CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression Martin Thomsen, Huiqiang Cai, Bin Zhang, Johanne Ahrenfeldt, Justin Joseph, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3013972/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Mar, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men, and treatment options for metastatic disease remain limited. In this study, we employed Adeno-associated virus (AAV) delivery and CRISPR-Cas9 technology to simultaneously mutate multiple genes in the mouse prostate, in order to investigate positive genetic interactions during PCa progression. By targeting five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL), we induced advanced prostate tumors in mice without metastasis, and mice reached humane endpoint at eight weeks. When three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16) were further depleted, tumor progression remained identical, but metastases were observed in the lung of all mice. Whole genome sequencing of metastatic tumors revealed few genetic alterations, confirming that mutations in the targeted genes are sufficient to transform prostatic cells into metastatic cancer. Mechanistic analysis uncovered multiple altered pathways, including impaired p-Src/p-Lyn-cMyc signaling, underscoring the importance of cMyc signaling in PCa metastasis. RNA sequencing revealed a significant dysregulation of overall transcriptome and increased expression of numerous genes in a genomic region at chr5qE1, which is highly conserved to human chr4q13.3 and regulated by KMT2C. Intriguingly, the depletion of Odam and Cabs1 in this gene cluster dismissed metastasis formation, indicating that they have critical functions for secondary tumor formation, which has not been reported before. Further genetic analysis demonstrated that Kmt2c deficiency is sufficient to facilitate lung metastasis, identifying Kmt2c as an essential gatekeeper for metastatic formation. Interestingly, the gene expression signature from mutations of the three epigenetic regulators were predictive of progression-free and overall survival and able to distinguish primary and metastatic human prostate cancer. Overall, this study highlights the positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in the progression and onset of metastatic disease, providing predictions and insights into possible treatments for metastatic PCa. Health sciences/Oncology/Cancer/Urological cancer/Prostate cancer Health sciences/Oncology/Cancer/Cancer models Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplemetarytablesCaietal.zip Supplementary tables CaietalsupNatureCom.pdf Supplementary figures NCOMMS2324310Ars.pdf Reporting Summary Cite Share Download PDF Status: Published Journal Publication published 07 Mar, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3013972","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":266480705,"identity":"366a4fcf-926c-4cf5-aa9f-6b89bb45c665","order_by":0,"name":"Martin Thomsen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYDACHgbGxwwGIFYCiGA2IEILM7MxyVrYpCEsYrXo9pw/Vl1QYBPNz57A+JmHwdqYoBazs81st2cYpOXO7HnALM3DkG5GWMt5ZrbbPAaHczfcSGAAajlsQ5SWYpCW/TcSmH8TpwXoMGawLRIJbCBbiHDYmcPG0iC/zDjzsM1yjkE6Ed4/k/jwc8Efm9z+9uTDN95UWBs2ENSDAIxAxURE5CgYBaNgFIwCIgAAstE2g1Vb78UAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5055-7531","institution":"Aarhus University","correspondingAuthor":true,"prefix":"","firstName":"Martin","middleName":"","lastName":"Thomsen","suffix":""},{"id":266480706,"identity":"a5ca0451-1775-4838-bfce-66b36813ebc1","order_by":1,"name":"Huiqiang Cai","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Huiqiang","middleName":"","lastName":"Cai","suffix":""},{"id":266480707,"identity":"b13511ea-0609-4b52-8480-6f776e34c0a8","order_by":2,"name":"Bin Zhang","email":"","orcid":"https://orcid.org/0000-0001-8835-8370","institution":"King Abdullah University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Zhang","suffix":""},{"id":266480708,"identity":"5d893f1c-54e4-46f1-a26b-ad9acc3bb38f","order_by":3,"name":"Johanne Ahrenfeldt","email":"","orcid":"","institution":"Aarhus University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Johanne","middleName":"","lastName":"Ahrenfeldt","suffix":""},{"id":266480709,"identity":"2f62315e-180d-457c-93f5-36f79c073a19","order_by":4,"name":"Justin