WO2016184793A1 - Méthodes de traitement de patients souffrant d'un adénocarcinome rénal métastatique résistant aux inhibiteurs du vegfr - Google Patents

Méthodes de traitement de patients souffrant d'un adénocarcinome rénal métastatique résistant aux inhibiteurs du vegfr Download PDF

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WO2016184793A1
WO2016184793A1 PCT/EP2016/060806 EP2016060806W WO2016184793A1 WO 2016184793 A1 WO2016184793 A1 WO 2016184793A1 EP 2016060806 W EP2016060806 W EP 2016060806W WO 2016184793 A1 WO2016184793 A1 WO 2016184793A1
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sunitinib
cells
patient
combination
weak base
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Gilles PAGES
Sandy GIULIANO
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Universite Nice Sophia Antipolis
Centre National De La Recherche Scientifique (Cnrs)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/365—Lactones
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
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    • A61K31/33—Heterocyclic compounds
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/451—Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • A—HUMAN NECESSITIES
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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    • A61K31/47—Quinolines; Isoquinolines
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the invention relates to methods for treating a patient with VEGFR inhibitor-resistant metastatic renal cell carcinoma (mRCC).
  • mRCC VEGFR inhibitor-resistant metastatic renal cell carcinoma
  • Metastatic Renal Cell Carcinomas are highly vascularized tumors that are a paradigm for the treatment with anti-angiogenesis drugs particularly with tyrosine kinase inhibitors targeting the Vascular Endothelial Growth Factor (VEGF) pathway.
  • VEGF Vascular Endothelial Growth Factor
  • the available drugs increase the time to progression but are not curative and the patients eventually relapse.
  • These inhibitors include notably sunitinib 1 , which since 2006 is considered as the standard first line treatment option for this disease.
  • Sunitinib has been designed to disrupt major signaling pathways (HRAS/RAF 1/MAP2K1 , 2/MAPK1, 3 and MTOR pathways) that are responsible for the abnormal proliferation of cancer cells and tumor angiogenesis.
  • sunitinib has not significantly improved the overall survival of the majority of patients compared to treatment with interferon alpha or interleukin 2 (median time of survival after the diagnosis of about 20 months) 1 2 , the standard treatments used before the development of anti-angiogenesis drugs. Moreover, the fact that mRCC patients gradually become refractory to sunitinib represents an important obstacle to better outcome for patients.
  • sunitinib induced autophagy in bladder cancer cells 5 and that inhibition of autophagy potentiated the anti-pro liferative effects of sunitinib 6 7 .
  • cells were exposed to high doses of sunitinib and the cells were not representative of cancers for which sunitinib is the treatment of reference 6 .
  • Lysosomal sequestration of sunitinib 8 may be explained by the fact that it is a hydrophobic weak base (pKa 8.95).
  • Sequestration in lysosomes may prevent access of the drug to the kinase domain of tyrosine kinase receptors present in the cytoplasm, thus participating in the loss of efficacy of the drug.
  • sunitinib has not been described for mRCC cells.
  • autophagy and lysosome trapping in the mechanisms of resistance has not been addressed.
  • the invention relates to a drug or a combination of drugs selected from the group consisting of a proteasome inhibitor, an efflux inhibitor or a combination of an efflux inhibitor and a lysosomotropic agent for use in a method for treating metastatic renal cell carcinoma (mRCC) in a patient with an acquired resistance to treatment with a multi- targeted weak base vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI).
  • mRCC metastatic renal cell carcinoma
  • VAGFR multi- targeted weak base vascular endothelial growth factor receptor
  • TKI tyrosine kinase inhibitor
  • the invention in a second aspect, relates to a method for predicting the survival time of a patient affected with RCC or mRCC comprising the steps of : (i) determining the expression level of at least one of the proteasome component genes selected from the group consisting of PSMB8, PSMB9, PSMB10 and PSMF1 genes in a primary renal tumor biopsy obtained from said patient; (ii) comparing the level determined at step (i) with a predetermined reference level; and (iii) concluding that the patient has a poor prognosis when the level determined at step (i) is higher than the predetermined reference level.
  • the invention relates to a combination comprising a multi-targeted weak base VEGFR TKI and a proteasome inhibitor or a combination comprising a multi- targeted weak base VEGFR TKI and an efflux inhibitor for use in a method for treating RCC or mRCC and/or preventing metastasis in a patient determined as having a poor prognosis according to the method of the invention.
  • the invention in a fourth aspect, relates to a method for determining whether a patient affected with RCC or mRCC has or is at risk of multi-targeted weak base VEGFR TKI- resistance, comprising a step of determining the expression level of at least one of the proteasome component genes selected from the group consisting of PSMB8, PSMB9, PSMB10 and PSMF1 genes in a primary renal tumor biopsy obtained from said patient; (ii) comparing the level determined at step (i) with a predetermined reference level; and (iii) concluding that the patient has or is at risk of multi-targeted weak base VEGFR TKI- resistance when the level determined at step (i) is higher than the predetermined reference level.
  • the invention relates to a combination comprising a multi-targeted weak base VEGFR TKI and a proteasome inhibitor or a combination comprising a multi- targeted weak base VEGFR TKI and an efflux inhibitor for use in a method for treating RCC or mRCC and/or preventing metastasis and/or preventing acquired resistance to treatment with a multi-targeted weak base VEGFR TKI in a patient determined as having or being at risk of multi-targeted weak base VEGFR TKI-resistance according to the method of the invention.
  • the invention addresses these needs, as it relates to methods and treatment approaches useful in the treatment of VEGFR inhibitor-resistant metastatic renal cell carcinoma (mRCC).
  • mRCC VEGFR inhibitor-resistant metastatic renal cell carcinoma
  • the inventors have indeed focused their attention on the molecular mechanisms leading to resistance to sunitinib, the first line treatment of mRCC. Because of the anarchic vascularization of tumors the core of mRCC tumors receives sub-optimal concentrations of the drug. To mimic this in vivo situation, which is encountered in a neo-adjuvant setting, they exposed sunitinib-sensitive mRCC cells to concentrations of sunitinib below the concentration of the drug that gives 50% inhibition of cell proliferation (IC50). At these concentrations, sunitinib accumulated in lysosomes, which down-regulated the activity of the lysosomal protease cathepsin B and led to incomplete autophagic flux.
  • Amino acid deprivation which initiates autophagy enhanced sunitinib resistance through the amplification of auto-lysosome formation.
  • Sunitinib stimulated the expression of ABCB1, which participates in the accumulation of the drug in auto-lysosomes and favor its cellular efflux. Inhibition of this transporter by elacridar and the permeabilization of lysosome membranes with Leu-Leu-O- Methyl (LLOM) re-sensitized mRCC cells that were resistant to concentrations of sunitinib superior to the IC50.
  • LLOM Leu-Leu-O- Methyl
  • Proteasome inhibitors also induced the death of resistant cells suggesting that the ubiquitin-proteasome system compensates inhibition of autophagy to maintain a cellular homeostasis. Based on the present results, they propose a new therapeutic approach combining sunitinib with molecules that prevent lysosomal accumulation or inhibit the proteasome.
  • a first aspect of the invention relates to a drug or a combination of drugs selected from the group consisting of a proteasome inhibitor, an efflux inhibitor or a combination of an efflux inhibitor and a lysosomotropic agent for use in a method for treating metastatic renal cell carcinoma (mRCC) in a patient with an acquired resistance to treatment with a multi-targeted weak base vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI).
  • mRCC metastatic renal cell carcinoma
  • VAGFR vascular endothelial growth factor receptor
  • TKI tyrosine kinase inhibitor
  • proteasome inhibitor or “inhibitor of the ubiquitin- proteasome system” (UPS) refers to compounds which inhibits the activity of the proteasome, more particularly its enzymatic activity. Inhibiting UPS enzymatic activity means reducing the ability of a UPS component to perform its activity.
  • Such compounds include, but are not limited to, bortezomib (also known as PS-341), MLN 341, carfilzomib, delanzomib (also known as CEP-18770) and MG132.
  • the proteasome inhibitor is bortezomib.
  • Bortezomib is commercially available from Millennium under the trade name Velcade® and may be prepared for example as described in EP788360, EP1312609, EP1627880, US 6066730 and US 6083903 or by processes analogous thereto.
  • efflux inhibitor refers to compound that inhibits the expression and/or activity of at least one transport protein (e.g., a P-Glycoprotein (P-GP)).
  • P-GP P-Glycoprotein
  • the efflux inhibitor is a P-Glycoprotein (ABCB1) inhibitor.
  • the P-Glycoprotein (ABCB1) inhibitor is selected from the group consisting of elacridar, biricodar, pantoprazole, and tariquidar.
  • lysosomotropic agent refers to a compound, which diffuses into cellular lysosomes, causes a decrease in the lysosome transmembrane proton gradient, and increases the pH inside the organelle.
  • the lysosomotropic agent is selected from the group consisting of Leu-Leu-OMe (LLME), chloroquine, hydroxychloroquine, 3- methyladenine, quinacrine, mefloquine, monensin and bafilomycin Al .
  • LLME Leu-Leu-OMe
  • chloroquine hydroxychloroquine
  • 3- methyladenine quinacrine
  • mefloquine monensin and bafilomycin Al
  • the terms “combination” refers to a "kit-of-parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e. simultaneously or at different time points.
  • the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
  • the ratio of the total amounts of the combination partners to be administered in the combined preparation can vary.
  • the combination partners can be administered by the same route or by different routes. When the administration is sequential, the first partner may be for instance administered 1 , 2, 3, 4, 5, 6, 7, days before the second partner.
  • treatment refers to an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • treatment may also mean prolonging survival as compared to expected survival if not receiving treatment.
  • vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor refers to a compound which lead to the intracellular inhibition of VEGF signaling pathway by targeting the intracellular kinase domains of the three VEGFRs (VEGF receptor [VEGFR]- 1, VEGFR-2, and VEGFR-3).
  • multi-targeted VEGFR TKI refers to a compound which inhibits several kinase targets in addition to the VEGFRs (e.g. platelet-derived growth factor receptor [PDGFR], stem cell factor receptor [c-kit] and FMS-like tyrosine kinase-3 [Flt3]).
  • the term “weak base” refers to a compound which in an aqueous environment exists in an equilibrium between a neutrally charged basic form which is a proton acceptor (also referred to as a "non-protonated” form) and a positively charged conjugate acid form which is a proton donor (also referred to as a "protonated” form).
  • the pKa is higher than a typical physiological pH, for example in a range from 7-12, and preferably from 8-10, such that the weak base exists predominantly in a positively charged (protonated) form at both a lysosomal pH and at the physiological pH.
  • the multi-targeted weak base VEGFR TKI is selected from the group consisting of sunitinib, axitinib and dovitinib.
  • said targeted weak base VEGFR TKI is sunitinib.
  • the term "sunitinib” (also known as SU-11248 and marked as Sutent®) refers to a member of a family of pyrrole substituted 2-indolinone compounds, reported as being receptor tyrosine kinase inhibitors (see, U.S. Pat. Nos. 6,573,293 and 7,211,600). Sunitinib inhibits cellular signaling via a variety of receptors which play a role in tumor angiogenesis and tumor cell proliferation. Hence, the simultaneous inhibition of these receptors promotes reduced tumor vascularization and cancer cell death.
