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Simple Summary Autophagy is a cell survival and recycling mechanism which protects cancer cells upon therapeutic drug treatment. Here we investigated the impact of autophagy inhibition in a cancer of lymphoid origin, namely ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We inhibited autophagy in two distinct cell subsets of ALK + ALCL, one of which we previously shown to possess more stem-like and tumorigenic properties. Our study found that blockage of autophagy in the stem-like subset resulted in marked drug-sensitization to crizotinib, a current therapeutic agent used to treat ALK + ALCL. We also found differential involvement of the Myconcogene in the autophagy process within the two subsets and identified its relative importance to the stem-like population. Our research suggests inhibition of autophagy alongside crizotinib preferentially targets stem-like cells, thus improving crizotinib therapy. Abstract Previously it was shown that autophagy contributes to crizotinib resistance in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We asked if autophagy is equally important in two distinct subsets of ALK + ALCL, namely Reporter Unresponsive (RU) and Reporter Responsive (RR), of which RR cells display stem-like properties. Autophagic flux was assessed with a fluorescence tagged LC3 reporter and immunoblots to detect endogenous LC3 alongside chloroquine, an autophagy inhibitor. The stem-like RR cells displayed significantly higher autophagic response upon crizotinib treatment. Their exaggerated autophagic response is cytoprotective against crizotinib, as inhibition of autophagy using chloroquine or shRNA against BECN1 or ATG7 led to a decrease in their viability. In contrast, autophagy inhibition in RU resulted in minimal changes. Since the differential protein expression of MYC is a regulator of the RU/RR dichotomy and is higher in RR cells, we asked if MYC regulates the autophagy-mediated cytoprotective effect. Inhibition of MYC in RR cells using shRNA significantly blunted crizotinib-induced autophagic response and effectively suppressed this cytoprotective effect. In conclusion, stem-like RR cells respond with rapid and intense autophagic flux which manifests with crizotinib resistance. For the first time, we have highlighted the direct role of MYC in regulating autophagy and its associated chemoresistance phenotype in ALK + ALCL stem-like cells.

International audience; Haematopoiesis is a tightly orchestrated process where a pool of hematopoietic stem and progenitor cells (HSPCs) with high self-renewal potential can give rise to both lymphoid and myeloid lineages. The HSPCs pool is reduced with ageing resulting in few HSPC clones maintaining haematopoiesis thereby reducing blood cell diversity, a phenomenon called clonal haematopoiesis. Clonal expansion of HSPCs carrying specific genetic mutations leads to increased risk for haematological malignancies. Therefore, it comes as no surprise that hematopoietic tumours develop in higher frequency in elderly people. Unfortunately, elderly patients with leukaemia or lymphoma still have an unsatisfactory prognosis compared to younger ones highlighting the need to develop more efficient therapies for this group of patients. Growing evidence indicates that macroautophagy (hereafter referred to as autophagy) is essential for health and longevity. This review is focusing on the role of autophagy in normal haematopoiesis as well as in leukaemia and lymphoma development. Attenuated autophagy may support early hematopoietic neoplasia whereas activation of autophagy in later stages of tumour development and in response to a variety of therapies rather triggers a pro-tumoral response. Novel insights into the role of autophagy in haematopoiesis will be discussed in light of designing new autophagy modulating therapies in hematopoietic cancers.

Our current understanding of oncogenic Anaplastic Lymphoma Kinase (ALK)-induced lymphomagenesis has relied for over 20 years on multiple and complementary studies performed on various experimental models, encompassing ALK oncogene expressing cells, their grafts into immune-compromised mice, the generation of genetically engineered mouse models (GEMMs) and, when available, the use of patient samples from Anaplastic Large Cell Lymphoma (ALCL) tumour banks. Of note, and to our knowledge, no ALK-positive ALCL 3D culture system has been described so far. In this review, we will first outline how these different cell and mouse models were designed, and what key findings they revealed (or confirmed) towards oncogenic ALK-induced lymphomagenesis. Secondly, we will discuss how recent and revolutionary advances in genetic engineering technology are likely to complete our understanding of ALK-related disease in an effort to improve current therapeutic approaches.

Autophagy is an evolutionarily conserved catabolic process, which is used by the cells for cytoplasmic quality control. This process is induced following different kinds of stresses e.g., metabolic, environmental, or therapeutic, and acts, in this framework, as a cell survival mechanism. However, under certain circumstances, autophagy has been associated with cell death. This duality has been extensively reported in solid and hematological cancers, and has been observed during both tumor development and cancer therapy. As autophagy plays a critical role at the crossroads between cell survival and cell death, its involvement and therapeutic modulation (either activation or inhibition) are currently intensively studied in cancer biology, to improve treatments and patient outcomes. Over the last few years, studies have demonstrated the occurrence of autophagy in different Anaplastic Lymphoma Kinase (ALK)-associated cancers, notably ALK-positive anaplastic large cell lymphoma (ALCL), non-small cell lung carcinoma (NSCLC), Neuroblastoma (NB), and Rhabdomyosarcoma (RMS). In this review, we will first briefly describe the autophagic process and how it can lead to opposite outcomes in anti-cancer therapies, and we will then focus on what is currently known regarding autophagy in ALK-associated cancers.

Autophagy is a self-cannibalism process essential for tissue homeostasis, which can be activated following different environmental stressful conditions. In normal cells, autophagy could act as a brake to prevent tumorigenesis, but cancer cells are able to hijack this process to their own benefit, to promote tumor growth and/or tumor resistance to anti-cancer therapies. Scientists and clinicians attempt to modulate this process to improve therapies, using autophagy inhibitors or activators, some of them being tested currently in clinical trials against several types of tumors. Thus, it appears that autophagy is at the center of a showdown between cancer cells and anti-cancer therapies. In this review, we focus on the mechanisms by which autophagy could be either the yin or the yang of cancers.

International audience; Autophagy is a self-cannibalism process essential for tissue homeostasis, which can be activated following different environmental stressful conditions. In normal cells, autophagy could act as a brake to prevent tumorigenesis, but cancer cells are able to hijack this process to their own benefit, to promote tumor growth and/or tumor resistance to anti-cancer therapies. Scientists and clinicians attempt to modulate this process to improve therapies, using autophagy inhibitors or activators, some of them being tested currently in clinical trials against several types of tumors. Thus, it appears that autophagy is at the center of a showdown between cancer cells and anti-cancer therapies. In this review, we focus on the mechanisms by which autophagy could be either the yin or the yang of cancers.

