BACKGROUND AND PURPOSE: Hypoxia in tumours is known to cause resistance to conventional chemotherapeutic drugs. In contrast, little is known about the effects of hypoxia on targeted anti-cancer drugs. This study evaluated the effect of hypoxia on a series of clinically approved tyrosine kinase inhibitors (TKIs). EXPERIMENTAL APPROACH: The effect of hypoxia (0.1% oxygen) on the activity of conventional cytotoxic drugs (5-fluorouracil, doxorubicin and vinblastine), the hypoxia-activated prodrug tirapazamine and 9 TKIs was determined in a panel of cell lines. Where hypoxia had a marked effect on chemosensitivity, Western blot analysis was conducted to determine the effect of hypoxia on target expression and the effect of TKIs on cell signalling response under aerobic and hypoxic conditions. KEY RESULTS: Three patterns of chemosensitivity were observed: resistance under hypoxia, equitoxic activity against hypoxic and aerobic cells, and preferential cytotoxicity to hypoxic cells. Significant hypoxia selectivity (independent of HIF1) was observed in the case of dasatinib and this correlated with the ability of dasatinib to inhibit phosphorylation of Src at tyrosine 530. Sorafenib was significantly less effective under hypoxic conditions but resistance did not correlate with hypoxia-induced changes in Raf/MEK/ERK signalling. CONCLUSIONS AND IMPLICATIONS: Hypoxia influences the activity of TKIs but in contrast to conventional cytotoxic drugs, preferential activity against hypoxic cells can occur. The search for hypoxia-targeted therapies has been long and fruitless and this study suggests that some clinically approved TKIs could preferentially target the hypoxic fraction of some tumour types.

Le traitement médical du cancer colorectal a connu ces dernières années d'importantes avancées avec l'arrivée notamment des thérapies ciblées anti-angiogéniques et anti-EGFR. Néanmoins ces molécules ne bénéficient pas à tous les patients et il est à ce jour impossible de bien individualiser leur utilisation. Mon travail de thèse s'est donc intéressé à d'une part à identifier de nouveaux facteurs prédictifs de réponse en particulier au bévacizumab, d'autre part à rechercher et antagoniser de nouvelles cibles ou des mécanismes participant à la résistance aux molécules disponibles notamment pour les patients non répondeurs aux anti-EGFR. Premièrement, nous avons montré que le sorafenib améliorait l'activité anti-tumorale de l'irinotecan in vitro et in vivo sur des lignées cellulaires de cancer du colon rendues résistantes au SN38 indépendamment de leur statut K-ras. Pour expliquer ce phénomène, nous avons mis en évidence que le sorafenib inhibait la pompe d'efflux ABCG2 et favorisait l'accumulation intra-cellulaire de l'irinotecan et donc sa cytotoxicité. De plus, nous avons vérifié la faisabilité d'une telle association chez l'homme et confirmé son efficacité chez des patients K-ras mutés lourdement prétraités. Deuxièmement, nous avons développé un nouveau score radiologique objectif combinant l'évolution de la taille et la densité tumorale normalisée à celle du foie qui pourrait être utilisé comme marqueur de substitution pour déterminer précocément les bons répondeurs à une chimiothérapie à base de bevacizumab chez des patients présentant des métastases hépatiques. / The medical treatment of colorectal cancer has made significant progresses in recent years including the arrival of targeted therapies: anti-angiogenic and anti-EGFR. However, these molecules don't benefit all patients and their use is not well personalized. My thesis is therefore aimed, on one hand, to identify new predictors of response especially to bevacizumab and, on the other hand, to find and antagonize new targets or mechanisms involved in chemo-resistance, especially for K-ras mutated patients. Firstly, we have showed that sorafenib improved the anti-tumoral activity of irinotecan, both in vitro and in vivo, in SN-38 resistant colon adenocarcinoma cell lines independently of their K-ras status. To explain this phenomenon, we have demonstrated that sorafenib inhibited the efflux pump ABCG2 and promoted the intracellular accumulation of irinotecan and thus its cytotoxicity. In addition, we have checked the feasibility of such an association in human and confirmed its efficacy in K-ras mutated heavily pretreated patients. Secondly, we have developed a new objective radiological score combining both tumor size and density normalized to the liver that could be used as objective surrogate markers to determine early good responders after bevacizumab-containing chemotherapy in patients with colorectal liver metastases.

Hepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC. / Hepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC.

Les formes réactives de l’oxygène (FRO) ont un rôle bien établi dans l’oncogénèse et le fonctionnement des cellules tumorales en augmentant les capacités de prolifération et d’invasion cellulaire. Les FRO présentent une moindre toxicité dans les cellules normales où le niveau de stress oxydant est bas et les systèmes d’oxydoréduction opérationnels. Une modulation pharmacologique de l’équilibre d’oxydoréduction permet d’améliorer l’efficacité des molécules cytotoxiques qui agissent sur les FRO. L’activité anti tumorale de la chimiothérapie étant médiée en partie par le stress oxydant, nous nous sommes dans un premier temps intéressé aux variations du stress oxydant lors d’un traitement par le sorafenib (Nexavar©), un inhibiteur de tyrosine kinases. Nous avons mis en évidence une activité anti tumorale du sorafenib liée à l’augmentation de la production d’anion superoxyde par les cellules. Ce phénomène nous a permis d’identifier un marqueur prédictif d’efficacité du sorafenib, le dosage des Produits d’Oxydation Avancée des Protéines (AOPP), dans le sérum des patients cirrhotiques développant un carcinome hépatocellulaire. Sachant que l’efficacité anti tumorale des chimiothérapies conventionnelles passe en partie par l’induction de FRO, nous nous sommes intéressés dans un deuxième temps au mangafodipir, un modulateur du stress oxydant, qui est connu pour augmenter l’index thérapeutique des agents cytotoxiques in vivo. Le mangafodipir diminue les effets toxiques du stress oxydatif dans des cellules non tumorales et potentialise l’activité anti tumorale de l’oxaliplatine. Ces données nous ont conduits à explorer in vivo et in vitro le mécanisme de la neurotoxicité induite par l’oxaliplatine et le rôle protecteur du mangafodipir. Nous avons ainsi observé dans un modèle murin que le mangafodipir prévient la neurotoxicité induite par l’oxaliplatine au niveau de la gaine de myéline. Nous avons confirmé ces résultats chez l’homme lors d’une étude de phase II. Compte tenu des taux plus élevés de FRO dans les cellules tumorales par rapport aux cellules normales, plusieurs stratégies ont été proposées pour cibler sélectivement les tumeurs sans endommager les tissus sains avec des modulateurs du stress oxydant. Notre troisième axe de travail a eu pour objectif d’identifier et de valider de nouvelles molécules liées à la modulation des FRO. Cette étude a été réalisée dans le cadre d’un projet du programme Européen Pierre et Marie Curie (International Training Network 7-FP7-2007-1-1-ITN201114), au sein du réseau REDCAT. Cette collaboration a permis de concevoir et de synthétiser de nouveaux agents thérapeutiques, les organochalcogenes. Nous avons mis en évidence in vitro et in vivo le potentiel anti tumoral de cette nouvelle classe thérapeutique et principalement celui du composé LAB027. Le LAB027 présente une activité anti tumorale seul ou associé à l’oxaliplatine. L’ensemble de ces travaux nous a permis d’envisager l’évaluation précoce de l’efficacité du sorafenib par un marqueur du stress oxydant, les AOPP, de mettre en évidence l’effet anti-neurotoxique d’un mimétique de la superoxyde dismutase, le mangafodipir et d’identifier une nouvelle classe médicamenteuse en oncologie, les organochalcogènes. Ces optimisations thérapeutiques permettent d’envisager une meilleure prise en charge des malades traités par chimiothérapie afin d’optimiser l’efficacité des traitements utilisés en oncologie. / Reactive oxygen species (ROS) forms have an established role in oncogenesis and tumor cell function by increasing the capacity of proliferation and cell invasion. ROS have a lower toxicity in normal cells where the level of oxidative stress is low and redox systems operational. Pharmacological modulation of redox balance enhances the effectiveness of cytotoxic molecules that act on the FRO. The anti-tumor activity of chemotherapy is mediated in part by oxidative stress, we initially interested in the changes of oxidative stress during treatment with sorafenib (Nexavar©), an inhibitor of tyrosine kinases. We have demonstrated anti-tumor activity of sorafenib due to increased production of superoxide anion by cells. This allowed us to identify a predictive marker of efficacy of sorafenib dosing Products Advanced Oxidation Protein (AOPP) in the serum of patients with cirrhosis develop hepatocellular carcinoma. Knowing that the antitumor efficacy of conventional chemotherapy is partly through the induction of ROS, we are interested in a second time mangafodipir, a modulator of oxidative stress, which is known to increase the therapeutic index of cytotoxic agents in vivo. Mangafodipir reduces the toxic effects of oxidative stress in non-tumor cells and potentiates the anti-tumor activity of oxaliplatin. These data led us to explore in vivo and in vitro the mechanism of neurotoxicity induced by oxaliplatin and the protective role of mangafodipir. We observed in a mouse model that mangafodipir prevents neurotoxicity induced by oxaliplatin in the myelin sheath. We confirmed these results in humans in a phase II study. Given the higher levels of ROS in tumor cells compared to normal cells, several strategies have been proposed to selectively target tumors without damaging healthy tissue with modulators of oxidative stress. Our third area of work has aimed to identify and validate novel molecules related to the modulation of ROS. This study was conducted as part of a project of the European Pierre et Marie Curie (International Training Network 7-FP7-2007-1-1-ITN201114) within the network REDCAT. This collaboration has led to the design and synthesis of new therapeutic agents, organochalcogenes. We have demonstrated in vitro and in vivo antitumor potential of this new therapeutic class and mainly composed of the LAB027. LAB027 the present anti-tumor activity alone or in combination with oxaliplatin. All of this work has allowed us to consider the early assessment of the effectiveness of sorafenib a marker of oxidative stress, the AOPP, highlight the anti-nerve of a superoxide dismutase mimetic, mangafodipir and identify a new class of drugs in oncology, organochalcogènes. These optimizations allow therapeutic consider better care of patients treated with chemotherapy to enhance the efficacy of treatments used in oncology.

