Cristina E. Tognon

Expression of the ETV6-NTRK3 gene fusion as a primary event in human secretory breast carcinoma

We report that human secretory breast carcinoma (SBC), a rare subtype of infiltrating ductal carcinoma, expresses the ETV6-NTRK3 gene fusion previously cloned in pediatric mesenchymal cancers. This gene fusion encodes a chimeric tyrosine kinase with potent transforming activity in fibroblasts. ETV6-NTRK3 expression was confirmed in 12 (92%) of 13 SBC cases, but not in other ductal carcinomas. Retroviral transfer of ETV6-NTRK3 (EN) into murine mammary epithelial cells resulted in transformed cells that readily formed tumors in nude mice. Phenotypically, tumors produced glands and expressed epithelial antigens, confirming that EN transformation is compatible with epithelial differentiation. This represents a recurrent chromosomal rearrangement and expression of a dominantly acting oncogene as a primary event in human breast carcinoma.

Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML

BACKGROUND The molecular causes of many hematologic cancers remain unclear. Among these cancers are chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL1-negative) chronic myeloid leukemia (CML), both of which are diagnosed on the basis of neoplastic expansion of granulocytic cells and exclusion of genetic drivers that are known to occur in other myeloproliferative neoplasms and myeloproliferative-myelodysplastic overlap neoplasms. METHODS To identify potential genetic drivers in these disorders, we used an integrated approach of deep sequencing coupled with the screening of primary leukemia cells obtained from patients with CNL or atypical CML against panels of tyrosine kinase-specific small interfering RNAs or small-molecule kinase inhibitors. We validated candidate oncogenes using in vitro transformation assays, and drug sensitivities were validated with the use of assays of primary-cell colonies. RESULTS We identified activating mutations in the gene encoding the receptor for colony-stimulating factor 3 (CSF3R) in 16 of 27 patients (59%) with CNL or atypical CML. These mutations segregate within two distinct regions of CSF3R and lead to preferential downstream kinase signaling through SRC family-TNK2 or JAK kinases and differential sensitivity to kinase inhibitors. A patient with CNL carrying a JAK-activating CSF3R mutation had marked clinical improvement after the administration of the JAK1/2 inhibitor ruxolitinib. CONCLUSIONS Mutations in CSF3R are common in patients with CNL or atypical CML and represent a potentially useful criterion for diagnosing these neoplasms. (Funded by the Leukemia and Lymphoma Society and others.).

Translational Activation of Snail1 and Other Developmentally Regulated Transcription Factors by YB-1 Promotes an Epithelial-Mesenchymal Transition

Increased expression of the transcription/translation regulatory protein Y-box binding protein-1 (YB-1) is associated with cancer aggressiveness, particularly in breast carcinoma. Here we establish that YB-1 levels are elevated in invasive breast cancer cells and correlate with reduced expression of E-cadherin and poor patient survival. Enforced expression of YB-1 in noninvasive breast epithelial cells induced an epithelial-mesenchymal transition (EMT) accompanied by enhanced metastatic potential and reduced proliferation rates. YB-1 directly activates cap-independent translation of messenger RNAs encoding Snail1 and other transcription factors implicated in downregulation of epithelial and growth-related genes and activation of mesenchymal genes. Hence, translational regulation by YB-1 is a restriction point enabling coordinated expression of a network of EMT-inducing transcription factors, likely acting together to promote metastatic spread.

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

ABSTRACT As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.