  • drug resistant refers to a condition which demonstrates acquired resistance.
  • “acquired resistance” is meant a multifactorial phenomenon occurring in tumor formation and progression that can influence the sensitivity of cancer cells to a drug. Acquired resistance may be due to several mechanisms such as but not limited to; alterations in drug-targets, decreased drug accumulation, alteration of intracellular drug distribution, reduced drug-target interaction, increased detoxification response, cell-cycle deregulation, increased damaged-DNA repair, and reduced apoptotic response. Several of said mechanisms can occur simultaneously and/or may interact with each other.
  • Various qualitative and/or quantitative methods may be used to determine if a patient has developed or is susceptible to developing a resistance to treatment with a multi-targeted weak base VEGFR TKI such as sunitinib. For example, a patient who showed initial improvement while taking a multi-targeted weak base VEGFR TKI, may display signs that the multi-targeted weak base VEGFR TKI has become less effective or is no longer effective.
  • Symptoms that may be associated with resistance to a multi-targeted weak base VEGFR TKI include, for example, a decline or plateau of the well-being of the patient, an increase in the size of a tumor, arrested or slowed decline in growth of a tumor, and/or the spread of cancerous cells in the body from one location to other organs, tissues or cells.
  • a decrease in the sensitivity of cancer cells to a multi-targeted weak base VEGFR TKI, an increase in the growth or proliferation of cancer cells, and/or a decrease in cancer cell apoptosis as compared to a control may also be indicative that the patient has developed or is susceptible to developing a resistance to a multi-targeted weak base VEGFR TKI. It is possible to determine cancer cell sensitivity, growth, proliferation or apoptosis using standard methods as described further herein. For example, cancer cell sensitivity, growth, proliferation or apoptosis may be determined either in situ or in vitro.
  • In situ measurements may involve, for example, observing the effect of a multi- targeted weak base VEGFR TKI therapy in a patient by examining cancer growth or metastasis.
  • RECIST criteria are analyzed.
  • RECIST Response Evaluation Criteria In Solid Tumors
  • CT computed tomography
  • a patient is considered as resistant when at least a 30 % increase of metastases is detected in said patient by [ 18 F]fluoro-2-deoxy-2-d-glucose (FDG) positron emission tomography (PET) imaging (FDG-PET scan).
  • FDG fluoro-2-deoxy-2-d-glucose
  • PET positron emission tomography
  • the patient with an acquired resistance is still under multi-targeted weak base VEGFR TKI treatment.
  • the invention relates to a method for treating metastatic renal cell carcinoma (mRCC) in a patient with an acquired resistance to treatment with a multi-targeted weak base vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI) comprising the followings steps of: a) selecting a patient with mRCC who has developed a resistance to treatment with a VEGFR, and b) administering to said patient an therapeutically effective amount of a drug or a combination of drugs selected from the group consisting of a proteasome inhibitor, an efflux inhibitor or a combination of an efflux inhibitor and a lysosomotropic agent.
  • mRCC metastatic renal cell carcinoma
  • VEGFR vascular endothelial growth factor receptor
  • TKI tyrosine kinase inhibitor
  • terapéuticaally effective amount is meant an amount sufficient to achieve a concentration of compound which is capable of preventing or slowing down the disease to be treated. Such concentrations can be routinely determined by those of skilled in the art.
  • the amount of the polypeptide actually administered will typically be determined by a physician or a veterinarian, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the patient, the severity of the subject's symptoms, and the like. It will also be appreciated by those of skilled in the art that the dosage may be dependent on the stability of the administered compound.
  • the compounds of the invention may be administered by any means that achieve the intended purpose.
  • administration may be achieved by a number of different routes including, but not limited to, subcutaneous, intravenous or parenteral, intramuscular, intraperitoneal or oral routes. Parenteral route is particularly preferred. Dosages to be administered depend on individual needs, on the desired effect and the chosen route of administration. It is understood that the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The total dose required for each treatment may be administered by multiple doses or in a single dose.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • the daily dosage of the polypeptides may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 10 mg/kg of body weight per day.
  • the invention in a second aspect, relates to a method for predicting the survival time of a patient affected with RCC or mRCC comprising the steps of : (i) determining the expression level of at least one of the proteasome component genes selected from the group consisting of PSMB8, PSMB9, PSMB10 and PSMF1 genes in a primary renal tumor biopsy obtained from said patient; (ii) comparing the level determined at step (i) with a predetermined reference level; and (iii) concluding that the patient has a poor prognosis when the level determined at step (i) is higher than the predetermined reference level.
  • the invention in a third aspect, relates to a method for determining whether a patient affected with RCC or mRCC has or is at risk of multi-targeted weak base VEGFR TKI- resistance, comprising (i) a step of determining the expression level of at least one of the proteasome component genes selected from the group consisting of PSMB8, PSMB9, PSMB10 and PSMF1 genes in a primary renal tumor biopsy obtained from said patient; (ii) comparing the level determined at step (i) with a predetermined reference level; and (iii) concluding that the patient has or is at risk of multi-targeted weak base VEGFR TKI- resistance when the level determined at step (i) is higher than the predetermined reference level.
  • the term "Proteasome subunit beta type-8 (PSMB8) gene” encodes a protein of 204 amino acids, member of the proteasome B-type family, also known as the TIB family, which is a 20S core beta subunit. This term includes naturally occurring PSMB8 and variants thereof.
  • the naturally occurring human PSMB8 protein has an amino acid sequence as shown in UniProt Accession number P28062 and is encoded by the nucleic acid sequence provided in the GenBank database under accession number NM 004159.
