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  • 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
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
  • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
• • 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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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• Cancer treatments especially those for difficult to treat cancers like melanoma, require further advancement in order to achieve the clinical benefits that patients require to resume a healthy life without significant morbidity and mortality from the disease.
• HSPs heat- shock proteins
• PSR proteotoxic stress, response
• HSFs heat shock transcription factors
• HSF1 -mediated PSR antagonizes many pathological conditions, including hyperthermia, heavy-metal toxification, ischemia and reperfusion, and oxidative damage, and impacts aging and neurodegeneration (Dai et al., 2012a).
• HSF1 acts as a longevity factor (Hsu et al., 2003).
• our and others' work has revealed a pro-oncogenic role of HSF1 (Dai et al., 2007; Dai et al., 2012b; Jin et al., 2011; Meng et al., 2010; Min et al., 2007).
• HSF1 is crucial for tumor cells' growth and survival (Dai et al., 2007).
• RAS-MEK-ERK signaling critically regulates the PSR. It is MEK that phosphorylates and activates HSF1. MEK inhibition destabilizes the proteome, provoking protein aggregation and amyloidogenesis. Combinatorial proteasome blockade potently augments this tumor-suppressive amyloidogenic effect.
• MEK inhibitors were known in the art, as were proteasome inhibitors, yet there was no reason or motivation to combine them prior to the present invention and each treatment had its limitations in efficacy, including drug resistance.
• a method of treating cancer comprises administering a MEK inhibitor in combination with a proteasome inhibitor.
• the cancer has at least one mutation chosen from a NF1, RAS (including N-, K-, and H-RAS), RAF (including A-, B-, and C-RAF), and MEK (including MEK1 and MEK2) mutation.
• RAS mutation may be in at least codon 12, 13, or 61.
• the RAF mutation is in at least codon 600.
• the MEK1 mutation is at least P124S or S203K or the MEK2 mutation is at least Q60P.
• the MEK inhibitor is selumetinib (AZD6244), trametinib (GSK1120212), binimetinib (MEK162), PD-325901, cobimetinib, PD184352 (CI-1040), U0126-EtOH, refametinib (RDEA119), PD98059, BIX 02189, pimasertib (AS- 703026), SL-327, BIX 02188, AZD8330, TAK-733, honokiol, or PD318088, PD0325901, WX-554, GDC-0623, E6201, R04987655, R05126766.
• the proteasome inhibitor is bortezomib, lactacystin, disulfiram, epigallocatcechin-3-gallate, salinosporamide A, carfilzomib, oprozomib (ONX 0912), delanzomib (CEP-18770), MLN9708, epoxomicin, MG132, ixazomib (MLN2238), PI- 1840, or celastrol.
• the proteasome inhibitor and the MEK inhibitor are administered at a dosage that does not create a therapeutic benefit when either agent is administered alone.
• selumetinib is administered at about 5 mg/Kg and bortezomib is administered at about 0.5mg/Kg.
• FIGS lA-O show that MEK and ERK oppositely regulate the PSR.
• C and D NIH3T3 cells were treated with 20 ⁇ U0126 or 20nM AZD6244 for 3 hr followed by HS and 4-hr recovery.
• HEK293T cells were transfected with dual HSF1 reporter plasmids, a heat-shock element (HSE)- driven SEAP plasmid and a CMV-GLuc plasmid.
• HSE heat-shock element
• FIGS 2A-J show that ERK, MEK, and HSF1 form a stress-inducible protein complex.
• a and B After HS at 43°C for 30min, endogenous HSF1 proteins were precipitated from HEK293T cells. WCL: whole cell lysate; HC: heavy chain.
• C Endogenous MEKl-HSFl interactions were detected by PLA in HeLa cells using a rabbit anti-MEKl antibody and a mouse anti-HSFl antibody. Scale bars: 50 ⁇ for LM, ⁇ for HM.
• D and E Endogenous MEK-HSF1 interactions were detected by IP in
• HEK293T cells stably expressing shRNAs.
• F Endogenous MEK-HSF1 interactions were detected in HEK293T cells transfected with siRNAs.
• G Endogenous HSF1-MEK and HSFl-GFP-ERKl interactions were detected in HEK293T cells transfected with indicated plasmids.
• H Schematic depictions of three possible scenarios. P: phosphorylation.
• I Immediately after HS, HSFl-ERK interactions were detected by co-IP.
• J Endogenous ERK-HSFl interactions were detected in HEK293T cells stably expressing shRNAs.
• K and L Endogenous ERK-MEK and ERK-HSFl interactions were detected by PLA in HeLa cells. Scale bar: 50 ⁇ for LM, ⁇ HM. See also Figure 9.
• FIG. 3A-0 shows that MEK phosphorylates Ser326 to activate HSF1.
• a and B HSF1 Ser326 phosphorylation was measured by immunoblotting in HEK293T cells stably expressing shRNAs or transfected with MEK1 DD plasmid.
• D Control or ERK-targeting siRNAs, A
• FIGS 4A-R show that MEK preserves proteostasis.
• GFP and GFP- GR plasmids were co-transfected into HEK293T cells followed by treatments with 20nM AZD6244, 20 ⁇ U0126, or 200nM 17-DMAG for 4 hr.
• B GFP-GR plasmids were co- transfected into HEK293T cells with HA-Ub-K48 plasmids, which encode a mutant ubiquitin that can be conjugated to protein substrates only via lysine 48.
• AZD6244 and ubiquitinated proteins were detected in both detergent-soluble and - insoluble fractions using Lys48-specific ubiquitin antibodies.
• E A2058 cells stably expressing LacZ or HSF1 S32 6D were treated with 20nM AZD6244 for 8 hr.
• F C57BL/6J mice were i.p. injected with DMSO or AZD6244 three times a week for 2 weeks. S: spleen; K: kidney; L: liver.
• G Experimental procedures of MS-based quantitation of
• V5-TOR1AIP2 or V5-RPL3 plasmids were co-transfected with HA-Ub-K48 plasmids into HEK293T cells. Following 20nM AZD6244 treatment for 8 hr, proteins were precipitated with anti-V5 antibodies.
• N Following AZD6244 treatment, endogenous c-MYC proteins were precipitated from A2058 cells and immunoblotted with anti-ubiquitin antibodies.
• V5-RPL15 and V5-RPL3 plasmids were co-transfected with HA-Ub-K48 plasmids into HEK293T cells stably expressing shRNAs.
• FIGS 5A-Y show MEK and proteasome inhibition provoke protein aggregation and amyloidogenesis.
• A-B WM115 cells treated with 20nM AZD6244, ⁇ Bortezomib, or both for 24 hr were stained with Lys48-specific ubiquitin antibodies. Arrowheads mark ubiquitin-positive aggregates. Scale bar: ⁇ . Amounts of aggregates per cell were quantitated using ImageJ (median, n>100, ANOVA).
• C-F C-F
• HEK293T cells were treated with inhibitors as described in (A-B). Cells were either analyzed for aggregate size or stained with 10 ⁇ ThT.
• G Treated tumor cell lines were stained with 10 ⁇ ThT. Geometric means were used to calculate fold changes in ThT fluorescence intensity and the log 2 (FC) values were presented as a heat map.
• L Detection of metastases by in vivo bioluminescence imaging.
• M Representative micrographs illustrate metastatic melanomas in the lung, skeletal muscle, pelvic adipose tissue, and ovary. T: tumors; L: lung; M: muscle; B: bone; A: adipose tissue; OF: ovarian follicle. Scale bar: 500 ⁇ .
• N Combined MEK and proteasome inhibition prevents melanoma metastasis (Barnard's exact test). See also Figure 13 and Table 2.
• Figures 7A-H show amyloidogenesis suppresses tumor growth.
• (C) Lysates of CR-treated tumors exhibited absorbance at 498nm, 3 tumors per group (mean ⁇ SD, n 3).
• Lysis buffer containing CR served as a positive control.
• D Kaplan-Meier survival curves were compared (Log-rank test).
• G Proteins were detected by immunoblotting, 3 tumors per group.
• H Schematic depiction of the interplay among MEK, ERK, and HSFl, and its role in regulating proteome stability. Balanced proteostasis suppresses toxic protein aggregation and amyloidogenesis, thereby facilitating tumorigenesis. See also Figure 14.
