Jean-Max Pasquet

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.

Overexpression of the heat-shock protein 70 is associated to imatinib resistance in chronic myeloid leukemia

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 μM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.

Aminophospholipid exposure, microvesiculation and abnormal protein tyrosine phosphorylation in the platelets of a patient with Scott syndrome: a study using physiologic agonists and local anaesthetics.

The Scott syndrome is a rare inherited haemorrhagic disorder characterized by the inability of blood cells to expose aminophospholipids and to shed microparticles. We have had the opportunity to study a recently reported French patient with this syndrome and have confirmed by means of a fluorescence assay for transbilayer lipid movement a reduced aminophospholipid exposure when platelets were stimulated with the calcium‐ionophore ionomycin, in spite of a normal elevation of intracellular Ca2+. Secretion and calpain activation were also shown to be normal. Significantly, the level of phosphotyrosine‐labelled proteins in platelets treated with thrombin or a thrombin + collagen mixture and in particular the phosphorylation of a 40 kD band were severely reduced. Furthermore, inhibition of thiol‐containing enzymes, including tyrosine‐phosphatases, by N‐ethyl maleimide did not lead to aminophospholipid exposure in the patients platelets, in spite of increased tyrosine protein phosphorylation. In contrast, amphiphilic membrane drugs such as tetracaine and propranolol induced both surface aminophospholipid exposure in Scott platelets and the shedding of microparticles, thereby showing that membrane perturbation can lead to loss of phospholipid asymmetry in this syndrome. Our results provide the first insight that the lack of expression of procoagulant phospholipids and microparticle formation in Scott syndrome platelets is associated with a defect of intracellular signalling.

Ultrastructural Studies of Platelet Aggregates From Human Subjects Receiving Clopidogrel and From a Patient With an Inherited Defect of an ADP-Dependent Pathway of Platelet Activation

Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti-ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.

Persistent Activation of the Fyn/ERK Kinase Signaling Axis Mediates Imatinib Resistance in Chronic Myelogenous Leukemia Cells through Upregulation of Intracellular SPARC

SPARC is an extracellular matrix protein that exerts pleiotropic effects on extracellular matrix organization, growth factor availability, cell adhesion, differentiation, and immunity in cancer. Chronic myelogenous leukemia (CML) cells resistant to the BCR-ABL inhibitor imatinib (IM-R cells) were found to overexpress SPARC mRNA. In this study, we show that imatinib triggers SPARC accumulation in a variety of tyrosine kinase inhibitor (TKI)-resistant CML cell lines. SPARC silencing in IM-R cells restored imatinib sensitivity, whereas enforced SPARC expression in imatinib-sensitive cells promoted viability as well as protection against imatinib-mediated apoptosis. Notably, we found that the protective effect of SPARC required intracellular retention inside cells. Accordingly, SPARC was not secreted into the culture medium of IM-R cells. Increased SPARC expression was intimately linked to persistent activation of the Fyn/ERK kinase signaling axis. Pharmacologic inhibition of this pathway or siRNA-mediated knockdown of Fyn kinase resensitized IM-R cells to imatinib. In support of our findings, increased levels of SPARC mRNA were documented in blood cells from CML patients after 1 year of imatinib therapy compared with initial diagnosis. Taken together, our results highlight an important role for the Fyn/ERK signaling pathway in imatinib-resistant cells that is driven by accumulation of intracellular SPARC.

Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy

AXL receptor tyrosine kinase (RTK) is implicated in proliferation and invasion of many cancers, particularly in pancreatic ductal adenocarcinoma (PDAC), for which new therapeutic options are urgently required. We investigated whether inhibition of AXL activity by specific monoclonal antibodies (mAbs) is efficient in limiting proliferation and migration of pancreatic cancer cells. Expression of AXL was evaluated by immunohistochemistry in 42 PDAC. The AXL role in oncogenesis was studied using the short hairpin RNA approach in a pancreatic carcinoma cell line. We further generated antihuman AXL mAbs and evaluated their inhibitory effects and the AXL downstream signaling pathways first in vitro, in a panel of pancreatic cancer cell lines and then in vivo, using subcutaneous or orthotopic pancreatic tumor xenografts. AXL receptor was found expressed in 76% (32/42) of PDAC and was predominantly present in invasive cells. The AXL-knockdown Panc-1 cells decreased in vitro cell migration, survival and proliferation, and reduced in vivo tumor growth. Two selected anti-AXL mAbs (D9 and E8), which inhibited phosphorylation of AXL and of its downstream target AKT without affecting growth arrest-specific factor 6 (GAS6) binding, induced downexpression of AXL by internalization, leading to an inhibition of proliferation and migration in the four pancreatic cancer cell lines studied. In vivo, treatment by anti-AXL mAbs significantly reduced growth of both subcutaneous and orthotopic pancreatic tumor xenografts independently of their KRAS mutation status. Our in vitro and preclinical in vivo data demonstrate that anti-human AXL mAbs could represent a new approach to the pancreatic cancer immunotherapy.

Proteomic analysis of an imatinib-resistant K562 cell line highlights opposing roles of heat shock cognate 70 and heat shock 70 proteins in resistance.

Understanding the molecular basis of resistance to imatinib, a tyrosine kinase inhibitor used as front‐line therapy in chronic myeloid leukemia, remains a challenge for successful treatment. In an attempt to identify new mechanisms of resistance, we performed a comparative proteomic analysis of an imatinib‐resistant cell line generated from the erythroblastic cell line K562 (K562‐r) for which no known mechanism of resistance has been detected. Bidimensional gel electrophoresis was carried out to compare the protein expression pattern of imatinib‐sensitive and of imatinib‐resistant K562 cells. Among the 400 matched spots on five pairs of gels, only 14 spots had a significantly increased or decreased expression leading to the identification of 24 proteins identified as scaffold proteins, metabolic enzymes, DNA translation and maturation, and chaperon proteins. Among the chaperon family, only Hsp70 and Hsc70 are overexpressed in K562‐r, results confirmed by Western blotting. We recently reported the participation of Hsp70 overexpression in imatinib resistance whereas a role for Hsc70 has yet to be determined. Hsc70 is not involved in imatinib resistance as the inhibition of its expression by siRNA does not restore sensitivity to imatinib. In contrast, the induced decreased expression of Hsc70 was accompanied by a greater overexpression of Hsp70. This proteomic study therefore suggests opposing roles of Hsp70 and Hsc70 in imatinib resistance.

Inhibition of imatinib-mediated apoptosis by the caspase-cleaved form of the tyrosine kinase Lyn in chronic myelogenous leukemia cells.

Once cleaved by caspases, the Lyn tyrosine kinase (LynΔN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynΔN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynΔN or its catalytically inactive counterpart LynΔNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynΔN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynΔN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynΔN was suppressed by two inhibitors of Lyn activity. LynΔN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR–ABL pathway.

The Necrotic Signal Induced by Mycophenolic Acid Overcomes Apoptosis-Resistance in Tumor Cells

Background The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42. Methodology/Principal Findings Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA–mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL–overexpressing cells). All tested cells remained sensitive to MPA–mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers. Conclusions/Significance These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells.