EP3846799A1 - Polythérapie inhibant l'expression, l'activité et/ou la fonction du récepteur du facteur de croissance épidermique et c-raf contre le cancer - Google Patents

Polythérapie inhibant l'expression, l'activité et/ou la fonction du récepteur du facteur de croissance épidermique et c-raf contre le cancer

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Publication number
EP3846799A1
EP3846799A1 EP19745113.1A EP19745113A EP3846799A1 EP 3846799 A1 EP3846799 A1 EP 3846799A1 EP 19745113 A EP19745113 A EP 19745113A EP 3846799 A1 EP3846799 A1 EP 3846799A1
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EP
European Patent Office
Prior art keywords
raf
egfr
expression
inhibitor
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19745113.1A
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German (de)
English (en)
Inventor
Mariano Barbacid
Carmen Guerra
María Teresa Blasco
Carolina Navas
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Fundacion del Sector Publico Estatal Centro Nacional de Investigaciones Oncologicas Carlos III FSP CNIO
Original Assignee
Fundacion del Sector Publico Estatal Centro Nacional de Investigaciones Oncologicas Carlos III FSP CNIO
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Priority claimed from EP18382555.3A external-priority patent/EP3598973A1/fr
Application filed by Fundacion del Sector Publico Estatal Centro Nacional de Investigaciones Oncologicas Carlos III FSP CNIO filed Critical Fundacion del Sector Publico Estatal Centro Nacional de Investigaciones Oncologicas Carlos III FSP CNIO
Publication of EP3846799A1 publication Critical patent/EP3846799A1/fr
Pending legal-status Critical Current

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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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    • 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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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
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    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/203—Animal model comprising inducible/conditional expression system, e.g. hormones, tet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy

Definitions

  • the present invention relates to a combined therapy against cancer which has proven to be particularly useful in the prevention and treatment of pancreatic cancer.
  • Pancreatic ductal adenocarcinoma is the third cause of cancer deaths in the US and is projected to be second after non-small cell lung cancer (NSCLC) by 2030 (Rahib et al., 2014) 1 .
  • NSCLC non-small cell lung cancer
  • Gemcitabine a nucleoside analogue approved in 1997, is still the standard of care 2 3 , and its combination with nab-paclitaxel or erlotinib has shown only modest improvements 4 5 .
  • Other therapies such as FOLFIRINOX are very toxic and can only be administered to selected patients 6 .
  • the main genetic drivers of PDAC have been identified 7 .
  • K-RAS oncogenic signaling is mediated by two signaling cascades made up of druggable kinases 11 .
  • Previous studies have shown that ablation of the EGF receptor (EGFR) delayed PDAC development in K-Ras/Tp53 driven GEM tumor models 12 13 .
  • c-Raf as a suitable therapeutic target for K-Ras/Tp53 driven lung adenocarcinoma 14 .
  • systemic ablation of EGFR and c-Raf expression in advanced K-Ras/Tp53 mutant PDAC tumors results in complete regression of a significant percentage of tumors with tolerable toxicities.
  • WO2015087279 A1 describes the triple combination of a B-RAF inhibitor (dabrafenib), a MEK inhibitor (trametinib) and a EGFR inhibitor (panitumumab) and its use in the treatment of cancer. It has been described that the inhibition of B-Raf is not useful because it inhibits the complete pathway and its toxicity is quite high (Blasco et al 201 1 Cancer Cell 19, 652- 663).
  • Inhibiting K-Ras and MEK also implies intolerable side effects due to the toxicity of the treatment.
  • the inventors have shown that the simultaneous deletion of Egfr and c-Raf in PDAC results in a significant therapeutic effect with an extremely low toxicity.
  • the present invention represents a very promising therapy in the treatment of pancreatic cancer since it leads to a complete tumor regression while its side effects are much smaller than other therapies described up to date.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of the expression, activity and/or function of c-Raf and an inhibitor of the expression, activity and/or function of EGF Receptor (EGFR).
  • EGFR EGF Receptor
  • the inhibitor of the expression, activity and/or function of c-Raf does not inhibit C-RAF kinase activity.
  • MAPK and Pi3K pathways are not affected by the pharmaceutical composition of the invention.
  • the expression“are not affected” as used herein means that the MAPK and Pi3K pathways are not inhibited by the pharmaceutical composition of the invention.
  • the inhibitor of the expression, activity and/or function of c-Raf comprises a c-Raf inhibitor compound, a c-Raf inhibitor antibody or an antigen binding fragment thereof, a peptide or a nucleotide sequence.
  • a c-Raf inhibitor compound As illustrated in the examples, the inventors have shown that inhibiting the expression of c-Raf in combination with inhibiting Egfr leads to a complete tumor regression. The inhibition of c-Raf expression achieved by shRNA is very effective and specific.
  • the inhibitor of the expression, activity and/or function of c-Raf does not inhibit C-RAF kinase activity. In a preferred embodiment, the inhibitor of the expression, activity and/or function of c-Raf inhibits the expression of c-Raf or promotes C- RAF degradation. In another preferred embodiment, the inhibitor of the expression, activity and/or function of c-Raf blocks C-RAF kinase independent activities.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of the expression, activity and/or function of c-Raf and an inhibitor of the expression, activity and/or function of EGF Receptor (EGFR), wherein the inhibitor of the expression, activity and/or function of c-Raf:
  • the inhibitor of the expression, activity and/or function of c-Raf is other than sorafenib, vemurafenib, dabrafenib y LY3009120.
  • the inhibitor of the expression, activity and/or function of c-Raf does not inhibit the expression, activity and/or function of b-Raf.
  • the inhibitor of the expression, activity and/or function of c-Raf promotes C-RAF degradation.
  • said inhibitor is a proteolysis targeting chimera (PROTAC).
  • the inhibitor of the expression, activity and/or function of EGFR comprises an EGFR inhibitor compound, an EGFR inhibitor antibody or an antigen binding fragment thereof, a peptide or a nucleotide sequence.
  • the nucleotide sequence is or codifies for a guide RNA, an interfering RNA or a micro RNA.
  • the inhibitor of the expression, activity and/or function of EGFR is selected from afatinib, erlotinib, gefitinib, brigatinib, icotinib, neratinib, lapatinib, vandetanib, osimertinib, cetuximab, panitumumab, necitumumab, nimotuzumab, zalutumumab, matuzumab and combinations thereof.
  • the pharmaceutical composition comprises a therapeutically effective amount of an inhibitor of the expression, activity and/or function of c-Raf, a therapeutically effective amount of an inhibitor of the expression, activity and/or function of EGFR and optionally a pharmaceutically acceptable excipient.
  • the present invention relates to the pharmaceutical composition according to any one of the preceding claims for use in the prevention and/or treatment of cancer.
  • the pharmaceutical composition is for use in tumor regression in a subject afflicted with cancer.
  • for use in tumor regression in a subject afflicted with pancreatic cancer More preferably, for use in tumor regression in a subject afflicted with pancreatic intraepithelial neoplasia or of pancreatic ductal adenocarcinoma.
  • An aspect of the present invention is a method for the prevention and/or treatment of cancer comprising the administration of a therapeutically effective amount of an inhibitor of the expression, activity and/or function of c-Raf and a therapeutically effective amount of an inhibitor of the expression, activity and/or function of EGF Receptor (EGFR).
  • the method of the present invention is for the treatment and/or prevention of pancreatic cancer, more preferably of pancreatic intraepithelial neoplasia (PanIN) or of PDAC.
  • mice All mice died of PDAC tumors at the indicated times d, PCR analysis of c -Raf and Egfr alleles using DNA extracted from K-Ras G12V expressing, X-Gal positive acinar cells isolated by Laser Capture Microdissection. Migration of c -Raf- and Egfr- null alleles (lane 1 ) c-Raf ox and Egff ox conditional floxed alleles (lane 2) and c-Raf + and Egfr wild type alleles (lane 3).
  • Lane 6 is a blank control. Lane M depicts a DNA ladder. Expected size for each DNA fragment is indicated.
  • FIG. 1 Ablation of c-Raf and Egfr expression induces acceptable toxicities.
  • a Western blot analysis of EGFR, c-RAF, pERK1 /2, ERK1/2, pAKT and AKT expression in tissues obtained from Egfr l+ Raf1 +l+ ,Tg.
  • GAPDH served as a loading control b
  • Scale bars represent 100 pm (H&E) and 20 pm (toluidine blue) d, Representative H&E and cleaved Caspase-3 staining in sections of small intestine of c-Rafr + ;Egfr +/+ ;Tg.h L/EC-CreERT2 +/T and c-Raf 0Xd0X ;Egfr 0X/l0X ;Tg.h L ⁇ C-CreERT2 +/T mice exposed to TMX for 15 weeks. Scale bars represent 100 pm (H&E) and 20 pm (cleaved Caspase-3).