Foretinib is a potent inhibitor of oncogenic ROS1 fusion proteins

Significance ROS1 fusion kinases are critical oncogenes in several malignancies, suggesting that ROS1 inhibitors are likely to be effective molecularly targeted therapies in these patients. Although phase I/II clinical trials using the ALK/ROS1 inhibitor crizotinib to treat ROS1 fusion-harboring non–small-cell lung cancer patients demonstrate early success, evidence of clinical resistance to crizotinib due to the acquired ROS1G2032R mutation was recently reported. Here, we demonstrate that foretinib is a more potent ROS1 inhibitor than crizotinib in vitro and in vivo and remains effective against crizotinib-resistant ROS1 kinase domain mutations, including ROS1 G2032R. Taken together, our findings establish foretinib as a highly promising therapeutic candidate for treating patients with ROS1-driven malignancies and provide rationale for rapid clinical translation. The rapidly growing recognition of the role of oncogenic ROS1 fusion proteins in the malignant transformation of multiple cancers, including lung adenocarcinoma, cholangiocarcinoma, and glioblastoma, is driving efforts to develop effective ROS1 inhibitors for use as molecularly targeted therapy. Using a multidisciplinary approach involving small molecule screening in combination with in vitro and in vivo tumor models, we show that foretinib (GSK1363089) is a more potent ROS1 inhibitor than crizotinib (PF-02341066), an ALK/ROS inhibitor currently in clinical evaluation for lung cancer patients harboring ROS1 rearrangements. Whereas crizotinib has demonstrated promising early results in patients with ROS1-rearranged non–small-cell lung carcinoma, recently emerging clinical evidence suggests that patients may develop crizotinib resistance due to acquired point mutations in the kinase domain of ROS1, thus necessitating identification of additional potent ROS1 inhibitors for therapeutic intervention. We confirm that the ROS1G2032R mutant, recently reported in clinical resistance to crizotinib, retains foretinib sensitivity at concentrations below safe, clinically achievable levels. Furthermore, we use an accelerated mutagenesis screen to preemptively identify mutations in the ROS1 kinase domain that confer resistance to crizotinib and demonstrate that these mutants also remain foretinib sensitive. Taken together, our data strongly suggest that foretinib is a highly effective ROS1 inhibitor, and further clinical investigation to evaluate its potential therapeutic benefit for patients with ROS1-driven malignancies is warranted.

Targeting the insulin-like growth factor 1 receptor (IGF1R) signaling pathway for cancer therapy

Introduction: The IGF system controls growth, differentiation, and development at the cellular, organ and organismal levels. IGF1 receptor (IGF1R) signaling is dysregulated in many cancers. Numerous clinical trials are currently assessing therapies that inhibit either growth factor binding or IGF1R itself. Therapeutic benefit, often in the form of stable disease, has been reported for many different cancer types. Areas covered: Canonical IGF signaling and non-canonical pathways involved in carcinogenesis. Three recent insights into IGF1R signaling, namely hybrid receptor formation with insulin receptor (INSR), insulin receptor substrate 1 nuclear translocation, and evidence for IGF1R/INSR as dependence receptors. Different approaches to targeting IGF1R and mechanisms of acquired resistance. Possible mechanisms by which IGF1R signaling supports carcinogenesis and specific examples in different human tumors. Expert opinion: Pre-clinical data justifies IGF1R as a target and early clinical trials have shown modest efficacy in selected tumor types. Future work will focus upon assessing the usefulness or disadvantages of simultaneously targeting the IGF1R and INSR, biomarker development to identify potentially responsive patients, and the use of IGF1R inhibitors in combination therapies or as an adjunct to conventional chemotherapy.

Translational Activation of HIF1α by YB-1 Promotes Sarcoma Metastasis

Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.

IGFBP7 Binds to the IGF-1 Receptor and Blocks Its Activation by Insulin-Like Growth Factors

By inactivating the insulin-like growth factor receptor, IGFBP7 suppresses growth and promotes cell death. Deadly Receptor Blockade When bound to insulin-like growth factor 1 or 2 (IGF-1/2), the IGF-1 receptor (IGF1R) promotes protein synthesis, cell growth, and survival. One mechanism by which the family of IGF-binding proteins (IGFBPs) interferes with IGF1R activation is by binding to IGF-1/2. Evdokimova et al. showed that, unlike other IGFBPs, IGFBP7 attenuated activation of IGF1R in various cell lines by interacting with the extracellular domain of IGF1R and preventing IGF-1/2 from binding to the receptor. IGFBP7 increased the abundance of IGF1R in various normal and cancer cells and xenograft tumors. However, in cultured cells, IGFBP7 treatment resulted in the retention of IGF1R at the cell surface in an inactive form and in apoptosis. Thus, IGFBP7 could be used to trigger cell death in tumors that depend on IGF-1/2 for growth or survival. Insulin-like growth factor–binding protein 7 (IGFBP7) is a secreted factor that suppresses growth, and the abundance of IGFBP7 inversely correlates with tumor progression. Here, we showed that pretreatment of normal and breast cancer cells with IGFBP7 interfered with the activation and internalization of insulin-like growth factor 1 receptor (IGF1R) in response to insulin-like growth factors 1 and 2 (IGF-1/2), resulting in the accumulation of inactive IGF1R on the cell surface and blockade of downstream phosphatidylinositol 3-kinase (PI3K)–AKT signaling. Binding of IGFBP7 and IGF-1 to IGF1R was mutually exclusive, and the N-terminal 97 amino acids of IGFBP7 were important for binding to the extracellular portion of IGF1R and for preventing its activation. Prolonged exposure to IGFBP7 resulted in activation of the translational repressor 4E-binding protein 1 (4E-BP1) and enhanced sensitivity to apoptosis in IGF1R-positive cells. These results support a model whereby IGFBP7 binds to unoccupied IGF1R and suppresses downstream signaling, thereby inhibiting protein synthesis, cell growth, and survival.