• FIGS 8A-H illustrate that MEK and ERK inversely regulate the PSR. This figure is related to Figure 1.
• B MEK blockade suppresses HSFl activation triggered by diverse proteotoxic stressors. Following transfection with dual HSFl reporter plasmids, HEK293T cells were treated with diverse stressors as described in Figure IB. After treatments, stressors were removed and cells were incubated with fresh medium for 24hrs before measuring reporter activities
• G and H ERK2 knockdown does not reduce ERK1 mRNAs.
• FIG. 9A-F demonstrates that MEK physically interacts with HSFl. This figure is related to Figure 2.
• HSFl is co-precipitated with MEK1 and MEK2.
• a FLAG-HSF1 plasmid was co-transfected with either HA-MEK1 or MEK2-V5 plasmid into HEK293T cells.
• HA-Raptor and LacZ-V5 plasmids served as negative controls.
• C and D Validation of MEK1 and HSFl antibodies.
• HeLa cells stably expressing scramble or MEK1 / 2- targeting shRNAs were immunostained with rabbit anti-MEKl antibodies.
• HeLa cells stably expressing scramble or fiiF/-targeting shRNAs were immunostained with mouse anti-HSFl antibodies. Scale bar: 50 ⁇ .
• E ERK suppresses HSFl activation. GFP or GFP-ERK1 plasmid was co-transfected with dual HSFl reporter plasmids into HEK293T cells.
• FIGS 10A-H illustrate that MEK phosphorylates HSFl at Ser326.
• This figure is related to Figure 3.
• (B) MEK blockade impairs HSFl Ser326 phosphorylation. NIH3T3 cells were treated with 20 ⁇ U0126 for 3hrs followed by heat shock at 43°C for 30mins.
• HSFl Ser326 phosphorylation was detected by immunoblotting.
• C MEK blockade impairs and ERK blockade enhances HSFl Ser326 phosphorylation. Following overnight treatments with 20 ⁇ U0126, 20nM AZD6244, ⁇ FR180204, or ⁇ Sch772984, HSFl Ser326 phosphorylation was detected by immunoblotting in HEK293T cells.
• D-E S326A mutation impairs HSFl nuclear translocation. HEK293T cells were transfected with FLAG-HSFl ⁇ 1" or FLAG- HSF1 S326A plasmids.
• HEK293T cells stably expressing HSF1- targeting shRNAs were transfected with FLAG-HSFI ⁇ T or FLAG-HSF1 S326D plasmids. Following 20nM AZD6244 treatment for 8hrs, HSFl Ser326 and Ser307 phosphorylation was detected by immunoblotting.
• FIGS 11A-P show that MEK regulates proteome stability. This figure is related to Figure 4.
• HSFl stabilizes GR-GFP proteins. Both GR-GFP and HA-Ub- K48 plasmids were co-transfected with LacZ or FLAG-HSF1 plasmids into HEK293T cells stably expressing scramble or fiiF/-targeting shRNAs. HA-Ub-K48 plasmids encode a mutant ubiquitin that can be conjugated to protein substrates only via lysine 48. GFP-GR proteins were precipitated and ubiquitination was detected by anti-HA immunoblotting.
• B HSFl maintains cellular chaperoning capacity.
• HSF1 suppresses protein ubiquitination.
• LacZ or FLAG-HSF1 plasmids were transfected into HEK293T cells stably expressing scramble or HSF1 -targeting shRNAs. Lys48-specific protein ubiquitination was detected by immunoblotting in both detergent-soluble and - insoluble fractions.
• H AZD6244 promotes HSF1 ubiquitination. A2058 cells were treated with 20nM AZD6244. Endogenous HSF1 proteins were immunoprecipitated and blotted with anti-ubiquitin antibodies.
• V5-tagged ribosomal proteins and HA-Ub-K48 were co-expressed in HEK293T cells. Following treatment with 20nM AZD6244 for 8hrs, ribosomal proteins were immunoprecipitated with anti-V5 antibodies and ubiquitination of these ribosomal proteins was detected by immunoblotting with anti-HA antibodies.
