Signal transduction in response to growth factors is a strictly controlled process with networks of feedback systems, highly selective interactions and finely tuned on-and-off switches. In the context of cancer, detailed signaling studies have resulted in the development of some of the most frequently used means of therapy, with several well established examples such as the small molecule inhibitors imatinib and dasatinib in the treatment of chronic myeloid leukemia. Impaired function of receptor tyrosine kinases is implicated in various types of tumors, and much effort is put into mapping the many interactions and downstream pathways. Here we discuss the hematopoietic growth factor receptors c-Kit and Flt3 and their downstream signaling in normal as well as malignant cells. Both receptors are members of the same family of tyrosine kinases and crucial mediators of stem-and progenitor-cell proliferation and survival in response to ligand stimuli from the surrounding microenvironment. Gain-of-function mutations/alterations render the receptors constitutively and ligand-independently activated, resulting in aberrant signaling which is a crucial driving force in tumorigenesis. Frequently found mutations in c-Kit and Flt3 are point mutations of aspartic acid 816 and 835 respectively, in the activation loop of the kinase domains. Several other point mutations have been identified, but in the case of Flt3, the most common alterations are internal tandem duplications (ITDs) in the juxtamembrane region, reported in approximately 30% of patients with acute myeloid leukemia (AML). During the last couple of years, the increasing understanding of c-Kit and Flt3 signaling has also revealed the complexity of these receptor systems. The impact of gain-of-function mutations of c-Kit and Flt3 in different malignancies is well established and shown to be of clinical relevance in both prognosis and therapy. Many inhibitors of both c-Kit or Flt3 or of their downstream substrates are in clinical trials with encouraging results, and targeted therapy using a combination of such inhibitors is considered a promising approach for future treatments.

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IntroductionFms-like tyrosine kinase 3 (Flt3) functions as a growth factor receptor and is expressed primarily in multipotential hematopoietic stem cells and progenitors as well as in placenta, gonads, and brain. Together with its activating ligand Flt3 ligand (FL) it is a crucial player in assuring normal function of stem cells and the immune system. [1][2][3] Moreover, approximately 30% to 35% of patients with acute myeloid leukemia (AML) carry a mutation in Flt3, rendering Flt3 the most frequently mutated gene in AML. 4,5 Flt3 mutations are generally grouped into 2 classes: point mutations in the vicinity of codon 835 or 842 within the tyrosine kinase domain (TKD); or internal tandem duplications (ITDs) of varying lengths within the juxtamembrane domain of Flt3, which sterically represses the intrinsic kinase activity of Flt3 in the absence of ligand. 4,6 Both classes of mutations result in constitutive activation of Flt3 but distinct signaling and transforming capacities. 7-10 Although debatable as prognostic markers by themselves, ITD and TKD mutations are correlated with poor prognostic features for AML patients, suggesting Flt3 or one of its downstream effectors as potential therapeutic targets. [11][12][13][14][15] Flt3 constitutes, together with the receptor for stem cell factor (c-Kit), the receptors for platelet-derived growth factors (PDGFRs), and colony-stimulating factor-1 (CSF-1), the type III family of receptor tyrosine kinases (RTKs). 5 Type III RTKs share a common modular structure consisting of 5 extracellular Ig-like domains, a short transmembrane stretch, a juxtamembrane region followed by a bipartite kinase domain interrupted by the kinase insert, and the carboxyterminal tail. 16 Ligand binding causes receptor dimerization, kinase activation, and transphosphorylation of RTK on multiple tyrosine residues. 17 These autophosphorylated tyrosine residues together with 3 to 6 adjacent amino acids form high-affinity docking sites for relay molecules possessing either phosphotyrosine binding (PTB) or Src homology 2 (SH2) domains. 18 Upon relocation to the receptor, these signaling or adapter molecules become activated in either a phosphorylation-dependent or -independent manner and are thereby capable of transducing the signal downstream. Relay molecules reported to be recruited and/or activated upon Flt3 activation include the p85 subunit of PI3K, Ras-GAP, PLC-␥, Vav, SHIP1, SHP2, ShcA, Grb2, Cbl, and Src family kinases (SFKs) as well as Stat5. [19][20][21][22][23][24][25][26] Whereas the immediate signaling steps following ligand binding (ie, binding of signaling molecules to autophosphorylated tyrosines) are well studied in the c-Kit, PDGFRs, and CSF-1 receptor systems, 16 For personal use only. on June 19, 2019. by guest www.bloodjournal.org From Y589, Y591, Y597, or Y599, which could theoretically add to the aberrant signal relay from the autoactivated receptor. 27 Here we report that Y572, Y589, Y591, and Y599 of Flt3 are phosphorylated in vivo in Flt3-expressing cells following ligand stimu...