ETV6-NTRK3 transformation requires insulin-like growth factor 1 receptor signaling and is associated with constitutive IRS-1 tyrosine phosphorylation

Congenital fibrosarcoma (CFS) and cellular mesoblastic nephroma (CMN) are pediatric spindle cell malignancies that share two specific cytogenetic abnormalities: trisomy of chromosome 11 and a t(12;15)(p13;q25) translocation. The t(12;15) rearrangement creates a transcriptionally active fusion gene that encodes a chimeric oncoprotein, ETV6-NTRK3 (EN). EN transforms NIH3T3 fibroblasts through constitutive activation of both the Ras-mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol-3′kinase (PI3K)-Akt pathway. However, the role of trisomy 11 in CFS and CMN remains unknown. In this study we demonstrate elevated expression of the chromosome 11p15.5 insulin-like growth factor 2 gene (IGF2) in CFS and CMN tumors. Moreover, we present evidence that an intact IGF signaling axis is essential for in vitro EN-mediated transformation. EN only very weakly transformed so-called R-murine fibroblasts derived from mice with a targeted disruption of the IGF1 receptor gene (IGFRI), but transformation activity was fully restored in R− cells engineered to re-express IGFRI (R+ cells). We also observed that the major IGFRI substrate, insulin-receptor substrate-1 (IRS-1), was constitutively tyrosine phosphorylated and could be co-immunoprecipitated with EN in either R− or R+ cells expressing the EN oncoprotein. IRS-1 association with Grb2 and PI3K p85, which link IGFRI to the Ras-MAPK and PI3K-Akt pathways, respectively, was enhanced in both cell types in the presence of EN. However, activation of the Ras-MAPK and PI3K-Akt pathways was markedly attenuated in EN-expressing R− cells compared to EN-transformed R+ cells. This suggests that IRS-1 may be functioning as an adaptor in EN signal transduction, but that a link to EN transformation pathways requires the presence of IGFRI. Our findings indicate that an intact IGF signaling axis is essential for EN transformation, and are consistent with a role for trisomy 11 in augmenting this pathway in EN expressing tumors.

ETV6-NTRK3–Mediated Breast Epithelial Cell Transformation Is Blocked by Targeting the IGF1R Signaling Pathway

The insulin-like growth factor (IGF) 1 receptor (IGF1R) is an important therapeutic target under study in many cancers. Here, we describe a breast cancer model based on expression of the ETV6-NTRK3 (EN) chimeric tyrosine kinase that suggests novel therapeutic applications of IGF1R inhibitors in secretory breast cancers. Originally discovered in congenital fibrosarcomas with t(12;15) translocations, EN was identified subsequently in secretory breast carcinoma (SBC) which represent a variant of invasive ductal carcinoma. Because fibroblast transformation by EN requires the IGF1R axis, we hypothesized a similar dependency may exist in mammary cells and, if so, that IGF1R inhibitors might be useful to block EN-driven breast oncogenesis. In this study, we analyzed EN expressing murine and human mammary epithelial cell lines for transformation properties. Various IGF1R signaling inhibitors, including the dual specificity IGF1R/insulin receptor (INSR) inhibitor BMS-536924, were then tested for effects on three-dimensional Matrigel cell growth, migration, and tumor formation. We found that EN expression increased acinar size and luminal filling in Matrigel cultures and promoted orthotopic tumor growth in mice. Tumors were well differentiated and nonmetastatic, similar to human SBC. The known EN effector pathway, PI3K-Akt, was activated in an IGF1- or insulin-dependent manner. BMS-536924 blocked EN transformation in vitro, whereas BMS-754807, another IGIFR/INSR kinase inhibitor currently in clinical trials, significantly reduced tumor growth in vivo. Importantly, EN model systems mimic the clinical phenotype observed in human SBC. Moreover, EN has a strict requirement for IGF1R or INSR in breast cell transformation. Thus, our findings strongly encourage the evaluation of IGF1R/INSR inhibitors to treat EN-driven breast cancers.