WO2012079578A1 - Destruction sélective de cellules cancéreuses - Google Patents

Destruction sélective de cellules cancéreuses Download PDF

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WO2012079578A1
WO2012079578A1 PCT/DK2011/050468 DK2011050468W WO2012079578A1 WO 2012079578 A1 WO2012079578 A1 WO 2012079578A1 DK 2011050468 W DK2011050468 W DK 2011050468W WO 2012079578 A1 WO2012079578 A1 WO 2012079578A1
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sirna
dusp6
agent
sense
raf
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WO2012079578A8 (fr
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Jan Mollenhauer
Steffen Schmidt
Dirk Schadendorf
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Syddansk Universitet
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • C12N2310/00—Structure or type of the nucleic acid
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    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention is directed to the targeting of a tumor cell with a hyperactivated BRAF kinase and/or with hyperactivated RAS kinases and increased DUSP6 activity.
  • the invention provides a method of contacting a tumor cell with a hyperactivated BRAF kinase and/or with hyperactivated RAS kinases with an agent that reduces DUSP6 activity.
  • the invention provides a diagnostic test for therapy responder prediction.
  • Raf/Ras protein serine/threonine kinase family consists of six members, A-Raf, B-Raf, C-Raf, H-Ras, K-Ras, and N-Ras.
  • Raf/Ras family members are signaling molecules in the MAPK (Ras/Raf/MAPK/ERK kinase (M EK)/extracellular signal-regulated kinase (ERK)) pathway, which is a signal transduction pathway that relays extracellular signals from the cell membrane to the nucleus via an ordered series of consecutive phosphorylation events.
  • MAPK Ras/Raf/MAPK/ERK kinase
  • ERK extracellular signal-regulated kinase
  • an extracellular ligand binds to its tyrosine kinase receptor, leading to Ras kinase activation and initiation of a cascade of phosphorylation events.
  • Activated Ras kinases cause phosphorylation and activation of Raf kinases, which in turn phosphorylate and activate M EK1/2.
  • M E K kinases i n turn phosphorylate and activate ERK1/2, which phosphorylates several cytoplasmic and nuclear targets that ultimately lead to expression of proteins playing important roles in cell growth and survival.
  • a hyperactivation of Raf/Ras family members is well known to play a role in diverse cancer types, and results in increased activity of downstream components of the Raf/Ras-MEK- ERK signalling pathway, cumulating in increased phosphorylation of ERK1/2.
  • Events that lead to Raf/Ras hyperactivation are well known to the art and can, for example, include hyperactivating mutations, gene amplifications, increased mRNA expression levels or
  • B-Raf hyperactivation The highest frequency of B-Raf hyperactivation is found in malignant melanoma (27%- 5 70%), papillary thyroid cancer (36%-53%), colorectal cancer (5%-22%) and serous ovarian cancer (30%), but it also occurs at a low frequency (1 %-3%) in a wide variety of other cancers. Many different mutations in BRAF have been identified, but most mutations are extremely rare. The V600E mutation predominates, representing 86% of BRAF mutations in cancer (reviewed by Garnett and Marais 2004).
  • This mutation increases basal kinase activity of B-Raf, resulting in hyperactivity of the MAPK D pathway evidenced by constitutively elevated activity of downstream kinases MEK and ERK.
  • B-RAF mutations are acquired. Somatic, post-zygotic events have not been identified in familial melanomas.
  • N-Ras is frequently hyperactivated in about 20% of the malignant 5 melanoma. In the majority of the cases, this is caused by the Q61 R mutation, resulting in hyperactivity of the MAPK pathway, as evidenced by constitutively elevated activity of downstream kinases MEK and ERK.
  • malignant melanoma carries the highest risk of D mortality from metastasis.
  • the prognosis for patients in the late stages of this disease remains very poor with average survival from six to ten months.
  • Patients with metastatic (Stage IV) malignant melanoma have a median survival of approximately one year.
  • Current standard treatment consists of combination chemotherapy with agents such as cisplatin, DTIC, and BCNU, with or without cytokines such as interleukin-2 (I L-2) or interferon-a (IFN-a).
  • I L-2 interleukin-2
  • IFN-a interferon-a
  • Response rates to chemotherapy have been 5 reported to be as high as 60%, yet only approximately 5% of patients experience long-term survival, regardless of the therapeutic regimen employed.
  • Conventional chemotherapy aims to control the growth of cancer by targeting rapidly growing cells.
  • B-Raf-inhibitors such as the drug PLX4032 are presently under investigation in clinical trials. 5 While the initial data point to good response rates in melanoma patients with hyperactivated B-Raf kinase (Flaherty et al. , 2010), there are also first hints that the tumor cells may develop resistance against such drugs via hyperactivation of N-Ras (Nazarian et al., 2010; Johannessen et al. , 2010), which defines a still persisting demand for novel targeted therapeutic approaches.
