WO2023094662A1 - Traitement du rhabdomyosarcome - Google Patents

Traitement du rhabdomyosarcome Download PDF

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WO2023094662A1
WO2023094662A1 PCT/EP2022/083503 EP2022083503W WO2023094662A1 WO 2023094662 A1 WO2023094662 A1 WO 2023094662A1 EP 2022083503 W EP2022083503 W EP 2022083503W WO 2023094662 A1 WO2023094662 A1 WO 2023094662A1
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rms
cells
tumour
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Enrico Pozzo
Maurilio SAMPAOLESI
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Katholieke Universiteit Leuven
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    • 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
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    • C12N2320/31—Combination therapy

Definitions

  • the invention relates to treatment of cancers with miRNAs.
  • Rhabdomyosarcoma occurs in 50% of paediatric soft tissue sarcomas and accounts for 3-4% of all paediatric cancers. The incidence is high below the age of 20, after which the incidence decreases to 1% of adult malignancies. In most cases, paediatric RMS tumours occur in the head and neck region.
  • a combination of oncosuppressor microRNAs is identified and their effects on the tumorigenicity and the metabolism of pyruvate in FN-RMS were characterized.
  • the present invention investigates the effect of mitochondrial pyruvate carrier (MPC) inhibition in human and murine FN-RMS and compare it to oncosuppressor miRNAs (hsa-miR-449a-5p and hsa-miR-340-5p).
  • MPC mitochondrial pyruvate carrier
  • Human FN-RMS (RD18 and RD) and murine FN-RMS (KMR46) were used as models, and the treatment effects were investigated via immunofluorescence staining, quantitative real-time polymerase chain reaction (RT-qPCR), and western blot in order to assess changes at both the morphological and molecular levels.
  • RT-qPCR quantitative real-time polymerase chain reaction
  • EVs extracellular vesicles
  • MPC inhibition led to a decrease in cell proliferation and formation of multinucleated structures, through the alteration of tumour metabolism by MPC inhibition.
  • miRNA treatment led to cell death and the formation of large multinucleated structures.
  • Both treatments showed an increase in the fusion percentage, i.e., increase in the amount of multiple nuclei cells, and with the feature of enlargement of cells after miRNA treatment, the results indicate that these cells are senescent-like cells after treatment.
  • MPCi MPC inhibitor
  • the MPCi showed similar effects as the oncosuppressor miRNAs in terms of reduction in cell proliferation in human and murine FN-RMS. These results allow development of therapies such as the in vivo delivery of miRNAs through the use of EVs.
  • Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to statement 3, in a human individual. 5. Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to statement 3 or 4, wherein the tumour is a soft tissue sarcoma, such as a paediatric soft tissue sarcoma.
  • Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to any one of statements 3 to 5, wherein the tumour is rhabdomyosarcoma.
  • rhabdomyosarcoma RMS
  • the rhabdomyosarcoma is selected from the group of embryonal RMS, alveolar RMS, spindle cell RMS, mixed-type RMS, pleomorphic RMS, and RMS with ganglionic differentiation and alveolar RMS.
  • rhabdomyosarcoma rhabdomyosarcoma
  • FP-RMS fusion-positive RMS
  • FN- RMS fusion-negative RMS
  • Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to any one of statements 3 to 6, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
  • a pharmaceutical composition comprising mir-340 and/or mir-449a.
  • composition according to statement 10 wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
  • a method of treating tumour in an individual comprising the step of administering mir-340 and/or mir-449a to said individual, thereby converting tumour or tumorigenic cells into cell with a non-tumorigenic phenotype.
  • mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
  • Figure 1 Differences between different samples of KMR46 day 3 and day 5 at lOx magnification
  • A Graphical representation of KMR46 day 3 and day 5 nuclei counting for different sample groups.
  • B Graphical representation of KMR46 day 3 and day 5 nuclei size (pm) of different sample groups.
  • Figure 2 Differences between different samples of human FN-RMS day3 and day 5 at lOx magnification
  • A Graphical representation of human FN-RMS day 3 and day 5 nuclei counting for different sample groups.
  • B Graphical representation of human FN-RMS day 3 and day 5 nuclei size (pm) of different sample groups.
  • Day 3 Changes in protein contents after treatment with MPCi and miRNA, compared to the DMSO control group
  • FIG. 6 In vitro and in vivo effects of miR-449a+340 in FN-RMS.
  • FIG. 7 miRNA effects on metabolism.
  • B Downregulated Gene Ontology pathways in FN-RMS patients compared to skeletal muscle;
  • FIG. 8 MPC inhibition and oncosuppressor miRNAs similarly affect oxygen consumption rate, cell cycle and proliferation.
  • B Seahorse measurement of oxygen consumption rate (n>4);
  • D Nuclei counting,
  • E Size, and
  • F Fusion percentage in human FN-RMS. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • (n 3)
  • MPCi and miRNAs similarly target in vivo migration and proliferation, and improve overall exercise capability in mice.
  • FIG. 10 Epigenetic effects of miR-449a+340 on FN-RMS.
  • 2-DG 2-deoxyglucose ;
  • AGO Argonaute protein;
  • AKT Protein kinase B;
  • ALK Anaplastic lymphoma kinase;
  • Alpha-SMA Alpha smooth muscle actin;
  • ARMS Alveolar rhabdomyosarcoma;
  • ATP Adenosine triphosphate;
  • BCOR BCL6 corepressor;
  • BMPs Bone morphogenetic proteins;
  • BSA Bovine serum albumin;
  • CCND2 Cyclin- dependent kinase cyclin D2 (CCND2);
  • CPT1A Carnitine palmitoyltransferase ;
  • CTNNB1 Catenin beta 1;
  • CXCR4 C-X-C chemokine receptor type 4;
  • DMEM Dulbecco's Modified Eagle Medium;
  • DMEM-HG High glucose Dulbecco's
  • Rhabdomyosarcoma is a soft tissue sarcoma with characteristics of skeletal muscle lineage, having high incidence in young adults below the age of 20.