  • c Representative ultrasound images of the regression of a large tumor (23.5 mm 3 ) present in the R3 mouse after 3 and 6 weeks of TMX exposure. Visible lesions are outlined. Tumor volumes are indicated. ND: not detectable d, (left column) Representative H&E stained paraffin sections of the pancreata of control KPeFC;c -Raf +l+ ,Egfr +/+ C1 and KPeFC;c- Ra/’ ox/lox ;Eg / ox/lox R1 and R3 mice after six weeks of TMX exposure. The tumor present in the C1 mouse is outlined by a dotted line. Scale bar represents 1000 pm.
  • Scale bar represents 20 pm.
  • the R2 mouse did not had a scar lesions g
  • b Representative ultrasound images of the progression of the tumor present in the NR2 mouse after 3 weeks of TMX exposure. Visible lesions are outlined in white.
  • Tumor volumes are indicated c, Western blot analysis of c- Raf and Egfr expression in lysates obtained from PDAC tumors present in control KPeFC;c- Raf +,+ ,Egfr ,+ mice depicted in Figure 3a (C1 -C3) and in NR1 -NR3 KPeFC;c -Raf ox/lm -,Egff ox,lox mice exposed to TMX for six weeks. Expression levels of Erk1/2 pErk1/2, Akt and pAkt are also shown.
  • Gapdh served as a loading control d
  • (left column) Representative H&E stained paraffin sections of the pancreata of control C2 KPeFC;c -Raf +l+ ,Egfr +/+ mouse ( depicted in Fig 3a> and of non-responder NR1 and NR2 KPeFC;c -Raf mnm Eg mnm mice after six weeks of TMX exposure. Tumors are outlined by dotted lines. Box insets, indicated by arrowheads, mark areas shown at higher magnification in the adjacent images shown to the right stained with H&E, Cytokeratin19 (CK19), pErk and Ki67. Scale bar represents 50 pm.
  • RNA expression profiles of PDAC tumor cells sensitive and resistant to c-Raf and Egfr ablation a Colony formation assay of tumor cell lines established from individual tumors of two KPeFC;c -Raf +l+ ,Egfr +l+ control mice (C1 and C2) and of six KPeFC;c-Raf ox/lox ;Egfr o, ⁇ /lo, ⁇ animals
  • These tumor cell lines are designated as“Responder cells” (RC1 -RC3) or Non responder cells (NRC1 -NRC3) based on their proliferative properties after ablation of c-Raf and Egfr expression with Ad-Cre particles.
  • e Heat map comparing the transcriptional profiles of RC and NRC cells with those of PDAC tumors as reported by Bailey and cols. e2, Genes selected from the two thousand genes differentially expressed between RC and NC cells based on their involvement in signaling pathways known to participate in the development and/or progression of PDAC. Genes are ordered according to the log2 fold change f, Western blot analysis of Egfr, c-Raf, pAkt, Akt, pErk1/2, Erk1/2, pCofilin, pStat3 and Stat3 protein expression in whole cell extracts of the indicated cell lines obtained 5 days after (top) Adeno-GFP or (bottom) Adeno-Cre infection.
  • Gapdh served as a loading control g, pStat3 staining in paraffin-embedded sections of PDAC tumors of KPeFC;c -Raf +l+ ,Egfr +l+ C1 mouse and KPeFC,c- Raf mllm Egff mnm mice that harbored tumors that regressed (R3), progressed (NR1 , NR3) or relapsed (RT1 , RT2) upon c-Raf and Egfr ablation. Scale bar represents 20 pm.
  • FIG. 6 c-Raf and Egfr expression are required for proliferation of patient-derived PDAC xenografts.
  • PDX1 -4 Western blot analysis of Egfr and c-Raf protein expression in whole cell extracts obtained from the indicated PDX cell line (PDX1 -4) expressing a scramble shRNA (-) as well as shRNAs against Egfr (E), c-Raf ⁇ R) and Egfr + c-Raf ⁇ E/R).
  • Gapdh served as a loading control.
  • FIG. 8 PDAC tumors of KPeFC mice become resistant to c-Raf and Egfr ablation a, Total tumor volume visualized by weakly ultrasound monitorization of KPeFC;c- ⁇ a tox/iox . g, ⁇ jox/iox mjce exposed to TMX.
  • Each color represents a different mouse b, Western blot analysis of c-Raf and Egfr expression in lysates derived from the PDAC tumors present in KPeFC;c-Rafr /+ ;Egfr /+ control C1 mouse depicted in Figure 3a and KPeFC;c-Raf l0X/l0X ;Egfr 0X/l0X RT2 mouse exposed to TMX for 1 1 weeks.
  • Gapdh served as a loading control c, (left column) Representative H&E stained paraffin sections of the pancreata of KPeFC;c- Ra ox/iox j Egfr ox/iox RT1 and RT2 mice after 10 and 1 1 weeks of TMX exposure, respectively. Tumors are outlined by a dotted line. Scale bar represents 1000 pm. Box insets, indicated by arrowheads, mark areas shown at higher magnification in the adjacent images to the right stained with H&E, CK19, pErk and Ki67. Scale bar represents 50 pm.