Joseph","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Justin","middleName":"","lastName":"Joseph","suffix":""},{"id":266480710,"identity":"57c77cda-83f1-4836-8a0f-0e713e7fb54f","order_by":5,"name":"Maria Riedel","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Riedel","suffix":""},{"id":266480711,"identity":"6f1f76fc-0d1b-4862-bf59-05ec0220e938","order_by":6,"name":"Zongliang Gao","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Zongliang","middleName":"","lastName":"Gao","suffix":""},{"id":266480712,"identity":"f8d1f8c0-34c5-48d6-bc82-c15d88a4cd5a","order_by":7,"name":"Ditte Christensen","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Ditte","middleName":"","lastName":"Christensen","suffix":""},{"id":266480713,"identity":"ea26eaf0-40bc-4ab1-8727-57d3f718be91","order_by":8,"name":"Rasmus Bak","email":"","orcid":"https://orcid.org/0000-0002-7383-0297","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Rasmus","middleName":"","lastName":"Bak","suffix":""},{"id":266480714,"identity":"e16e4dd9-264e-4857-8ef7-f60680854514","order_by":9,"name":"Henrik Hager","email":"","orcid":"","institution":"Department of Pathology, Vejle Hospital","correspondingAuthor":false,"prefix":"","firstName":"Henrik","middleName":"","lastName":"Hager","suffix":""},{"id":266480715,"identity":"78fd0601-e1d1-4519-ab5d-a434ff6e38d7","order_by":10,"name":"Mikkel Vendelbo","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Mikkel","middleName":"","lastName":"Vendelbo","suffix":""},{"id":266480716,"identity":"2ddcc289-8d1d-4611-8e5b-6fa9e71e87a0","order_by":11,"name":"Xin Gao","email":"","orcid":"https://orcid.org/0000-0002-7108-3574","institution":"King Abdullah University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Gao","suffix":""},{"id":266480717,"identity":"6a9992da-c655-4443-9f47-2c36b8f8e465","order_by":12,"name":"Nicolai Birkbak","email":"","orcid":"https://orcid.org/0000-0003-1613-9587","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Nicolai","middleName":"","lastName":"Birkbak","suffix":""},{"id":266480718,"identity":"d60a833b-c286-4e6d-b610-210f6b77736b","order_by":13,"name":"Sofie Thomsen","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Sofie","middleName":"","lastName":"Thomsen","suffix":""}],"badges":[],"createdAt":"2023-06-02 10:02:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3013972/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3013972/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-46370-0","type":"published","date":"2024-03-07T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49641264,"identity":"5b9cd275-25ba-485e-8993-f1943c2d98b3","added_by":"auto","created_at":"2024-01-15 19:15:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2541553,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration of an aggressive primary tumor by loss of five tumor repressor genes.\u003c/p\u003e\n\u003cp\u003eA) Three AAV constructs were cloned to generate AAV particles. A construct with a non-targeting (NT) sgRNA, a construct with a sgRNA for Pten and a construct with sgRNAs for Pten, Trp53, Rb1, Stk11 and RnaseL (5g). B) Mice were injected with AAV particles targeting the five genes and macroscopic pictures were taken 4 and 8 weeks after injection with bright field and GFP channel. DLVP: Dorsal, lateral and ventral prostate. AP: Anterior prostate. C) The weight of the prostates was measured at different time points after administration of the AAV particles (*= p\u0026lt;0,05; n=3-6). D) Overall survival for the three groups of mice. E) Indel formation in the tumors at 8 weeks after delivery of AAV particles containing five sgRNAs (n=7, each color represents the same sample). F) H\u0026amp;E staining of prostate tissues from the three groups at different time points. A representative picture is shown (n=10). G) IHC on prostatic sections at 8 weeks after the mice receiving either sgPten or the 5g construct (n\u0026gt;5, a representative picture is shown). H) Quantification of Ki67 positive cells in tissue sections from sgPten control and 5g tumor samples at 8 weeks (**= p\u0026lt;0,01; n= 15, 57 fields respectively). I) A group of mice were castrated at 5 weeks after delivery of AAV particles containing 5 sgRNAs and sacrificed at 8 weeks. Images were taken with bright field (BF) and GFP channel. J) The weight of the prostatic tissues from castrated and intact mice 8 weeks after injection of AAV particles with 5 sgRNAs (n=6-8; n.s.