The targeted inactivation of oncogenes offers a rational therapeutic approach for the treatment of cancer. However, the therapeutic inactivation of a single oncogene has been associated with tumor recurrence. Therefore, it is necessary to develop strategies to override mechanisms of tumor escape from oncogene dependence. We report here that the targeted inactivation of MYC is sufficient to induce sustained regression of hematopoietic tumors in transgenic mice, except in tumors that had lost p53 function. p53 negative tumors were unable to be completely eliminated, as demonstrated by the kinetics of tumor cell elimination revealed by bioluminescence imaging. Histological examination revealed that upon MYC inactivation, the loss of p53 led to a deficiency in thrombospondin-1 (TSP-1) expression, a potent antiangiogenic protein, and the subsequent inability to shut off angiogenesis. Restoration of p53 expression in these tumors re-established TSP-1 expression. This permitted the suppression of angiogenesis and subsequent sustained tumor regression upon MYC inactivation. Similarly, the restoration of TSP-1 alone in p53 negative tumors resulted in the shut down of angiogenesis and led to sustained tumor regression upon MYC inactivation. Hence, the complete regression of tumor mass driven by inactivation of the MYC oncogene requires the p53-dependent induction of TSP-1 and the shut down of angiogenesis. Notably, overexpression of TSP-1 alone did not influence tumor growth. Therefore, the combined inactivation of oncogenes and angiogenesis may be a more clinically effective treatment of cancer. We conclude that angiogenesis is an essential component of oncogene addiction.

Microarray-based formats offer a high-resolution alternative to conventional, chromosome-based comparative genomic hybridization (CGH) methods for assessing DNA copy number alteration (CNA) genome-wide in human cancer. For murine tumors, array CGH should provide even greater advantage, since murine chromosomes are more difficult to individually discern. We report here the adaptation and evaluation of a cDNA microarray-based CGH method for the routine characterization of CNAs in murine tumors, using mouse cDNA microarrays representing approximately 14,000 different genes, thereby providing an average mapping resolution of 109 kb. As a first application, we have characterized CNAs in a set of 10 primary and recurrent lymphomas derived from a Myc-induced murine lymphoma model. In primary lymphomas and more commonly in Myc-independent relapses, we identified a recurrent genomic DNA loss at chromosome 3G3-3H4, and recurrent amplifications at chromosome 3F2.1-3G3 and chromosome 15E1/E2-15F3, the boundaries of which we defined with high resolution. Further, by profiling gene expression using the same microarray platform, we identified within CNAs the relevant subset of candidate cancer genes displaying comparably altered expression, including Mcl1 (myeloid cell leukemia sequence 1), a highly expressed antiapoptotic gene residing within the chr 3 amplicon peak. CGH on mouse cDNA microarrays therefore represents a reliable method for the high-resolution characterization of CNAs in murine tumors, and a powerful approach for elucidating the molecular events in tumor development and progression in murine models.

The ability to model cancer in the mouse has provided a robust methodology to dissect the molecular etiology of cancer. These models serve as potentially powerful platforms to preclinically evaluate novel therapeutics. In particular, the recent development of strategies to conditionally induce the or knockout the function of genes in a tissue specific manner has enabled investigators to engineer mice to demonstrate that the targeted inactivation of specific oncogenes can be effective in inducing sustained regression of tumors. Thus, these animal models will be useful to define the specific genes that will be therapeutically useful to target for the treatment of particular human cancers.

Many recent reports demonstrate that at least initially, the inactivation of an oncogene can induce sustained regression of even a highly invasive and genetically complex cancer. However, upon prolonged oncogene inactivation, some cancers ultimately relapse, becoming independent of the very oncogene that initiated the process of tumorigenesis. Understanding the specific mechanisms by which cancers can escape dependence upon a particular oncogene will be critical to anticipate mechanisms by which human cancers will evade therapies that target individual oncogenes. Thereby, more effective strategies will be developed to clinically treat cancer.

The targeted inactivation of oncogenes may be a specific and effective treatment for cancer. However, because human cancers are the consequence of multiple genetic changes, the inactivation of one oncogene may not be sufficient to cause sustained tumor regression. Moreover, cancers are genomically unstable and may readily compensate for the inactivation of a single oncogene. Here we confirm by spectral karyotypic analysis that MYC-induced hematopoietic tumors are highly genetically complex and genomically unstable. Nevertheless, the inactivation of MYC alone was found to be sufficient to induce sustained tumor regression. After prolonged MYC inactivation, some tumors exhibited a distinct propensity to relapse. When tumors relapsed, they no longer required the overexpression of MYC but instead acquired novel chromosomal translocations. We conclude that even highly genetically complex cancers are reversible on the inactivation of MYC, unless they acquire novel genetic alterations that can sustain a neoplastic phenotype. (Blood. 2003;101:2797-2803)

SH2-containing inositol-5-phosphatase 1 (SHIP1) was originally identified as a 145 kDa protein that became tyrosine-phosphorylated in response to multiple cytokines. It is now well established that SHIP1 is specifically expressed in haemopoietic cells and is important as a negative regulator of signalling. We found recently that SHIP1 was present in human blood platelets as an Ins(1,3,4,5)P4-phosphatase and a PtdIns(3,4,5)P3-5-phosphatase that became tyrosine-phosphorylated and was relocated to the cytoskeleton in an integrin-dependent manner. Here we report biochemical and pharmacological evidence that the tyrosine kinase pp60c-src is constitutively associated with SHIP1 and is involved in its tyrosine phosphorylation downstream of integrin engagement in thrombin-activated human platelets. The use of cytochalasin D allowed us to demonstrate that the actin cytoskeleton reorganization induced on thrombin stimulation was not required for its integrin-mediated phosphorylation. Moreover, the integrin-dependent relocation of SHIP1 to the cytoskeleton did not require its tyrosine phosphorylation. These results suggest that SHIP1 is first recruited to the integrin-linked signalling complexes and then becomes tyrosine-phosphorylated through a Src-kinase-dependent mechanism but independently of the actin cytoskeleton reorganization.