• MEK and HSF1 deficiency destabilize ribosomal proteins. Endogenous RPL15 and RPL3 proteins were detected in HEK293T cells stably expressing scramble, MEK// -targeting, or HSF1- targeting shRNAs with and without 500nM MG132 treatment for 24hrs.
• FIG. 12A-U shows that MEK and proteasome inhibition disrupt proteostasis. This figure is related to Figure 5.
• A-B AZD6244 depletes HSF1 proteins in melanoma cells. WM115 and A2058 cells were treated with 20nM AZD6244, ⁇ Bortezomib, or both for 24hrs.
• C Quantitation of protein aggregate size. Four days after transfection with LacZ or polyQ79 plasmids, detergent-insoluble fractions of HEK293T cells were extracted to quantitate particle sizes using a MultisizerTM 3 coulter counter.
• D HSF1 suppresses protein aggregation.
• HEK293T cells stably expressing scramble or H5F7 -targeting shRNAs were co-transfected with polyQ79 and LacZ or FLAG-HSF1 plasmids for 2 days. Aggregate sizes were measured.
• E and F Detection of amyloids in intact cells by ThT and CR.
• HEK293T cells transfected with LacZ or polyQ79 plasmid were stained with ⁇ ThT (E) or 50nM CR (F) and analyzed by flow cytometry.
• G Following treatments, endogenous amyloids in WM115 cells were detected by CR staining.
• H and I HSF1 suppresses amyloidogenesis.
• N (N)-(R) Genetic inhibition of MEK and proteasome disrupts proteostasis and provokes amyloidogenesis.
• HEK293T cells were transduced with lentiviral shRNAs targeting MEK1 / 2, PSMB5, or both. Proteostasis disruption was evidenced by increased protein ubiquitination (N).
• Figure 13A-J illustrates combined MEK and proteasome inhibition disrupts proteostasis and impedes in vivo tumor growth. This figure is related to Figure 6.
• G Amyloid oligomer levels are inversely correlated with tumor weights.
• H Combined AZD6244 and Bortezomib treatment markedly enhances ThT staining of tumors. Nuclei were stained with SYT062. Scale bar: 50 ⁇ .
• J J
• Combined AZD6244 and Bortezomib treatment aggravates protein ubiquitination but fails to induce apoptosis in mouse spleens. Lys48-specific protein ubiquitination and caspase 3 cleavage were measured by immunoblotting in 3 spleens per group.
• Figure 14A-B shows that CR treatment promotes tumor growth and antagonizes tumor suppression imposed by combined MEK and proteasome inhibition. This figure is related to Figure 7.
• Table 1 provides a listing of certain sequences referenced herein.
• the cancer is a solid tumor.
• the solid tumor is biliary (cholangiocarcinoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, epidermoid carcinoma, esophageal carcinoma, gallbladder cancer, gastric (stomach) cancer, glioblastoma, glioma, head and neck cancers, hepatocellular (liver) carcinoma, kidney cancer, lung cancer, mesothelioma, non-small cell lung cancer, ovarian, pancreatic cancer, pediatric malignancies, prostate cancer, renal cancer, sarcomas, skin cancer (including melanoma), small bowel cancer, and the like.
• the solid tumor is melanoma.
• the method may provide additional advantages when the cancer has at least one mutation.
• the cancer may have at least one mutation chosen from a NFl, RAS (including N-, K-, and H-RAS), RAF (including A-, B-, and C-RAF), and MEK (including MEK1 and MEK2) mutation.
• a RAS mutation may be present in at least codon 12, 13, or 61.
• a RAF mutation may be in at least codon 600.
• a MEK1 mutation may be in at least P124S or S203K.
• the MEK inhibitor may be chosen from, but is not limited to, selumetinib (AZD6244), trametinib (GSK1120212), binimetinib (MEK162), PD-325901, cobimetinib, PD184352 (CI-1040), U0126-EtOH, refametinib (RDEA119), PD98059, BIX 02189, pimasertib (AS-703026), SL-327, BIX 02188, AZD8330, TAK-733, honokiol, or
• the proteasome inhibitor may be chosen from, but is not limited to, bortezomib, lactacystin, disulfiram, epigallocatcechin-3-gallate, salinosporamide A, carfikomib, oprozomib (ONX 0912), delanzomib (CEP-18770), MLN9708, epoxomicin, MG132, ixazomib (MLN2238), PI-1840, or celastrol.
