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WO2025208215A1 - Thérapie ciblant malat1 - Google Patents

Thérapie ciblant malat1

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Publication number
WO2025208215A1
WO2025208215A1 PCT/CA2025/050471 CA2025050471W WO2025208215A1 WO 2025208215 A1 WO2025208215 A1 WO 2025208215A1 CA 2025050471 W CA2025050471 W CA 2025050471W WO 2025208215 A1 WO2025208215 A1 WO 2025208215A1
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inhibitor
combination
seq
cancer
malat1
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Inventor
Gobi THILLAINADESAN
Olivia R. GRAFINGER
Hon S. LEONG
Gang Zheng
Hansen H. HE
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University Health Network
Sunnybrook Research Institute
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University Health Network
Sunnybrook Research Institute
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein

Definitions

  • cancer cells acquire various invasive qualities that enable them to pass through layers of extracellular matrix (ECM) and stromal cells in order to intravasate and extravasate through vasculature (1).
  • ECM extracellular matrix
  • stromal cells in order to intravasate and extravasate through vasculature (1).
  • Invadopodia formation by metastatic cancer cells is crucial during these dynamic manoeuvres around vessel walls.
  • Invadopodia are subcellular protrusions formed by metastatic cancer cells which extend 2-5 pm into extracellular space during cell invasion in vitro and cancer cell extravasation in vivo (2). Mechanical and chemical stimuli often cue invadopodia formation, leading to cancer cell extravasation which precedes metastatic colony formation (3, 4).
  • MT1-MMP is a membrane bound protease that plays a significant role in cancer progression due its localization at invadopodia (5).
  • MT1-MMP is continuously recycled at invadopodia to maintain proteolysis needed for metastasis (6).
  • pharmacologic targeting of MT1-MMP is ineffective and produces significant negative side effects (7). Given its central role in invasion and within invadopodia, identifying cancer-specific interactors with MT1-MMP could be more therapeutically effective.
  • RBPs are a large constituent of invadopodia (60 out of 230 proteins) alongside canonical invadopodia proteins such as cortactin, Arp2/3, Tks4/5, and vimentin (12).
  • RBPs were speculated to be facilitative for invadopodia-specific translation of proteins.
  • an aggregation of RBPs of this magnitude suggests something other than protein translation is involved in invadopodia formation.
  • specific ncRNA bound to RBPs at invadopodia could have a regulatory role in invadopodia formation. If ncRNA is a linchpin for RPB congregation at invadopodia, it would be a previously untested target for invadopodia inhibition and a straightforward therapy prospect.
  • an inhibitor to MALAT1 wherein the inhibitor targets positions 965-1934 of SEQ ID No.1. In some embodiments, the inhibitor targets positions 1255-1269 of SEQ ID NO.1., positions 1311-1316 of SEQ ID NO.1 , or positions 1884-1899 of SEQ ID NO.1.
  • a method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of the combination described herein or the inhibitor described herein.
  • the combination described herein or the inhibitor described herein for use in the treatment of cancer or for the prevention of cancer metastasis in a patient.
  • Figure 1 MT1-MMP Associates with RNA Binding Proteins and RNA to Assemble “R-Bodies” in the Cytoplasm of Metastatic Breast Cancer Cells.
  • H-J Immunofluorescence detection of R-bodies in integrin-activated metastatic breast cancer cells (arrowheads) by various staining methods: (H) RNA (green), hnRNPC (red), MT1-MMP (cyan); (I) RNA (green), hnRNPC (red), RALY (cyan); (J) RNA (green), MT1-MMP (red), RALY (cyan).
  • FIG. 2 RBPs and MALAT IncRNA are Required for “R-body” Formation, Functional Invadopodia, and Cancer Cell Extravasation in Metastatic Breast Cancer Cells.
  • RNAase Benzonase
  • K Intravital image quantification of cells following extravasation (K) and metastatic colony formation (M).
  • M metastatic colony formation
  • L Quantification of cells forming invadopodia following siMALI treatment. aE7 and aG11 indicates control and b1 activation respectively.
  • Figure 3 m6A Epigenetically Regulates MALAT1 and its Incorporation into R- Bodies.
  • FIG. 4 RNA Therapy for Targeting MALAT1 Abrogates Cancer Cell Extravasation and Metastasis Dynamics.
  • C -25 denotes 25 pg/chick embryo, -50 denotes 50 pg/embryo.