  • RNA interference is a polynucleotide sequence-specific, post transcriptional gene silencing mechanism elicited by double-stranded formation of a small RNA-oligonucleotide with the messenger RNA (mRNA) of a target gene that results in degradation or in inhibition of translation of a specific mRNA, thereby reducing the expression of a desired target
  • RNAi is mediated by single-stranded RNA- polynucleotides that are commonly administered to the cells as double-stranded molecules as also described herein below, for example, double-stranded RNA (dsRNA), having sequences that correspond to exonic sequences encoding portions of the polypeptides for which expression is to be compromised or dsRNAs that correspond to the 5 ' - or 3 ' -
  • dsRNA double-stranded RNA
  • siRNA polynucleotides may offer certain advantages over other polynucleotides known to the art for use in sequence-specific alteration or modulation of gene expression to yield altered levels of an encoded polypeptide product. These advantages include lower effective 5 siRNA polynucleotide concentrations, enhanced siRNA polynucleotide stability, and shorter siRNA polynucleotide oligonucleotide lengths relative to other polynucleotides (e.g. , antisense, ribozyme or triplex polynucleotides).
  • antisense polynucleotides bind in a sequence-specific manner to target nucleic acids, such as mRNA or DNA, to prevent transcription of DNA or translation of the mRNA (see, e.g., U.S. Pat. No. 5, 168,053).
  • Ribozyme polynucleotides can be targeted to any RNA transcript and are capable of catalytically cleaving such transcripts, thus impairing translation of mRNA (see, e.g. , U .S. Pat. No. 5,272,262).
  • Triplex DNA molecules refer to single DNA strands that bind duplex DNA to form a collinear triplex molecule, thereby preventing transcription (see, e.g., U.S. Pat. No. 5, 176,996, describing methods for making synthetic oligonucleotides that bind to target sites on duplex DNA). Such triple-stranded structures are unstable and form only transiently under physiological conditions. Because single stranded polynucleotides do not readi ly diffuse into cells and are therefore susceptible to nuclease digestion, development of single-stranded DNA for antisense or triplex technologies often requires chemically modified nucleotides to improve stability and absorption by cells.
  • siRNAs by contrast, are readily taken up by intact cells, are effective at interfering with the expression of specific polypeptides at concentrations that are several orders of magnitude lower than those required for either antisense or ribozyme polynucleotides, and do not require the use of chemically modified nucleotides.
  • malignant melanoma is the skin cancer with the most significant impact on man carrying the highest risk of death from metastases. Both incidence and mortality rates continue to rise each year, with no effective long-term treatment on the horizon. In part, this reflects the lack of knowledge about critical genes involved in melanoma and specific therapies targeted to correct these defects. Accordingly, a need exists in the art for identification of critical genes involved in melanoma and specific therapies targeted to correct these defects, for exam ple by a targeted reduction of the activity of key genes/effectors in the relevant signalling pathways.
  • Identifying a gene as a selective target provides new therapeutic opportunities for cancer patients. Simultaneously, it provides methods for the identification of a patient that would profit from such therapy in the form of diagnostic kits. A distinction between patients that profit and patients that do not profit from a given treatment strategy is of critical importance for the treatment success and for avoiding unnecessary treatment of patients.
  • US 2008/0131885 A1 discloses a method for selecting an individual for treatment with a Ras/Raf/MEK ERK pathway inhibitor, comprising: (a) obtaining a cancerous tumor sample from said individual; (b) measuring the amount of a biomarker in said tumor sample, wherein said biomarker can be DUSP6; and (c) selecting said individual for treatment with a 5 Ras/Raf/MEK/ERK pathway inhibitor based on the measured amount of said biomarker.
  • the cancerous tumor can be a skin tumor, e.g. a melanoma and said tumor can comprise a B- Raf mutation.
  • WO 2009/140409 A1 discloses a method for predicting a likelihood that a human patient D having a K-Ras-negative EGFR-expressing cancer will exhibit a beneficial response to an EGFR inhibitor comprising: (a) measuring, in a tumor sample obtained from the patient, an expression level of at least one response indicator gene, or its expression product, e.g. DUSP6; (b) normalizing the expression level to obtain a normalized expression level; (c) using the normalized expression level to determine the likelihood that the patient will exhibit 5 a beneficial response to the EGFR inhibitor; and (d) generating a report based on the determined likelihood.
  • WO 2009/140409 A1 further discloses an array comprising a plurality of polynucleotides immobilized on a surface of a solid support suitable for carrying out the method and further comprising a probe that hybridizes under stringent hybridization conditions to a nucleic acid comprising an activating K-Ras mutation.