  • Figure 9 Histological characterization of the residual scar lesions present in“Responder” mice after TMX exposure a, Low and b, high magnification of representative sections of a PDAC tumor present in control PeFC ⁇ c- RaP l+ ,Egfr l+ C1 mouse and in the scar lesions of KPeFC;c-Raf 0X/l0X ;Egfr 0X/l0X R3 and R4 mice stained with Masson ' s trichrome (T. Masson), Hyaluronic Acid Binding Protein (HABP), F4/80 and CD3. Scale bars represents a. 100 pm and b, 20 pm.
  • Masson ' s trichrome T. Masson
  • HABP Hyaluronic Acid Binding Protein
  • FIG. 10 PanIN lesions present in “Responder” mice. Representative H&E stained paraffin sections of the pancreata of KPeFC;c-/3 ⁇ 4/ 0X/l0X ;Egfr 0X/l0X R2 mouse after six weeks of TMX exposure. Scale bar represents 1000 pm. Box inset, indicated by an arrowhead, marks the area shown at higher magnification in the adjacent images to the right stained with H&E, Egfr, CK19, pErk and Ki67. Scale bar represents 50 pm.
  • FIG. 11 Differential expression of CK19 and Gata6 in PDAC tumors used to generate the“Responder” and Non Responder” cell lines.
  • c-Raf and Egfr are essential for proliferation of PDX cell lines in vitro a, Left, Western blot analysis of Egfr and c-Raf expression in whole cell extracts obtained from the corresponding PDX cell line (PDX1 -4) using a scramble shRNA (-), two shRNA against Egfr (E1 , E2), two shRNAs against c-Raf (R1 , R2) and the combination of shRNAs against Egfr and c-Raf (E/R). Gapdh served as loading control.
  • n.s no significant c, EGFR and c-RAF expression is essential for in vitro proliferation of PDAC cells-derived from PDX tumor models.
  • (Left) Cell proliferation of the indicated PDX-derived cells infected with a scramble shRNA (black) or with shRNAs against EGFR (light and dark blue), RAF1 (red and pink) and EGFR plus RAF1 (green).
  • (Right) Western blot analysis of EGFR and c-RAF expression in whole cell extracts obtained from the indicated PDX-derived cells using either a scramble shRNA (-), shRNAs against EGFR (E1 , E2), RAF1 (R1 , R2) and EGFR plus RAF1 (E1/R1 ) (right). Proliferation was determined by MTT and expressed as fold increase in the number of cells determined at each of the indicated days. Error bars indicate mean ⁇ SD. GAPDH served as loading control.
  • FIG. 13 Pharmacologic inhibition of EGFR in combination with c-RAF knockdown inhibits proliferation of PDAC cells derived from PDX tumor models.
  • FIG. 14 Mice not included in the trial a, Spurious expression of the FlpO recombinase. Fluorescent labeling in representative sections of PDAC, healthy pancreas, skin and intestine of KPeFC, Rosa26 /CAG dJom3lo ⁇ EGFP mice exposed to TMX for 4 weeks. Dotted white line indicates a small EGFP + papilloma. Arrows indicate two recombinant cells in intestine. Scale bar represents 50 pm.
  • Figure 15 Generation of a conditional c-Raf kinase dead allele b, Lack of therapeutic effect of a c-Raf D468A kinase dead isoform on K-Ras /TP53 mutant tumors c, In vitro c-Raf kinase assay, c-Raf active kinase domain was expressed in baculoviral systems. Negative control 1 : only MEK. Negative control 2: only c-Raf.
  • FIG. 16 Lack of significant inhibition of PDX tumor cell growth in vitro by four independent c-Raf kinase inhibitors: a, MLN2480, b, GW5074, c, PLX8394 and d, LSN3074753.
  • PDX- derived cell lines (PDX dd and PDX dc2) were treated with 2 pan-Raf Kinase inhibitors (MLN2480 and LSN3074753), a c-Raf Kinase inhibitor (GW5074) and a paradox breaker inhibitor (PLX8394) for 72 hours to determine their respective IC50. Only the pan-Raf inhibitor LSN3074753 has a significant effect in cell viability.
  • Figure 17 Only the pan-Raf inhibitor LSN3074753 has a significant effect in cell viability.
  • FIG. 18 Comparative effect of treating K-Ras/TP53 genetically engineered mice with a c-Raf kinase inhibitor (a) and genetically ablating c-Raf expression (b).