  • RMS Rhabdomyosarcoma
  • FP-RMS fusion-positive RMS
  • FN-RMS fusion-negative RMS
  • RMS can be subdivided into six histological groups, i.e. embryonal, alveolar, spindle cell, mixed-type, pleomorphic, and RMS with ganglionic differentiation.
  • ERMS embryonal RMS
  • RMS alveolar RMS
  • ERMS embryonal RMS
  • RMS alveolar RMS
  • FN-RMS typically have a high rate of single-nucleotide alterations, and the recurrent mutation typically occurs in cancer genes such as harvey-ras (HRAS), neuroblastoma-ras (NRAS), kirsten-ras (KRAS), anaplastic lymphoma kinase (ALK), fibroblast growth factor receptor 4 (FGFR4), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), f-box and wd repeat domain containing 7 (FBXW7), neurofibromatosis type 1 (NF1), TP53, catenin beta 1 (CTNNB1) or BCL6 corepressor (BCOR).
  • HRAS harvey-ras
  • NRAS neuroblastoma-ras
  • KRAS kirsten-ras
  • ALK fibroblast growth factor receptor 4
  • PIK3CA phosphatidyl
  • FN-RMS tumours are characterized by the loss of function TP53 mutations, and this is due to focal amplification of the mouse double minute 2 homolog (MDM2), a TP53 negative regulator.
  • PAX3-FOXO1 has the ability to alter the transcription of genes such as FGFR4 and insulin-like growth factor 2 (IGF-2).
  • IGF-2 overexpression occurs in FN-RMS due to loss of heterozygosity (LOH) at the llpl5.5 locus.
  • FN-RMS mutations occur in genes that affect the activation of the receptor tyrosine kinase (RTK)/RAS/phosphatidylinositol-3- kinase (PI3K) signaling axis, and somatic mutations occur in cell cycle genes, including CTNNB1, FBXW7, BCOR and p53, which commonly occur with the gain of chromosomes 2,8 and 13.
  • RTK receptor tyrosine kinase
  • RTK receptor tyrosine kinase
  • PI3K phosphatidylinositol-3- kinase
  • FP-RMS The characteristics of FP-RMS are sustained activation of the RTK/RAS/PI3K signaling axis, tumours with a high copy number of the N-MYC gene (due to the PAX3-FOXO1 fusion protein regulating the oncogenic transcription of N-MYC) and a loss of imprinting at the llpl5.5 locus which leads to IGF-2 overexpression. Therefore, inhibiting the expression and/or activity of PAX3-FOXO1 by using specific inhibitors is one of the main goals of current studies for treatment of FP-RMS.
  • FN-RMS fusion-negative RMS
  • RMS cells express PAX3, PAX7 and myogenic regulatory transcription factors MRFs).
  • PAX3 and PAX7 indicate both muscle-forming and neural crest cells.
  • MSCs mesenchymal stem cells
  • FN-RMS low-passage myoblasts
  • sarcomas from both low-passage MSCs and myoblasts.
  • MRFs myogenic factor 5 (MYF5) and myoblast determination protein (MyoD) [Clark et al. (1991) Br. J. Cancer 64(6), 1039-1042].
  • MYF5 and MyoD have important roles in regulating growth, proliferation and TPC activity in RMS. Not only is MYF5 a marker of TPCs in the zebrafish FN-RMS model, but also in vivo it can transmit tumour propagating potential to differentiated FN-RMS cells. Both MYF5 and MyoD are essential for RMS proliferation where together they regulate molecular programs that are commonly found in healthy muscle development and regeneration.
  • MYF5 and MyoD are located on the promoter and enhancer regions of certain genes, such as cyclin-dependent kinase cyclin D2 (CCND2), myogenin (MYOG), and cadherin 15 (m-cadherin, CDH15'). These genes regulate cell cycle progression and muscle differentiation. This is also the reason why when MYH5 and MyoD are knocked down, the aforementioned genes are commonly downregulated. In general, MyoD and MYF5 have essential roles in muscle cell identity regulation and cell cycle regulation. Both are needed for tumour growth and may regulate stem cell self-renewal in healthy muscles.
  • MPC Mitochondrial pyruvate carrier
  • the blockade of MPC activity promotes cytosolic accumulation of pyruvate which stops the intracellular production of pyruvate through lactate conversion and prevents the uptake and consumption of extracellular lactate by cancer cells. Therefore, when lactate was the only available nutrient, the blockage of MPC activity prevented cancer cell growth. Also, the MPC inhibition could force cells to go from partially aerobic glucose metabolism to strict anaerobic glycolysis which could also cause cytotoxic effects. Finally, when blocking lactate and glucose from entering the TCA cycle as fuel, the inhibition of MPC activity also massively decreases the oxygen consumption and thus sensitizes tumours to radiotherapy via local reoxygenation. However, the blockade of MPC activity leads to the inhibition of lactate uptake, but also alters glucose oxidation.
  • MPC activity could lead to aerobic glycolysis and induce growth of tumour cells.
  • a lower expression of MPC proteins has been shown to lead to poor survival rates in patients with different types of cancers, such as colon cancer, prostate cancer, and oesophageal cancer.
  • cancer cells undergo metabolic status changes where cells adapt to prevent cell death and to sustain continuous cell division.
  • One important characteristic of cancer metabolism is that tumour cells tend to metabolize glucose anaerobically, even in aerobic conditions. Compare to the aerobic glycolysis, this type of metabolism leads to increase in glucose uptake and production of lactate via fermentation of glucose..