  • DUSP6 inhibition confers anticancer properties. This is surprising since up-regulated DUSP6 is understood as a natural defence towards cancer. DUSP6 is a tumor suppressor, wherefore it is very surprising that reducing its activity confers anti-cancer activity. The present inventors have
  • the present invention is based on the present inventors' discovery that inhibiting DUSP6 selectively confers cell death and/or growth reduction to cancer cel ls with DUSP6 hyperactivation in combination with either B-Raf or Ras hyperactivation.
  • the invention provides a method for killing a tumor cell having a mutation or hyperactivity of B-Raf and/or Ras kinases and preferably increased DUSP6 activity by reducing DUSP6 activity.
  • the invention provides a method for killing a tumor cell having a mutation or hyperactivity of B-Raf and/or Ras kinases and preferably increased DUSP6 activity, comprising contacting a tumor cell with an agent that reduces DUSP6 activity.
  • the tumor cell is a melanoma cell.
  • this provides a diagnostic kit for identifying a patient that would profit from such therapy, comprising a detection method for increased DUSP6 levels alone or in combination with detection of mutation or hyperactivity of B-Raf and/or Ras kinases.
  • the present inventors contemplate a method for destroying a tumor with a B-Raf and/or Ras kinase hyperactivation and preferably with increased DUSP6 activity in a patient, comprising the steps of administering to a cancer patient an effective amount of an agent to inactivate DUSP6, and optionally administering additionally to the cancer patient an effective amount of another therapeutic agent to achieve a positive therapeutic effect.
  • Also disclosed herein is a method for destroying cancer cells with B-Raf and/or Raf kinase hyperactivation and preferably with increased DUSP6 activity in a patient comprising the steps of: administering to a tumor in a mammal an effective amount of an agent that reduces DUSP6 activity in conjunction with administering to the tumor an effective amount of an agent that reduces B-Raf activity, thereby treating the tumor.
  • Also disclosed herein is a method for destroying cancer cells with B-Raf hyperactivation that has become resistant to B-Raf inhibitors via N-Ras hyperactivation.
  • Also disclosed herein is a method for avoiding resistance against B-Raf inhibitors of cancer cells with B-Raf hyperactivation by administering to a tumor in a mammal an effective amount of an agent that reduces DUSP6 activity in conjunction with administering to the tumor an effective amount of an agent that reduces B-Raf activity, thereby treating the tumor. Also disclosed herein is a method for avoiding resistance against Ras inhibitors of cancer cells with Ras hyperactivation by administering to a tumor in a mammal an effective amount of an agent that reduces DUSP6 activity in conjunction with administering to the tumor an effective amount of an agent that reduces Ras activity, thereby treating the tumor.
  • Also disclosed herein is a method for avoiding resistance against N-Ras inhibitors of cancer cells with N-Ras hyperactivation by administering to a tumor in a mammal an effective amount of an agent that reduces DUSP6 activity in conjunction with administering to the tumor an effective amount of an agent that reduces N-Ras activity, thereby treating the D tumor.
  • the methods of the present invention involves contacting of said cancer cells with a DUSP6 inhibiting agent, where the agent is a siRNA molecule, an antisense molecule, an antagonist, a ribozyme, an inhibitor, a small molecules or a peptide, that acts as a
  • the agent is an agent that acts as a competitive inhibitor for DUSP6, such as a competitive inhibitor for a catalytic domain of DUSP6 or an agent that results in downregulation of DUSP6 protein levels.
  • the agent may be a siRNA selected from Table 1 or Table 2. In the most preferred embodiment of the present invention
  • the agent is a siRNA molecule that comprises a polynucleotide selected from the group having a sequence of GUGCAACAGACUCGGAUGGUA, AGCUCAAUCUGUCGAUGAA, and the complements thereof.
  • the methods of the present invention involves contacting of said cancer cells 5 using one of more the following vehicles: a liposome, a nanoliposome, a ceramide- containing nanoliposome, a proteoliposome, a nanoparticulate, a calcium phosphor-silicate nanoparticulate, a calcium phosphate nanoparticulate, a silicon dioxide nanoparticulate, a nanocrystal i ne particulate, a sem iconductor nanoparticulate, poly(D-arginine), a nanodendrimer, a virus, calcium phosphate nucleotide-mediated nucleotide delivery, D electroporation, and microinjection.
  • a liposome a nanoliposome, a ceramide- containing nanoliposome, a proteoliposome
  • a nanoparticulate a calcium phosphor-silicate nanoparticulate
  • a calcium phosphate nanoparticulate a silicon dioxide nanoparticulate
  • the invention provides a pharmaceutical composition for eliminating cancer cells comprising: an agent that reduces DUSP6 activity and a carrier.
  • the sample is a sample of melanoma tissue.
  • the sample is selected from the group consisting of cancers with hyperactivated B-Raf kinase (eg. by the V600E mutation) and/or hyperactivated Ras kinases (eg. by the Q61 R mutation in N-Ras) and elevated levels of DUSP6 expression such as malignant melanoma, papillary thyroid cancer, lung cancer, colorectal cancer, ovarian cancer, liver cancer, leukemia and a wide variety of other cancers.