Phosphorylation of wild type FLT3 and AML-associated mutant FLT3 was recently analyzed using site-specific phosphotyrosine antibodies (15). Interestingly, the phosphorylation pattern of the different FLT3 variants showed quantitative and also qualitative differences. Although FLT3-ITD or mutations in the kinase domain resulted in ligand-independent FLT3 autophosphorylation and signaling activity, the wild type receptor is only autophosphorylated in response to stimulation with its cytokine FL.Signaling of receptor tyrosine kinases is modulated by protein-tyrosine phosphatases (PTP) (16), and aberrations in PTP function play a role in carcinogenesis (17). Some PTP, notably SHP-2, have been found to positively influence growth-stimulatory signaling pathways, and mutations leading to gain-offunction of these PTP can potentially be oncogenic. It has been demonstrated that SHP-2 directly interacts with FLT3 in a phosphorylation-dependent manner via phosphotyrosine 599. Table S1. 1 To whom correspondence should be addressed. Tel.: 49-3641-9395634; Fax:49-3641-9395602; E-mail: joerg.mueller2@med.uni-jena.de.2 The abbreviations used are: AML, acute myeloid leukemia; PTP, proteintyrosine phosphatase; FL, FLT3 ligand; PLC␥, phospholipase C␥; ITD, internal tandem duplication; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

The ubiquitin E3 ligase Cbl has been shown to negatively regulate tyrosine kinase receptors, including the stem cell factor receptor/c-Kit. Impaired recruitment of Cbl to c-Kit results in a deregulated positive signalling that eventually can contribute to carcinogenesis. Here, we present results showing that Cbl is activated by the SFKs (Src family kinases) and recruited to c-Kit in order to trigger receptor ubiquitination. We demonstrate that phosphorylated Tyr568 and Tyr936 in c-Kit are involved in direct binding and activation of Cbl and that binding of the TKB domain (tyrosine kinase binding domain) of Cbl to c-Kit is specified by the presence of an isoleucine or leucine residue in position +3 to the phosphorylated tyrosine residue on c-Kit. Apart from the direct association between Cbl and c-Kit, we show that phosphorylation of Cbl by SFK members is required for activation of Cbl to occur. Moreover, we demonstrate that Cbl mediates monoubiquitination of c-Kit and that the receptor is subsequently targeted for lysosomal degradation. Taken together, our findings reveal novel insights into the mechanisms by which Cbl negatively regulates c-Kit-mediated signalling.

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR/ErbB1), human EGFR 2 (HER2/ErbB2), ErbB3/HER3, and ErbB4/HER4. The first two members of this family, EGFR and HER2, have been implicated in tumorigenesis and cancer progression for several decades, and numerous drugs have now been approved that target these two proteins. Less attention, however, has been paid to the role of this family in mediating cancer cell survival and drug tolerance. To better understand the complex signal transduction network triggered by the ErbB receptor family, we built a computational model that quantitatively captures the dynamics of ErbB signaling. Sensitivity analysis identified ErbB3 as the most critical activator of phosphoinositide 3-kinase (PI3K) and Akt signaling, a key pro-survival pathway in cancer cells. Based on this insight, we designed a fully human monoclonal antibody, seribantumab (MM-121), that binds to ErbB3 and blocks signaling induced by the extracellular growth factors heregulin (HRG) and betacellulin (BTC). In this article, we present some of the key preclinical simulations and experimental data that formed the scientific foundation for three Phase 2 clinical trials in metastatic cancer. These trials were designed to determine if patients with advanced malignancies would derive benefit from the addition of seribantumab to standard-of-care drugs in platinum-resistant/refractory ovarian cancer, hormone receptor-positive HER2-negative breast cancer, and EGFR wild-type non-small cell lung cancer (NSCLC). From preclinical studies we learned that basal levels of ErbB3 phosphorylation correlate with response to seribantumab monotherapy in mouse xenograft models. As ErbB3 is rapidly dephosphorylated and hence difficult to measure clinically, we used the computational model to identify a set of five surrogate biomarkers that most directly affect the levels of p-ErbB3: HRG, BTC, EGFR, HER2, and ErbB3. Preclinically, the combined information from these five markers was sufficient to accurately predict which xenograft models would respond to seribantumab, and the single-most accurate predictor was HRG. When tested clinically in ovarian, breast and lung cancer, HRG mRNA expression was found to be both potentially prognostic of insensitivity to standard therapy and potentially predictive of benefit from the addition of seribantumab to standard of care therapy in all three indications. In addition, it was found that seribantumab was most active in cancers with low levels of HER2, consistent with preclinical predictions. Overall, our clinical studies and studies of others suggest that HRG expression defines a drug-tolerant cancer cell phenotype that persists in most solid tumor indications and may contribute to rapid clinical progression. To our knowledge, this is the first example of a drug designed and clinically tested using the principles of Systems Biology.