  • the term “Proteasome subunit beta type-9 (PSMB9) gene” encodes a protein of 199 amino acids, member of the proteasome B-type family. This term includes naturally occurring PSMB9 and variants thereof.
  • the naturally occurring human PSMB9 protein has an amino acid sequence as shown in UniProt Accession number P28065 and is encoded by the nucleic acid sequence provided in the GenBank database under accession number NM 002800.
  • PSMBIO Protein subunit beta type-9
  • PSMFI Protein inhibitor PI31 subunit
  • predetermined reference level refers to the expression levels of PSMB8, PSMB9, PSMBIO and PSMFI genes in biological samples obtained from the general population or from a selected population of patients.
  • the selected population may be comprised of apparently RCC patients who had any sign or symptoms indicating the presence of a multi-targeted weak base VEGFR TKI-resistance.
  • a predetermined reference level may be determined, for example, by determining the expression level ot PSMBS, PSMB9, PSMBIO and PSMFI nucleic acids or encoded polypeptides, in a corresponding biological sample obtained from one or more control patient(s) (e.g., no resistance or known not to be resistant).
  • a higher or increased levels determined in a biological sample is indicative for example that said patient has or is at risk of having has or is at risk of multi-targeted weak base VEGFR TKI-resistance.
  • the predetermined reference level may be established based upon comparative measurements between patients classified with no resistance and patients with established resistance.
  • the predetermined reference value can be a threshold value or a range.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the expression level of the gene(s) of interest in properly banked historical subject samples may be used in establishing the predetermined reference value.
  • the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • a "higher” or “increased” level refers to a expression level in a biological sample (i.e. a primary renal tumor biopsy obtained from the patient) which is at least 20% higher, in an embodiment at least 30% higher, in a further embodiment at least 40% higher; in a further embodiment at least 50% higher, in a further embodiment at least 100% higher (i.e. 2-fold), in a further embodiment at least 200% higher (i.e. 3-fold), in a further embodiment at least 300% higher (i.e. 4-fold), relative to the predetermined reference level.
  • Determination of the expression level of PSMB8, PSMB9, PSMB10 and/or PSMF1 genes may be performed by a variety of techniques.
  • the expression level as determined is a relative expression level.
  • the determination comprises contacting the biological sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptide or nucleic acids of interest originally in said biological sample. Contacting may be performed in any suitable device, such as a plate, micro titer dish, test tube, well, glass, column, and so forth. In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array.
  • the substrate may be a solid or semi- so lid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the biological sample.
  • determining includes qualitative and/or quantitative detection (i.e. detecting and/or measuring the expression level) with or without reference to a control or a predetermined value.
  • detecting means determining if PSMB8, PSMB9, PSMBIO and/or PSMFI is present or not in a biological sample and “measuring” means determining the amount of PSMB8, PSMB9, PSMBIO and/or PSMFI in a biological sample.
  • the expression level may be determined for example by RT-PCR or immunohistochemistry (IHC) performed on a primary renal tumor biopsy.
  • the expression level of PSMB8, PSMB9, PSMBIO and/or PSMFI genes may be assessed by determining the quantity of mRNA.
  • nucleic acid contained in the biological samples e.g., cell or tissue prepared from the patient
  • the nucleic acid contained in the biological samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions.
  • the extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and/or amplification (e.g., RT-PCR).
  • Quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.
  • nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above-mentioned methods may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a particular kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the invention also relates to a kit for performing the above-mentioned methods, wherein said kit comprises means for determining the expression level of the PSMB8, PSMB9, PSMB10 and/or PSMF1 genes in a biological sample of interest.
  • the methods of the invention comprise the steps of providing total R As extracted from a primary renal tumor biopsy and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi-quantitative RT-PCR.
  • the expression level of PSMB8, PSMB9, PSMB10 and/or PSMF1 genes may be assessed by determining the quantity of proteins encoded by said genes.
  • Such methods comprise a step of contacting the biological sample with a binding partner capable of selectively interacting with a marker protein present in the biological sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • Monoclonal antibodies directed against PSMB8, PSMB9, PSMB10 and/or PSMF1 are well known from the skilled man in the art such as the antibodies commercialized by
  • PSMB8, PSMB9, PSMB10 and/or PSMF1 proteins may be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays include, but are not limited to, Western blots; agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • Labels are known in the art that generally provide (either directly or indirectly) a signal (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE)).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • an immunohistochemistry (IHC) method may be used on a primary renal tumor biopsy. IHC specifically provides a method of detecting a target protein in a biological sample or tissue specimen in situ. The overall cellular integrity of the biological sample is maintained in IHC, thus allowing detection of both the presence and location of the target of interest.
  • IHC Intra-human immunosorbent assays
  • a biological sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy.
  • Current methods of IHC use either direct labelling or secondary antibody-based or hapten-based labelling.
  • Examples of known IHC systems include, for example, En Vision TM (DakoCytomation), Powervision® (Immunovision, Springdale, AZ), the NBATM kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine ® (Nichirei Corp, Tokyo, Japan).
  • the invention relates to a combination comprising a multi-targeted weak base VEGFR TKI and a proteasome inhibitor or a combination comprising a multi- targeted weak base VEGFR TKI and an efflux inhibitor for use in a method for treating RCC or mRCC and/or preventing metastasis in a patient determined as having a poor prognosis according to a method of the invention as above-defined.
  • the combination further comprises a lysosomotropic agent as previously described.