  • hyperactivated B-Raf kinase eg. by the V600E mutation
  • Ras kinases eg. by the Q61 R mutation in N-Ras
  • elevated levels of DUSP6 expression such as malignant melanoma, papillary thyroid cancer, lung cancer, colorectal cancer, ovarian cancer
  • the present invention provides a method for inducing apoptosis in a melanoma tumor cell having a B-Raf mutation or exhibiting B-Raf hyperactivity and/or having a Ras kinase mutation or exhibiting Ras kinase hyperactivity by reducing DUSP6 activity.
  • the melanoma tumor cell is contacted with an agent that reduces DUSP6 activity.
  • an agent is preferably a siRNA molecule, an antisense molecule, an antagonist, a ribozyme, an inhibitor, a peptide, or a small molecule.
  • the agent may be a siRNA selected from Table 1 or Table 2.
  • the agent is a siRNA molecule that comprises a polynucleotide selected from the group having a sequence of GUGCAACAGACUCGGAUGGUA, AGCUCAAUCUGUCGAUGAA, and the complements thereof.
  • the agent is preferably administered in a liposome, a nanoliposome, a ceramide-containing nanol i posome, a proteol iposome, a nanoparticulate, a calcium phosphor-silicate nanoparticulate, a calcium phosphate nanoparticulate, a silicon dioxide nanoparticulate, a nanocrystali ne particulate, a sem iconductor nanoparticulate, poly(D-arginine), a nanodendrimer, a virus, calcium phosphate nucleotide-mediated nucleotide delivery, by electroporation, or by microinjection.
  • the agent may be administered along with a chemotherapeutic agent selected from the group consisting of alkylating agents, antimetabolites, antibiotics, natural or plant derived products, hormones and steroids, and platinum drugs.
  • a chemotherapeutic agent selected from the group consisting of alkylating agents, antimetabolites, antibiotics, natural or plant derived products, hormones and steroids, and platinum drugs.
  • the present invention also provides a pharmaceutical composition for treating a melanoma tumor, having a B-Raf mutation or exhibiting B-Raf hyperactivity and/or having a Ras kinase mutation or exhibiting Ras kinase hyperactivity, comprising: an agent that reduces DUSP6 activity; and a carrier.
  • the carrier is selected from a group consisting of: a liposome, a nanoliposome, a ceramide-containing nanoliposome, a proteoliposome, a nanoparticulate, a calcium phosphor-silicate nanoparticulate, a calcium phosphate nanoparticulate, a silicon dioxide nanoparticulate, a nanocrystaline particulate, a semiconductor nanoparticulate, poly(Darginine), a nanodendtimer, a virus, and calcium phosphate nucleotide-mediated nucleotide delivery.
  • the DUSP6 inhibiting agent is preferably a si RNA molecule, an antisense molecule, an antagonist, a ribozyme, an inhibitor, a small molecules or a peptide, that acts as a pseudosubstrate for DUSP6, such as a pseudosubstrate for a catalytic domain or a regulatory domain of DUSP6.
  • the agent is an agent that acts as a competitive inhibitor for DUSP6, such as a competitive inhibitor for a catalytic domain of DUSP6 or an agent that results in downregulation of DUSP6 protein levels.
  • the agent may be a siRNA selected from Table 1 or Table 2.
  • the agent is a siRNA molecule that comprises a polynucleotide selected from the group having a sequence of GUGCAACAGACUCGGAUGGUA, AGCUCAAUCUGUCGAUGAA, and the complements thereof.
  • the present invention provides a method for detecting a cancer cell that is susceptible to this treatment, said method comprising: detecting a level of DUSP6 or fragment thereof in a test sample obtained from a cell of a subject, comparing the level of DUSP6 to a control level of DUSP6, wherein the presence of a cancerous cell is indicated by detection of an increased level of DUSP6 relative to the control.
  • BRAF and/or RAS mutations or increased levels of BRAF and/or RAS in conjunction with the increased DUSP6 level may be performed.
  • Fig. 1 shows exon structure of the two DUSP6 variants and location of the siRNAs.
  • Fig. 2 shows mRNA sequence of a long DUSP6 variant (NM_001946.2).
  • Fig. 3 shows mRNA sequence of a short DUSP6 variant (NM_022652.2).
  • Fig. 4 shows protein sequence of a long DUSP6 variant (NM_001946.2).
  • Fig. 5 shows protein sequence of a short DUSP6 variant (NM_022652.2).
  • Fig. 6 shows double stranded siRNA sequences.
  • Fig. 7 shows that DUSP6 mRNA levels are elevated in cancer cells (melanoma) with mutations resulting in hyperactivation of B-Raf and Ras kinases.
