Circulating ensembles of tumor-associated cells (C-ETACs) which comprise tumor emboli, immune cells and fibroblasts pose well-recognized risks of thrombosis and aggressive metastasis. However, the detection, prevalence and characterization of C-ETACs have been impaired due to methodological difficulties. Our findings show extensive pan-cancer prevalence of C-ETACs on a hitherto unreported scale in cancer patients and virtual undetectability in asymptomatic individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of 16,134 subjects including 5,509 patients with epithelial malignancies in various organs and 10,625 asymptomatic individuals with age related higher cancer risk. PBMCs were treated with stabilizing reagents to protect and harvest apoptosis-resistant C-ETACs, which are defined as cell clusters comprising at least three EpCAM + and CK + cells irrespective of leucocyte common antigen (CD45) status. All asymptomatic individuals underwent screening investigations for malignancy including PAP smear, mammography, low-dose computed tomography, evaluation of cancer antigen 125, cancer antigen 19-9, alpha fetoprotein, carcinoembryonic antigen, prostate specific antigen (PSA) levels and clinical examination to identify healthy individuals with no indication of cancer. C-ETACs were detected in 4,944 (89.8%, 95% CI: 89.0-90.7%) out of 5,509 cases of cancer. C-ETACs were detected in 255 (3%, 95% CI: 2.7-3.4%) of the 8,493 individuals with no abnormal findings in screening. C-ETACs were detected in 137 (6.4%, 95% CI: 5.4-7.4%) of the 2,132 asymptomatic individuals with abnormal results in one or more screening tests. Our study shows that heterotypic C-ETACs are ubiquitous in epithelial cancers irrespective of radiological, metastatic or therapy status. C-ETACs thus qualify to be a systemic hallmark of cancer.Additional Supporting Information may be found in the online version of this article.

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FYN is a non-receptor tyrosine kinase belonging to the SRC family of kinases, which are frequently over-expressed in human cancers, and play key roles in cancer biology. SRC has long been recognized as an important oncogene, but little attention has been given to its other family members. In this report, we have studied the role of FYN in FLT3 signaling in respect to acute myeloid leukemia (AML). We observed that FYN displays a strong association with wild-type FLT3 as well as oncogenic FLT3-ITD and is dependent on the kinase activity of FLT3 and the SH2 domain of FYN. We identified multiple FYN binding sites in FLT3, which partially overlapped with SRC binding sites. To understand the role of FYN in FLT3 signaling, we generated FYN overexpressing cells. We observed that expression of FYN resulted in slightly enhanced phosphorylation of AKT, ERK1/2 and p38 in response to ligand stimulation. Furthermore, FYN expression led to a slight increase in FLT3-ITD-dependent cell proliferation, but potent enhancement of STAT5 phosphorylation as well as colony formation. We also observed that FYN expression is deregulated in AML patient samples and that higher expression of FYN, in combination with FLT3-ITD mutation, resulted in enrichment of the STAT5 signaling pathway and correlated with poor prognosis in AML. Taken together our data suggest that FYN cooperates with oncogenic FLT3-ITD in cellular transformation by selective activation of the STAT5 pathway. Therefore, inhibition of FYN, in combination with FLT3 inhibition, will most likely be beneficial for this group of AML patients.

The response of childhood acute lymphoblastic leukemia (ALL) to dexamethasone predicts the long-term remission outcome. To explore the mechanisms of dexamethasone resistance in B cell ALL (B-ALL), we generated dexamethasone-resistant clones by prolonged treatment with dexamethasone. Using RNA-sequencing and high-throughput screening, we found that dexamethasone-resistant cells are dependent on receptor tyrosine kinases. Further analysis with phosphokinase arrays showed that the type III receptor tyrosine kinase FLT3 is constitutively active in resistant cells. Targeted next-generation and Sanger sequencing identified an internal tandem duplication mutation and a point mutation (R845G) in FLT3 in dexamethasone-resistant cells, which were not present in the corresponding sensitive clones. Finally, we showed that resistant cells displayed sensitivity to second-generation FLT3 inhibitors both in vitro and in vivo. Collectively, our data suggest that long-term dexamethasone treatment selects cells with a distinct genetic background, in this case oncogenic FLT3, and therefore therapies targeting FLT3 might be useful for the treatment of relapsed B-ALL patients.