= not significant). K) H\u0026amp;E staining and IHC for Ki67 on prostate tissue sections from castrated mice injected with 5g AAV particles (n=5).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/db46bea435f171a5998e21ad.jpg"},{"id":49641257,"identity":"1839c0f3-4216-4029-afa3-91c556f47f3d","added_by":"auto","created_at":"2024-01-15 19:15:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2324858,"visible":true,"origin":"","legend":"\u003cp\u003eLoss of epigenetic factors facilitated metastasis in the lung\u003c/p\u003e\n\u003cp\u003eA) Two new AAV constructs were cloned to generate AAV particles. A construct containing sgRNAs targeting Kmt2c, Kmt2d and Ztbt16 together with a sgRNA for Pten (Epi). The sgRNAs cassettes for Kmt2c, Kmt2d and Ztbt16 were added to the 5g vector, resulting in a construct named 8g. B) Mice were injected with AAV particles of 8g and macroscopic pictures were taken 4 and 8 weeks after injection with bright field (BF) and GFP channel. C) The weight of the prostates was measured at different time points after administration of the AAV particles. A group of mice were castrated 5 weeks after AAV delivery (Cast) (*=p\u0026lt;0,05; n=5-7). D) Overall survival for mice reaching humane endpoint for three groups of mice. E) Indels formation in tumors at 8 weeks after delivery of AAV particles containing eight sgRNAs (n=8, each color represents the same sample). F) H\u0026amp;E staining of prostate tissues from mice targeted by sgPten or 8g at different time points. A representative picture is shown (n=10). G) IHC for Ki67 on tissues sections from 8g-transformed prostates at 8 weeks (n\u0026gt;5, a representative picture is shown). H) Quantification of Ki67 positive cells in tissue sections from sgPten control, 5g and 8g tumor samples at 8 weeks (*=p\u0026lt;0,05; **= p\u0026lt;0,01; n=15, 57, 41 fields for each group). I) PET/MRi scanning of mice injected with 8g particles at 8 weeks with tracer for glucose metabolism (FDG) and Sodium Fluoride (NaF) for bone metastasis. Representative pictures are shown (n=5-10). J) Images of bright field and GFP of lungs from mice receiving 8g AAV particles at 8 weeks after injection. Representative pictures are shown; black arrows mark metastases (n\u0026gt;10). K) Presence of metastasis in lung tissues at different time points from mice injected with either 5g or 8g AAV particles. L) H\u0026amp;E staining of lung metastasis. Black box (left) marks the area in high magnification (right). Representative pictures are shown (n\u0026gt;5). M) IHC staining for Ki67, E-cad and SYP on lung metastasis. N) IF staining of lung metastasis with Ck8 (green) and Ck5 (red). Arrows mark Ck5 positive basal cells and arrowheads indicate double positive cells. Representative pictures are shown (n\u0026gt;5). O) Lungs isolated at 8 weeks from castrated mice injected with 8g AAV particles. Representative bright field (BF) and GFP channel images are shown. Arrows mark metastasis (n=5).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/fc0cae12f757862859517f4d.jpg"},{"id":49641261,"identity":"1e304c48-d811-4736-90b3-8eb7feed9859","added_by":"auto","created_at":"2024-01-15 19:15:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":542898,"visible":true,"origin":"","legend":"\u003cp\u003eMutations landscape in metastatic prostate cancer samples\u003c/p\u003e\n\u003cp\u003eLung metastases samples from four mice injected with 8g AAV particles were subjected to whole genome sequencing and mapped to the mouse reference genome (mm39). A) Indel analysis for the sgRNAs at the specific eight genomic locations targeted by CRISPR/Cas9. B) Analysis of off-target induced by CRISPR/Cas9 for Trp53 sgRNA. C) The distribution of genomic alterations was analyzed for each sample and grouped into: exon, UTR3, UTR5, intron or intergenic regions. D) Total number of detected genomic mutations based on chromosomes. E) The contributions of single-nucleotide variants (SNV), deletions and insertions were analyzed for each sample. F) A Venn diagram showing either unique or shared exonic mutations across the metastatic samples. Genes with underline were mutated by CRISPR/Cas9 (n=4).