  • Fig. 8 confirms the knockdown of the DUSP6 protein due to treatment with siRNA-du1 and siRNA-du2.
  • Fig. 9 is a diagram displays the relative expression-levels of DUSP6 mRNA in the same cancer cell line MaMel73b (melanoma) treated under identical conditions (25nM siRNA-du1 , siRNA-du2 and siRNA Ctrl.
  • Fig.10 shows differential effects on the viabil ity of cancer cells (melanoma) with hyperactivation of Raf/Ras-MEK-ERK signalling pathways.
  • Fig 1 1 shows selective effect of DUSP6 inactivation in cancer cells (melanoma) with hyperactivation of Raf/Ras-MEK-ERK signalling pathways and inccreased DUSP6 activity.
  • Fig. 12 shows that the reduction of cell viability is based on apoptosis in cancer cells (melanoma) with hyperactivation of Raf/Ras-MEK-ERK signalling pathways and inccreased DUSP6 activity.
  • Fig. 13 shows a time course of the apoptotic effect of DUSP6 inactivation in cancer cells (melanoma) with hyperactivation of Raf/Ras-MEK-ERK signalling pathways and inccreased DUSP6 activity.
  • Fig. 14 shows the effect of exemplarily selected siRNAs from table 1 on the viability of cells with B-Raf and DUSP6 hyperactivation (UKRV Ma-Mel 6b) and without B-Raf D hyperactivation (MM Bank Ma-Mel 73b).
  • Dual specificity phosphatase 6 (DUSP6, also known as MKP3 or PYST1) is well known as tumor suppressor (Zhang et al. 2010; Furukawa et al, 2003), thus at the present state of the art, the inactivation of DUSP6 is thought to promote tumorigenesis, which disqualifies a DUSP6 inactivation for use in cancer therapy.
  • DUSP6 transcripts There are presently two DUSP6 transcripts known (Access, nos. NM_001946.2 and NM_022652.2; Fig 1-3), emerging by alternative
  • siRNA-du1 corresponded to duplex2, previously published to efficiently reduce DUSP6 protein levels (Zeliadt et al., 2008).
  • siRNA-du2 corresponds to the commercially available siRNA s4380 (Silencer® Pre-Designed & Validated siRNAs) from Applied Biosystems, verified to reduce target mRNA levels by 70% or greater.
  • a set of 81 melanoma (45 with hyperactivating B-Raf mutation, 18 with hyperactivating N-Ras mutation, 18 with neither hyperactivating B-Raf nor hyperactivating N-Ras mutation) were analyzed for elevated mRNA levels by quantitative RT-PCR (Fig. 7).
  • Relative transcript levels of DUSP6 were assayed with the Hs00169257_m 1 TaqMan® Gene Expression Assay (Applied Biosystems) and normalized to the Endogenous Control Expression Assays for Human GAPDH (Applied Biosystems). The following thermal cycling parameters were used: 50°C for 2 min and 95°C for 15 min followed by 40 cycles of 95°C for 15 sec.
  • the levels of the inidividual tumors were referred to the level of the matched normal tissue (NH EM ; Normal Human Epidermal Melanocytes, adult donor; PromoCell), which was set to 1.
  • NH EM Normal Human Epidermal Melanocytes, adult donor; PromoCell
  • the results from the inidividual tumors were averaged, which reveals a significant upregulation of DUSP6 mRNA levels in both, tumors (melanoma) with hyperactivated B-Raf kinase and/or hyperactivated Ras kinases (N-Ras).
  • the cells were cultured in RPM I medium (Invitrogen) with 10% fetal calf serum, 2mM L-Glutamin, penicillin (100 U/mL) and streptomycin (100 mg/mL) in a humidified atmosphere with 5% C0 2 at 37°C.
  • RPM I medium Invitrogen
  • 12.500 cells per well were seeded into 24-well plates and cultured over night. Afterwards, the medium was replaced with fresh medium and cells transfected with 25nM si RNA usi ng Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to manufacturer's protocol.
  • the media was replaced 24h after transfection and cells cultured for additional 96h before viability was assayed. Relative numbers of viable cells were determined with the fluorometric CellTiter-Blue reagent (Promega) and the fluorescence signal detected at the wavelengths 560/590 nm with a Victor3 multilabel counter (PerkinElmer).
  • Fig. 8 is a confirmation of DUSP6 knockdown via siRNA-du1 and siRNA-du2.
  • Protein extracts were prepared from the cancer cell line MaMel73b (melanoma) after treatment with 25nM siRNA-du 1 or si R NA-du2 and corresponding control treatment with siRNA-Ctrl. (Allstars control siRNA from Qiagen not targeting a cellular gene) and in addition also untreated cells without any treatment grown under normal conditions. Proteins were separated by SDS-PAGE and transferred by Western blotting. Afterwards, detection of
  • 5 DUSP6 was performed using the antibody MKP-3 (C-20) sc-8599 (goat polyclonal) from Santa Cruz Biotechnology.