• the proteasome inhibitor and the MEK inhibitor are administered at a dosage that does not create a therapeutic benefit when either agent is administered alone.
• the selumetinib may be administered at 5mg/Kg and the Bortezomib may be administered at 0.5mg/Kg.
• the method may present additional advantages when the cancer is resistant to treatment with at least one of a proteasome inhibitor or a MEK inhibitor.
• the combination therapy may produce a synergistic effect.
• the cancer is resistant to treatment with at least one of a proteasome inhibitor or a MEK inhibitor (meaning a proteasome inhibitor administered without a MEK inhibitor and/ or a MEK inhibitor administered without a proteasome inhibitor), yet the combination of the two agents overcomes the resistance that may be associated with one or both alone.
• the MEK inhibitor and the proteasome inhibitor may be prepared in separate compositions or they may be formulated into a single combined dosage form.
• the inhibitors may be prepared as a tablet or capsule. Both agents may be coformulated in a single tablet or capsule, as separate sections in a bilayer tablet or capsule, or in separate tablets or capsules.
• the inhibitors may be prepared in a dry powdered form to be mixed with water for injection prior to administration through a parenteral route of administration. In such an embodiment, they may be coformulated in the same vial or they may be prepared separately for administration to the patient.
• inhibitors are formulated separately, they may be administered at the same time or in sequential order, including on either the same day or different days.
• tumor sections were stained with 0.5% CR in PBS at RT for 20min followed by differentiation in alkaline solutions (0.01% NaOH, 50% alcohol). Nuclei were stained with either Hoechst 33342 or hematoxylin. Fluorescence was visualized using a Leica TCS SP5 confocal microscope and the birefringence visualized using a Leica DM5000B upright microscope equipped with polarized light filters. For ThT staining, sections were stained with 0.2% ThT in PBS at RT for lOmin, rinsed in 1% acetic acid for 2min, and washed with ddH 2 0 for 3 times. Nuclei were stained with SYTO® 62 (Life Technologies).
• A2058 cells were s.c. injected into the left flanks of 9-week-old female NOD.CB17-Prkdc ⁇ scid>/J (NOD/SCID) mice (The Jackson Laboratory).
• NOD/SCID mice The Jackson Laboratory
• mice were i.p. injected with PBS or CR one day prior to combined AZD6244 and Bortezomib treatments. Tumor volumes were calculated following the formula 4/3 ⁇ R 3 .
• engineered A2058 cells were transplanted into 10- week-old female NOD/SCID mice via tail vein injections. All mouse experiments were performed under a protocol approved by The Jackson Laboratory Animal Care and Use Committee. D. Statistical methods
• Primer sequences for qRT-PCR are provided in Table 1 (SEQ ID NOS: 1- 22) and primer sequences for ChIP are provided in Table 1 (SEQ ID NOS: 23-32).
• T202/Y204 (D13.14.4E), phospho-MSKl T581, MEKl (61B12), cleaved caspase 3 Aspl75 (D3E9), GFP (D5.1), and GST tag (91G1) were from Cell SignaHng Technology; antibodies against phospho-MEKl T292, phospho-MEKl T386, and Lys48-specific ubiquitin (Apu2) were from Millipore; and Total MSK1 antibody, PActin antibody-HRP, GAPDH antibody-HRP, HA-antibody-HRP, and FLAG antibody-HRP conjugates were from GenScript. Antibodies against p-HSFl Ser326 (EP1713Y), 6xHis tag (GT359), RPL15, and RPL3 were from GeneTex. Mouse monoclonal anti-V5 antibody was from Life Technologies.
• VER155008 Tocris Bioscience
• 17-DMAG LC Laboratories
• Bortezomib LC Laboratories
• AZD6244 ChemieTek
• tubastatin A ChemieTek
• azetidine Bachem Americas
• Q-VD-OPH APExBio
• Cytoplasmic and nuclear fractions were prepared using the NE-PER Nuclear protein Extraction Kit from Thermo Scientific.