  • This type of molecular adaptation is called the Warburg effect where glucose-derived carbon skeletons are used for macromolecule biosynthesis instead of oxidation via the TCA cycle in the mitochondria.
  • cancer cells alternatively use the glucose backbone to produce ribose and nicotinamide adenine dinucleotide phosphate (NADPH) for nucleotide synthesis, lipid biosynthesis and the maintenance of the cells' redox state.
  • NADPH nicotinamide adenine dinucleotide phosphate
  • Glutamine is a non- essential amino acid precursor, a substrate for gluconeogenesis, and also an alternative energy source for rapidly dividing cells.
  • FAS fatty acid synthase
  • PK pyruvate kinase
  • Rh30 cells the balance between energy production and the biosynthetic precursor production may be regulated by an activation of anaplerotic carboxylation and the malate/aspartate shuttle which promotes nicotinamide adenine dinucleotide hydrogen (NADH) hydride to migrate from the cytoplasm to the mitochondrion for oxidative phosphorylation.
  • NADH nicotinamide adenine dinucleotide hydrogen
  • PAX3-FOXO1 coordinates the expression of several PAX3 downstream genes, such as hepatocyte growth factor receptor (HGFR or c-MET), FGFR4, IGF-2 and C-X-C chemokine receptor type 4 CXCR4), which are key genes involved in the increase in tumour aggressiveness and metastasis recurrence of FP-RMS.
  • PAX3-F0X01 regulates the transcription of the GLUT4 gene which leads to an increase of glucose consumption in FP-RMS cells.
  • PAX3-FOXO1 also causes the downregulation of the phosphate and tensin homolog deleted on chromosome 10 PTEN) gene [101], thus resulting in the activity alteration of the PI3K/(protein kinase B)AKT/mammalian target of the rapamycin (mTOR) pathway).
  • acyl carnitines which facilitate lipid degradation. This could be the source of energy for FP-RMS cells for migration and metastasis.
  • Both FN-RMS and FP-RMS are related to the activation of RTK-dependent pathways, which include the PI3K/AKT and RAS pathways.
  • the PI3K/AKT pathway triggers the consumption of glucose, amino acids and other nutrients.
  • mTOR glycolysis and lactate production, lipogenesis and other protein synthesis are promoted.
  • HIF Hypoxia inducible factors
  • IL-8 proangiogenic interleukin-8
  • CXCR1 and CXCR2 7-transmembrane G-protein-coupled receptors
  • p53 has a role in inhibiting the expression of glucose transporters (GLUTs), for example GLUT1 and GLUT3/GLUT4, and it reduces glucose consumption in the glycolytic pathway.
  • GLUTs glucose transporters
  • PTEN which negatively regulates glycolysis and the PI3K/AKT/mTOR and HIF pathways. Therefore, for cells with no p53 activity, typically high sugar transport across the cell membrane and high glucose consumption are found. The chances to have a loss of p53 activity are higher in FN-RMS than in FP- RMS, with an incidence rate of 5-19%.
  • glucose consumption is higher since the expression of the GLUT1 and GLUT4 increase due to the loss of p53 activity. Additionally, increasing the expression of GLUT1 transporter increases glucose consumption and glycolytic use which eventually is the reason for high cell proliferation and invasiveness.
  • Activation of the RAS pathway is closely related to FN-RMS aggressiveness.
  • RAS-positive FN-RMS cells are related to increased oxidative stress.
  • FN-RMS tumours are found to have a high rate of G to T transversions due to oxidative damage.
  • FN-RMS promote methylation of certain genes which regulate metabolism, mitochondrial function and oxidative stress.
  • ROS mitochondrial reactive oxygen species
  • MAPK mitogen activated protein kinase
  • FN-RMS tumours could benefit from increased ROS levels, but at the same time, increasing accumulation of ROS-induced gene mutations could lead to cell death as well. Therefore, oxidative stress inducer, actinomycin-D could be the key to control tumour growth.
  • MicroRNAs belong to the class of non-coding RNAs (ncRNAs), with an average size of 22 nucleotides and having roles in regulating gene expression. Most miRNAs are first transcribed from DNA into primary miRNAs (pri-miRNAs), then inside the nucleus pri-miRNA is further processed into a precursor miRNA (pre-miRNA) by a class 2 RNase III enzyme called Drosha. After this, exportin-5 (EXP-5) helps with the transportation of pre-miRNA to the cytoplasm via nuclear pore complexes, a large proteinaceous channel located in the nuclear membrane. Inside the cytoplasm, pre- miRNAs are processed by Dicer to become mature miRNAs.
  • Dicer an RNase III type protein
  • Dicer-1 is responsible for miRNA maturation
  • Dicer-2 is responsible for siRNA maturation.
  • AGO Argonaute
  • RISC effector RNA-induced silencing complex
  • miRNAs interact with the target mRNAs via the 3' untranslated region (3' UTR), or in some cases, via other sites such as the 5' UTR, a coding sequence, and gene promoters. Depending on the situation, miRNAs can activate translation or regulate transcription as well. However, interaction of miRNAs with target genes can be affected by many factors, for example, the subcellular location of miRNAs, the abundancy of miRNAs and mRNAs and the affinity of their interactions. miRNAs secreted into extracellular fluids can be transported to target cells by vesicles. Extracellular miRNAs act as chemical messengers which can mediate cell-cell communications.
  • the eIF4F complex is related to the initiation of translation.
  • This complex consists of subunits, such as eIF4A, eIF4E, and eIF4G.
  • the eIF4E subunit recognizes the mRNA 5' terminal cap and starts the initiation process.
  • another initiation factor, eIF3 reacts with eIF4G and helps with the 40S ribosomal subunit assembly at the 5'end of the mRNA.