  • a beta-tubulin antibody ( ⁇ -Tubulin (H-235) sc-9104; rabbit polyclonal from Santa Cruz Biotechnology) was used on the same membrane to confirm equal loading in the lanes.
  • the following secondary antibodies were used: HRP-conjugated (Fab2)-fragment donkey anti-goat I gG (H + L) and Rabbit a-mouse IgG (H+L), both
  • the same cancer cell line MaMel73b (melanoma) was confirmed for siRNA mediated silencing of DUSP6 mRNA via quantitative RT-PCR (Fig. 9).
  • the diagram in Fig. 9 displays the relative DUSP6 expression-levels of the same cancer cell line MaMel73b (melanoma) treated under identical conditions (25nM siRNA-du1 , siRNA-du2 and siRNA Ctrl, respectively as well as untreated sample).
  • Total RNA was purified from the transfected cells and reversely transcribed. Relative transcript levels of DUSP6 were
  • Viability data are based on at least 3 independent assays.
  • Fig 1 1 The selective effect of DUSP6 inactivation is shown in Fig 1 1 .
  • the diagram displays the 5 average relative DUSP6 expression levels of the different tumors (in this example melanoma) in relation to the matched normal tissue (NH EM; Normal Human Epidermal Melanocytes, adult donor; PromoCell). GAPDH served as the reference gene for the determination of the mRNA levels.
  • the top panel depicts the BRAF-mutation status (red/1 : activating BRAF mutation, e.g. V600E mutation; green/0: no activating BRAF mutation), and the effects of siRNA-du1 and siRNA-du2 on the viability of the cancer cells.
  • the numbers depict relative remaining cell viability compared to control-treated cells after treatment with the respective siRNA.
  • the cells are cultured in RPMI medium (Invitrogen) with 10% fetal calf serum, 2mM L-Glutamin, penicillin (100 U/mL) and streptomycin (100 mg/mL) in a humidified atmosphere with 5% C0 2 at 37°C.
  • RPMI medium Invitrogen
  • 12.500 cells per well are seeded into 24-well plates and cultured over night. Afterwards, the medium is replaced with fresh medium and cells transfected with 25nM siRNA using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to manufacturer's protocol.
  • the media are replaced 24h after transfection and cells cultured for additional 96h before viability are assayed.
  • Relative numbers of viable cells are determined with the fluorometric CellTiter-Blue reagent (Promega) and the fluorescence signal is detected at the wavelengths 560/590 nm with a Victor3 multilabel counter (PerkinElmer).
  • DUSP6 The reduction of DUSP6 at the protein and mRNA level is determined as described above for the examples shown in Fig. 8 and 9.
  • DUSP6 inactivation in tumors with Ras (N-Ras in this example) kinase hyperactivation is determined in analogy to Fig 1 1 .
  • a reduction of cell viability equivalent to the one observed for tumors with B-Raf kinase hyperactivation and DUSP6 hyperactivation is observed for tumors with Ras (N-Ras) kinase hyperactivation and DUSP6 hyperactivation.
  • a selective elimination of tumors with Ras kinase and DUSP6 hyperactivation can also be achieved.
  • the diagram in fig. 12 displays the caspase-3 and -7 activity of in relation to the viable cells 120 h after treatment with the D US P6-inhibiting siRNA-du 1 and si RNA-du2 in the responsive MaMel7 cells as an example for a tumor with B-Raf or Ras ki nase hyperactivation (in this example B-Raf hyperactivation). From fig. 12, it can be seen that inhibition of DUSP6 eliminates cancer cells by inducing apoptosis.
  • FIG. 13 shows the caspase-3 and -7 activity of in relation to the viable cells 48h, 72h and 120 h after treatment with the DUSP6-inhibiting siRNA-du2 in the responsive U KRVM el6b and M aM el 7 cel l s exam ples for tu mors with B-Raf or Ras kinase 5 hyperactivation (in this example B-Raf hyperactivation). From fig. 13, it can be seen that inhibition of DUSP6 eliminates cancer cells by inducing apoptosis.
  • the cells are cultured in RPM I medium (Invitrogen) with 10% fetal calf serum, 2mM L- Glutamin, penicillin (100 U/mL) and streptomycin (100 mg/mL) in a humidified atmosphere with 5% C02 at 37°C.
  • RPM I medium Invitrogen
  • penicillin 100 U/mL
  • streptomycin 100 mg/mL
  • 2.000 cells per well are seeded into 96-well plates and cells are transfected with 20 nM siRNA using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to manufacturer's protocol and using 5 one of the siRNAs listed in table 1.
  • the media are replaced 24h after transfection and cells cultured for additional 96h before viability are assayed.