  • LNPs lipid nanoparticles
  • E In vitro imaging of LNP pre-loaded cells (siRNA - green signal; LNPs - red signal; nuclei - cyan signal) that arrested within the capillary bed prior to extravasation. Intravital imaging of LNP pre-loaded cells with siCON (F) and siMAL (G) reveals increase in siRNA signal within the cytoplasm over time. RNA therapy with LNP:siMAL or LNP:siCON reveals significant internalization and surface binding (H), resulting in inhibition on cancer cell extravasation rates (I), and metastatic colony formation (J), ‘denotes p ⁇ 0.05. K) Intravital imaging of LNPs on the surface and within the cytoplasm of cells, demonstrating uptake of LNPs within 1 hour of injection.
  • Figure 5 Pharmacologic and Genetic Targeting of METTL3 Abrogates Cancer Cell Extravasation and Metastasis Dynamics.
  • Figure 6 shows MALAT 1 mRNA annotation.
  • Figure 7 Total RIPseq Analysis with m6A Antibody on MALAT1. Analysis of PC3 and V16A cell lines with a focus on immunoprecipitated MALAT1 IncRNA. This figure corresponds to Fig. 3A. Red shaded region contains the m6A and hnRNPC binding sites..
  • FIG. 8 Secondary Structure of MALAT1. Secondary structure analysis of the broad peak region encompassing the m6A methylation site and hnRNPC binding site. This figure corresponds to Fig. 3B. Shaded region (grey box) indicates m6A site and hnRNPC binding site.
  • FIG. 9 M6A Deletion Abrogates R-body Formation and Invadopodia. Shows the deletion of M6A and it’s consequence.
  • A) Confirmation of the 300bp deletion in the mutant strains compared to WT.
  • C) RNA immunoprecipitation of the MT1-MMP14 protein followed by QPCR for MALAT 1 of MDA-231 cells treated with control antibody (E7), MDA-231 cells treated with activating antibody G11 and the MALAT1-M6A deleted cell lines (1G4 and 1 F4).
  • D) Same as (C) but immunoprecipitated using hnRNPC.
  • Metastatic cancer cells form invadopodia which physically mediate cancer cell extravasation but their “call to action” mechanisms during metastasis were unclear until now.
  • MT1-MMP an invadopodia specific protease
  • R-bodies RNA binding proteins
  • m6A- epigenetically marked MALAT 1 was exclusively found in the R-bodies. Depletion of any component of R-bodies and inhibition of m6A epigenetic marking of MALAT 1 halted R-body formation. This subsequently disabled invadopodia formation, culminating in major losses of cancer cell extravasation rates.
  • a combination of an inhibitor of MALAT1 with a delivery vehicle for targeting the inhibitor to the cytoplasm in an aspect, there is provided a combination of an inhibitor of MALAT1 with a delivery vehicle for targeting the inhibitor to the cytoplasm.
  • R-bodies reside in the cytoplasm and promote their effects on cancer cell extravasation.
  • the formation of RNA, RNA binding proteins, MT1-MMP, and other components of R-bodies is prevented.
  • Inhibition of R- bodies with this approach can inhibit cancer cell extravasation, which is a key step of the cancer metastatic cascade.
  • MALAT1 metastasis associated lung adenocarcinoma transcript 1 also known as NEAT2 (noncoding nuclear-enriched abundant transcript 2) is a large, infrequently spliced non-coding RNA, which is highly conserved amongst mammals and highly expressed in the nucleus.
  • the inhibitor is an antisense oligonucleotide, siRNA, miRNA or shRNA.
  • the inhibitor is an siRNA.
  • the MALAT1 has SEQ ID NO. 1 :
  • the inhibitor targets regions of MALAT1 with adenosine methylation potential.
  • the M6A peak is at positions 965-1934 of SEQ ID NO. 1.
  • the DRACH methylation site is at positions 1255-1269 of SEQ ID NO.1.
  • the hnRNPC binding site is at positions 131 1-1316 of SEQ ID NO.1.
  • the siRNA binding site is at positions 1884-1899 of SEQ ID NO.1.
  • the inhibitor targets positions 965-1934 of SEQ ID NO.1.
  • the inhibitor targets positions 1255-1269 of SEQ ID NO.1 ; positions 1311-1316 of SEQ ID NO.1 ; or positions 1884-1899 of SEQ ID NO.1.
  • the inhibitor is 5' GGCUUAUACUCAUGAAUCUtt 3' (SEQ ID NO. 2).
  • the delivery vehicle is a lipid nanoparticle.
  • Lipid nanoparticles are described in the art and may be suitable for use in the presently claimed combination.