  • the invention relates to a combination comprising a multi- targeted weak base VEGFR TKI and a proteasome inhibitor or a combination comprising a multi-targeted weak base VEGFR TKI and an efflux inhibitor for use in a method for treating RCC or mRCC and/or preventing metastasis and/or preventing acquired resistance to treatment with a multi-targeted weak base VEGFR TKI in a patient determined as having or being at risk of multi-targeted weak base VEGFR TKI-resistance according to a method of the invention as above-defined.
  • the combination further comprises a lysosomotropic agent as previously described.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 A sunitinib concentration below the IC50 slowed down cell proliferation but did not induce cell death.
  • A General scheme illustrating the different concentrations to which RCC cells may be exposed to in a tumor.
  • B The proliferative capacity of 786-0 cells in the absence (Ct) or presence of increasing concentrations of sunitinib (sun) was evaluated by counting the cells at the indicated times. Data are the mean fold increase ⁇ SD. The fold increase of untreated cells was taken as the reference value for statistics. Statistical significances of the results compared to untreated cells are indicated; */? ⁇ 0.05; **p ⁇ 0.01; ***/? ⁇ 0.001.
  • ABC transporters reverted sunitinib resistance of 786-OR cells. Determination of the percentage of dead 786-OS and 786-OR cells after incubation for 24 hours with the indicated combinations of drugs (sunitinib (sun) 2.5 ⁇ /L; LLOM (L) 1 ⁇ /L; elacridar (E) 5 ⁇ /L). * p ⁇ 0.05; *** p ⁇ 0.001.
  • Figure 3 Primary tumor cells derived from a patient who progressed on sunitinib were sensitive to sunitinib when in the presence of elacridar or LLOM.
  • proteasome inhibitors MG132 (mg, 10 ⁇ /L) or bortezomib (borte, 5 ⁇ /L) alone or in combination with sunitinib (sun, 2.5 ⁇ /L) decreased the viability of 786-OS and 786- OR cells after incubation for 24 hours. ** p ⁇ 0.01; *** p ⁇ 0.001, NS (non significant).
  • Figure 5 Description of the different phases of events justifying a combinatorial approach for treatment.
  • the proteasome degrades misfolded proteins and participates in recycling of amino acids.
  • the lysosome destabilizing agent Leu-Leu-O- Methyl (LLOM) prevents the trapping of sunitinib in lysosomes so the drug is localizes to the cytoplasm but is "taken in charge” by the ABCG1, which transport the drug out of the cell thus leading to intermediate resistance (++).
  • LLOM Leu-Leu-O- Methyl
  • Maximal sensitivity to sunitinib can be obtained by destabilization of lysosomes with LLOM combined with inhibitors of ABC transporters (Elacridar) or with proteasome inhibitors (MG132 (mg) or bortezomib (borte)).
  • Figure 6 LLOM, elacridar, bafilomycin or a combination of these different drugs alone or with sunitinib induced a higher mortality in 786-OS cells than in 786-OR cells.
  • A Effect of increasing concentrations of elacridar (E) on cell viability after incubation for 24 hours. * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
  • B Effect of increasing concentrations of LLOM (L) on cell viability after incubation for 24 hours. ** p ⁇ 0.01.
  • Figure 7 The combination of a lysosomal destabilizing agent and an inhibitor of ABC transporters reverts sunitinib resistance of RCC 10 cells. Determination of the percentage viable of RCC 1 OS and RCC 1 OR cells after incubation for 24 hours with the indicated combinations of drugs sunitinib (sun 2.5 mmol/L), LLOM (L 1 mmol/L), elacridar (E 5 mmol/L). * p ⁇ 0.05; ** p ⁇ 0.01.
  • Figure 8 Proteasome inhibitors induced the death of cells resistant to sunitinib.
  • proteasome inhibitors MG132 (mg, 10 ⁇ /L) or bortezomib (borte, 5 ⁇ /L) alone or in combination with sunitinib (sun, 2.5 ⁇ /L for RCC 10 cells and 5 ⁇ /L for CC cells) decreased the viability of RCC 1 OS, RCC 1 OR (A) and CC cells (B) after incubation for 24 hours.
  • Figure 9 Analysis of cbioportal databases highlighted the prognostic value of a cluster of proteasome associated genes.
  • A Heatmaps of three microarrays (GSE 14494, 11151 and 22541) for the relative expression of PSMB8, PSMB9, PSMBIO and PSMFl).
  • B Kaplan-Meier analysis of overall survival of patients with RCC at cbioportal. Overall survival was calculated from patient subgroups with mRNA levels for PSMB8, PSMB9, PSMBIO and PSMFl that were 1.4 less or greater than the median value. Statistical significance (p value) is indicated.
  • C Kaplan-Meier analysis of disease free survival or overall survival of patients with non metastatic (M0) or metastatic (M+) RCC. Statistical significance (p value) is indicated.
  • EXAMPLE Resistance to sunitinib in renal clear cell carcinoma results from sequestration in lysosomes and inhibition of the autophagic flux.
  • Anti- LAMP1 (H4A3) and anti-EEAl (N-19) were from Santa-Cruz
  • anti SQSTM1 was from BD Bioscience
  • CTSB (Ab-1) was purchased from Merck
  • anti LAMP2 was from Abeam (H4B4)
  • anti-ARDl antibodies were produced and characterized in our laboratory 50
  • anti-actin (1-19) was from Santa-Cruz
  • anti-AKTl (9272) anti-phospho MAPK1/3 at Thrl85/Tyrl87 and Thr202/Tyr204 (4370)
  • anti-MAPKl/3 (137F5) antibodies were all obtained from Cell Signaling Technology.
  • Hank's Balanced Salt Solution (HBSS) was from Life technology.
  • Cell culture Human 786-0 cells were purchased from the American Tissue Culture
  • RCC10 cells were a kind gift from W.H. Kaelin (Dana-Farber Cancer Institute, Boston, MA) and were used in one of our published studies 51 .