  • Relative numbers of viable cells are determined with the fluorometric CellTiter-Blue reagent (Promega) and the fluorescence signal is detected at the wavelengths 560/590 nm with a Victor3 multilabel counter (PerkinElmer).
  • DUSP6 The reduction of DUSP6 at the protein and mRNA level is determined as described above for the examples shown in Fig. 8 and 9.
  • FIG. 13 shows exemplarily the effects of siRNA-du84, siRNA-du85, siRNA-du86, siRNA-du87 from table 1 and of a pool of these four siRNAs on the cell viability of the UKRV Ma-Mel 6b (with B-Raf kinase and DUSP6 hyperactivation) and MM Bank Ma- 5 Mel 73b (without B-Raf hyperactivation) demonstrating that any siRNA randomly selected from table 1 would exert the effect.
  • the cells are cultured in RPMI medium (Invitrogen) with 10% fetal calf serum, 2mM L-Glutamin, penicillin (100 U/mL) and streptomycin (100 mg/mL) in a humidified atmosphere with 5% C02 at 37°C.
  • RPMI medium Invitrogen
  • penicillin 100 U/mL
  • streptomycin 100 mg/mL
  • DUSP6 The reduction of DUSP6 at the protein and mRNA level is determined as described above for the examples shown in Fig. 8 and 9.
  • DUSP6 inactivation in tumors with Ras (N-Ras in this example) kinase hyperactivation is determined in analogy to Fig. 1 1 and Fig. 14.
  • a reduction of cell viability equivalent to the one observed for tumors with B-Raf kinase hyperactivation and DUSP6 hyperactivation is observed for tumors with Ras (N-Ras) kinase hyperactivation and D DUSP6 hyperactivation.
  • a selective elimination of tumors with Ras kinase and DUSP6 hyperactivation can also be achieved with any of the siRNAs listed in table 1.
  • siRNA-du1 sense: 5 " -GUGCAACAGACUCGGAUGGUA ⁇ .i-3 3 ⁇ 4 SEQ ID NO 1 anti-sense: S ' -UACCAUCCGAGUCUGUUGCACtt-S ' siRNA-du2 sense: 5 3 ⁇ 4 -AGCUCAAUCUGUCGAUGAAtt-3 3 ⁇ 4 SEQ ID NO 2 anti-sense: 5 ' -UUCAUCGACAGAUUGAGCUtc-3 ' siRNA-du3 sense: 5'-GAGACGCUCGCUGUUUGUAUCCAUUtt-3' SEQ I D NO 3 anti-sense: 5'-AAUGGAUACAAACAGCGAGCGUCUCtt-3' siRNA-du4 sense: 5'-CAAGAGAUAGCAAAUCGAGUCUUAAtt-3' SEQ I D NO 4 anti-sense: 5'-UUAAGACUCGAUUUGCUAUCUUGtt-3' siRNA-du5 sense: 5'-CAGUAGCACG
  • siRNA-du19 sense: 5'-GAGAUAGCAAAUCGAGUCUUAtt-3' SEQ ID NO 19 anti-sense: 5'-UAAGACUCGAUUUGCUAUCUCtt-3' siRNA-du20 sense: 5'-GGGAUUAGAAGCCGCUAGACUtt-3' SEQ ID NO 20 anti-sense: 5'-AGUCUAGCGGCUUCUAAUCCCtt-3' siRNA-du21 sense: 5'-GGAUUAGAAGCCGCUAGACUUtt-3' SEQ ID NO 21 anti-sense: 5'-AAGUCUAGCGGCUUCUAAUCCtt-3' siRNA-du22 sense: 5'-GAGAGAACCUCCGGCUUUACUtt-3' SEQ ID NO 22 anti-sense: 5'-AGUAAAGCCGGAGGUUCUCUCUCtt-3' siRNA-du23 sense: 5'-GGUAAGGCGAGGCGGAAUUAAtt-3
  • siRNA-du37 sense: 5'-GAUUAGAAGCCGCUAGACUtt-3' SEQ ID NO 37 anti-sense: 5'-AGUCUAGCGGCUUCUAAUCtt-3' siRNA-du38 sense: 5'-AGUCCGAAUUAAUUGGAUUtt-3' SEQ ID NO 38 anti-sense: 5'-AAUCCAAUUAAUUCGGACUtt-3' siRNA-du39 sense: 5'-GGGCAGCUUCAUUGAGAGAtt-3' SEQ ID NO 39 anti-sense: 5'-UCUCUCAAUGAAGCUGCCCtt-3' siRNA-du40 sense: 5'-GCAGCUUCAUUGAGAGAGAtt-3' SEQ ID NO 40 anti-sense: 5'-UCUCUCUCAAUGAAGCUGCtt-3' siRNA-du41 sense: 5'-CUUCAUUGAGAGAGAUUCAtt-3' SEQ ID NO 41 anti-sense: 5'-UGAAUCUCUCAAUGAAGtt-3
