Cédric Leroy

Evidence that Resistance to Nilotinib May Be Due to BCR-ABL, Pgp, or Src Kinase Overexpression

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in chronic myeloid leukemia (CML) and in Bcr-Abl-positive acute lymphoblastic leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second-generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance.

Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells.

In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML.

Quantitative Phosphoproteomics Reveals a Cluster of Tyrosine Kinases That Mediates Src Invasive Activity in Advanced Colon Carcinoma Cells

The nonreceptor tyrosine kinase Src is frequently overexpressed and/or activated in human colorectal carcinoma (CRC), and its increased activity has been associated with a poor clinical outcome. Src has been implicated in growth and invasion of these cancer cells by still not well-known mechanisms. Here, we addressed Src oncogenic signaling using quantitative phosphoproteomics. Src overexpression increased growth and invasiveness of metastatic SW620 CRC cells. Stable isotope labeling with amino acids in cell culture in combination with liquid chromatography tandem mass spectrometry allowed the identification of 136 proteins which exhibited a significant increase in and/or association with tyrosine phosphorylation upon Src expression. These mainly include signaling, cytoskeleton, and vesicular-associated proteins. Interestingly, Src also phosphorylated a cluster of tyrosine kinases, i.e., the receptors Met and EphA2, the cytoplasmic tyrosine kinase Fak, and pseudo-tyrosine kinase SgK223, which were required for its invasive activity. Similar results were obtained with metastatic Colo205 CRC cells that exhibit high endogenous Src activity. We concluded that Src uses a tyrosine kinases network to promote its invasive activity in CRC and this implicates a reverse signaling via tyrosine kinase receptors. Targeting these tyrosine kinases may be of significant therapeutic value in this cancer.

The tyrosine kinase Abl is required for Src-transforming activity in mouse fibroblasts and human breast cancer cells

The cytoplasmic tyrosine kinase Src has been implicated in signal transduction induced by growth factors and integrins. Src also shows oncogenic activity when deregulated. Accumulating evidence indicates that the tyrosine kinase Abl is an important substrate for Src signalling in normal cells. Here we show that Abl is also required for Src-induced transformation of mouse fibroblasts. Abl does not mediate tyrosine phosphorylation of Stat3 and Shc, two important regulators of Src oncogenic activity. In contrast, Abl controls the activation of the small GTPase Rac for oncogenic signalling and active Rac partly rescued Src transformation in cells with inactive Abl. Moreover, Abl mediates Src-induced extracellular regulated kinase 5 (ERK5) activation to drive cell transformation. Finally, we find that Abl/Rac and Abl/ERK5 pathways also operate in human MCF7 and BT549 breast cancer cells, where neoplastic transformation depends on Src-like activities. Therefore, Abl is an important regulator of Src oncogenic activity both in mouse fibroblasts and in human cancer cells. Targeting these Abl-dependent signalling cascades may be of therapeutic value in breast cancers where Src-like function is important.

Tyrosine phosphatase SHP2 increases cell motility in triple-negative breast cancer through the activation of SRC-family kinases.

Tumor cell migration has a fundamental role in early steps of metastasis, the fatal hallmark of cancer. In the present study, we investigated the effects of the tyrosine phosphatase, SRC-homology 2 domain-containing phosphatase 2 (SHP2), on cell migration in metastatic triple-negative breast cancer (TNBC), an aggressive disease associated with a poor prognosis for which a targeted therapy is not yet available. Using mouse models and multiphoton intravital imaging, we have identified a crucial effect of SHP2 on TNBC cell motility in vivo. Further, analysis of TNBC cells revealed that SHP2 also influences cell migration, chemotaxis and invasion in vitro. Unbiased phosphoproteomics and biochemical analysis showed that SHP2 activates several SRC-family kinases and downstream targets, most of which are inducers of migration and invasion. In particular, direct interaction between SHP2 and c-SRC was revealed by a fluorescence resonance energy transfer assay. These results suggest that SHP2 is a crucial factor during early steps of TNBC migration to distant organs.

The Hippo kinases LATS1 and 2 control human breast cell fate via crosstalk with ERα

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1–cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.

SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2

The adaptor SLAP is a negative regulator of receptor signalling in immune cells but its role in human cancer is ill defined. Here we report that SLAP is abundantly expressed in healthy epithelial intestine but strongly downregulated in 50% of colorectal cancer. SLAP overexpression suppresses cell tumorigenicity and invasiveness while SLAP silencing enhances these transforming properties. Mechanistically, SLAP controls SRC/EPHA2/AKT signalling via destabilization of the SRC substrate and receptor tyrosine kinase EPHA2. This activity is independent from CBL but requires SLAP SH3 interaction with the ubiquitination factor UBE4A and SLAP SH2 interaction with pTyr594-EPHA2. SRC phosphorylates EPHA2 on Tyr594, thus creating a feedback loop that promotes EPHA2 destruction and thereby self-regulates its transforming potential. SLAP silencing enhances SRC oncogenicity and sensitizes colorectal tumour cells to SRC inhibitors. Collectively, these data establish a tumour-suppressive role for SLAP in colorectal cancer and a mechanism of SRC oncogenic induction through stabilization of its cognate substrates.