We have demonstrated previously that microtubule depolymerization by colchicine in human monocytes induces selective production of interleukin-1 (IL-1) (Manie, S., Schmid-Alliana, A., Kubar, J., Ferrua, B., and Rossi, B. (1993) J. Biol. Chem. 268, 13675–13681). Here, we provide evidence that disruption of the microtubule structure rapidly triggers extracellular signal-regulated kinase (ERK) activation, whereas it was without effect on SAPK2 activity, which is commonly acknowledged to control pro-inflammatory cytokine production. This process involves the activation of the entire cascade including Ras, Raf-1, MEK1/2, ERK1, and ERK2. Activation of ERKs is followed by their nuclear translocation. Although other SAPK congeners might be activated upon microtubule depolymerization, the activation of ERK1 and ERK2 is mandatory for IL-1 production as shown by the blocking effect of PD 98059, a specific MEK1/2 inhibitor. Additionally, we provide evidence that microtubule disruption also induces the activation of c-Src and Hck activities. The importance of Src kinases in the mediation of the colchicine effect is underscored by the fact that CP 118556, a specific inhibitor of Src-like kinase, abrogates both the colchicine-induced ERK activation and IL-1 production. This is the first evidence that ERK activation is an absolute prerequisite for induction of this cytokine. Altogether, our data lend support to a model where the status of microtubule integrity controls the level of Src activities that subsequently activate the ERK kinase cascade, thus leading to IL-1 production.

The SH2 domain-containing inositol 5-phosphatase, SHIP, known to dephosphorylate inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-trisphosphate has recently been shown to be expressed in a variety of hemopoietic cells. This 145-kDa protein is induced to associate with Shc by multiple cytokines and may play an important role in the negative regulation of immunocompetent cells mediated by FcgammaRIIB receptor. We report here that SHIP is present in human blood platelets and may be involved in platelet activation evoked by thrombin. Platelet SHIP was identified by Western blotting as a single 145-kDa protein. Both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4, 5-tetrakisphosphate 5-phosphatase activities could be demonstrated in anti-SHIP immunoprecipitates of platelet lysate. Thrombin stimulation induced a tyrosine phosphorylation of SHIP, this effect being prevented if platelets were not shaken or if RGD-containing peptides were present, indicating an aggregation-dependent, integrin-mediated event. Moreover, although the intrinsic phosphatase activity of SHIP did not appear to be significantly increased, tyrosine-phosphorylated SHIP was relocated to the actin cytoskeleton upon activation in an aggregation- and integrin engagement-dependent manner. Finally, the striking correlation observed between phosphatidylinositol 3,4-bisphosphate production and the tyrosine phosphorylation of SHIP, as well as its relocation to the cytoskeleton upon thrombin stimulation, suggest a role for SHIP in the aggregation-dependent and GpIIb-IIIa-mediated accumulation of this important phosphoinositide.

Anaplastic large-cell lymphomas (ALCLs) bearing the t(2;5) translocation (ALK+ALCLs) are frequently characterized by skin colonization and associated with a poor prognosis. Using conditional transgenic models of anaplastic lymphoma kinase–positive (ALK+) lymphomas and human ALK+ALCL cell lines, in the present study, we show that high-mobility-group box-1 (HMGB-1), a proinflammatory cytokine, is released by ALK+ cells, and demonstrate extracellular HMGB-1–stimulated secretion of the IL-8 chemokine by HaCaT keratinocytes through the involvement of MMP-9, PAR-2, and the NF-κB pathway. Furthermore, we demonstrate that, in vitro, IL-8 is able to induce the invasiveness of ALK+ cells, which express the IL-8 receptors CXCR1 and CXCR2. In vitro and in vivo, HMGB-1 inhibition achieved by glycyrrhizin treatment led to a drastic reduction in ALK+ cell invasiveness. The pathophysiological relevance of our observations was confirmed by demonstrating that the HMGB-1 and IL-8 receptors are expressed in ALK+ALCL biopsies. We have also shown that IL-8 secretion is correlated with leukemic dissemination of ALK+ cells in a significant number of patients. The results of the present study demonstrate for the first time a relationship among the pro-inflammatory mediators HMGB-1, MMP-9, PAR-2, and IL-8. We propose that these mediators create a premetastatic niche within the skin, thereby participating in ALK+ lymphoma epidermotropism.

International audience; Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a tyrosine kinase oncogene responsible for the pathogenesis of the majority of human ALK-positive lymphomas. We recently reported that it activated the Rac1 GTPase in anaplastic large-cell lymphoma (ALCL), leading to Rac-dependent formation of active invadopodia required for invasiveness. Herein, we went further into the study of this pathway and used the inhibitor of Rac, NSC23766, to validate its potential as a molecular target in ALCL in vitro and in vivo in a xenograft model and in a conditional model of NPM-ALK transgenic mice. Our data demonstrate that Rac regulates important effectors of NPM-ALK-induced transformation such as Erk1/2, p38 and Akt. Moreover, inhibition of Rac signaling abrogates NPM-ALK-elicited disease progression and metastasis in mice, highlighting the potential of small GTPases and their regulators as additional therapic targets in lymphomas.

A monoclonal anti-CD160 antibody inhibits the growth of new vessels in pathological ocular and tumor neoangiogenesis but not in healthy tissues., Angiogenesis plays an essential role in several diseases of the eye and in the growth of solid tumors, but existing antiangiogenic therapies have limited benefits in several cases. We report the antiangiogenic effects of a monoclonal antibody, CL1-R2, in several animal models of neovascularization. CL1-R2 recognizes human CD160, a membrane receptor which is conserved in various mammal species. We show that CD160 is expressed on the endothelial cells of newly formed blood vessels in human colon carcinoma and mouse B16 melanoma but not in vessels of healthy tissues. CL1-R2 reduced fibroblast growth factor 2–induced neovascularization in the rabbit cornea, in a mouse model of oxygen-induced retinopathy, and in a mouse Matrigel plug assay. Treatment of B16 melanoma-bearing mice with CL1-R2 combined with cyclophosphamide chemotherapy caused regression of the tumor vasculature and normalization of the remaining vessels as shown by Doppler ultrasonography, intravital microscopy, and histology. These studies validate CD160 as a potential new target in cases of human pathological ocular and tumor neoangiogenesis that do not respond or become resistant to existing antiangiogenic drugs.

Background Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as "oncogene-addiction." However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment. Methodology/principal findings To examine how the MYC and K-ras(G12D) oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-ras(G12D) to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-ras(G12D)- or MYC/K-ras(G12D)-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-ras(G12D)-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-ras(G12D) resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-ras(G12D) in maintenance of lung tumors, we found that the down-stream mediators of K-ras(G12D) signaling, Stat3 and Stat5, are dephosphorylated following conditional K-ras(G12D) but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-ras(G12D). Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation. Conclusions/significance Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas.