  • siRNA-du54 sense: 5'-CUGCAAUCUACGUGAAAGAtt-3' SEQ ID NO 54 anti-sense: 5'-UCUUUCACGUAGAUUGCAGtt-3' siRNA-du55 sense: 5'-CGGAAUUGGUUAAUACUAAtt-3' SEQ ID NO 55 anti-sense: 5'-UUAGUAUUAACCAAUUCCGtt-3' siRNA-du56 sense: 5'-AGGACAUGCUGUAUAGAUAtt-3' SEQ ID NO 56 anti-sense: 5'-UAUCUAUACAGCAUGUCCUtt-3' siRNA-du57 sense: 5'-AG AU ACAGGCAG U AGG U U Utt-3' SEQ ID NO 57 anti-sense: 5'-AAACCUACUGCCUGUAUCUtt-3' siRNA-du58 sense: 5'-CCAUGCAGGGACUGGGAUUtt-3' SEQ ID NO 58 anti-sense: 5'-AAUCCCAGUCCCUGC
  • siRNA-du71 sense: 5'-AAUCAUGGGCUCACUUUAAtt-3' SEQ ID NO 71 anti-sense: 5'-UUAAAGUGAGCCCAUGAUUtt-3' siRNA-du72 sense: 5'-ACCCAUUUGAUAAGAGAAAtt-3' SEQ ID NO 72 anti-sense: 5'-UUUCUCUUAUCAAAUGGGUtt-3' siRNA-du73 sense: 5'-UGCAUUUGAUUGUGAAGAAtt-3' SEQ ID NO 73 anti-sense: 5'-UUCUUCACAAUCAAAUGCAtt-3' siRNA-du74 sense: 5'-UGAAGAAGGGAGAGUUAAAtt-3' SEQ ID NO 74 anti-sense: 5'-UUUAACUCUCCCUUCUUCAtt-3' siRNA-du75 sense: 5'-CCAUUAUGUUCGUGGUGUAtt-3' SEQ ID NO 75 anti-sense: 5'-UACACCAC
  • Flaherty KT Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, O'Dwyer PJ, Lee RJ, Grippo JF, Nolop K, Chapman PB. I nhibition of mutated, activated BRAF in metastatic D melanoma. N Engl J Med. 2010 Aug 26;363(9):809-19.
  • Fukushima T Takenoshita S. Roles of RAS and BRAF mutations in thyroid carcinogenesis. Fukushima J Med Sci. 2005 Dec;51 (2):67-75.
  • B-RAF is a human oncogene. Cancer Cell. 2004 D Oct;6(4):313-9.
  • Dual specificity phosphatase 6 is an ETS-regulated negative feedback mediator of oncogenic ERK signaling in lung cancer cells. Carcinogenesis. 2010 Apr;31 (4):577-86.

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Abstract

L'invention concerne un procédé pour éliminer une cellule tumorale présentant une kinase B-Raf ou une kinase Ras hyperactivées et une activité DUSP6 augmentée. En particulier, l'invention concerne un procédé de mise en contact d'une cellule tumorale présentant une kinase B-Raf ou une kinase Ras hyperactivées avec un agent qui réduit l'activité de DUSP6. De plus, elle concerne un test diagnostique pour la prédiction de la réponse à la thérapie.
PCT/DK2011/050468 2010-12-15 2011-12-07 Destruction sélective de cellules cancéreuses WO2012079578A1 (fr)

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US10006027B2 (en) 2014-03-19 2018-06-26 Ionis Pharmaceuticals, Inc. Methods for modulating Ataxin 2 expression
US11078486B2 (en) 2018-07-25 2021-08-03 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing ATXN2 expression

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3119888A4 (fr) * 2014-03-19 2018-02-28 Ionis Pharmaceuticals, Inc. Compositions permettant de moduler l'expression de l'ataxine 2
US10006027B2 (en) 2014-03-19 2018-06-26 Ionis Pharmaceuticals, Inc. Methods for modulating Ataxin 2 expression
US10308934B2 (en) 2014-03-19 2019-06-04 Ionis Pharmaceuticals, Inc. Compositions for modulating Ataxin 2 expression
US10533178B2 (en) 2014-03-19 2020-01-14 Ionis Pharmaceuticals, Inc. Methods for modulating Ataxin 2 expression
US11111494B2 (en) 2014-03-19 2021-09-07 Ionis Pharmaceuticals, Inc. Compositions for modulating Ataxin 2 expression
US11834660B2 (en) 2014-03-19 2023-12-05 Ionis Pharmaceuticals, Inc. Compositions for modulating Ataxin 2 expression
US11078486B2 (en) 2018-07-25 2021-08-03 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing ATXN2 expression
US11926825B2 (en) 2018-07-25 2024-03-12 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing ATXN2 expression

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