The Src-like adaptor protein regulates PDGF-induced actin dorsal ruffles in a c-Cbl-dependent manner

The Src-like adaptor protein (SLAP) belongs to the subfamily of adapter proteins that negatively regulate cellular signalling initiated by tyrosine kinases. SLAP has a unique, myristylated N-terminus, followed by SH3 and SH2 domains with high homology to Src family tyrosine kinases (SFK) and a unique C-terminal tail, which is important for c-Cbl binding. We have previously shown that SLAP negatively regulates platelet-derived growth factor (PDGF)-induced mitogenesis in fibroblasts and we now report that it regulates F-actin assembly for dorsal ruffles formation. c-Cbl mediated SLAP inhibition towards actin remodelling. Moreover, SLAP enhanced PDGF-induced c-Cbl phosphorylation by SFK. In contrast, SLAP mitogenic inhibition was not mediated by c-Cbl, but it rather involved a competitive mechanism with SFK for PDGF-receptor (PDGFR) association and mitogenic signalling. Accordingly, phosphorylation of the Src mitogenic substrates Stat3 and Shc were reduced by SLAP. Thus, we concluded that SLAP regulates PDGFR signalling by two independent mechanisms: a competitive mechanism for PDGF-induced Src mitogenic signalling and a non-competitive mechanism for dorsal ruffles formation mediated by c-Cbl.

Inhibition of DDR1‐BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer

The clinical management of metastatic colorectal cancer (mCRC) faces major challenges. Here, we show that nilotinib, a clinically approved drug for chronic myeloid leukaemia, strongly inhibits human CRC cell invasion in vitro and reduces their metastatic potential in intrasplenic tumour mouse models. Nilotinib acts by inhibiting the kinase activity of DDR1, a receptor tyrosine kinase for collagens, which we identified as a RAS‐independent inducer of CRC metastasis. Using quantitative phosphoproteomics, we identified BCR as a new DDR1 substrate and demonstrated that nilotinib prevents DDR1‐mediated BCR phosphorylation on Tyr177, which is important for maintaining β‐catenin transcriptional activity necessary for tumour cell invasion. DDR1 kinase inhibition also reduced the invasion of patient‐derived metastatic and circulating CRC cell lines. Collectively, our results indicate that the targeting DDR1 kinase activity with nilotinib may be beneficial for patients with mCRC.

Abstract 2085: The antileukemic drug nilotinib inhibits the invasive activity and the metastatic potential of colorectal cancer cells by targeting the receptor tyrosine kinase DDR1.

Tyrosine kinases (TK) are frequently deregulated in human cancer and they play important roles in tumor progression. Since then, they have become valuable therapeutic targets and several inhibitors are currently used in the clinic. For example, the BCR-ABL inhibitor nilotinib is currently used for the treatment of patients with chronic myeloid leukemia (CML). Here we show that this antileukemic drug also inhibits invasive properties of colorectal cancer (CRC) cells. Inhibition was observed at the same dose-range for growth inhibition of CML cells. Nilotinib also strongly reduced liver metastasis induced by injection of CRC cells in the spleen of nude mice. Since we could not detect Abl deregulation in CRC cells, we speculated the involvement of an alternative target to be identified. Interestingly, a previous chemical-proteomic approach identified DDR1 as a novel target of nilotinib (Rix et al, Blood, 2007). DDR1 is a poorly-characterized TK and a receptor for collagen, a major component of the extracellular matrix. Accordingly, we found that DDR1 catalytic activity regulates cell invasion and metastasis of CRC cells. In addition, we demonstrate that nilotinib pharmacological activity is mediated by the inhibition of DDR1 in CRC cells: expression of the nilotinib resistant DDR1/T701I mutant renders CRC cells resistant to nilotinib treatment. Finally, we found that DDR1 catalytic activity is significantly increased in metastatic nodules compared to the primary tumor and the healthy tissue of the same patient. Altogether, these data suggest that the targeting of DDR1 by nilotinib may be of therapeutic value in metastatic CRC. Citation Format: Priscillia Tosti, Cedric Leroy, Valerie Simon, Bruno Robert, Josiane Pierre, Audrey Sirvent, Serge Roche. The antileukemic drug nilotinib inhibits the invasive activity and the metastatic potential of colorectal cancer cells by targeting the receptor tyrosine kinase DDR1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2085. doi:10.1158/1538-7445.AM2013-2085 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.