SummaryThe haematopoietic growth factor receptor Flt3 has been implicated as major cause of transformation in acute myeloid leukaemia. Intracellular signals mediated by wild-type Flt3 are involved in cell differentiation and survival whereas signalling via the mutant Flt3 ITD (internal tandem duplication) promotes enhanced cell growth. In this study, we identified tyrosines 768, 955 and 969 of Flt3 as phosphorylation sites and mediators of growth factor receptor binding protein 2 (Grb2) interaction, leading to the association of Grb2 associated binder 2 (Gab2) and contributing to proliferation and survival. Ba/F3 cells were transfected with either the wild-type Flt3 or the ITD, with or without a triple mutation of the Grb2 binding sites, and characterised in terms of proliferation and viability. Interestingly, the Flt3 ITD promoted increased survival but after introducing the triple mutation, this phenotype was lost. When looking into different downstream pathways, this effect was mainly caused by decreased phosphoinositide 3-kinase and Stat5 signalling, and the Flt3 ITD carrying the Grb2 binding mutations showed less Akt and Stat5 activation compared to the regular Flt3 ITD receptor. These findings not only reveal novel phosphorylation sites in Flt3 but contribute to the understanding of the molecular mechanism by which Flt3 ITD functions in pathological conditions.

Approximately 30% of human cancers harbor oncogenic gain-offunction mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.O ncogenic mutations in the RAS family member KRAS are among the most common mutations in human cancer (1). For example, KRAS-mutation frequencies in lung, colon, and pancreas adenocarcinoma are 30%, 50%, and 90% respectively (2). These observations, coupled with functional studies, suggest that KRAS is a highly attractive therapeutic target for many cancers. Unfortunately, small-molecule targeting of KRAS has not yet been achieved, and no effective KRAS inhibitors have been described. Blockade of prenylation of the KRAS C-terminal membrane anchoring domain with farnesyltransferase inhibitors has met with limited success, due at least in part to increased expression of geranylgeranyl transferase (3). Similarly, targeting other steps in the processing of the KRAS C-terminal region through the inhibition of Ras-converting enzyme or isoprenylcysteine-carboxymethyltransferase has yet to be clinically validated (2). Efforts to target the downstream effector pathways of KRAS with MEK inhibitors alone or in combination with PI3K inhibitors have shown promising preclinical results and are currently being evaluated in the clinic (4). Nevertheless, the therapeutic targeting of KRAS remains one of the grand challenges in cancer research.Recently, an alternative approach to targeting KRAS has been proposed-namely, the RNA interference (RNAi)-based screening for synthetic lethal gene/RNA interactions that might then suggest protein targets more "druggable" than the targeted mRNA or the KRAS protein itself (5-9). A recently reported RNAi screen suggested the serine-threonine kinase STK33 as such a target (9). Knock down of STK33 was reported to induce apoptosis in KRAS-dependent AML cancer cell lines but spare KRAS wildtype cells. While the normal function of the STK33 protein is unknown, the results led the authors to propose that a small-molecule inhibitor of STK33's protein kinase activity would selectively kill KRAS-mutant cancer cells. As no such small-molecule inhibitors exist, we set out to discover them, and to characterize their activity as anti-KRAS agents. ResultsHigh Throughput Screening for STK33 Kinase Inhibitors. We first established an a...