  • a tumor volume of vehicle (9 mice/17 CT+ tumors, black bars) and c-Raf inhibitor LSN3074753 inhibitor (10 mice/20 CT+ tumors, white bars) treated mice b, _fusion of figures 3b and 3c of Sanclemente et al.
  • the inventors demonstrate that combined inhibition of EGFR and c-RAF expression is a very effective therapy against PDAC, both in mutant Kras/Trp53- driven GEM tumor models as well as in human PDXs. Also, they have found the surprising fact that systemic elimination of these targets results in tolerable toxicities, primarily resulting from the lack of EGFR activity.
  • Human PDACs are genetically more complex than those of GEM tumor models. Yet, the inhibitory effect of EGFR and c-RAF knockdown in nine out of ten independent PDX tumor models illustrates that this therapeutic strategy is plausible to be also effective in the clinic. It is adopted to highlight the surprising fact that human PDX-derived tumor cells are more sensitive than the corresponding mouse tumors in spite of their more complex mutational profile.
  • conditional Raf1 kinase alleles in a lung“therapeutic model” that show that the therapeutic effect observed upon loss of c-RAF expression is not be mediated by its kinase activity.
  • Pan-RAF kinase inhibitors have limited anti-tumor activity (they do not induce tumor stasis or tumor regression) in several assays, including: in vitro PDX cell lines, in vivo PDX tumor model, in vivo K-Ras/TP53 genetically engineered mouse models.
  • in vitro PDX cell lines in vitro PDX cell lines
  • in vivo PDX tumor model in vivo K-Ras/TP53 genetically engineered mouse models.
  • expression of a kinase dead isoform of c-Raf c-Raf D468A
  • Figures 3B and 3C of Sanclemente et al 2017 show genetically engineered mouse tumors induced by K-Ras and TP53 mutations and the therapeutic effect of c-RAF ablation on K- Ras/TP53 mutant tumors.
  • Figure 15A depicts the strategy that was used by the inventors for generating a conditional c-Raf kinase dead allele.
  • Said c-RafD468A kinase dead isoform lacked therapeutic effect on K-Ras/TP53 mutant tumors (Fig. 15B).
  • the c-Raf D468A isoform does not have kinase activity neither in vitro as determined by measuring its kinase activity in a baculovirus expression system or in vivo as determined by its ability to block the“paradoxical effect” caused by expression of a B- Raf kinase mutant (Fig. 15C).
  • c-Raf kinase inhibitors Four independent c-Raf kinase inhibitors: MLN2480, GW5074, PLX8394 and LSN3074753 were used to treat PDX-derived cell lines (PDX dd and PDX dc2). Two of them are pan- Raf Kinase inhibitors (MLN2480 and LSN3074753), one is a c-Raf Kinase inhibitor (GW5074) and the fourth one is a paradox breaker inhibitor (PLX8394). The treatment lasted 72 hours and their respective IC 5 o was determined. Only the pan-Raf inhibitor LSN3074753 has a significant effect in cell viability (Fig. 16).
  • pan-Raf (LSN3074753) treatment of K-Ras G12C mutant lung adenocarcinoma PDX model did not lead to tumor regression in vivo.
  • K- Ras +/LSLG12V ;p53 l0X/l0X mice were infected with 10 6 pfu of Ad-Cre at 8 weeks of age and tumor development was monitored by CT measurements. Once the animals developed at least one tumor bigger than 3 mm 3 they were treated with the pan-Raf LSN3074753 inhibitor (25 mg/kg, PO, BID for one month).
  • Figure 18 A represents the tumor volume of individual CT+ tumors treated with vehicle (black bars) and LSN3074753 (grey bars).
  • figure 18 B is presented.
  • This figure is a combination of the data presented in figures 3B and 3C of Sanclemente et al 2017, showing the tumor volume in K-Ras +IFSFG TM, TP53F IF , c-Raf +/+ mice (black bars) and in K-Ras +IFSFG ⁇ , TP5 ⁇ IF , c-Raf lox/lox mice (grey bars).
  • This strain has been designated as KPeC to indicate that the driver mutations are selectively induced by the elastase gene promoter in the acinar cell compartment instead of in all pancreatic cell lineages as in the classical KPC model (Hingorani et al., 2005).
  • CDK4 cell cycle kinase and the c-RAF kinase based on our prior observations that they are essential for the development of K-RAS G12V driven lung tumors (Blasco et al., 201 1 ; Puyol et al., 2010; Sanclemente et al., 2018).