Overexpression and activation of TPM3-ALK tyrosine kinase fusion protein is a causal oncogenic event in the development of Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic ALK-positive tumours. Thus, the development of ALK specific tyrosine kinase inhibitors is a current therapeutic challenge. Animal models are essential to assess, in vivo, the efficiency of ALK-oncogene inhibitors and to identify new and/or additional therapeutic targets in the ALK tumorigenesis pathway. Using the tetracycline system to allow conditional and concomitant TPM3-ALK and luciferase expression, we have developed a unique transplant model for bioluminescent TPM3-ALK-induced fibroblastic tumours in athymic nude mice. The reversible TPM3-ALK expression allowed us to demonstrate that this oncogene is essential for the tumour growth and its maintenance. In addition, we showed that this model could be used to precisely assess tumour growth inhibition upon ALK chemical inactivation. As proof of principle, we used the general tyrosine kinase inhibitor herbimycin A to inhibit ALK oncoprotein activity. As expected, herbimycin A treatment reduced tumour growth as assessed both by tumour volume measurement and bioluminescent imaging. We conclude that this transplant model for TPM3-ALK-induced tumours represents a valuable tool not only to accurately and rapidly evaluate in vivo ALK-targeted therapies but also to gain insight into the mechanism of ALK-positive tumour development.

ALK (Anaplastic Lymphoma Kinase)-positive Anaplastic Large Cell Lymphoma (ALCL) occurs predominantly in children and young adults. Their chemotherapeutic treatment leads to a 5-year overall survival amounted to 70-80%. The tumor relapses are often very aggressive and lethal and their underlying mechanisms are unknown. Therefore, there is still a need to improve current therapy. Crizotinib is the most advanced ALK tyrosine kinase inhibitor already used in clinics for ALK-associated lung cancers. However, mechanisms of escape to crizotinib have been reported in cell lines and patients submitted to continuous crizotinib treatment. Thus, its use in frontline treatment for ALCL is hampered by the emergence of resistance. As autophagy has been proposed as a cell survival mechanism potentially involved in the acquisition of resistance to tyrosine kinase inhibitor, we investigated here whether autophagy was activated during ALCL treatment. We demonstrated in ALCL that autophagy is induced upon ALK inactivation, as a pro-survival response. We found that different ALK inhibition approaches (crizotinib or ALK-targeting siRNA) combined with autophagy inhibition (chloroquine, 3-methyladenine or ATG7-targeting siRNA) compromised cell survival and cell growth. Altogether, our results suggest that crizotinib and chloroquine (two drugs already used in clinics) co-treatment could be beneficial for ALK-positive ALCL patients. Citation Format: Géraldine MITOU, Julie FRENTZEL, Laurence LAMANT, Fabienne MEGGETTO, Estelle ESPINOS, Patrice CODOGNO, Pierre BROUSSET, Sylvie GIURIATO. Targeting autophagy potentiates the anti-tumoral action of crizotinib in ALK-positive anaplastic large cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 663. doi:10.1158/1538-7445.AM2015-663

With the use of microarray gene-expression profiling, we analyzed a homogeneous series of 32 patients with systemic anaplastic large-cell lymphoma (ALCL) and 5 ALCL cell lines. Unsupervised analysis classified ALCL in 2 clusters, corresponding essentially to morphologic subgroups (ie, common type vs small cell and “mixed” variants) and clinical variables. Patients with a morphologic variant of ALCL had advanced-stage disease. This group included a significant number of patients who experienced early relapse. Supervised analysis showed that ALK+ALCL and ALK− ALCL have different gene-expression profiles, further confirming that they are different entities. Among the most significantly differentially expressed genes between ALK+ and ALK− samples, we found BCL6, PTPN12, CEBPB, and SERPINA1 genes to be overexpressed in ALK+ ALCL. This result was confirmed at the protein level for BCL-6, C/EBPβ and serpinA1 through tissue microarrays. The molecular signature of ALK− ALCL included overexpression of CCR7, CNTFR, IL22, and IL21 genes but did not provide any obvious clues to the molecular mechanism underlying this tumor subtype. Once confirmed on a larger number of patients, the results of the present study could be used for clinical and therapeutic management of patients at the time of diagnosis.

Src homology domain 2-containing inositol 5-phosphatases 1 and 2 (SHIP1 and SHIP2) are capable of dephosphorylating the second messenger PtdIns(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) and interacting with several signalling proteins. SHIP1 is essentially expressed in haematopoietic cells, whereas SHIP2, a closely related enzyme, is ubiquitous. In the present study, we show that SHIP1 and SHIP2 are expressed as functional PtdIns(3,4,5)P3 5-phosphatases in human blood platelets and are capable of interacting when these two lipid phosphatases are co-expressed, either naturally (platelets and A20 B lymphoma cells) or artificially (COS-7 cells). Using COS-7 cells transfected with deletion mutants of SHIP2, we demonstrate that the Src homology domain 2 of SHIP2 is the minimal and sufficient protein motif responsible for the interaction between the two phosphatases. These results prompted us to investigate the relative importance of SHIP1 and SHIP2 in the control of PtdIns(3,4,5)P3 levels in platelets using homozygous or heterozygous SHIP1- or SHIP2-deficient mice. Our results strongly suggest that SHIP1, rather than SHIP2, plays a major role in controlling PtdIns(3,4,5)P3 levels in response to thrombin or collagen activation of mouse blood platelets.

Many recent reports demonstrate that at least initially, the inactivation of an oncogene can induce sustained regression of even a highly invasive and genetically complex cancer. However, upon prolonged oncogene inactivation, some cancers ultimately relapse, becoming independent of the very oncogene that initiated the process of tumorigenesis. Understanding the specific mechanisms by which cancers can escape dependence upon a particular oncogene will be critical to anticipate mechanisms by which human cancers will evade therapies that target individual oncogenes. Thereby, more effective strategies will be developed to clinically treat cancer.

SHIP2 belongs to the inositol 5-phosphatase family and is characterized by a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) 5-phosphatase activity. Evidence based on mice lacking the SHIP2 gene has demonstrated its predominant role in the control of insulin sensitivity. However, SHIP2 expression in both hematopoietic and non-hematopoietic cells suggests additional functions. SHIP2 was previously identified in chronic myelogenous progenitor cells, in which its constitutive tyrosine phosphorylation was reported by Wisniewski et al., [Blood 93 (1999) 2707-2720]. Here, we further investigated the function of SHIP2 in this hematopoietic and malignant context. A detailed analysis of the substrate specificity of SHIP2 indicated that this phosphatase is primarily directed towards PI(3,4,5)P(3) both in vitro and in K562 chronic myeloid leukemia cells. The SHIP2-mediated decrease in PI(3,4,5)P(3) levels and increase in phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) was accompanied by a reduction of cell proliferation, characterized by an accumulation of the cells in the G2/M phase of the cell cycle. Thus, in addition to its role in the control of insulin sensitivity, SHIP2 may also play a role in cell proliferation, at least in chronic myelogenous progenitor cells.