  • RCC cells were grown in DMEM supplemented with 7% FCS at 37°C in a humidified atmosphere containing 5% C0 2 .
  • HBSS experiments cells were pre-incubated in HBSS for 30 minutes before sunitinib treatment for 24 hours for the determination of cell viability.
  • clonogenic assays cells were incubated for seven days in fresh medium after the same procedure. Resistant cells were obtained by chronic exposure to increasing concentrations of sunitinib up to 8 ⁇ /L.
  • An INVIVO2 200 workstation (Ruskinn Technology Biotrace International Pic) set at 1% oxygen, 94% nitrogen and 5% carbon dioxide was used for hypoxic conditions.
  • Cells were seeded in six-well dishes and transiently treated with sunitinib the following day. Cells were next detached from days 2 to 6 and counted with a Coulter counter (Beckman) in duplicate to assess cell proliferation. Cell viability and cell death was assessed using the ADAM-MC apparatus (NanoEnTek) based on fluorescent propidium iodide staining according to the manufacturer's instructions.
  • Colony formation assay RCC cells (500 cells per condition) were treated or not with sunitinib. Colonies were detected after 10 days of culture. Cells were then washed, fixed at room temperature for 20 minutes with 3% paraformaldehyde and colored by crystal violet.
  • RTCA Real-Time Cell Analyzer
  • Immunoblotting Cells treated with sunitinib and/or exposed to pharmacological inhibitors, were lysed in buffer containing 3% SDS, 10% glycerol, 0.825mM Na 2 HP04. Samples (30 ⁇ g) were separated by 10%> SDS-PAGE, transferred onto a PVDF membrane (Immobilon, Millipore, France) and then exposed to the appropriate antibodies: anti-LC3, anti-p62, anti-LAMPl, anti-ARDl, anti-cathepsin B or anti-actin. Proteins were visualized with the ECL system using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies.
  • Sub-cellular fractionation was performed using proteo- extract sub-cellular proteome extraction kit according to the manufacturer's instructions (Calbiochem).
  • Flow Cytometry - Cell cycle distribution Cells were trypsinized, washed and re- suspended in cold 70% ethanol overnight. After 2 washes with PBS, cells were re-suspended in propidium iodide (40 ⁇ g/ml) containing ribonuclease A (10 ⁇ g/ml) for 15 min at room temperature and were analyzed using a fluorescence-activated cell sorter (BD healthcare F AC S C ALIBUR, analyzer).
  • BD healthcare F AC S C ALIBUR fluorescence-activated cell sorter
  • CTSB activity RCC cells treated with sunitinib for 24 h were lysed for 30 min at 4°C in lysis buffer (400 mmol/L Na Phosphate pH 6, 150 mmol/L NaCl, 4 mmol/L ethylene- diaminetetraacetic acid, 1 mmol/L phenylmethylsulfonyl fluoride, 10 ⁇ g/ml aprotinin and 1% Triton X-100) and lysates were cleared at lOOOOg for 15 min at 4°C. Each assay (in quadruplicate) was performed with 50 ⁇ g of protein prepared from control or sunitinib treated cells.
  • lysis buffer 400 mmol/L Na Phosphate pH 6, 150 mmol/L NaCl, 4 mmol/L ethylene- diaminetetraacetic acid, 1 mmol/L phenylmethylsulfonyl fluoride, 10 ⁇ g/ml aprotinin
  • cellular extracts were incubated in a 96-well plate, with 60 ⁇ of z-RR-AMC (7-amino-4-methylcoumarin) as substrate for various times at 37°C.
  • the CTSB activity was measured by following the emission at 460 nm (excitation at 390 nm) in the presence or absence of 1 ⁇ of CA-074Me (an inhibitor of CTSB activity). Enzyme activities were expressed in arbitrary units per mg of protein.
  • mRCC cells showed reduced proliferation in the presence of concentrations of sunitinib below the IC50 (sub-optimal concentration):
  • Fig. 1A Because of the abnormal vascularization of tumors, the core of primary mRCC or metastases is not exposed to optimal concentrations of the drug (Fig. 1A).
  • Fig. 1A We first determined the in vitro concentrations that resulted in progressive adaptation to sunitinib and final selection of resistant cells.
  • the plasma concentrations of patients or mice exposed to sunitinib was low (0.1-1 ⁇ /L range) compared to the intra-tumor amount, which was ten times higher (10 ⁇ /L range).
  • the IC50 of endothelial cells for sunitinib was approximately 0.1 ⁇ ⁇ / ⁇ 9
  • the IC50 of mRCC cells was approximately 5 ⁇ /L 3 ' 8
  • mRCC cells (Fig. IB, C) have a reduced proliferation rate, which was linked to prolonged S and G2/M phases of the cell cycle (Fig. ID).
  • a sub-optimal concentration of sunitinib (2.5 ⁇ /L) did not affect cell viability (Fig. ID, E) whereas exposure to a higher concentration resulted in cell death, as measured by cell counting or a clonogenic assay.
  • Phase contrast microscopy highlighted a modification of the cell shape and the appearance of a yellowish color inside the cells after incubation with sunitinib for two days.
  • the intracellular localization of sunitinib was confirmed by visualization of its auto- fluorescence.
  • Sunitinib auto-fluorescence co-localized with a specific lysosomal staining (Lysosome- Associated Membrane Protein 1 (LAMPl)) confirming that sunitinib accumulated in acidic lysosomal structures. Accumulation in lysosomes was also observed in two independent cell lines (RCC10 and A498) and two RCC primary cell lines (CC and TFE3) that we previously described 3 .