No description available.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults, affecting thousands of people worldwide every year, with a life expectancy, post diagnosis of 12 months. Surgery, radiotherapy and chemotherapy together, result in an overall mean survival not exceeding 15 months. Targeted therapeutic agents sorafenib, an oral multi kinase inhibitor, and lapatinib, an epidermal growth factor receptor (EGFR) inhibitor, used in combination have been shown to kill GBM cells be through inhibition of major growth mediating signaling pathways that are frequently over expressed in gliomas, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/ protein kinase B (PI3K/AKT). Sorafenib can restore lapatinib induced cytotoxicity by down regulation of myeloid cell leukaemia-1 (Mcl-1) expression. Prior studies have shown Mcl-1 to play an important role in resistance to lapatinib. Furthermore, data indicated that this drug combination is able to trigger activation of autophagic and apoptotic pathways and induce endoplasmic reticulum (ER) stress response in GBM cells, collectively resulting in cell death. In conclusion, data presented here demonstrates that the combination of sorafenib and lapatinib can kill GBM cells in a greater than additive fashion, through induction of autophagy, apoptotic events (extrinsic and intrinsic) and ER stress.

We investigated the mechanism of toxicity and resistance development of small molecule tyrosine kinase inhibitor lapatinib in HCT 116 colon cancer cells. Lapatinib mediated cell death in HCT 116 cells was caspase independent and involved cytosolic release of apoptosis inducing factor. Treatment of HCT 116 cells with 10µM Lapatinib lead to the outgrowth of lapatinib resistant HCT 116 cells. Our studies show that alterations in the expression and activation of Bcl-2 family proteins allow lapatinib resistant HCT 116 cells to resist cytotoxic effects of lapatinib as well as of other commonly used chemotherapeutic agents. In hepatoma and pancreatic cancer cells, the effects of combining multi-kinase inhibitor sorafenib with histone deacetylase inhibitors (HDACIs) namely, vorinostat and sodium valproate were investigated. It was found that sorafenib synergizes with HDACIs resulting in enhanced cell death compared to death induced by the drugs individually. The mechanism of action of sorafenib and vorinostat combination treatment as well as sorafenib and sodium valproate combined treatment was shown to involve activation of the CD95 death receptor pathway. Alterations in the CD95 pathway can render cancer cells resistant to chemotherapeutic agents. Hence, we combined sorafenib+sodium valproate with a BH-3 domain mimetic named obatoclax (GX-15-070) which resulted in enhanced toxicity to cancer cells. More importantly, knock-down of CD95 (to mimic non-functional CD95 pathway) reduced cell death induced by sorafenib+sodium valproate combined but failed to protect cells from cell death induced by sorafenib+sodium valproate+obatoclax combined. This suggests that combining sorafenib+HDACI with obatoclax may not only enhance toxicity to cancer cells but may also reduce chances of resistance development via alterations in the CD95 pathway. These studies enhance our knowledge of existing treatment strategies for cancer as well as throw light on how current approaches can be improved in order to better diagnose and treat cancer. Understanding mechanisms of drug action as well as resistance development will allow us to combine existing therapies effectively in order best target cancer cells as well as provide us with information that can help us design new cancer treatment strategies.