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.

Molecular profiling of Anaplastic Lymphoma Kinase (ALK)-positive versus negative cell lines and patients’ samples has unravelled critical roles for transcriptional factors (TF) (C/EBPbeta, Bcl-6) in Anaplastic Large Cell Lymphomas (ALCL), suggesting that targeting these TF with ALK tyrosine kinase inhibitors should yield better results (Lamant L et al., Blood 2008). In this latter work of our group, beta-catenin and its transcriptional partner T Cell Factor 4 (TCF4) were also found upregulated at a transcriptional level in ALK+ vs ALK− patients’ samples and cell lines. We sought to determine the relevance of such a finding. At the protein level, ALK+ cell lines (SU-DHL-1, Karpas299 and COST) and ALK− cell line (FEPD) express detectable beta-catenin. TCF-4 is also expressed at similar levels among cell lines, whatever ALK status. Since beta-catenin is mainly regulated through post-transcriptional mechanism, we assessed its phosphorylation status. ALK+ (SU-DHL-1 and Karpas299), but not ALK− cell lines displayed increased tyrosine phosphorylation at both Tyr142 and Tyr654 residues, as well as association between beta-catenin and ALK (in immuno-precipitation assays). Moreover, both total beta-catenin and phosphoTyr-beta-catenin were found associated with TCF4, and therefore transcriptionally active. Using the tetracycline system to allow conditional expression of NPM-ALK in MEF cell line (murine embryonic fibroblasts), we found that beta-catenin phosphorylation became barely detectable upon loss of NPM-ALK expression. Based on these findings, we investigated the functional consequences of the disruption of beta-catenin/TCF4 complexes using small molecules such as PKF115–584 and CGP049090 (a generous gift from Novartis). Interestingly, both compounds induced dissociation of beta-catenin/TCF4 complexes at 0.5μM for 24h, and also induced apoptosis in SU-DHL-1 cells. But, since compounds could not dissociate beta-catenin/ALK complexes, they should be combined to ALK inhibitors to fully exert their anti-lymphoma effects. Moreover, the pool of beta-catenin linked to Glycogene Synthase Kinase 3beta (regulated by external Wnt-dependant signals) is neither affected by small compounds, indicating a specific beta-catenin/TCF4 disruption with these drugs. To conclude, this study shows that, in ALCL, ALK activates a Wnt-independent pathway, which appears to be critical for cell survival. This study offers a rationale for investigating the potential of molecules designed to interfere with beta-catenin/TCF/LEF proteins and currently evaluated in colon carcinomas, alone or in combination with ALK tyrosine kinase inhibitors.

The blood is a perpetually renewing tissue seeded by a rare population of adult bone marrow hematopoietic stem cells (HSC). During steady-state hematopoiesis, the HSC population is relatively quiescent but constantly maintains a low numbers of cycling cells that differentiate to produce the various lineage of mature blood cells. However, in response to hematological stress, the entire HSC population can be recruited into cycle to self-renew and regenerate the blood-forming system. HSC proliferation is therefore highly adaptative and requires appropriate regulation of cell cycle progression to drive both differentiation-associated and self-renewal-associated proliferation, without depletion of the stem cell pool. Although the molecular events controlling HSC proliferation are still poorly understood, they are likely determined, at least in part, by regulated expression and/or function of components and regulators of the cell cycle machinery. Here, we demonstrate that the long-term self-renewing HSC (defined as Lin−/c-Kit+/Sca-1+/Thy1.1int/Flk2−) exists in two distinct states that are both equally important for their in vivo functions as stem cells: a numerically dominant quiescent state, which is critical for HSC function in hematopoietic reconstitution; and a proliferative state, which represents almost a fourth of this population and is essential for HSC functions in differentiation and self-renewal. We show that when HSC exit quiescence and enter G1 as a prelude to cell division, at least two critical events occur: first, during the G1 and subsequent S-G2/M phases, they temporarily lose efficient in vivo engraftment activity, while retaining in vitro differentiation potential; and second, they select the particular cell cycle proteins that are associated with specific developmental outcomes (self-renewal vs. differentiation) and developmental fates (myeloid vs. lymphoid). Together, these findings provide a direct link between HSC proliferation, cell cycle regulation and cell fate decisions that have critical implications for both the therapeutic use of HSC and the understanding of leukemic transformation.

L'inositol polyphosphate 5-phosphatase SHIP (SH2 demain containing inositol polyphosphate 5-phosphatase), est une phosphatase tres particuliere agissant sur un inositol tetrakisphosphate ou sur un phosphatidylinositol trisphosphate. Elle possede un domaine SH2 amino-terminal, des sequences riches en proline et deux motifs NPXY (Asn-Pro-X-Tyr) pouvant se lier, apres phosphorylation, a des domaines PTB (phosphotyrosine binding domain). Le controle de ses fonctions par phosphorylation en fait une proteine de signalisation typique. On retrouve SHIP dans le systeme immunitaire, dans les mastocytes et les cellules B, et dans les plaquettes humaines. Dans les cellules B, SHIP est recrutee a la membrane par son domaine SH2 sur un motif ITIM (immunoreceptor tyrosine-based inhibitor motif) des recepteurs Fcγ RIIB, puis phosphorylee. Dans les plaquettes, la stimulation par la thrombine provoque une phosphorylation et une translocation de SHIP vers le cytosquelette suivant une cinetique identique, selon des mecanismes qui dependent de l'agregation et de l'engagement des integrines.

The hallmark (8:14) translocation of Burkitt’s lymphoma causes MYC overexpression, a primary event in the development of Burkitt’s lymphoma. While Burkitt’s lymphoma comprises nearly one third of childhood malignancies, it is rare in adults. Among many explanations is the possibility that the capacity for a lymphocyte to undergo malignant transformation by MYC activation is influenced by factors related to the developmental state of the host. To address this possibility, we used transgenic mice in which the MYC proto-oncogene is conditionally regulated via the Tetracycline Regulatory System (Tet system). When MYC was activated in cohorts of mice less than 3 weeks of age, mice universally succumbed to lymphoma within 23 weeks. However, when MYC was activated in adult mice (over 7 weeks of age) the mice failed to develop lymphoma within 50 weeks. We were not able to detect differences in the effects of MYC on the proliferation or apoptosis in lymphocytes from adult versus neonatal hosts. We next explored whether the concomitant activation of MYC with a second oncogene could induce lymphomagenesis in adult mice. When we conditionally induced both MYC and BCL2 or MYC and RAS in adult hosts, mice succumbed to lymphoma with a mean latency of 24–27 weeks. Our results suggest that in adult hosts, concurrent activation of at least two oncogenes is required for MYC to initiate tumorigenesis.