  • Lipid nanoparticles are nanoparticles composed of lipids and typically spherical with an average diameter between 10 and 1000 nanometers.
  • Solid lipid nanoparticles possess a solid lipid core matrix that can solubilize lipophilic molecules.
  • the lipid core may be stabilized by surfactants (emulsifiers).
  • the emulsifier used depends on administration routes and is more limited for parenteral administrations.
  • the term lipid is used herein in a broader sense and includes triglycerides (e.g. tristearin), diglycerides (e.g.
  • LNPs can contain porphyrin, such as those described in U.S. Provisional Patent Application No. 63/445773, which is incorporated herein in its entirety.
  • the lipid nanoparticle is a porphyrin-lipid nanoparticle.
  • the porphyrin lipid nanoparticle comprises DLin-MC3-DMA, Porphyrin-lipid, cholesterol, and DMG-PEG2000.
  • the combination is in combination further with a METTL3 inhibitor.
  • an inhibitor to MALAT1 wherein the inhibitor targets positions 965-1934 of SEQ ID No.1. In some embodiments, the inhibitor targets positions 1255-1269 of SEQ ID NO.1., positions 1311-1316 of SEQ ID NO.1 , or positions 1884-1899 of SEQ ID NO.1.
  • the inhibitor is 5' GGCUUAUACUCAUGAAUCUtt 3' (SEQ ID NO. 2).
  • a pharmaceutical composition comprising the combination described herein or the inhibitor described herein along with a pharmaceutically acceptable carrier.
  • a method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of the combination described herein or the inhibitor described herein.
  • therapeutically effective amount refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.
  • a method of preventing cancer metastasis in a patient comprising administering to the patient a therapeutically effective amount of the combination described herein or the inhibitor described herein.
  • the combination described herein or the inhibitor described herein for use in the treatment of cancer or for the prevention of cancer metastasis in a patient.
  • MT1-MMP (ab51074; Abeam, Toronto, Canada); P4G11 pi Integrin, E7-s (Developmental Systems Hybridoma Bank, Iowa City, IA); RALY (PA5-83671 ; Thermo Fisher Scientific; Nepean, ON); hnRNPC1/C2 (4F4) (sc-32308; Santa Cruz Biotechnology; Santa Cruz, CA); m6A (ab151230; Abeam, Toronto, Canada).
  • MDA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, A) and cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Growth conditions were 37°C with humidity and 5% CO 2 . Cells were lifted using 5 mM EDTA/PBS (pH 7.4). Cells were plated in serum-free DMEM for 16 h prior to plating onto 0.2%-gelatin-coated coverslips or culture plates in serum-free medium. Cells were treated with P4G11 (10 mg/mL) where indicated. Control conditions for all experiments were cells treated with the same concentration of a nonspecific IgG (E7-s).
  • Immunoprecipitation was performed as described previously (6). Antibodies were coupled to protein G Dynabeads (Invitrogen) according to the manufacturer’s instructions. Cells were lysed in situ with cold RIPA buffer (150 mM NaCI, 2 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.5) containing protease inhibitor cocktail, and lysate was incubated with antibodybound Dynabeads overnight at 4°C, then washed three times with ice-cold PBS. Proteins bound to the beads were eluted with 2.5* Laemmli loading buffer heated to 95°C. Immunoprecipitates were separated using SDS-PAGE and analyzed by western blot.
  • Cell culture inserts were prepared as described previously (32). Briefly, the bottoms of transwell inserts (8 mm pore diameter, Corning Inc.) were coated with 20 mg/mL fibronectin/PBS. The top of the chamber was coated with 0.125 mg/mL growth factor- reduced Matrigel (Corning). MDA-MB-231 cells were serum-starved for 24 h, lifted, seeded into chambers with simultaneous antibody treatment, and allowed to migrate for 20 h. The cells that invaded towards the lower chamber (10% FBS/0.1% BSA in DMEM) were fixed in 4% paraformaldehyde, stained with Hoechst for nuclear visualization, and counted. Cells that did not migrate through the membrane were removed with a cotton swab prior to fixation. Ten fields of cells per membrane were counted per treatment. Data is presented as percent of control.
  • Invadopodium formation was performed as described previously (32). Glass coverslips were coated with 50 mg/mL poly-L-lysine/PBS, followed by 0.5% glutaraldehyde/PBS. Coverslips were then inverted onto a 70 mL drop of Alexa Fluor 488-labeled gelatin (Thermo Fisher Scientific). The coverslips were then incubated with 5 mg/mL sodium borohydride (Sigma-Aldrich) and then washed extensively in PBS. Tissue culture plates were coated similarly, with the exception being that 0.2% unlabelled gelatin/PBS was used. Cells were plated onto coverslips at 50% confluency and incubated for 4 h.