  • ablation of CDK4 or c-RAF does not induce unacceptable toxic effects such as those observed upon ablation of the MEK1 /2 and ERK1/2 kinases (Blasco et al., 201 1 ; Puyol et al., 2010).
  • control KPeC mice succumbed to PDAC at the average of 15 weeks of age.
  • Expression of the kinase dead CDK4 K35M did not prevent PDAC development, but it increased the median survival of the tumor-bearing mice to similar to that observed in the absence of EGFR (Ardito et al., 2012; Navas et al., 2012).
  • mice retained K-RAS G12V expression in their acinar cell compartment as determined by the presence of b-galactosidase, a surrogate marker for K- RAS G12V expression (Guerra et al., 2003). Isolation of these cells by laser-capture microdissection confirmed efficient recombination of Raff ox and Egfr ox alleles ( Figure 1 D). No such recombination was observed in adjacent acinar cells negative for b-galactosidase expression ( Figure 1 D). Thus, EGFR and c-RAF, but not CDK4, must signal through independent pathways essential for initiation and development of pancreatic tumors.
  • NGS next generation sequencing
  • Target ablation at the time of tumor initiation does not reflect therapeutic intervention in the clinic. Moreover, in most studies, targets are selectively ablated in selected tissues or in those cells that express the oncogenic insult(s) (Drosten et al., 2017; Perez-Mancera et al., 2012). These strategies fail to provide information regarding the toxic effects that might occur in the clinic when the targets are inhibited via systemic administration of the corresponding inhibitors. Therefore, we have developed a GEM strain that separates temporally and spatially tumor development from target ablation/inhibition. This strain, ras + FSFG12V : Trp5 M : E/as- tT A/ 7efO- FI pO :Tg .
  • KPeFC incorporates two distinct recombinases, FlpO and CreERT2.
  • FlpO responsible for tumor induction, is expressed by the same Tet-Off system used in the KPeC strain.
  • KPeFC and KPeC mice develop PanIN lesions and PDACs with complete penetrance and similar kinetics ( Figure 7A, 7B).
  • Expression of the tamoxifen (TMX)-inducible CreERT2 recombinase is driven by the promoter of the human Ubiquitin C gene ( UBC ), a locus expressed in all adult tissues (Ruzankina et al., 2007).
  • UBC human Ubiquitin C gene
  • pancreata of these“Regressor” mice contained low-grade PanINs (3 to 10 per mouse) including the R2 mouse in which the original PDAC had completely disappeared. Most of these lesions expressed EGFR ( Figure 10). Whether these PanINs are derived from cells that were not able to progress or represent late events during the course of the study, remains to be determined.
  • Western blot analysis of tumor tissue revealed the absence of EGFR and c-RAF, indicating that tumor progression was not due to incomplete recombination of the conditional Egfr and/or Raf1 alleles ( Figure 8B). Therefore, we have designated these mice as “Resistant” (T). Whether tumor progression was due to the acquisition of new mutations or to the emergence of clones that did not require EGFR and c-RAF signaling, remains to be determined. Indeed, the tumor present in the T2“Resistant” mouse had a distinct sarcomatoid phenotype as illustrated by the lack of expression of CK19 and pERK ( Figure 8C).
  • RNAseq analysis was performed by RNAseq analysis of three RC cell lines and three NC cell lines.
  • RC and NC cells displayed distinct transcriptional profiles that included more than two thousand differentially expressed genes.
  • GSEA Gene Set Enrichment Analysis
  • the most significantly enriched gene signatures in RC cells included those corresponding to“bile acid, cholesterol, xenobiotic and fatty acid metabolism”,“apoptosis” and“p53 pathway” ( Figure 5B).
  • Significantly enriched gene sets in the NC cells were those corresponding to“E2F targets”,“EMT” and“MYC targets”.
  • L/SC-CreERT2 and Rosa26 +AmF3(CAGddTomat ° rEGFP)Pien/J strains have been previously described (Guerra et al., 2007; Jesenberger et al., 2001 ; Jonkers et al., 2001 ; Lee et al., 2012; Natarajan et al., 2007; Nieto et al., 2017; Plummer et al., 2015; Ruzankina et al., 2007).
  • the Cc//c4 K35M/K35M strain was obtained by a Cre-dependent FLEx switch strategy that replaced expression of the wild-type CDK4 protein by a CDK4 K35M kinase dead isoform (Schnutgen et al., 2003) (Schnutgen et al., 2003).
  • the transgenic Te/O-FlpO strain was generated by pronuclear injection of CMV- Te/O-FlpO DNA into B6.CBA zygotes (Pease and Saunders, 201 1 ). All mice were maintained in a mixed 129/Sv-C57BL/6 background.