• the plasmids used in this study include: pLenti6-LacZ-V5 and pLenti6- MEK2-V5 (generated from pDONR223 vectors via Gateway® LR reaction), pMCL-HA- MEK1 from Natalie Ahn (Addgene#40808), pMCL-HA-MEKl T292A, T386A (generated by HA-MEK1 site-directed mutageneses), pGFP-ERKl from Rony Seger
• pBabe-FLAG-HSFl S326A and S326D generated from FLAG-HSF1 by site-directed mutagenesis
• pLenti6-FLAG-HSFl generated via Gateway® LR reaction
• RNAs were extracted using RNA STAT-60 reagent (Tel-Test, Inc.), and RNAs were used for reverse transcription using a Verso cDNA Synthesis kit (Thermo Fisher Scientific). Equal amounts of cDNA were used for quantitative PCR reaction using a DyNAmo SYBR Green qPCR kit (Thermo Fisher Scientific). Signals were detected by an ABI 7500 Real-Time PCR System (Applied Biosystems). The sequences of individual primers for each gene are listed in the Supplemental Materials.
• Plasmids were transfected with TurboFect transfection reagent (Thermo Scientific). SEAP and luciferase activities in culture supernatants were quantitated using a Ziva® Ultra SEAP Plus Detection Kit (Jaden BioScience) and a Gaussia Luciferase Glow Assay Kit (Thermo Scientific), respectively. Luminescence signals were measured by a VICTOR 3 Multilabel plate reader (PerkinElmer).
• Biotinylated ideal HSE (5 '-CTAGAAGCTTCTAGAAGCTTCTAG-3 ' (holding indicates the nucleotide sequences recognized by HSFl, biotin added to the 5' end)) oligonucleotides were self-annealed to form double-stranded DNA probes in annealing buffer (lOmM Tris, pH 7.5, 50mM NaCl, ImM EDTA).
• annealing buffer lOmM Tris, pH 7.5, 50mM NaCl, ImM EDTA.
• ⁇ of 500nM biotinylated HSE probes diluted in PBS were added to Neutravidin-coated 96-well plates (Thermo Fisher Scientific) and incubated at 4°C overnight.
• each well was incubated with ⁇ rabbit HSFl antibodies (B7109, Assay Biotechnology Company, Inc.) diluted 1:1000 in SuperBlock blocking buffer at RT for 2 hrs. After TBS-T washing, each well was incubated with HRP-conjugated anti-mouse IgG secondary antibodies diluted in the blocking buffer at RT for 1 hr. Following extensive TBS-washing, colorimetric signals were developed using 1-Step Ultra TMB-ELISA substrate (Thermo Fisher Scientific). 6. siRNA and shRNA knockdown
• siRNA which targets no known genes in human and mouse, was purchased from Thermo Fisher Scientific (D-001810-01). siERKl_l
• Recombinant firefly luciferase proteins (Promega) were denatured by incubating with denaturing buffer (25mM HEPES, pH7.5, 50mM KCl, 5mM MgCl 2 , 5mM ⁇ -mercaptoethanol, and 6M guanidine HC1) at 37°C for 20min.
• denaturing buffer 25mM HEPES, pH7.5, 50mM KCl, 5mM MgCl 2 , 5mM ⁇ -mercaptoethanol, and 6M guanidine HC1
• luciferases diluted in refolding buffer (25mM HEPES, pH7.5, 50mM KCl, 5mM MgCl 2 , lOmM DTT, and ImM ATP) were incubated with 5mg/ml cell lysates extracted in passive lysis buffer (lOmM Tris-HCl pH7.5, 2mM DTT, 1% Triton X- 100, and 2mM EDTA).
• lOmM Tris-HCl pH7.5, 2mM DTT, 1% Triton X- 100, and 2mM EDTA passive lysis buffer
• 20 ⁇ 1 refolding mixtures were removed and incubated with D-Luciferin (PerkinElmer) diluted in refolding buffer.
• Luminescence signals were measured by a VICTOR 3 Multilabel plate reader (PerkinElmer).