  • Invadopodia e.g. F-actin
  • Invadopodia were counted as spots of gelatin degradation overlayed by F-actin punctae as visualized by epifluorescence microscopy.
  • Fifty cells per coverslip per treatment were scored for their ability to form invadopodia.
  • Image processing and analysis was carried out using Imaged software (National Institutes of Health). In cases of uneven background illumination, the “Subtract background” plugin was run with a rolling ball radius of 50.0 pixels.
  • Cells were serum-starved overnight and plated for 4 hrs onto 0.2% gelatin-coated glass coverslips (as described in “Invadopodia Formation Assay”). Cells were fixed in 4% formalin (ACP Chemicals) and then washed in 150 mM glycine/PBS. Cells were permeabilized in 0.1% Triton X-100/PBS and blocked in 5% (w/v) BSA/PBS prior to antibody staining. Samples were imaged using a Nikon Fast A1 R upright confocal microscope. Images were captured using Nikon confocal software, and image processing and analysis was done using Imaged software (National Institutes of Health, Bethesda, MD). The ViewRNA Cell Plus Assay kit (Thermo Fisher Scientific) was used for simultaneous fluorescent in situ hybridization (FISH) and immunofluorescent staining, according to the protocol of the manufacturer.
  • FISH fluorescent in situ hybridization
  • Antibodies were coupled to protein G Dynabeads (Invitrogen) according to the manufacturer’s instructions.
  • Cells were lysed in situ with cold RIPA buffer (20 mM Tris- HCI [pH 7.4], 150 mM NaCI, 0.1% SDS, 1% Triton X-100, 1% Deoxycholate, 1% NP40) with 1x protease inhibitor cocktail (Thermo Fisher Scientific). Lysate was incubated with antibody-bound Dynabeads overnight at 4°C, then washed three times with ice-cold PBS. Proteins bound to the beads were eluted with 2x Laemmli loading buffer (BioRad) heated to 95°C. Immunoprecipitates were separated using SDS-PAGE and analyzed by Western Blot.
  • cytoplasmic lysis buffer 50 mM HEPES-KOH pH 7.5, 140 mM NaCI, 1 mM EDTA, 10% glycerol, 0.5% NP-40/lgepal CA-630 and 0.25% Triton X-100
  • Thermo complete EDTA-free proteinase inhibitor cocktail
  • Thermo Fisher Scientific RNase inhibitors
  • Beads were eluted twice in 75 pL of RIP elution buffer (50 mM Tris pH 8, 10 mM EDTA, 300 mM NaCI, 1 % SDS) at 37 °C for 10 min.
  • 100 pL of RIP elution buffer was added to a final volume of 150 pL.
  • 20 pg of proteinase K was added to both IP and input samples, and the mixtures were incubated at 37°C for 1 h and then at 65°C for 1 h to de-crosslink.
  • the samples were extracted one time with phenol-chloroform and one time with chloroform and precipitated with ethanol.
  • Maximum injection time was set to 50 ms with an automatic gain control of 4e5.
  • the fragment ion scan was done in the Orbitrap using a Quadrupole isolation window of 1 .6 m/z and HOD fragmentation energy of 30 eV.
  • Orbitrap resolution was set to 30,000 with a maximum ion injection time of 50 ms and an automatic gain control target set to 5e4.
  • Raw data processing was performed as described previously (14).
  • Raw files were analyzed together using MaxQuant software (version1.6.0.26) (33).
  • the derived peak list was searched with the built-in Andromeda search engine against the reference Homo sapiens proteome (July 2019; 74,811 sequences) from Uniprot (http://www.uniprot.org) (34).
  • the parameters were as follows: strict trypsin specificity, allowing up to two missed cleavages, minimum peptide length was seven amino acids.
  • a minimum of two peptides required for protein identification and peptide spectral matches and protein identifications were filtered using a target-decoy approach at a false discovery rate (FDR) of 5%.
  • FDR false discovery rate
  • ‘Match between runs’ was enabled with a match time window of 0.7 min and an alignment time window of 20 min.
  • Relative, label-free quantification (LFQ) of proteins used the MaxLFQ algorithm integrated into MaxQuant using a minimum
  • a Student’s t-test identified proteins with significant changes in abundance (p-value ⁇ 0.05) with multiple hypothesis testing correction using the Benjamini-Hochberg FDR cutoff at 0.05.