  • the elongation process is then initiated by the 60S ribosomal subunit and 40S preinitiation complex binding at the AUG codon of the mRNA.
  • miRNA-induced silencing complex may repress the elongation process, and it could promote early ribosome dissociation from mRNAs.
  • miRISC miRNA-induced silencing complex
  • miRISC prevents the mRNA from turning into a circular shape which causes translational inhibition.
  • miRISC can inhibit the 60S ribosomal subunit from joining with the 40S subunit, leading to translational repression.
  • miRNA or miRISC can have an effect on translational repression due to the increase in target mRNAs in the processing bodies.
  • miRNAs The degradation of target mRNAs is induced by the AGO protein slicer activity. miRNAs also affect this process. Some other mechanisms are also involved, such as deadenylation, decapping and exonucleolytic digestion of mRNA. However, the number, type, and position of mismatches in miRNA and mRNA duplexes are essential for the selection of the degradation or translational repression mechanisms. miRNAs and myogenesis
  • MRFs Muscle development is regulated by the MRFs: MYF5, MyoD, MYOG and MRF4 which promote the formation of muscle fibres.
  • miRNAs in this case have an important role in the development of skeletal muscle tissues [Buckingham (2001) Curr Opin Genet Dev 11, 440-448].
  • some typically upregulated miRNAs such as miR-133a-l, miR-133a-2, miR-133b and miR-206, have an important role in myogenic differentiation [Chen et al (2006) Nat Genet 38, 228-233; Kim et al. (2006) J Cell Biol 174, 677-687].
  • miRNAs in muscle tissue can be subdivided into two types, muscle-specific myo- miRNAs and non-myomiRNAs which are found in both skeletal and cardiac muscles [Gasparini et al. (2019) Int J Mol Sci 20, 5818].
  • muscle-specific myo-miRNAs can be either uniformly expressed over muscles (i.e., miR-1 and miR-133a), or can be highly expressed in slow-twitch/type I muscles (i.e., miR-206, miR-208b and miR-499).
  • Myo-miRNAs correspond to only 3% of expressed miRNAs in quiescent satellite cells, but in differentiated cells myo-miRNAs correspond to 74% of expressed miRNAs.
  • regulation is needed to activate specific miRNAs at different stages of myogenesis to achieve optimal balance between proliferation and myoblast differentiation.
  • miRNAs as a biomarker and the role in RMS development miRNAs can regulate post-transcription of different phases of muscle-specific processes, which shows their ability as biomarkers in the development of RMS [Bersani et al. (2016) cited above].
  • the myo-miRNAs are the key to skeletal muscle tissue differentiation, where their downregulation is related to the dedifferentiation of the phenotype in RMS [Novak et al (2013) Pediatr Blood Cancer 60, 1739-1746].
  • the overexpression of miR-1 in RD cell lines regulates the gene expression of muscle tissue and cell cycle arrest, whereas miR-133a decreases the expression of muscle markers.
  • miR-1, miR-22 and miR-206 are downregulated while miR-29 is deregulated in a few FP-RMS [Bersani et al. (2016) Cancer Res 76, 6095-6106]) .
  • miRNAs have important effects on the development and progression of RMS, these molecules could be interesting as therapeutic targets in pediatric tumours.
  • overexpression of miR-378 can cause changes in apoptosis, migration, and viability.
  • miR-183 has an oncogenic role and it targets two tumour suppressor genes, specifically early growth response protein 1 EGR1) and PTEN.
  • EGR1 early growth response protein 1
  • Extracellular vesicles are mediators of cell-cell communication and play an important role in cancer metastasis. Therefore, their use as biomarkers for clinical purposes is being explored extensively .
  • EVs are lipid bilayer vesicles with a diameter size of 40-1000 nm which are produced and released by cells or detached from the plasma membrane. EVs can be divided into subtypes based on their biogenesis, release pathway, size, content, and function. The three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies.
  • Exosomes or intraluminal vesicles have a single outer membrane and are secreted by all types of cells. Exosomes can be found in many fluids such as plasma, urine, saliva, or tears. Exosomes have a diameter size of 30-150 nm and are formed via an endosomal route. Early endosomes formed from inward budding of the cells' plasma membrane, later mature into multivesicular bodies (MVBs) which are involved in protein sorting, recycling, storage, transport, and release. Then, MVBs are either degraded by the lysosomes or fuse with the cells' plasma membrane to secrete exosomes and other contents into the extracellular space. The fate of a MVB depends on the level of cholesterol it contains.
  • Vesicles rich in cholesterol are secreted while vesicles with a low cholesterol level are degraded by the lysosomes.
  • the formation of MVBs is promoted by growth factors.
  • the cell adapts its production of exosomes based on its needs.
  • exosomal formation and MVB transportation are regulated by endosomal sorting complexes required for transport (ESCRT) proteins and with accessory proteins. These are commonly considered as “exosomal marker proteins”. These "marker proteins” can be found in all exosomes, independent from cell type.
  • the transmembrane proteins CD63, CD9 and CD81 belong to the tetraspanin family, which are also often found in exosomes.
  • exosomes The biological purpose of exosomes includes cell-cell communication, cell maintenance and tumour progression. Furthermore, when exosomes act as antigen- presenting vesicles, immune responses can be stimulated. Exosomes in the nervous system can promote myelin formation, neurite growth, and neuronal survival, which in the end also help with tissue repair and regeneration.
  • exosomes are also suitable for immunological applications since they have a relatively long circulating half-life, are tolerated by the human body, are able to penetrate cellular membranes, and have the potential to target specific cell types.
  • exosomes One of the important properties of exosomes is its inherent property, which can act inherently as antigen presenting vesicles, and makes exosomes ideal to use as a drug delivery system. Nucleic acids and proteins can be introduced into exosomes which can be further modified to target specific cells.