  • Tumors were measured with a micro-ultrasound system (Vevo 770, Visualsonics) with an ultrasound transducer of 40 MHz (RMV704, Visualsonics). To this end, mice were anesthetized with a continuous flow of 1 % to 3% isoflurane in 100% oxygen at a rate of 1 .5 liter/min. Hypothermia associated with anesthesia was avoided using a bed-heater. Abdominal hair was removed by depilation cream to prepare the examination area. Tumor size was calculated as Length x Width 2 /2.
  • mice were fed with a TMX-containing diet (Teklad CRD TAM400 diet, Harlan) ad libitum. Control mice carrying the corresponding wild-type alleles were also fed with the same diet.
  • TMX-containing diet Teklad CRD TAM400 diet, Harlan
  • c-Raf wM type, floxed and null alleles were identified with forward c-fiaf 1 F (SEQ ID NO: 1 : 5 ' -CTGATTGCCCAACTGCCATAA-3 ' ), c-Raf 3F (SEQ ID NO: 2: 5 ' - GAGTCAGCAAATGCACTGAAATG-3 ' ) and reverse c-Raf 1 R (SEQ ID NO: 3: 5 ' - ACTGATCTGGAGCACAGCAAT-3 ' ) primers at 94 °C for 1 minute, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 60 °C for 30 seconds and extension at 72 °C for 30 s, and finally, followed by a long extension at 72 °C for 10 minutes.
  • These primers yielded DNA products of 196 bp
  • Tissues were fixed overnight by immersion in 4% paraformaldehyde (PFA) in 0.01 M phosphate-buffered saline (PBS) at 4°C and rinsed in PBS before equilibration in 30% sucrose in PBS for 48 h at 4°C. Samples were thereafter included in O.C.T.TM compound (Sakura) and frozen. Cryosections of the samples were stained with Dapi for nuclei detection (ThermoFisher), mounted with Prolong Gold antifade reagent (ThermoFisher) and visualized with a TCS-SP5 laser scanning confocal microscope (Leica) equipped with AOBS and both 10X/0.4NA and 20X/0.7NA dry objectives. A z-stack was acquired and the maximum projection is shown.
  • PFA paraformaldehyde
  • PBS phosphate-buffered saline
  • Samples were thereafter included in O.C.T.TM compound (Sakura) and frozen.
  • mice PDAC explants To generate mouse PDAC explants, freshly isolated tumors were minced with sterile razor blades, digested with collagenase P (1 .5pg/ml) in Hank’s Balanced Salt Solution (HBSS) for 30 min at 37 Q C, and cultured in DMEM with 10% of fetal bovine serum (FBS) and 1 % Penicillin/Streptomycin. All studies were done on cells maintained in culture for less than ten passages. Their corresponding genotypes were verified by PCR analysis. PDAC cells explants were infected with Adeno-Cre particles (multiplicity of infection, 100) and seeded for colony formation assay 5 days after. Adeno-GFP particles were used as negative controls.
  • Adeno-Cre particles multiplicity of infection, 100
  • Cells were seeded in equal cell numbers (5x 10 3 ) and allowed to form colonies for 2 weeks. Plates were fixed with 0.1 % glutaraldehyde (Sigma) and stained with 0.5% Crystal Violet (Merck). Colonies were counted and quantified.
  • PDX tumors models were used include Panc-1 , Panc-2, Panc-4, Panc-185, Panc-198, H- PDAC-H-X132, H-PDAC-M-X3 and H-PDAC-M-X7 (Table 4). Table 4. KRAS and TP53 mutations in PDX tumor models.
  • Panc-1 , Panc-2, and Panc-4 were obtained from patients who underwent surgical resection at the Kog University Hospital, Istanbul, Turkey with approval by the Ethical Committee (CEI 60-1057-A068).
  • Panc-185, Panc-198, H-PDAC-H-X132 were obtained from Hospital HM Sanchinaro, Madrid, Spain, with approval by the Ethical Committee (CEIC HM Hospitales, FHM.06.10).
  • H-PDAC-M-X3 and H-PDAC-M-X7 were obtained from the Hospital Virgen de la Arrixaca, Murcia, Spain, with approval by the Ethical Committee (CEIC HCUVA-2013/01 ).
  • Specific informed consent for PDX model generation was obtained from all patients.
  • PDAC003T and PDAC013T tumor models have been already described (Nicolle et al., 2017).
  • E1 , TRCN0000121203 and E2, TRCN0000121206 lentiviral supernatants expressing shRNAs against EGFR
  • the E1 , E2 and R2 shRNAs were cloned in a plasmid that carries a Puromycin resistant cassette. Instead, the R1 shRNA was cloned in a plasmid that conferred Blasticidine resistance.