• lxlO 7 cells were fixed with 1% formaldehyde for 8 min, and 125mM glycine was added to stop the crosslinking. After washing with cold PBS, cells were collected and lysed in cytoplasm extraction buffer (20mM Tris-HCl, 85mM KCl, 0.5% Triton X-100, pH8.0) for 10 min followed by centrifugation at 5,000 rpm for 5 min. Pellets were further lysed in nuclei extraction buffer (50mM Tris-HCl, 1% SDS, lOmM EDTA, pH8.0) for 10 min and sonicated to shear chromatin to fragments with an average length of 500bp.
• nuclei extraction buffer 50mM Tris-HCl, 1% SDS, lOmM EDTA, pH8.0
• soluble amyloid prefibrillar oligomers 20 ⁇ g soluble cellular proteins diluted in PBS were incubated for each well in a 96-well ELISA plate at 4°C overnight followed by blocking (5% non-fat milk in PBS-T) at RT for lhr. Each well was incubated with ⁇ amyloid oligomer antibodies (Al l, 1:1000 diluted in blocking buffer) at RT for 2hr. After washing with PBS-T, goat anti-rabbit Ab HRP conjugates (1:5000 diluted in blocking buffer) were added to each well and incubated at RT for lhr. Following washing, ⁇ 1-StepTM Ultra TMB-ELISA substrates (Thermo Fisher Scientific) were added to each well.
• amyloid fibrils To quantitate amyloid fibrils, detergent-insoluble proteins were extracted. Briefly, whole-cell lysates were centrifuged at 500xg for 2 min at 4°C. The supernatants were further centrifuged at 20,000xg for 20 min at 4°C. The final pellets were collected as detergent-insoluble fractions and solubilized by sonication for lOmin in PBS with 2% SDS. Following protein quantitation, 10 ⁇ g of solubilized proteins diluted in PBS were added to each well and incubated at 37°C without cover overnight to dry the wells. The following steps were identical to the oligomer detection with the exception of the use of amyloid fibril antibodies (OC) as the primary Ab.
• OC amyloid fibril antibodies
• amyloid fibrils were pelleted and re-suspended in distilled H2O. One drop of fibril solution was placed on a 200-mesh carbon-coated nickel grid (Electron Microscopy Sciences). After 1 min, the remaining liquid was wicked.
• Equal numbers of cells from different samples were lysed with cold cell lysis buffer. Following centrifugation at 20,000xg for 15 min at 4°C, detergent-insoluble pellets were further extracted with RIPA buffer 3 times. The final insoluble pellets were re- suspended in 10% SDS by pipetting and immediately subjected to aggregate sizing using a MultisizerTM 3 Coulter Counter equipped with a 20 ⁇ aperture (Beckman Coulter).
• A2058 cells were grown in 150mm culture dishes and treated with DMSO or 20nM AZD6244 for 8hrs. 2x10 8 cells receiving the same treatment were pooled and snap frozen in liquid nitrogen.
• Global quantitative analysis of cellular ubiquitination was conducted through the UbiScan® service (Cell Signaling Technology), which combines enrichment of ubiquitinated peptides by an ubiquitin branch ( ⁇ - ⁇ -GG) monoclonal antibody with liquid chromatograph tandem mass spectrometry (LC-MS/MS). Two technical replicates were analyzed for each treatment.
• ERK inhibitors FRl 80204 and Sch772984 (Ohori et al., 2005; Morris et al., 2013), activated HSF1 (Figure IF and 8C). Both MEK and ERK inhibitors impaired two ERK-mediated events— MSK1 phosphorylation and ELK1 activation ( Figure 1G and 8D; Roux and Blenis, 2004). While MEK inhibitors reduced ERK phosphorylation, two ERK inhibitors showed distinct effects (Figure 1G).
• MEKl and MEK2 form either homo- or heterodimers in vivo (Catalanotti et al., 2009).
• MEKl -HSFl interactions in the deficiency of MEK2. Under HS more MEKl proteins were precipitated with HSFl in MEX2-deficient cells ( Figure 2D).