  • sunitinib did not accumulate in early endosomes (no co-localization with the early endosome antigen 1 (EEAl)). This result suggests accumulation of sunitinib in intracellular compartments with no major consequences to cell viability. This characteristic defines sunitinib as a lysomotropic agent 10 . FACS analysis showed that sunitinib accumulated in lysosomes in a time-dependent manner and that there was an increase in the lysosomal mass, which coincided with increased expression of LAMP 1. Such accumulation of sunitinib was not dependent on the oxygen concentration since sunitinib sequestration was equivalent in normoxia or hypoxia.
  • sunitinib neutralized the pH of lysosomes and inhibited CTSB/cathepsin B:
  • Sunitinib is a weak base (pKa 8.95), which accumulates in lysosomes where it is protonated by a pH-partitioning process 10 . Once ionized, sunitinib becomes membrane impermeable with the impossibility of diffusing out of the organelle, which results in lysosome trapping. Accumulation continues as long as the low pH is maintained by the vacuolar proton pump (V-type H+ATPase) but ultimately results in buffering of the acidic pH of lysosomes.
  • V-type H+ATPase vacuolar proton pump
  • the lysosensor DND-153 fluorescence (pKa 7.5) was intense in control conditions (this dye fluoresces in an acidic environment) but almost disappeared in the presence of sunitinib suggesting that the acidic pH of lysosomes has been neutralized. This correlated with decreased expression and activity of one of the major lysosome-associated proteases CTSB.
  • autophagy is responsible for the degradation of dysfunctional organelles and proteins and allows cell survival during nutrient deprivation n . So, autophagy is important in maintaining cell homeostasis, but if exacerbated, it can lead to cell death 12 . Sunitinib treatment resulted in an increase in the lysosomal pH and inhibition of the lysosomal protease activity. Hence, we investigated the consequences of these modifications on autophagy. Autophagy is characterized by the accumulation of lipidated forms of LC3 (LC3-II) and the degradation of SQSTMl/p62 protein.
  • LC3-II lipidated forms of LC3
  • Phase contrast microscopy clearly showed an enhanced accumulation of yellow granules when cells are cultured in HBSS medium. Quantification by FACS confirmed this qualitative observation. A high concentration (10 ⁇ /L) of sunitinib induces cell death, but at a lower concentrations (2.5 ⁇ /L), it slows proliferation without inducing cell death, as expected. However, if the cells were first cultured in HBSS medium, cell proliferation was minimally affected by 2.5 ⁇ /L sunitinib and cell death was substantially decreased, even at a high concentration of the drug (10 ⁇ /L). These results were confirmed with two other independent cell lines (RCC10 and A498 cells).
  • sunitinib-resistant cells by chronic exposure of cells to the drug (786-OR and RCCIOR). Incomplete autophagy in these cells was attested by accumulation of LC3-II and sustained expression of SQSTMl/p62.
  • Primary sunitinib-resistant cells were also derived from a RCC removed surgically from a patient, as we previously described (TFE3 cells) 3 . The 786-OR and RCC10R cells survive and proliferate in the presence of a high concentration of the drug, which is sufficient to induce parental cell death.
  • TFE3 cells are highly resistant to sunitinib in vitro even at high concentrations of the drug (IC50 10 ⁇ /L) 3 .
  • the ability to accumulate sunitinib inside 786-OR was increased compare to parental cells. As shown previously by Gotink et al. 8 , resistance (maintained several weeks) was not genetically acquired since it could be reverted by culturing the cells in the absence of the drug for a few passages (not shown). Electron microscopy showed that 786-OR cells accumulate bigger vacuolar structures, identified above as auto-lysosomes, compared to 786-OS cells when incubated in the presence of 2.5 ⁇ /L of sunitinib, a finding in favor of an exacerbated incomplete autophagy.
  • elacridar an inhibitor of ABC transporters, induced slightly 786-OS cell death at a low concentration (1 ⁇ /L) (Fig. 6A), it potentiated sunitinib activity on 786-OS cells (Fig. 2).
  • Elacridar did not significantly mediate 786-OR cell death when alone (Fig. 6A) but potentiated sunitinib activity (Fig. 2).
  • the lysomotropic agent LLOM had little effect on cell death at a low concentration (1 ⁇ /L). Higher concentrations are needed to induce cell death probably through the release of cathepsins and induction of lysosome membrane permeabilization leading finally to apoptosis 17 (Fig. 6B).
  • bafilomycin an inhibitor of the V-ATPase pump, which is responsible for the maintenance of the low pH of the lysosomes, exerted a comparable effect to LLOM (Fig. 6D).
  • the triple combination sunitinib/bafilomycin/elacridar was less potent than the sunitinib/LLOM/elacridar mix.
  • Equivalent results were obtained with an independent cell line (RCC10, Fig. 7).
  • Elacridar and LLOM alone or in combination had no effect on TFE3 cell viability.
  • Elacridar was more active in the presence of a concentration of 2.5 ⁇ /L sunitinib, (55% cell death) but this was not the case for the LLOM/sunitinib combination.
  • massive TFE3 cell death was obtained with the triple combination (Fig. 3).
  • Lysosomal trapping reduces the activity of sunitinib, since its targets, the kinase domain of tyrosine kinase receptors are located in the cytoplasm. This mechanism has been described in chronic myeloid leukemia for which agents that destabilize lysosomes revert resistance to imatinib 17 .
  • axitinib and dovitinib can be protonated at physiological pH and subsequently trapped in the lysosomes. Hence, resistance mechanisms equivalent to that described herein for sunitinib may be the cause of reduced efficacy of these drugs.
  • Pazopanib another ATP mimetic approved for the treatment of mRCC is the only drug that cannot be protonated and trapped in the lysosomes, hence not concerned by this mechanism of resistance.