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/9f1d558f0d715675c5c55c59.jpg"},{"id":49641260,"identity":"ddbd531b-90e3-4df9-be0a-c8940a098447","added_by":"auto","created_at":"2024-01-15 19:15:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2381004,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome analysis identified alterations in cancer-associated pathways.\u003c/p\u003e\n\u003cp\u003eRNA sequencing (RNAseq) was performed on control prostate tissues, primary tumors from 5g and 8g smaples, and lung metastases (met) (n=3,4,4,4). A) Proportion of the abnormal transcripts of the CRISPR/Cas9 targeted genes across all samples. B) Principal-component analysis of the samples. C) Pathways enrichment of the dysregulated genes for the three tumor groups compared to control. D) qPCR on selected genes on RNA from control tissues, 5g and 8g primary tumors at 8 weeks (n=4; *=p\u0026lt; 0,05; **=p\u0026lt; 0,01; ***=p\u0026lt; 0,001). (E, F) Analysis of AR signaling and neuroendocrine prostate cancer (NEPC) signature based on RNAseq data from the four groups. Each dot represents a sample. (G, H) IHC staining for androgen receptor (AR) or synaptophysin (SYP) on prostatic tissues from sgPten, 5g and 8g tumors together with lung metastases at 8 weeks after cancer initiation. Enrichment analysis for (I) prostatic basal and luminal cell signatures and (K) epithelial and mesenchymal gene signatures on the RNAseq samples. (J) IF staining for Ck8 (green) and Ck5 (red), or (L) IHC staining for e-cadherin (e-cad) on prostatic tissues from sgPten, 5g and 8g tumors together with lung metastasis at 8 weeks after cancer initiation. Representative pictures are shown (n≥5).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/edcd5e6950fb4d4ed81d9d8d.jpg"},{"id":49642535,"identity":"e4e2f8b0-b9c7-4eb7-8f21-19c8e6ee8ecd","added_by":"auto","created_at":"2024-01-15 19:31:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1846633,"visible":true,"origin":"","legend":"\u003cp\u003eEpigenetic factors altered expression of a unique chromosomal region and driving metastatic formation.\u003c/p\u003e\n\u003cp\u003eA) Volcano plot showing differential expressed genes between 5g and 8g primary tumors at 6 weeks post tumor induction (n=4). Genes underlined in green or names in green were located in a short genomic region. Myc and its targets genes are marked with an orange box. B) Dot plot indicating the association analysis between chromosomal regions and distribution of DEGs. C) Gene alignment of the mouse dysregulated region at chr5qE1 and its corresponding region in human, chr4q13.3. D) mRNA expression analysis for four genes located in the human chr4q13.3 in BPH1 cells. Cells with CRISPR/Cas9 induced mutations in either KMT2C or KMT2D or both (DKO) were analyzed and compared to control cells (NonT) (n=4; *= p\u0026lt;0,05, ***= p\u0026lt;0,001). E) Schematic strategy for engineering the mouse Chr5qE1 with four specific sgRNAs in a primary lung metastatic cell line derived from 8g PCa. As control, a clone transduced with four non-targeting sgRNAs was applied. F) KO scores for the four genes are shown for NonT and clone H8. G) Expression of three target genes in the control cells and clone H8 (n=3, ** = p\u0026lt;0,01, **** = p\u0026lt;0,0001). H) MRi scanning of mice 6 weeks after orthotopically implantation of the H8 clone to the\u003c/p\u003e\n\u003cp\u003eprostate. Yellow dotted line marked the primary tumor and the red dotted line marked metastasis. Representative pictures are shown (n=4). I) The weight of the primary tumor at 6 weeks after implantation (n=3, ** = p\u0026lt;0,01). J) Table for formation of primary and secondary tumors by control and two KO clones. K, L) Images of bright field and GFP of primary prostate tumors, lymph nodes, abdominal fat and lungs from mice receiving either control or KO clone for Odam and Cabs1 6 weeks after injection. Representative pictures are shown; black or white dotted lines marks lung metastases (n=6). M) H\u0026amp;E staining of primary and secondary tumors at 6 weeks after implantation. A representative picture is shown (n=3).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/79d6902077053be2ef262e9e.jpg"},{"id":49641262,"identity":"ac0853e4-da4b-49cd-9c8e-3b6dc2b46d0a","added_by":"auto","created_at":"2024-01-15 19:15:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1192085,"visible":true,"origin":"","legend":"\u003cp\u003ec-Myc pathway was increased with loss of epigenetic factors in Kinome assay and RNAseq.