  • oncosuppressor microRNAs (miR-449a+miR-340) was identified, which showed downregulation of MPCI and MPC2 upon treatment of FN-RMS models with these miRNAs.
  • RMS is a type of STS, and the most common type in children. RMS tumours are histologically similar to normal foetal skeletal muscle. As mentioned, there are two major subtypes of RMS: FP-RMS and FN-RMS. Looking at the morphology, FP-RMS typically are small round densely packed cells, whereas FN-RMS typically consist of spindle-shaped cells and are rich in stroma.
  • MPCi mitochondrial pyruvate carrier inhibitor
  • MPCi- and miRNA-treated groups For both murine KMR46 and human RD18 FN-RMS cells, a decrease in the confluency was found in the MPCi- and miRNA-treated groups. Furthermore, the MPCi-treated group showed elongation of cells and intention to form multinucleated-like structures, whereas in the miRNA-treated group, there were formations of gigantic mono-, bi-, tri- or multinucleated cells instead more visible in murine FN-RMS. This could be due to the senescence effects, which led to a lower cell confluency and formation of larger cells. From these results, a possible preliminary conclusion could be that the MPC inhibition has effects of inhibiting tumour growth or proliferation, by altering cancer metabolism.
  • Literature shows that the decrease in MPC activity can lead to aerobic glycolysis and induce the growth of tumour cells, metastasis, and poor survival of patients with different types of cancers, such as colon cancer, prostate cancer, and esophageal cancer.
  • an planation for MPCi having possible anti-cancer effects instead of pro-cancer effects is that the role of MPC is highly dependent on the tissue type and metabolic inclinations of cancers. Since colon, prostate, and esophageal cancers arise from epithelial cells, whereas RMS can arise from mesenchymal stem cells or muscle progenitor cells, there might be an effect on the response of MPC inhibition.
  • cancers that use mitochondrial pyruvate for tumour growth and glutamine for glutathione production are typically negatively affected by MPCi.
  • RMS tumours express myogenic regulatory transcription factors, such as MYF5 and MyoD, which have important roles in regulating growth, proliferation and tumour propagating cell activity in RMS. Furthermore, the binding sites of MYF5 and MyoD are located on the promoter and enhancer regions of some genes, such as MYOG, which regulate cell cycle and muscle differentiation. Indeed, the effect of MPCi treatments on both KMR46 and human FN-RMS samples affected MYF5/ Myf5, MyoD, and MYOG/ Myog expressions as indicated by the RT-qPCR analysis. In the KMR46 samples, after MPCi treatment, Myf5 was upregulated whereas MyoD and Myog were downregulated.
  • MYF5 and MyoD myogenic regulatory transcription factors
  • MYF5 was undetermined, MyoD was upregulated, and MYOG was again downregulated.
  • MYOG was again downregulated.
  • MYF5 and MyoD share the same binding site but due to functional divergence, each established a separate muscle lineage. Therefore, regulation of either MYF5 or MyoD could lead to changes in skeletal muscle differentiation.
  • regulation of MRFs due to MPC inhibition may promote cells to differentiate into skeletal muscle-like cells whereas the miRNA-treated cells are more likely to differentiate into smooth muscle-like cells.
  • MPCi treatment can be that it saves the cells from being trapped in the MYF5 path, by regulating the expression of MYF5 and MyoD. Thus, the cells either go down the same path or by another path, which eventually leads to a more successful myogenesis.
  • the tumour microenvironment consists, besides tumour cells, of fibroblasts, endothelial cells, immune cells, and non-cellular components (including exosomes and cytokines) which form an internal environment where tumour cells survive.
  • the TME plays an important role in all stages of tumourigenesis.
  • miRNAs can be carried by exosomes, which have an important role in intercellular transport and signalling.
  • the heterogeneity of the TME is mainly due to miRNAs' activation of cancer-associated fibroblasts which also reshape the extracellular matrix (ECM) and thus promote the spreading of tumour cells. Therefore, using extracellular vesicles (EVs) or exosomes as carriers for oncosuppressor miRNAs could be an interesting way to alter the proliferation and metabolism of tumour cells.
  • ECM extracellular matrix
  • Human RD18 and murine KMR46 FN-RMS cell lines were cultured in high glucose Dulbecco's Modified Eagles Medium (DMEM-HG) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S). Cells were maintained under standard incubator conditions humidified atmosphere (95% air, 5% CO2, 37°C) and passaged twice weekly with 0.25% Trypsin-EDTA.
  • DMEM-HG high glucose Dulbecco's Modified Eagles Medium
  • FBS fetal bovine serum
  • P/S Penicillin/Streptomycin
  • cells were seeded at 1 :20 KMR46/human MABs ratio on collagen-coated vessels in IMDM 15% FBS medium, 1% L-glutamine, 1% non- essential amino acids, 1% sodium pyruvate, 1% ITS, 1 : 10,000 basic FGF, 0.2% betamercaptoethanol. After 24 h, medium was changed and OptiMEM with miRNA- lipofectamine or lipofectamine was added to the wells. A total of 24 h after miRNA addition, cells were differentiated in DMEM 2% horse serum medium for 96-120 h in 5% 02/5% CO2 at 37°C.
  • Cells were transfected with lentiviral particles expressing EFla-eGFP-P2A-fLuc and selected with 1 pg/mL puromycin for 2 weeks.
  • GFP was used as a standard gene expression tracer in vitro.
  • Firefly luciferase (fLuc) was used as an optical reporter gene, upon coelenterazine administration, to detect the cell line engraftment in vivo via IVIS bioluminescence imaging (BLI).
  • cells were seeded at 12-15.000 or 50.000 cell number in 24- wells or 6-wells, respectively.
