of Braf‐knockout or paradoxically acting knock‐in alleles. However, BrafAVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B‐Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B‐RafAVKA. Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non‐V600E B‐Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP‐competitive inhibitors.

Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles

B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase.

Abstract 2708: New approaches to overcome tyrosine kinase inhibitor resistances in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is driven by the hyperactive fusion kinase Bcr-Abl, generated by a chromosomal translocation between chromosome 9 and 22. This oncogenic tyrosine kinase builds up its own signaling network with various proteins such as the docking protein Gab2 or the Src kinase Lyn. As a result, the cells become addicted to the constant signals derived from this fusion kinase. This is the reason why selective Bcr Abl tyrosine kinase inhibitors (TKIs), such as Imatinib mesylate or dasatinib, became so successful in the therapy of CML. Despite the great success of these TKIs in CML treatment, TKI resistance remains a serious clinical problem. These resistances can be caused by mutations in the Bcr-Abl oncogene, like the clinical relevant T315I mutation or by aberrant activity of components of the Bcr-Abl signaling network. In that regard, we could show that the overexpression of Gab2 or the expression of a hyperactive mutant of Lyn can confer TKI resistance. Using different CML cell lines and models we aimed to identify new approaches to overcome TKI resistances caused by Bcr-Abl mutations or aberrant downstream signaling. Therefore, we screened inhibitors for their ability to overcome Gab2 mediated resistance or to inhibit the activity of T315I mutated Bcr-Abl. We identified the multikinase inhibitors sorafenib and axitinib, both clinical approved as second line drugs in renal cell or hepatocellular carcinoma, as compounds reducing the viability of Bcr-AblT315I transformed cells. Interestingly, these inhibitors were highly active against TKI resistant Bcr-Abl positive cells in contrast to non-transformed cells. These results invite for the further evaluation of sorafenib and axitinib in the treatment of TKI-resistant CML. Citation Format: Sebastian Halbach, Franziska U. Wohrle, Sandra Braun, Tilman Brummer. New approaches to overcome tyrosine kinase inhibitor resistances in chronic myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2708. doi:10.1158/1538-7445.AM2015-2708