\u003c/p\u003e\n\u003cp\u003eA) Venn diagram showing the upregulated KEGG pathways in the three different tumor groups comparing to control tissues, based on RNAseq data. B) Heatmap for the genes involved in transcriptional misregulation in cancer (mmu05202), which were dysregulated and shared by 8g primary and metastatic samples. Transcriptional associated genes are marked in red, EMT related genes are marked in blue. C-E) Volcano plot for differential phosphorylated peptides targeted by serine/threonine and protein tyrosine kinases in the primary tumors and Pten control samples (n=4). Graphs indicating the down-regulated (F) and up-regulated kinases (G) activity between 5g and 8g primary tumor tissues (n=4; cutoff=1.2). Black arrows mark the kinases of interest, Lyn and Src. H) They were further visualized in a Coral tree. I) Western blot for p-Src and p-Lyn on protein lysate from sgPten, 5g and 8g samples at 8 weeks after AAV delivery. Vinculin (Vin) was used as loading control (n=3). J) Gene-set enrichment analysis for Myc hallmarks between 5g and 8g primary tumors at 6 weeks post AAV delivery (n=4).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/bf027df7bc7dcf870daebfbf.jpg"},{"id":49641266,"identity":"50382577-25d6-4b7b-861a-f3884a3f9d20","added_by":"auto","created_at":"2024-01-15 19:15:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":737649,"visible":true,"origin":"","legend":"\u003cp\u003eDepletion of Kmt2c drove metastasis formation in the lung\u003c/p\u003e\n\u003cp\u003eA) Schematic of AAV constructs cloning the sgRNAs for either Ztbt16, Kmt2d or Kmt2c together with 5g construct for AAV production. B) Mice were injected with the AAV particles to the prostatic lobes and after 8 weeks was bright field and GFP images taken of the lungs. Representative pictures are shown; white arrowheads mark the metastasis (n\u0026gt;5). C) Incidents of lung metastasis in three groups of mice (n=3-7). D) Weight of the primary tumor at 8 weeks after AAV delivery from three groups of mice (n=3-7; *=p\u0026lt;0,05). E) Indel analysis of Kmt2c, Kmt2d and Zbtb16 in primary tumors induced by respective AAV particles targeting 5g with sgKmt2c, sgKmt2d or sgZbtb16 (n=4). Indel analysis in primary (F) and metastasis (G) samples at 8 weeks after injection with AAV particles containing 5g and sgKmt2c (n=3).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/b7ddea1dfa5584e57b08e0d8.jpg"},{"id":49641267,"identity":"1c2d5a80-cf7d-47f2-a19c-5fd47b7e51d9","added_by":"auto","created_at":"2024-01-15 19:15:19","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2413531,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential expressed genes can predict clinical outcome of PCa progression.\u003c/p\u003e\n\u003cp\u003e(A) The commonly down-regulated 240 genes of 8g-derived primary tumor and metastases differentiate human PCa into primary and secondary tumors (GSE35988, n=54, 35). B-C) The down-regulated genes (n=394) of 8g-derived primary tumor but not present in 5g tumors differentiate human PCa into primary and secondary tumors (GSE35988, n=54, 35). D) A 10-gene set from DEG between 5g and 8g tumors distinguish human primary tumors from metastatic tumors (GSE6919, n=61, 25). E) A 20-gene set from DEG between 5g and 8g was used to predict progression free survival for TCGA dataset (n=494). F) Illustration of tumor initiation by five tumor suppressor genes and progression to metastatic disease by loss of Kmt2c at 6 weeks after tumor initiation. Mice reach humane endpoint at 8 weeks after initiation.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/20fbb642869f31c0dcd58d55.jpg"},{"id":52243116,"identity":"356dc484-f8de-4e78-b2fd-d6c723c8ec1c","added_by":"auto","created_at":"2024-03-08 08:07:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2080037,"visible":true,"origin":"","legend":"","description":"","filename":"CaietalNaturecomrevisionmktredtext.