Anaplastic Lymphoma Kinase-positive Anaplastic Large Cell Lymphomas (ALK+ ALCL) occur predominantly in children and young adults. Their treatment, based on aggressive chemotherapy, is not optimal since ALCL patients can still expect a 30% 2-year relapse rate. Tumor relapses are very aggressive and their underlying mechanisms are unknown. Crizotinib is the most advanced ALK tyrosine kinase inhibitor and is already used in clinics to treat ALK-associated cancers. However, crizotinib escape mechanisms have emerged, thus preventing its use in frontline ALCL therapy. The process of autophagy has been proposed as the next target for elimination of the resistance to tyrosine kinase inhibitors. In this study, we investigated whether autophagy is activated in ALCL cells submitted to ALK inactivation (using crizotinib or ALK-targeting siRNA). Classical autophagy read-outs such as autophagosome visualization/quantification by electron microscopy and LC3-B marker turn-over assays were used to demonstrate autophagy induction and flux activation upon ALK inactivation. This was demonstrated to have a cytoprotective role on cell viability and clonogenic assays following combined ALK and autophagy inhibition. Altogether, our results suggest that co-treatment with crizotinib and chloroquine (two drugs already used in clinics) could be beneficial for ALK-positive ALCL patients.

Cancer is caused by genetic events that result in the activation of oncogenes or the inactivation of tumor suppressor genes. Using the Tetracycline system, our laboratory has generated a transgenic mouse model in which MYC is conditionally overexpressed in hematopoietic cells, allowing us to turn MYC expression on and off at will. We have previously demonstrated that the inactivation of this single oncogene in an established and even highly invasive and metastatic lymphoma is sufficient to reverse cancer, suggesting that MYC may be an effective therapeutic target. In a variety of conditional oncogene models, we and others have found that tumor regression following oncogene inactivation involves similar phenomena, including cell cycle arrest, apoptosis, and differentiation of the tumor cells. The similarity in the regression process across a variety of different oncogenes and types of cancer strongly suggests the existence of a common signaling pathway following oncogene inactivation, which culminates in the cessation of cell proliferation and the induction of apoptosis and differentiation. Recently, we have demonstrated that MYC activation disrupts the repair of DNA breaks and results in genomic instability. We speculated that MYC inactivation may cause tumor regression by restoring the ability of tumor cells to recognize that they are genomically damaged, which could subsequently lead to the cell cycle arrest, differentiation and apoptosis that is generally observed. Indeed, we now report that upon MYC inactivation, tumor cells activate DNA damage signaling pathways and begin to repair their DNA breaks. We have found evidence for activation of a functional DNA damage response both by immunofluorescent staining for phosphorylated ATM and Mre11, which demonstrates foci formation following MYC inactivation; and by the Comet assay, which shows a quantitative decrease in severity of DNA breaks following MYC inactivation. Our results suggest that MYC inactivation may induce tumor regression at least in part through the restoration of a DNA damage checkpoint response.

The lipid phosphatase SHIP2 (Src homology 2 domain containing inositol 5-phosphatase 2) has been shown to be expressed in nonhemopoietic and hemopoietic cells. It has been implicated in signaling events initiated by several extracellular signals, such as epidermal growth factor (EGF) and insulin. In COS-7 cells, SHIP2 was tyrosine-phosphorylated at least at two separated tyrosine phosphorylation sites in response to EGF. SHIP2 was coimmunoprecipitated with the EGF receptor (EGFR) and also with the adaptor protein Shc. A C-terminal truncated form of SHIP2 that lacks the 366 last amino acids, referred to as tSHIP2, was also precipitated with the EGFR when transfected in COS-7 cells. The Src homology 2 domain of SHIP2 was unable to precipitate the EGFR in EGF-stimulated cells. Moreover, when transfected in COS-7 cells, it could not be detected in immunoprecipitates of the EGFR. When the His-tagged full-length enzyme was expressed in COS-7 cells and stained with anti-His6 monoclonal antibody, a signal was observed at plasma membranes in EGF-stimulated cells that colocalize with the EGFR by double staining. Upon stimulation by EGF, phosphatidylinositol 3,4,5-trisphosphate and protein kinase B activity were decreased in SHIP2-transfected COS-7 cells as compared with the vector alone. SHIP2 appears therefore in a tyrosine-phosphorylated complex with at least two other proteins, the EGFR and Shc.

Phosphoinositides play a central role in the control of several cellular events including actin cytoskeleton organization. Here we show that, upon infection of epithelial cells with the Gram-negative pathogen Shigella flexneri, the virulence factor IpgD is translocated directly into eukaryotic cells and acts as a potent inositol 4-phosphatase that specifically dephosphorylates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] into phosphatidylinositol 5-monophosphate [PtdIns(5)P] that then accumulates. Transfection experiments indicate that the transformation of PtdIns(4,5)P(2) into PtdIns(5)P by IpgD is responsible for dramatic morphological changes of the host cell, leading to a decrease in membrane tether force associated with membrane blebbing and actin filament remodelling. These data provide the molecular basis for a new mechanism employed by a pathogenic bacterium to promote membrane ruffling at the entry site.

Over the last few years, many reports have extended our knowledge of the inositol lipid metabolism and brought out some exciting information about the location, the variety and the role of phosphoinositides (PIs). Besides the so-called “canonical PI pathway” leading to the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), the precursor of the intracellular second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG), many other metabolic pathways have been identified to produce seven different polyphosphoinositides. Several of these quantitatively minor lipid molecules appear to be specifically involved in the control of cellular events, such as the spatial and temporal organisation of key signalling pathways, the rearrangement of the actin cytoskeleton or the intracellular vesicle trafficking. This is consistent with the fact that many of the enzymes, such as kinases and phosphatases, involved in the tight control of the intracellular level of polyphosphoinositides, are regulated and/or relocated through cell surface receptors for extracellular ligands. The remarkable feature of PIs, which can be rapidly synthesised and degraded in discrete membrane domains or even subnuclear structures, places them as ideal regulators and integrators of very dynamic mechanisms of cell regulation. In this review, we will summarise recent studies on the potential location, the metabolic pathways and the role of the different PIs. Some aspects of the temporal synthesis of D3 PIs will also be discussed.