  • sunitinib and pazopanib show the same overall survival 31 , but pazopanib is preferred by physicians and patients mainly for its better quality of life 32 .
  • drugs that are lysosomotropic shared certain physicochemical properties, possessing a ClogP>2 and a basic pKa between 6.5 and 11, predictably influenced their intracellular localization 33 .
  • the inventors also observed that the amine group of sunitinib, added to improve the solubility of the drug is responsible for the high pKa value.
  • the synthesis of an analog of sunitinib devoid of this amine group may prevent its accumulation in lysosomes.
  • ATF4 is a major transcription factor implicated in the adaptation to nutrient stress of tumor cells 35 .
  • ATF4 has also been implicated in resistance to cisplatin and cells overexpressing ATF4 showed multidrug resistance 36 .
  • ATF4 may be the driver of a transcriptional program leading to expression of ABCG1, as previously shown 37 38 .
  • ABCG1 may be due to a lack of its degradation.
  • membrane proteins including receptors and transporters, recycle to the plasma membrane through the recycling endosomal system.
  • Some cargo proteins sort cell membranes and discarded proteins into internal luminal vesicles of multi-vesicular bodies (early endosomes), and mature multi-vesicular bodies (late endosomes) that can fuse with lysosomes for proteolysis by lysosomal enzymes.
  • the lysosomal degradation pathway is impaired because of the modification of the lysosomal pH and could explain the decrease in ABCG1 degradation and its subsequent accumulation. Similar consequences were observed with chloroquine treatment, which resulted in Notch 1 accumulation due to a decrease in the activity of lysosomes 39 .
  • Lysosomal sequestration is rapid, occurring as soon as the drug is in contact with the target cells, and does not modify the genetic program.
  • Recent studies have also demonstrated that numerous cancer cells have defective acidification of their lysosomes.
  • lysomotropic agents would be in contact with their targets in the cytoplasm of cancer cells devoid of lysosome trapping 40 .
  • This elegant approach would limit toxicity to normal cell and would concentrate the cytotoxic/cytostatic effects on tumor cells.
  • they showed that the acidification of the lysosomes of mRCC cells was not defective. They observed that the TFE3 cells were resistant to a high concentration of sunitinib (IC50 10 ⁇ /L).
  • proteasome associated genes that are over- expressed in primary and mRCC but also in paired pulmonary metastasis (Fig. 9 A,) 44-45 .
  • the proteins encoded by these genes comprised a subset of the proteasome beta sub-units that affect the generation of peptides to promote efficient antigen recognition (PSMB8/ Proteasome subunit beta type-8, PSMB9/ Proteasome subunit beta type-9, PSMB10/ Proteasome subunit beta type- 10) and a cellular regulator of proteasome formation and of proteasome-mediated antigen processing (PSMFl /Proteasome inhibitor PI31 subunit) 47 .
  • Sunitinib acts primarily on tumor endothelium rather than tumor cells to inhibit the growth of renal cell carcinoma. Cancer Res 2010; 70: 1053-62.
  • tanton MJ Dutta S, Zhang H, Polavaram NS, Leontovich AA, Honscheid P, Sinicrope
  • Bevacizumab can induce reactivity to VEGF-C and -D in human brain and tumour derived endothelial cells. J Neurooncol 2011; 104: 103-12.
  • Ferrara N Role of myeloid cells in vascular endothelial growth factor-independent tumor angiogenesis. Curr Opin Hematol 2010; 17:219-24.
  • Activating transcription factor 4 confers a multidrug resistance phenotype to gastric cancer cells through transactivation of SIRT1 expression.
  • Zaiss DM, Standera S, Kloetzel PM, Sijts AJ. PI31 is a modulator of proteasome formation and antigen processing. Proc Natl Acad Sci U S A 2002; 99: 14344-9.

Abstract

L'invention concerne des méthodes de traitement de patients souffrant d'un adénocarcinome métastatique (mRCC) résistant aux inhibiteurs du VEGFR. Plus particulièrement, l'invention concerne un médicament ou une combinaison de médicaments sélectionnés dans le groupe constitué d'un inhibiteur de protéasome, d'un inhibiteur d'efflux ou d'une combinaison d'un inhibiteur d'efflux et d'un agent lysosomotrope, destinés à être utilisés dans une méthode de traitement du mRCC chez le patient présentant une résistance acquise au traitement par un inhibiteur multi-cible à base faible des tyrosines kinases (TKI) associées au VEGFR.
PCT/EP2016/060806 2015-05-15 2016-05-13 Méthodes de traitement de patients souffrant d'un adénocarcinome rénal métastatique résistant aux inhibiteurs du vegfr WO2016184793A1 (fr)

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WO2018189403A1 (fr) 2017-04-14 2018-10-18 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pharmaceutiques destinées au traitement du cancer
CN110575540A (zh) * 2018-06-07 2019-12-17 中山大学附属第六医院 Pdgf抑制剂用于制备治疗肠道炎症疾病的药物方面的用途
CN112423765A (zh) * 2018-04-25 2021-02-26 儿童医学中心公司 Abcb5配体和底物

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018189403A1 (fr) 2017-04-14 2018-10-18 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pharmaceutiques destinées au traitement du cancer
CN112423765A (zh) * 2018-04-25 2021-02-26 儿童医学中心公司 Abcb5配体和底物
EP3784249A4 (fr) * 2018-04-25 2022-03-02 Children's Medical Center Corporation Ligands abcb5 et substrats
CN110575540A (zh) * 2018-06-07 2019-12-17 中山大学附属第六医院 Pdgf抑制剂用于制备治疗肠道炎症疾病的药物方面的用途

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