  • a scrambled shRNA control vector was used as a negative control.
  • Infected PDX cells were seeded in 96-well plates at a density of 1 ,500 cells per well and proliferation was assessed using the MTT assay.
  • infected cells 0.5x10 6
  • PDAC003T PDAC013T
  • Panc-1 and Panc-4 tumor models were injected 1 : 1 in PBS:Matrigel Matrix (Corning, 354234) into dorsal flanks of immunodeficient mice. Tumor growth was measured every 3 days with a caliper and calculated as Length x Width 2 /2 until humane end point.
  • Protein extracts obtained from tumor tissue or cell lines were separated on SDS/PAGE gels (Thermo Fisher Scientific), transferred to a nitrocellulose membrane and blotted with antibodies raised against Egfr (Abeam, ab52894), c-Raf (BD Biosciences, 610151 ), Erk-1 (BD Biosciences 554100), Erk-2 (BD Biosciences, 610103), pErk1/2 (Cell Signaling, 9101 ), Akt (Cell Signaling, 9272), pAkt (Cell Signaling, 4060), Stat3 (Cell Signaling, 9132), pStat3 (Cell Signaling, 9131 ), pCofilin (Santa Cruz, sc-21867-R) and Gapdh (Sigma, G8795).
  • Egfr Abeam, ab52894
  • c-Raf BD Biosciences, 610151
  • Erk-1 BD Biosciences 554100
  • Erk-2 BD Biosciences
  • Genomic DNA obtained from 1 1 paired tumor and tail tissue was enriched in protein-coding sequences using the SureSelect Mouse All Exon kit (Agilent Technologies).
  • the resulting target-enriched pool was amplified and subjected to paired-end sequencing (2 c 100 bp) using HiSeq2000 sequencing instruments at the Beijing Genomics Institute (BGI).
  • Sequencing reads were mapped to the reference genome (mm9) using the Burrows- Wheeler Aligner (BWA) (Li and Durbin, 2010) alignment tool version 0.5.9. Sites that differed from the reference genome (variants) were identified and empirical priors were constructed for the distribution of variant frequencies in each sample independently.
  • BWA Burrows- Wheeler Aligner
  • candidate protein altering somatic variants were identified when variants were absent in the normal and present in the tumor with at least 1 % change in frequency from normal with high posterior probability (> 1 - 1 e-5).
  • RNA from PDAC cell explants was extracted with Qiagen RNeasy Mini Kit. 1 pg of total RNA was used for further analysis. PolyA + fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in lllumina's "TruSeq Stranded mRNA LT Sample Prep Kit". The adapter-ligated library was completed by PCR with lllumina PE primers. The resulting purified cDNA library was applied to an lllumina flow cell for cluster generation and sequenced on an lllumina NovaSeq 6000 instrument by following manufacturer's guidelines.
  • PDX cell lines were plated at 5,000 cells per well in triplicates in 96-well plates and grown for 24 hours. Cells were treated with a dilution series of Gefitinib (Cymit Quimica SL), Erlotinib (LC laboratories). Control cells were incubated with media containing DMSO. Cell viability was assessed with CellTiter Glo Luminescent Cell Viability Assay after 72 hours of treatment. Luminescence counts were read in a Victor Instrument (Perkin Elmer) with the recommended settings. To calculate the IC 50 , values were plotted against the inhibitor concentrations and fit to a sigmoid dose-response curve using GraphPad Software.
  • PDX-derived cells were seeded in 96-well plates at 1 ,500 and 3,000 cells/well in triplicates, and incubated for 24 hours in DMEM media supplemented with 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin and 50 pg/ml streptomycin (GIBCO- Invitrogen) before adding the IC 50 of the corresponding IC50 concentration of inhibitor in DMSO. The same concentration of DMSO was used as a control. Cells were exposed to the corresponding inhibitor for 12 days, in the presence or absence of a c-RAF shRNA (R1 ) changing medium and drug every two days. Cell viability was assessed with CellTiter Glo Luminescent Cell Viability.

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Abstract

La présente invention concerne une composition pharmaceutique comprenant un inhibiteur de l'expression, de l'activité et/ou de la fonction de c-Raf et d'un inhibiteur de l'expression, de l'activité et/ou de la fonction du récepteur de l'EGF (EGFR) et son utilisation dans le traitement du cancer du pancréas.
EP19745113.1A 2018-07-24 2019-07-24 Polythérapie inhibant l'expression, l'activité et/ou la fonction du récepteur du facteur de croissance épidermique et c-raf contre le cancer Pending EP3846799A1 (fr)

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