  • cells were transfected using 3 pl/mL Lipofectamine 2000 and 1.6 pl/mL of hsa-miR-449a-5p/-340-5p, or hsa-miR- 181a-5p/-212-3p (all MISSION® microRNA Mimics, Sigma-Aldrich), at 10 pM concentration resuspended in OptiMEM-medium.
  • MPCi treatment cells were treated with 8 pl (final concentration is 40 pM) of Mitochondrial pyruvate carrier inhibitor (UK-5099, Sigma-Aldrich) in 2 ml of Opti-MEMTM I Reduced Serum Medium.
  • DMSO Dimethyl sulfoxide
  • mouse anti-MyHC (DSHB #MF20), 1 :3; rabbit antilamin A/C (Epitomics #2966-1), 1:600; mouse anti-Myogenin (Invitrogen #AB_10977211), 1: 100; rabbit anti-MyoD (Thermo Fisher Scientific #MA5-12902), 1: 100.
  • Imaging was performed at Eclipse Ti microscope (Nikon) by means of Image- Pro Plus 6.0 software (Nikon).
  • 20,000 pretreated cells were seeded into 6-well Ultra-Low Attachment plates (Corning, USA) in serum-free DMEM/F12 (Invitrogen, USA), supplemented with B27 (lx, Invitrogen, USA), 20 ng/ml EGF (Peprotech, USA), 20 ng/ml bFGF (Invitrogen), and 1% Penicillin/Streptomycin (Sigma, USA). Pictures were taken every day for 11 days.
  • Seahorse XFp Extracellular Flux Analyzer was employed to measure oxygen consumption rates (OCR) using the Mito Stress Test Kit following the manufacturer's protocol. Briefly, pretreated 40,000 cells/well were plated Seahorse XFp cartridge for 4 hours in growth medium. Following attachment to the well, medium was changed with XF media pH 7.4 supplemented with 2mM L-Glutamine, 10 mM glucose and 1 mM sodium pyruvate. Basal OCR was measured for 4 cycles, then 8 pM oligomycin was injected to inhibit ATP-linked respiration, followed by 4.5 pM FCCP to measure maximal respiration, and finally 10 pM rotenone/antimycin A was injected to completely inhibit all mitochondrial respiration. After each experiment, protein concentration was measured and wells were normalized using the Wave software.
  • EdU staining was performed using Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay according to manufacturer's advice. Briefly, after EdU incubation of l-2h, cell pellets were fixed with 4% PFA, permeabilized with 0.5% Triton X-100 in PBS for 20 min, and subsequently treated with Click-iT reaction cocktail at room temperature for 10 minutes. Subsequently, cells were washed and incubated in propidium iodide (PI) staining buffer (20 pg ml-1 PI, 0.2 mg ml-1 RNase A, PBS) for 30 min at room temperature. Cell cycle distribution was analysed by flow cytometry (BD FACSCanto HTS) and FlowJo software.
  • PI propidium iodide
  • RNA-sequencing libraries were constructed with the Lexogen library. Samples were indexed with unique adapters and pooled for single read (50 bp) sequencing in Illumina HiSeq2000. RNA-seq reads were aligned with TopHat v2.0.2 to the mouse genome mmlO. Transcripts were assessed and quantities were determined by Cufflinks38. Differential expression levels were assessed using DESeq2.
  • Sequencing reads were trimmed and aligned to assembly GRCh38 using bwa mem. Duplicate reads and reads mapping to mitochondrial sequences were subsequently removed. Chromatin accessibility peaks were called using MACS2 and annotated using HOMER and/or ChipSeeker. Differential accessibility analysis was performed using the R/Bioconductor package DiffBind.
  • mice C57/BI6 mice were injected in the femoral artery with either 1 x 105 KMR46 Fluc+ cells (untreated group) or with 1 x 105 KMR46 Fluc+ cells pretreated for 3 days with s-PMC (treated group).
  • cells were suspended in 50 pl saline water.
  • the mice were monitored through BLI every other day starting from day 7 after tumour injection for 10 days.
  • mice were placed in the flow chamber of IVIS® Spectrum. Subsequently, 126 mg/kg of D-luciferin was injected subcutaneousl. Hence, consecutive frames were acquired until the maximum signal intensity was reached. Pulse/sec intensities were calculated by comparing the same ROI for all the animals, after subtracting the background signal coming from not injected mice.
  • Sample size for in vitro/in vivo experiments was calculated by means of Sample Size Calculator (parameters: power, 0.80; alpha, 0.05). When applicable, sample size analysis was based on average values obtained from preliminary optimization/validation trials. Depending on the experiment, two-tailed unpaired Student's T-tests analysis or two-way ANOVA test was performed. All statistical analyses were conducted using Prism v9.1.0 (GraphPad).
  • RD and RD18 Human FN-RMS (RD and RD18) and murine FN-RMS (KMR46) cell lines were used.
  • RD and RD18 are different cell lines but both are human FN-RMS with characteristics of expressing MYOG, p21 and Desmin.
  • KMR46 is a cell line derived from the mouse model MyoD-cre x KRAS G12D x TP53F/-).
  • the KMR46 cell line has similar characteristics to FN- RMS patients, which indicates it is a suitable model to use for this research.
  • RD18/RD and murine KMR46 FN-RMS cell lines were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM-HG) (GibcoTM, Thermo ScientificTM, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) (GibcoTM). The medium was filtered with 0.22
  • DMEM-HG high glucose Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • P/S Penicillin/Streptomycin
  • MPCi Mitochondrial pyruvate carrier inhibitor
  • DMSO Dimethyl sulfoxide
  • a Nikon Eclipse Ti-S microscope (Nikon, Minato, Tokyo, Japan) was used and the collected images were further processed with the Image J software (National Institutes of Health (NIH, Bethesda, MD, USA).