MYC overexpression is thought to induce tumorigenesis through a variety of different mechanisms including the induction of proliferation, inhibition of differentiation and disruption of genomic stability. Conventional transgenic systems that have been used to study the role of oncogenes in tumorigenesis continuously overexpress transgenes and hence preclude the investigation of the initial and age specific consequences of oncogene activation. To investigate the developmental specific consequences of MYC overexpression in the pathogenesis of lymphoma in vivo, we used transgenic mice in which the MYC proto-oncogene is conditionally regulated via the Tetracycline Regulatory System (Tet system). The ability of MYC to induce lymphomagenesis was found to be inversely correlated with the age of the host at the time of MYC activation. When MYC was activated constitutively, the mean time until tumor development was 13 weeks. When MYC was activated at increasing developmental ages of 3 and 5 weeks, identical tumors arose, but with an increased mean tumor latency. When MYC was activated at 7 weeks, an age equivalent to an adult mouse, no tumors developed even after 40 weeks of observation. However, we found that in adult mice, if both MYC and BCL2 are overexpressed at 7 weeks of age, mice succumbed to lymphoma with a mean latency of 27 weeks. Surprisingly, we could not find evidence that MYC overexpression induces apoptosis or that BCL2 overexpression reduced apoptosis. We conclude that the ability of MYC to induce lymphomagenesis is highly dependent on the developmental context. MYC and BCL2 cooperate to permit tumorigenesis in adult mice. Since lymphoma occurs generally as a disease in adults, this suggests that previous reports may have greatly overestimated the ability of MYC activation to induce tumorigenesis and underestimated the potential cooperation between MYC and BCL2 oncogenes. Our results also suggest that BCL2 may cooperate with MYC to induce lymphomagenesis through additional mechanisms other than preventing MYC from inducing apoptosis.

Anaplastic Lymphoma Kinase positive Anaplastic Large Cell Lymphomas (ALK+ ALCL) are an aggressive pediatric disease. They are characterized by chromosomal translocations involving the ALK gene with various translocation partner genes. NPM-ALK is the most prominent fusion protein observed. It results from the t(2;5) (p23;q35) chromosomal translocation and leads to the constitutive activation of the tyrosine kinase domain, which is driving lymphomagenesis through the activation of multiple survival/proliferation pathways. Therapeutic options comprise chemotherapy, which is efficient in about 70% of the patients, and targeted therapies, such as crizotinib (an ALK tyrosine kinase inhibitor) used in refractory/relapsed cases. Efforts converged also towards the development of combined therapies to improve treatment. In this context, we studied whether autophagy could be modulated to improve crizotinib therapy. Autophagy is a vesicular recycling pathway, known to be associated either with cell survival or cell death depending on cancers and therapies. The Unc-51-like kinase-1 (ULK1) protein plays a critical role in the autophagy initiation stage and is regulated mainly through AMPK- or mTOR- mediated serine/threonine phosphorylations on key residues. In NPM-ALK+ ALCL cell lines, previous work of my host team had demonstrated that autophagy was induced upon NPM-ALK inactivation, and was endowed with cytoprotective functions. During my PhD, I first reviewed the cytoprotective, or inversely, cytotoxic roles of autophagy upon therapy in various other ALK-dependent cancers. Then, my main thesis project was to identify microRNAs (miRNAs) and their potential targets that could be therapeutically modulated, in addition to crizotinib treatment, to drive autophagy towards cytotoxic functions and the outcome of tumor cells death. Indeed, deregulation of miRNA expression levels have been extensively described in cancers, including in NPM-ALK+ ALCL, and have also been shown to modulate the autophagy responses upon therapies. My work led to the demonstration that miRNA-7-5p, known primarily to harbor tumor suppressive functions in diverse cancer types, and RAF1, one of its targets, play essential roles in NPM-ALK+ ALCL, by controlling autophagy flux and tumor cell fate. RAF1 is a serine/threonine kinase, best known to connect RAS to the MEK/ERK pathway. However, the mechanism by which RAF1 inhibition, specifically, could induce autophagy had not been described so far. Our work points out for the first time the possible phosphorylation of ULK1 on its serine757 inhibitory residue by RAF1 (and not MEK or ERK), which opens up a new therapeutic avenue to modulate the autophagy flux in ALK+ ALCL. In a second part of my project, and to better understand the potential link between RAF1 and ULK1, I gave several attempts to purify autophagosomes from NPM-ALK+ ALCL cells. While our results suggest that NPM-ALK could be detected in an autophagosome-enriched fraction, further investigations will determine whether RAF1 and ULK1 could co-localized at autophagosomal membranes. Altogether, our results strengthens that autophagy lays at a center place for NPM-ALK+ ALCL tumor cells fate upon crizotinib treatment, and stressed that NPM-ALK and RAF1 combined inactivation, by increasing autophagy flux, might be beneficial for ALK+ ALCL patients.; Le lymphome anaplasique à grandes cellules ALK positif (LAGC ALK+) est un cancer pédiatrique très agressif. Il se caractérise par des translocations chromosomiques impliquant toujours le gène ALK et différents autres gènes, partenaires de translocation. NPM-ALK (Nucleophosmine- Anaplastic Lymphoma Kinase) est la protéine de fusion à activité tyrosine kinase la plus fréquemment observée. Elle résulte de la translocation chromosomique t(2;5)(p23 ;q35) et l'activation constitutive de son domaine catalytique permet le développement du lymphome, par l'activation de nombreuses voies de signalisation de survie et de prolifération. Les thérapies actuelles sont la chimiothérapie, qui est efficace dans 70% des cas, ou une thérapie ciblant l'oncogène ALK (notamment l'inhibiteur tyrosine kinase Crizotinib), qui est utilisée chez les patients réfractaires ou en rechute. Les efforts de la communauté scientifique convergent également au développement de thérapies combinées, pour améliorer le traitement de ces lymphomes. Dans ce contexte, nous étudions si l'autophagie peut être modulée pour améliorer la réponse au crizotinib. L'autophagie est un processus vésiculaire d'autodigestion, connu pour être associé soit à la survie, soit à la mort cellulaire, selon le type de cancer et de thérapie. La protéine ULK1 (Unc-51-like kinase-1) joue un rôle majeur dans l'initiation de ce processus et est régulée principalement par des phosphorylations sur des résidus sérine ou thréonine, qui sont assurées par les kinases mTOR ou AMPK. Dans les lignées de LAGC ALK+, des travaux antérieurs de l'équipe ont montré qu'une autophagie cytoprotectrice était induite lors de l'inactivation de l'oncogène NPM-ALK. Durant ma thèse, j'ai tout d'abord répertorié le rôle cytoprotecteur ou, à l'inverse, cytotoxique de l'autophagie dans différents cancers dépendants de l'oncogène ALK, et soumis à différentes thérapies. Mon projet de thèse principal a ensuite été d'identifier des microARNs ainsi que leurs cibles potentielles, dont la manipulation thérapeutique pouvait, en association avec le crizotinib, conduire à une autophagie associée à la mort des cellules tumorales. En effet, des dérégulations du niveau d'expression des microARNs ont été abondamment décrites dans différents cancers, incluant les LAGC ALK+, et leurs capacités à moduler la réponse autophagique sous thérapie a également été démontrée. Mon travail de thèse a permis de démontrer que le microARN-7-5p, connu pour ses propriétés anti-tumorales, et RAF1, une de ses cibles, jouaient un rôle crucial dans les LAGC ALK+, en contrôlant le flux autophagique et le devenir cellulaire. RAF1 est une sérine /thréonine kinase, essentiellement connue pour connecter RAS à la voie de prolifération MEK/ERK. Cependant, le mécanisme par lequel l'inhibition spécifique de RAF1 peut influer sur l'autophagie n'a encore jamais été décrit. Nos travaux montrent pour la première fois la capacité pour RAF1 (et non MEK ou ERK) de phosphoryler ULK1 sur le résidu inhibiteur sérine 757, ce qui offre une nouvelle voie thérapeutique pour moduler le flux autophagique dans les LAGC ALK+. Dans une seconde partie de ma thèse, et afin de mieux comprendre ce lien potentiel entre RAF1 et ULK1, nous nous sommes attachés, selon deux protocoles différents, à purifier les autophagosomes à partir d'une lignée cellulaire de LAGC ALK+. Nos résultats suggèrent la présence de NPM-ALK dans nos fractions enrichies en autophagosomes, et ces travaux seront poursuivis pour détecter RAF1 et ULK1 dans ces mêmes fractions. Ainsi, nos résultats démontrent l'importance de l'autophagie dans la réponse des LAGC ALK+ au crizotinib et mettent au jour que la combinaison thérapeutique consistant à inhiber NPM-ALK et RAF1, en potentialisant le flux autophagique, pourrait être bénéfique pour les patients atteints de ce lymphome.