• MEK1 deficiency heightened MEK2-HSF1 interactions ( Figure 2E), revealing a competition between the two MEK isoforms for HSFl binding and suggesting that MEK homodimers can interact with HSFl.
• HSF1 could maintain cellular proteostasis via HSPs.
• GR glucocorticoid receptor
• AZD6244 failed to deplete HSF1 and provoke ubiquitination in cells stably overexpressing HSF1 S326D ( Figure 4E and HJ), indicating a causative role of HSF1 inactivation in protein instability due to MEK inhibition.
• MEK inhibition also depleted HSPs and HSF1, and provoked ubiquitination in primary tissues ( Figure 4F).
• Aggregation-prone proteins can form amyloid fibrils (AFs) enriched for ⁇ - sheet structures (Eisenberg and Jucker, 2012).
• AFs amyloid fibrils
• Thioflavin T Thioflavin T
• CR Congo red
• PolyQ79 expression enhanced ThT and CR staining ( Figure 12Eand 12F), as expected.
• AZD6244, Bortezomib, and combined treatment further intensified this staining; and, HSF1 S326D expression antagonized the effect of AZD6244 ( Figure 5E-F).
• HSF1 depletion induced endogenous AOs and AFs Figure 12H and 121.
• a previously characterized antibody, OC was used to detect AFs (Kayed et al., 2007).
• HSF1 S326D expression suppressed AZD6244-induced amyloidogenesis ( Figure 5J and 5K).
• a unique feature of amyloids is their ability to seed AFs (Chiti and Dobson, 2006).
• lysates of fiiF/-depleted cells accelerated formation of ⁇ AFs ( Figure 12J).
• lysates of cells treated with AZD6244, Bortezomib, and combination exhibited augmented seeding efficacy (Figure 5L), which was confirmed using OC antibodies ( Figure 12K).
• HSF1 S326D expression abolished the effect of AZD6244 ( Figure 5L).
• transmission electron microscopy revealed that while seeding with DMSO-treated cell lysates resulted into disordered rod-like protofibrils, a dense lattice of fiber-like structures emerged following seeding with
• AZD6244 did not induce AOs in primary mouse embryonic fibroblasts (MEFs) and tissues (Figure 5P and 12T). This is not due to inability to detect murine amyloids, as severe stress did induce AOs in murine cells ( Figure 12U). These results suggest that non-transformed cells may be more refractory to amyloidogenesis than malignant cells.
• PHMC primary human mammary epithelial cells
• MCF10A immortalized human mammary epithelial
• MCF7 tumorigenic mammary epithelial
• Amyloidogenesis is tumor-suppressive
• HSF1 is a new MEK substrate
• RAS-RAF-MEK signaling may antagonize protein-misfolding diseases, such as amyloidosis, via guarding proteostasis.
• a patient having metastatic melanoma is treated with a combination of a MEK inhibitor and a proteasome inhibitor.
• the patient is treated with selumetinib as the MEK inhibitor and bortezomib as the proteasome inhibitor.
• the selumetinib is administered at 0.32 mg/Kg after reconstitution of a dry powder with water for daily intravenous injection or 0.64 mg/Kg with food through daily oral administration.
• the bortezomib is administered at 0.04 mg/Kg after reconstitution of a dry power with water for daily intravenous injection.
• the patient is expected to show a reduction in the growth of the tumor, the size of the tumor, or other clinical signs and symptoms of melanoma.
• Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan. Nature 472, 226-229.
• Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis.
• Heat shock transcription factor 1 is a key determinant of HCC development by regulating hepatic steatosis and metabolic syndrome. Cell Metab. 14, 91-103.
• the deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress.
• Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol. Neurodegener. 2, 18.
• Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells. Cancer Chemother. Pharmacol. 66, 535-545.
• Heat-shock transcription factor HSF1 has a critical role in human epidermal growth factor receptors- induced cellular transformation and tumorigenesis. Oncogene 29, 5204-5213.
• PSMB5 proteasome subunit beta5
• HSF1 is required for extra-embryonic development, postnatal growth and protection during inflammatory responses in mice. EMBO J. 18, 5943-5952.
• ARRY-142886 a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin. Cancer Res. 13, 1576-1583.

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