  • RT-qPCR Quantitative real-time polymerase chain reaction
  • RNA/supernatant was collected.
  • RNA collected after the DNase I treatment was then used for cDNA synthesis, by using a cDNA Reverse Transcription Kit (High-Capacity cDNA Reverse Transcription Kit, Applied BiosystemsTM).
  • a cDNA Reverse Transcription Kit High-Capacity cDNA Reverse Transcription Kit, Applied BiosystemsTM.
  • 1 pg of RNA was utilized and reverse- transcribed using 10 pl of cDNA synthesis buffer, 2 pl of reverse transcriptase and a total of 8 pl of sample RNA and DEPC-treated water.
  • the synthesis of cDNA was done in a thermocycler (Biometra T3000 Thermocycler, Biometra, Analytik Jena, Jena, Germany).
  • 1 pl of E. coli RNase H was added to each sample and placed back into the thermocycler.
  • SNAI1 PTEN, MYLK, MYBL2 and MEF2C. All primers were synthesized and purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA).
  • pellets were lysed in either 60 pl or 90 pl of RIPA buffer (Thermo ScientificTM, #89900).
  • the solutions were sonicated twice for 10 seconds at an amplitude of 10% (Sonifier® Cell Disruptor, Branson Ultrasonics Corporation, Danbury, CT, USA), incubated at room temperature for 10 minutes and centrifuged at 16000 rpm, for 15 minutes at 4°C.
  • the supernatants were collected and quantitatively measured by using the NanoDrop spectrophotometer (NanoDrop® ND-1000, Isogen life science B.V., De Meern, NL).
  • NanoDrop® ND-1000 Isogen life science B.V., De Meern, NL
  • the gel was run at 120V (PowerPacTM Basic Power Supply, Bio-Rad Laboratories, Inc.) until the dye front was close to the end of the gel. Then, the proteins were electro-transferred onto a nitrocellulose membrane with transfer buffer, at 200 amperes for 2 hours and 30 minutes. All of the equipment was purchased from Bio-Rad Laboratories, Inc. (Mini-PROTEAN Tetra Cell). The membranes were then stained with Ponceau (Sigma- Aldrich), to visualize proteins and to check if the transfer was successful.
  • Ponceau Sigma- Aldrich
  • the membranes were washed with TBS-Tween (Thermo ScientificTM) and were blocked with 5% (w/v) skim milk (Sigma-Aldrich) in TBS-Tween for one hour at room temperature. After being washed with TBS-Tween twice for 10 minutes on the shaker, the membranes were incubated with primary antibody in 2.5% (w/v) skim milk in TBS-Tween overnight at 4°C. On the second day, the primary antibodies were recollected, and then membranes were washed 3 times with TBS-Tween for 5 minutes. Membranes were incubated with secondary antibody in 2.5% (w/v) skim milk in TBS- Tween for one hour at room temperature.
  • the membranes were incubated with (1:3300 - in 2.5% (w/v) milk in TBS-Tween) anti-rabbit secondary antibody (Abeam, ab6721) (Cell Signaling Technology, #7074) and (1:3300 - in 2.5% (w/v) milk in TBS-Tween) and anti-mouse secondary antibody (Abeam, ab97046).
  • EVs were extracted from the LHCN-M2 (human immortalized myoblasts) cells.
  • cells were cultured in medium (1 vol. 199 medium, 4 vols. DMEM, 20% FBS and 50
  • the cells were left in the differentiation medium (DMEM, 10 pg/ml insulin, and 50 pg/ml gentamycin) for 4 days. Finally, the medium was collected for EV extraction.
  • the collected medium was centrifuged at 500g for 10 minutes (Eppendorf Centrifuge, Model 5810, Eppendorf), 3000g for 20 minutes (Avanti®J-E high-speed centrifuge, Beckman Coulter life sciences, IN, USA), 12,000g for 20 minutes (Beckman coulter), and then 100,000g for 70 minutes (Optima L-90K Ultracentrifuge, Beckman Coulter). After removal of the supernatant, the pellets were resuspended in 5 ml of PBS, centrifuged again at 100,000g for 70 minutes. Finally, the supernatant was removed, and the pellets were resuspended in 400 pl of PBS and stored at -80°C. All of the centrifugation steps were done at 4°C.
  • PBS was used to dilute the isolated EVs to reach a final volume of 500 pl.
  • the suspension was filtered with a 0.2 pm filter (Corning® syringe filters, CLS431212, Corning).
  • 50 pl of lOx Exo-Red (ExoGlowTM - Protein EV labelling kit, System Biosciences, Palo Alto, CA) were added to the purified EVs, incubated at 37°C for 10 minutes and placed on ice for 30 minutes. Finally, the labelled EVs were placed in a 24-well plate.
  • PBS was used to dilute the isolated EVs with a dilution factor of 1: 5 (i.e., 400 pl of EVs in 1.6 ml of PBS), and the suspension was filtered with a 0.2 pm filter.
  • the purified EV samples were then loaded into the sample chamber of a Nanosight machine (NanoSight LM10, Malvern Panalytical Ltd, Malvern, UK) and the quantification and size measurement were done by using the Nanosight NTA 2.3 software (Malvern Panalytical Ltd).
  • the miRNA signature was determined of patients diagnosed with embryonal RMS. Data from Bersani et al. (2016) Cancer Res. 76, 6095-6106. (accession number ID PRJNA326118) were used to identify miRNAs not expressed in FN-RMS patients. The predicted genes targeted by the downregulated miRNAs were cross-checked and identified the pathways using g: Profiler. These pathways mainly involved include cell cycle, hippo signalling, fatty acid metabolism and central carbon metabolism. The combination of the key microRNAs that could be used as novel therapeutic agents was identified by checking the single pathways targeted by the microRNAs using DIANA software, and miR-449a and miR-340 were identified as the two key microRNAs in the downregulated microRNA pool.