The cancers of liver, colon and breast are amongst the top five most prevalent and most fatal worldwide. As the Raf/MEK/ERK pathway is frequently deregulated in hepatocellular carcinoma (HCC), sorafenib, a Raf kinase inhibitor, became the first systemic therapy approved for the treatment of patients with HCC. However, sorafenib only produced modest effects with low response rates in the clinic. Similarly, regorafenib, which was approved for the treatment of metastatic colorectal cancer (CRC), has had a poor response rate in the clinic. Since phosphodiesterase type 5 has been reported to be overexpressed in HCC and CRC, we hypothesized that sildenafil, a phosphodiesterase type 5 inhibitor, could enhance the toxicities of sorafenib and regorafenib in HCC and CRC cells, respectively. Our in vitro data indicated that the drugs interacted strongly to kill cancer cells via induction of ER stress, autophagy and apoptosis. In accordance with these findings, our in vivo data demonstrated a significant reduction in tumor growth. The second study in this manuscript was conducted based on the growing body of evidence about the significant contribution of EGFR and JAK/STAT signaling to the breast tumorigenesis. Our preliminary in vitro data demonstrated that the concurrent inhibition of these two pathways by lapatinib, a dual ERBB1/2 inhibitor, and ruxolitinib, a JAK1/2 inhibitor, synergistically killed breast cancer cells of all types, including the resistant triple negative subtype. Our mechanistic studies showed that the combination of ruxolitinib and lapatinib triggered cytotoxic mitophagy, and autophagy-dependent activation of BAX and BAK leading to the mitochondrial dysfunction.

The FMS-like tyrosine kinase 3- internal tandem duplication (FLT3/ITD) aberration is common in acute myeloid leukemia (AML) and associated with poor patient outcome. Inhibitors targeting FLT3/ITD are in development, but clinical responses are transient. This project focussed on elucidating molecular signalling consequences of FLT3/ITD inhibition, to identify rational drug combinations for future development. A Multicolour Phospho Flow Cytometry (MPFC) assay was developed to assess signalling events downstream of FLT3/ITD in primary patient samples, focusing on alterations in ERK, STAT5, Akt, and S6. STAT5 signalling appeared to be important exclusively in FLT3/ITD samples. MPFC accurately predicted the presence of FLT3/ITD, inhibitor sensitivity and the initial positive clinical response of a trial patient receiving a FLT3/ITD inhibitor. PI3K pathway upregulation was observed in a Sorafenib-resistant FLT3/ITD cell line established to study resistance mechanisms of FLT3 inhibition. Further, combination FLT3 and PI3K inhibition demonstrated synergy, suggesting potential clinical relevance to this therapeutic strategy.

The FMS-like tyrosine kinase 3- internal tandem duplication (FLT3/ITD) aberration is common in acute myeloid leukemia (AML) and associated with poor patient outcome. Inhibitors targeting FLT3/ITD are in development, but clinical responses are transient. This project focussed on elucidating molecular signalling consequences of FLT3/ITD inhibition, to identify rational drug combinations for future development. A Multicolour Phospho Flow Cytometry (MPFC) assay was developed to assess signalling events downstream of FLT3/ITD in primary patient samples, focusing on alterations in ERK, STAT5, Akt, and S6. STAT5 signalling appeared to be important exclusively in FLT3/ITD samples. MPFC accurately predicted the presence of FLT3/ITD, inhibitor sensitivity and the initial positive clinical response of a trial patient receiving a FLT3/ITD inhibitor. PI3K pathway upregulation was observed in a Sorafenib-resistant FLT3/ITD cell line established to study resistance mechanisms of FLT3 inhibition. Further, combination FLT3 and PI3K inhibition demonstrated synergy, suggesting potential clinical relevance to this therapeutic strategy.