  • PCA principal component analysis
  • RNA immunoprecipitated sequences were aligned to the genome using HISAT2 (35).
  • Analysis and normalization of the sequencing data was done as previously described (27). Analysis was partly done on the high- performance computing platforms Graham (https://docs.alliancecan.ca/wiki/Graham) and Niagra (https://docs.alliancecan.ca/wiki/Niagra) in support with Compute Canada (www.computecanada.ca).
  • siRNA concentration and encapsulation efficiency were measured by Quant-itTM RiboGreen RNA Assay based on manufacturer’s protocol (Thermofisher), typically the encapsulation efficiency is >90%.
  • the hydrodynamic size and poly dispersity of porphyrin-LNPs were characterized using a Zetasizer Nano ZS (Malvern Instruments) and Hitachi HT7800 transmission electron microscopy (Nanoscale Biomedical Imaging facility, Peter Gilgan Centre for Research and Learning, Toronto).
  • siRNA si-Control Thermo Fisch er silencer select: 4390843 si-MALAT1 (siMAL) 5’ GGCUUAUACUCAUGAAUCUtt 3‘ si-MALAT1 (si MALI )
  • MT1-MMP is a protease involved in invadopodia function and is involved in cancer cell extravasation (2), also known as transendothelial migration of cancer cells across the vessel wall at distant sites (1).
  • pi integrin activation by the P4G11 antibody clone (13) is a potent stimulant for downstream invadopodia formation, in particular through regulation of MT1-MMP phosphorylation and intracellular recycling (6, 14). This antibody-mediated approach allows us to analyze invadopodia formation dynamics in a highly reliable and reproducible manner.
  • RNA binding association between MT1-MMP and the RBPs hnRNPC and RALY were confirmed through co-immunoprecipitation (Fig. 1G) as well as immunofluorescence microscopy (Fig. 1 H,I,J). RNA was also observed within these aggregates of RBPs (hnRNPC, RALY) and MT1-MMP; leading us to coin the term “R- bodies” (RNA, RBPs, protease). Interestingly, R-bodies were observed in patient- derived metastatic breast cancer cells (Fig. H,l, J bottom panels). Quantification of R- bodies in MDA-MB-231 cells indicated their elevated presence in 40.6 ⁇ 3.1% of cells treated with the pi integrin-activating antibody (Fig.
  • RNAse treatment led to a significant decrease in hnRPNC that immunoprecipitated with MT1-MMP (Fig. 2E) revealing a linchpin-like requirement of RNA for the formation of R-bodies. This prompted RIPseq of immunoprecipitants with the anti-MT1-MMP antibody but with only cytoplasmic portions of [31 integrin activated cells, a modified technique called ‘cytoplasmic RIPseq’.
  • Cytoplasmic RIPseq revealed an enrichment of MALAT1 (8 kb), RPVU1 , and RPU1 (both 164 bp) over mock antibody control, all of which are ncRNAs (Fig. 2F,G).
  • MALAT1 IncRNA binding to MT1-MMP was confirmed via cytoplasmic RIP-qPCR for MALAT1 when using the same anti-MT1-MMP antibody immunoprecipitates.
  • MALAT1 levels were elevated in pi integrin activated cell cytosols compared to control (Fig. 2H).
  • FISH probes for MALAT 1 revealed extra-nuclear punctate signal that co-localized with RNA/DNA, hnRNPC, and RALY signal (Fig. 2I) that did not exist in controls.
  • MALAT1 is known to have m6A epigenetic marks on various adenosine residues (15). These m6A methyl groups on RNA have been shown to alter secondary structure of the methylated IncRNA, which opens up various binding sites for RPBs (16-18).
  • Total cell RIPseq was performed on various cancer cell lines such as PC3, LnCAP, MDA-MB-468, etc. using the anti-m6A antibody revealing MALAT1 IncRNA regions possessing the same m6A epigenetic patterning (Fig. 7). When viewing these regions at higher resolution, specific portions of MALAT1 immunoprecipitated with the anti-m6A antibody revealing the exact regions that possess the m6A mark (Fig. 3A, Fig. 7).
  • m6A methylation on RNA is added by methyl transferase METTL3 and removed by the demethylase ALKBH5/FTO (20-22).
  • lentiviral shRNA vectors were used to knockdown (KD) these enzymes (90-95% knockdown) followed by meRIP.