Fms-like tyrosine kinase (FLT3) is a frequently mutated oncogene in acute myeloid leukemia (AML). FLT3 inhibitors display promising results in a clinical setting, but patients relapse after short-term treatment due to the development of resistant disease. Therefore, a better understanding of FLT3 downstream signal transduction pathways will help to identify an alternative target for the treatment of AML patients carrying oncogenic FLT3. Activation of FLT3 results in phosphorylation of FLT3 on several tyrosine residues that recruit SH2 domain-containing signaling proteins. We screened a panel of SH2 domain-containing proteins and identified SLAP2 as a potent interacting partner of FLT3. We demonstrated that interaction occurs when FLT3 is activated, and also, an intact SH2 domain of SLAP2 is required for binding. SLAP2 binding sites in FLT3 mainly overlap with those of SRC. SLAP2 over expression in murine proB cells or myeloid cells inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation in vitro, and tumor formation in vivo. Microarray analysis suggests that higher SLAP2 expression correlates with a gene signature similar to that of loss of oncogene function. Furthermore, FLT3-ITD positive AML patients with higher SLAP2 expression displayed better prognosis compared to those with lower expression of SLAP2. Expression of SLAP2 blocked FLT3 downstream signaling cascades including AKT, ERK, p38 and STAT5. Finally, SLAP2 accelerated FLT3 degradation through enhanced ubiquitination. Collectively, our data suggest that SLAP2 acts as a negative regulator of FLT3 signaling and therefore, modulation of SLAP2 expression levels may provide an alternative therapeutic approach for FLT3-ITD positive AML.

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-017-2494-0) contains supplementary material, which is available to authorized users.

GADS is a member of a family of SH2 and SH3 domain-containing adaptors that functions in tyrosine kinase-mediated signaling cascades. Its expression is largely restricted to hematopoietic tissues and cell lines. Therefore, GADS is mainly involved in leukocyte-specific protein tyrosine kinase signaling. GADS is known to interact with tyrosine-phosphorylated SHC, BCR-ABL and KIT. The SH2 domain of GADS has a similar binding specificity to that of GRB2 but its SH3 domain displays a different binding specificity, and thus it is involved in other downstream signaling pathways than GRB2. In the present study, we examined the role of GADS in FLT3 signaling. FLT3 is a type III receptor tyrosine kinase, which is mutated in more than 30% of acute myeloid leukemia (AML) and the most common mutations is the internal tandem duplication (ITD) mutations. We observed that expression of GADS enhanced oncogenic FLT3-ITD-induced cell proliferation and colony formation in vitro. In a mouse xenograft model, GADS accelerated FLT3-ITD-dependent tumor formation. Furthermore, expression of GADS induced a transcriptional program leading to upregulation of MYC and mTORC1 target genes. GADS localizes to the cell membrane and strongly binds to ligand-stimulated wild-type FLT3 or is constitutively associated with the oncogenic mutant FLT3-ITD. We mapped the binding sites in FLT3 to pY955 and pY969 which overlaps with the GRB2 binding sites. Expression of GADS enhanced FLT3-mediated phosphorylation of AKT, ERK1/2, p38 and STAT5. Taken together, our data suggests that GADS is an important downstream component of FLT3 signaling and expression of GADS potentiates FLT3-mediated mitogenic signaling.

The type III receptor tyrosine kinase FLT3 is one of the most commonly mutated oncogenes in acute myeloid leukemia (AML). Inhibition of mutated FLT3 in combination with chemotherapy has displayed promising results in clinical trials. However, one of the major obstacles in targeting FLT3 is the development of resistant disease due to secondary mutations in FLT3 that lead to relapse. FLT3 and its oncogenic mutants signal through associating proteins that activate downstream signaling. Thus, targeting proteins that interact with FLT3 and their downstream signaling cascades can be an alternative approach to treat FLT3-dependent AML. We used an SH2 domain array screen to identify novel FLT3 interacting proteins and identified ABL2 as a potent interacting partner of FLT3. To understand the role of ABL2 in FLT3-mediated biological and cellular events, we used the murine pro-B cell line Ba/F3 as a model system. Overexpression of ABL2 in Ba/F3 cells expressing an oncogenic mutant of FLT3 (FLT3-ITD) resulted in partial inhibition of FLT3-ITD-dependent cell proliferation and colony formation. ABL2 expression did not alter the kinase activity of FLT3, its ubiquitination or its stability. However, it partially blocked FLT3-induced AKT phosphorylation without affecting ERK1/2 and p38 activation. Taken together our data suggest that ABL2 acts as negative regulator of signaling downstream of FLT3.