  • Example 3.miR-449 and miR-340 dramatically affect tumourigenicity of FN- RMS
  • the role of the downregulated miRNAs in FN-RMS was investigated, by transfecting both human and mouse in vitro FN-RMS models with miR-449a and miR-340 and subsequently checking the perturbation effects at the transcriptome level with RNAseq analysis. Both the principal component analysis and a volcano plot showed a remarkable difference in miRNA-treated cells compared to vehicle-treated ones.
  • the downregulated genes are mainly involved in cell cycle, while among the top upregulated pathways in miRNA-treated samples cilium organization and cilium assembly were identified. This is in line with previously reported data regarding quiescent muscle cells that upon exit from the cell cycle undergo cilium formation.
  • oncosuppressor miRNAs were tested and the potential effects in vitro and in vivo were checked.
  • the proliferation level there was a reduction in proliferation in FN-RMS treated cells (figure 6A).
  • the effects on proliferation were checked in an in vitro hybrid system by co-culturing FN-RMS and mesoangioblasts (MABs) (Figure 6B). After transfection there was no tangible effect in terms of reduced number of cells in healthy skeletal muscle cells but solely in FN-RMS, which further confirmed the hypothesis of the specificity of these miRNAs in targeting the tumorigenic apparatus of FN-RMS.
  • RNA-treated tumour cells were treated using the miRNA combination, and subsequently injected the treated cells in C57BI/6 mice (Figure 6E). After 15 days, the reduction in proliferation of miRNA-treated tumour cells after a single treatment was still statistically significant compared to vehicle-treated implanted cells ( Figure 6F). Finally, as the main issue with respect to FN-RMS involves the metastasis to the lungs, the question arises whether RNA-treated tumour cells would have a reduced capacity at migrating compared to vehicle-treated ones. Indeed, both in vitro ( Figure 6G) and in vivo (Figure 6H) analysis showed a reduced migration potential of miRNA- treated cells.
  • Example 4 miR-449a+340 and mitochondrial pyruvate carrier (MPC) inhibition similarly affect proliferation and metastasis
  • MPCi mice performed significantly greater than the vehicle mice in terms of power on day 7, 9 and 13.
  • the power exerted was significantly greater than the one exerted by the vehicle mice on all days.
  • MPCi and miRNA treated tumour bearing mice were able to perform more similarly to sham mice, in contrast to the poor performance of vehicle mice, affected by the tumour.
  • MYOG reduced levels of MYOG were found both after miRNA and MPCi treatment ( Figure 9H). miRNA modulates and reverses the tumorigenic phenotype at the epigenetic level.
  • PBX1 was identified as the transcription factor with the highest number of enriched features ( Figure 5.7D-E).
  • snRNAseq single-nuclei RNAseq shows upregulation of myoblast feature CREB5, as well as senescence feature OXR1, in miRNA-treated FN- RMS
  • EZH2, TOP2A and MYBL2 were further investigated. These genes were found to be upregulated in FN-RMS patients compared to skeletal muscle, and they are downregulated by the miRNA combination in FN-RMS. the effects of drugs targeting these genes either alone or in combination were investigates.
  • Tazemetostat (EZH2 inhibitor), Doxorubicin (TOP2A inhibitor) and Topotecan (MYBL2 inhibitor) were determined using XTT proliferation assay which assesses cell viability as a function of redox potential.
  • Tazemetostat alone did not have effects, while at 96h Doxorubicin reduced the activity to 22.8% and Topotecan to 62.1% on FN-RMS cells compared to DMSO. As these targets are downregulated at the same time with miRNA treatment, the effect of these drugs combined was tested. Tazemetostat synergistically worked with Doxorubicin (18.9%) more than with Topotecan (42.3%) in reducing cell activity, while Doxorubicin combined with Topotecan has a dramatic effect on cell proliferation (2%). However, the synergy of the three drugs together has the most spectacular effect, with activity being reduced to 0.4% compared to vehicle-treated cells.
  • Example 6 MPCi-treated cells show similar effects as mi NA-treated samples when looking at the morphology of the cells
  • Example 7 MPCi-treated cells show similar effects as miRNA-treated samples in number, size, and multinucleation in the murine KMR46 cell lines When looking at the molecular level of different sample groups, three aspects were evaluated. First, the number of nuclei on day 3 and day 5 of different groups were compared. This was done to check if there was any effect on the proliferation rate of tumour cells after treatment with MPCi or miRNAs. The results showed that on day 3 there was already a decrease in the proliferation rate of the MPCi-treated group compared to the DMSO group. On day 5, the reduction in the rate of proliferation was even more significant (P ⁇ 0.0001) in the MPCi-treated samples.
  • Example 8 Western blot results show changes of MYF5 and MYOG protein contents after MPCi- and miRNA-treatment
  • exosomes isolated from the LHCN-M2 cells were visualized by using Exo-Red and checked under the microscope. The results show the presence of exosomal clusters at lOx magnification.
  • Nanosight was used for quantification of EVs and evaluation of the particle diameter.
  • the results showed EVs isolated from the LHCN-M2 cells had an average size of 143 +/- 92 nm, with a concentration of 0.14 +/- 0.13 E8 particles/ml (Figure 5).

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Abstract

L'invention concerne l'utilisation de mir-340 et mir-449a pour traiter ou prévenir des sarcomes de tissus mous, tels que le rhabdomyosarcome.
PCT/EP2022/083503 2021-11-26 2022-11-28 Traitement du rhabdomyosarcome WO2023094662A1 (fr)

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