  • KD knockdown
  • meRIP meRIP
  • RNA Therapy for Targeting MALAT1 Abrogates Cancer Cell Extravasation and Metastasis Dynamics
  • RNA Therapy of MALAT 1 we sought to only target its m6A region as opposed to a pooled approach used in Fig. 2 and Fig. 3.
  • siMAL was used for RNA Therapy which consisted of porphyrin labelled lipid nanoparticles (LNPs) (23-25); some versions possessed a fluorescent 6-FAM label for confirmation of cargo loading and unloading into cells during treatment (Fig. 4A).
  • LNPs porphyrin labelled lipid nanoparticles
  • Fig. 4A To confirm the genetic targeting efficacy of this RNA therapy, cells were pre-treated with various doses and then submitted to cytoplasmic RIP qPCR for MALAT1 using anti-MT1-MMP antibody.
  • the 6-FAM fluorescent version of siMAL and siCON was used to understand the offloading kinetics of RNA therapy cargo into the cell cytoplasm in vitro and during intravital imaging of cancer cell extravasation in vivo.
  • In vitro incubation revealed rapid uptake of LNPs (porphyrin fluorescent label) and the majority of endocytosed LNPs contained the 6-FAM labelled RNA (Fig. 4D). This was consistent with other measurements that confirmed efficient loading of siRNA into the LNPs.
  • Intravenous injection of vacant LNPs and LNP-preloaded cancer cells was submitted to intravital imaging to confirm their detection with this method.
  • LNP-preloaded cancer cells were arrested in the lumen of the CAM microvasculature immediately after intravenous injection, revealing cytoplasmic accumulation of 6-FAM signal released by internalized LNPs. Majority of the preloaded cells revealed increasing levels of siMAL or siCON in the cytoplasm over time presumably due to cargo release by the LNPs (Fig. 4E,F).
  • RNA therapy LNPs containing siMAL or siCON; LNP:siMAL and LNP:siCON respectively
  • RNA therapy treatment with LNPs containing siMAL cargo produced a 33% decrease in cancer cell extravasation rates (Fig. 4H), followed by a 77% decrease in metastatic colony formation (Fig. 4I).
  • RNA therapy for MALAT1 led to a cumulative 7.5 fold increase in metastatic inefficiency rates compared to LNP:siCON.
  • Intravital imaging yielded important pharmacological insights regarding the nature of the RNA therapy and its affinity for cancer cells.
  • all if not the majority of intravascular cancer cells prior to cancer cell extravasation exhibited an abundant coating of LNPs on their cell surface and internalization of LNPs (Fig. 4G; Fig. 4J).
  • No LNPs were observed to be within the nucleus.
  • LNPs were also observed to be in the microcirculation at high velocities whilst minimal uptake of LNPs by the stroma was observed. LNPs were only internalized by cancer cells or immune cells.
  • RNA immunoprecipitation of MT1-MMP14 followed by qPCR for MALAT 1 demonstrated increased enrichment in activated cells (G11 antibody- treated), whereas this signal was completely abrogated in the M6A-deleted lines (1G4 and 1 F4) (Fig. 9C). Similar results were observed with RIP using hnRNPC (Fig. 9D). Cytoplasmic R-body formation, visualized by microscopy, was evident in WT cells but absent in the M6A deletion mutants (Fig. 9E). Furthermore, invadopodia formation, both in quantification and in confocal images, was observed in WT cells but not in the MSA- deleted lines (Fig. 9F).
  • invadopodia formation upcycles cytoplasmic MT1-MMP to form large ternary complexes consisting of: 1) RNA binding proteins (i.e., hnRNPC, etc.), and 2) MALAT 1 IncRNA. These grow in size to form large aggregates called R-bodies that are extranuclear and not adjacent to invadopodia in 2D.
  • R-bodies that are extranuclear and not adjacent to invadopodia in 2D.
  • Our findings point to the role of the epitranscriptome, wherein m6A methylation of MALAT 1 is required for R-body formation (Fig. 5G). Disruption of any component of R-bodies (hnRNPC/MALATI) leads to loss of invadopodia formation, which has cascade-like consequences on metastasis dynamics.
  • RNA binding proteins (RBPs) play a significant role in R-bodies.

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Abstract

L'invention concerne une association d'un inhibiteur de MALAT1 avec un vecteur d'administration pour cibler l'inhibiteur sur le cytoplasme.
PCT/CA2025/050471 2024-04-03 2025-04-02 Thérapie ciblant malat1 Pending WO2025208215A1 (fr)

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