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1_covered_6b8f921c-9173-457a-851a-99fa13150ad6.pdf"},{"id":49641258,"identity":"e9d794ec-7531-4133-b5b4-97505927bf71","added_by":"auto","created_at":"2024-01-15 19:15:19","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1786332,"visible":true,"origin":"","legend":"Supplementary tables","description":"","filename":"SupplemetarytablesCaietal.zip","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/5eae26c0de1941eabb60088c.zip"},{"id":49642183,"identity":"caf5fc5d-0a39-4600-89b8-48170a2346b8","added_by":"auto","created_at":"2024-01-15 19:23:19","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17599375,"visible":true,"origin":"","legend":"Supplementary figures","description":"","filename":"CaietalsupNatureCom.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/7bc9b5bb05967667da62f140.pdf"},{"id":49642182,"identity":"c7716523-d9de-4d20-80d7-d159524200f0","added_by":"auto","created_at":"2024-01-15 19:23:19","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1928725,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"NCOMMS2324310Ars.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3013972/v1/2dca68dd7a2b9167d1a72f80.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3013972/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3013972/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men, and treatment options for metastatic disease remain limited. In this study, we employed Adeno-associated virus (AAV) delivery and CRISPR-Cas9 technology to simultaneously mutate multiple genes in the mouse prostate, in order to investigate positive genetic interactions during PCa progression. By targeting five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL), we induced advanced prostate tumors in mice without metastasis, and mice reached humane endpoint at eight weeks. When three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16) were further depleted, tumor progression remained identical, but metastases were observed in the lung of all mice. Whole genome sequencing of metastatic tumors revealed few genetic alterations, confirming that mutations in the targeted genes are sufficient to transform prostatic cells into metastatic cancer. Mechanistic analysis uncovered multiple altered pathways, including impaired p-Src/p-Lyn-cMyc signaling, underscoring the importance of cMyc signaling in PCa metastasis. RNA sequencing revealed a significant dysregulation of overall transcriptome and increased expression of numerous genes in a genomic region at chr5qE1, which is highly conserved to human chr4q13.3 and regulated by KMT2C. Intriguingly, the depletion of Odam and Cabs1 in this gene cluster dismissed metastasis formation, indicating that they have critical functions for secondary tumor formation, which has not been reported before. Further genetic analysis demonstrated that Kmt2c deficiency is sufficient to facilitate lung metastasis, identifying Kmt2c as an essential gatekeeper for metastatic formation. Interestingly, the gene expression signature from mutations of the three epigenetic regulators were predictive of progression-free and overall survival and able to distinguish primary and metastatic human prostate cancer. Overall, this study highlights the positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in the progression and onset of metastatic disease, providing predictions and insights into possible treatments for metastatic PCa.","manuscriptTitle":"CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-15 19:15:14","doi":"10.21203/rs.3.rs-3013972/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"edf3d672-de44-4d73-840e-27a84b2b3e95","owner":[],"postedDate":"January 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28085434,"name":"Health sciences/Oncology/Cancer/Urological cancer/Prostate cancer"},{"id":28085435,"name":"Health sciences/Oncology/Cancer/Cancer models"}],"tags":[],"updatedAt":"2024-03-08T08:07:34+00:00","versionOfRecord":{"articleIdentity":"rs-3013972","link":"https://doi.org/10.1038/s41467-024-46370-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-03-07 05:00:00","publishedOnDateReadable":"March 7th, 2024"},"versionCreatedAt":"2024-01-15 19:15:14","video":"","vorDoi":"10.1038/s41467-024-46370-0","vorDoiUrl":"https://doi.org/10.1038/s41467-024-46370-0","workflowStages":[]},"version":"v1","identity":"rs-3013972","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3013972","identity":"rs-3013972","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}