The ALK oncogene (Anaplastic Lymphoma Kinase) is a constitutively activated tyrosine kinase implied in various cancers including Anaplastic Large Cell Lymphomas (ALCL), or some lung adenocarcinomas. The current operative treatment is standard chemotherapy, which is not optimal (30% of relapses, low quality of life). In this context, new specific ALK inhibitors such as Crizotinib have been developed, and have showed their efficiency in vitro, in vivo and in patients. However, the emergence of resistant mutations has been described. Thus, the identification of alternative therapies targeting new pathways appears as mandatory to counteract those resistances. In this context, autophagy, an intracellular catabolic lysosomal process, has been described as a new therapeutic target in the treatment of cancers resistant to tyrosine kinase inhibitors. The first aim of my project was to characterize the autophagic process in ALK+ ALCL, upon different treatments. We showed that (1) autophagy was activated in ALK+ ALCL cell lines in response to ALK inhibition (2) that this autophagy played a cytoprotective role in our model and (3) that treatment with chemotherapies did not trigger an autophagic response. In a second part of the project, we focused on the potential regulation of autophagy by microRNAs. We showed that several microRNAs including miR-7 were down-regulated upon Crizotinib treatment and that ectopic re-expression of this miR-7 potentiates the effects of Crizotinib by induction of cytotoxic autophagy in our model. We hypothesized that this switch in the role of autophagy from cytoprotection to cytotoxicity observed in our model, could be explained by the regulation of several protein targets of miR-7 such as Bcl-2 or c-Raf. Altogether, these results enable a better understanding of the role and regulation of autophagy induced upon ALK inhibition in ALCL, and could in the longer term, contribute to improvement of current therapies of cancers involving the ALK oncogene.; L'oncogène ALK (Anaplastic Lymphoma Kinase) est une tyrosine kinase constitutivement active, impliquée dans divers cancers, tels que les lymphomes anaplasiques à grandes cellules (LAGC), ou certains carcinomes bronchiques. Ces tumeurs sont traitées par chimiothérapie, ce qui n'est pas un traitement optimal (30% de rechutes, abaissement de la qualité de vie). Dans ce contexte, de nombreux inhibiteurs spécifiques de la tyrosine kinase ALK tels que le Crizotinib ont été développés et ont prouvé leur efficacité à la fois dans des modèles in vitro, in vivo ainsi que chez les patients. Néanmoins, le succès de cette thérapie ciblée est limité par l'apparition de résistances. Il est donc essentiel de mettre au jour de nouvelles stratégies thérapeutiques permettant de contrecarrer ces résistances. Récemment, l'autophagie, un processus catabolique intracellulaire de dégradation lysosomale, a été proposée comme nouvelle cible thérapeutique dans le traitement des cancers résistants aux inhibiteurs de tyrosine kinase. Le premier objectif de mon projet de thèse a été de caractériser ce processus autophagique dans les LAGC ALK+, en réponse à différents traitements. Nous avons montré que (1) l'autophagie était activée dans des lignées de LAGC en réponse à l'inhibition de ALK, (2) que cette autophagie jouait un rôle cytoprotecteur dans ce modèle et (3) que les traitements par chimiothérapies de ces cellules n'induisaient pas de réponse autophagique. Dans un deuxième temps, nous nous sommes intéressés à la régulation potentielle de l'autophagie par les microARNs. Nous avons montré que plusieurs microARNs, dont le miR-7, étaient sous-exprimés en réponse au traitement par le Crizotinib et que la réexpression ectopique de ce miR-7 permettait une potentialisation de l'effet du Crizotinib, par l'induction d'autophagie cytotoxique dans notre modèle. Nous avançons également l'hypothèse que le " switch " autophagique de la cytoprotection à la cytotoxicité, que nous observons pourrait s'expliquer par la régulation de l'expression de plusieurs protéines cibles de miR-7 telles que Bcl-2 ou c-Raf. Ainsi, l'ensemble de nos résultats nous permettent de mieux comprendre le rôle et la régulation de l'autophagie induite en réponse à l'inhibition de ALK dans les LAGC ALK+, et pourrait à terme contribuer à l'amélioration des thérapies actuelles de divers cancers dépendants de l'oncogène ALK.