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Definitions

• RNA molecules are defined as having a length greater than 200 nucleotides (nt) and a lack of protein-coding potential, and therefore represent a diverse array of functional entities (Ulitsky et al., Cell 154:26-46 (2013)).
• LncRNAs are often classified in four separate subgroups based on their location relative to protein-coding genes: exonic, intronic, overlapping, intergenic, sense and antisense IncRNAs; alternatively, they can be functionally classified into cis- and trans-acting IncRNAs. Ulitsky et al., Cell 154:26-46 (2013).
• Trans- acting IncRNAs are of special interest for their diverse mechanisms of action, such as their role as precursor molecules for the biogenesis of mature microRNAs (miRNAs) (Lu et al., Nat. Med. 23: 1331-1341 (2017)) or through their direct interactions with proteins and nucleic acids to regulate protein function and/or stability (Tseng et al., Nature 572:82-6 (2014)). Aberrant expression and function of IncRNAs have been implicated in the progressive gain of a malignant phenotype by tumor cells (Gutschner and Diederichs, RNA. Biol. 9:703-19 (2012)). SUMMARY OF THE DISCLOSURE
• a first aspect of the present disclosure is directed to an antisense oligonucleotide (ASO) that binds the MIR-17-92a-l Cluster Host Gene (MIR17HG) pre-RNA under physiological conditions, wherein the ASO is 15 to about 30 nucleotides in length and the MIR-17-92a-l Cluster Host Gene (MIR17HG) pre-RNA has the nucleic acid sequence of SEQ ID NO: 1.
• ASO antisense oligonucleotide
• composition containing a therapeutically effective amount of the antisense oligonucleotide and a pharmaceutically acceptable carrier.
• Yet another aspect of the present disclosure is a method of treating a disease in which aberrant expression and function of the MIR17HG pre-RNA plays a role.
• the method entailing administering to the subject the pharmaceutical composition.
• kits containing a therapeutically effective amount of an ASO that binds MIR17HG pre-RNA , and printed instructions for using the first active agent in the treatment of a disease in which aberrant expression and function of MIR17HG pre-RNA plays a role in a subject.
• FIGs. 1A - ID are a set of schematics and dot and line plots that illustrate the genome-wide CRISPRi viability screen that identified MIR17HG as a leading dependency in multiple melanoma (MM).
• FIG. 1 A schematically illustrates the viability screen.
• FIG. IB is a set of line plots showing the RRA-based ranked analysis of IncRNA dependencies in the secondary screen.
• FIG. 1C is a set of line plots showing a CCK-8 proliferation assay of MM cell lines.
• FIG. ID is a set of line plots showing a CCK-8 proliferation assay of MM cell lines transfected with ASOs targeting the MIR17HG pre-RNA or a non-targeting ASO (NC).
• NC non-targeting ASO
• FIGs. 2A - 2G are a set of schematics, line, bar, and survival plots that show how the IncRNA lnc-17-92 TV1 , also referred herein as the Regulator of Lipogenesis (RROL) mediates dependency in microRNA-independent manner.
• FIG. 2A schematically illustrates an overview of the MIR17HG locus, including both IncRNA (RROL) and miRNA (miR-17- 92) derived transcripts.
• FIG. 2B is a set of line plots that show the prognostic significance (PFS and OS) of high RROL expression (top quartile) in 3 large cohorts of MM patients.
• FIG. 2C is a set of bar plots that illustrate a CCK-8 proliferation assay in AM01 and H929 cells transduced with pri-mir-17-92 (pri-miR) or GFP control.
• FIG. 2D is a set of bar plots and images that illustrate a CCK-8 proliferation assay of DROSHA WT or KO AM01 and H929 cells exposed to ASO1.
• FIG. 2E is a line plot that illustrates the effects of RROL depletion in a matrigel -based AMO1 DR ' KO xenograft in NOD SCID mice.
• FIG. 2F is a survival plot that illustrates a survival analysis of tumor injected mice.
• FIG. 2G is a set of bar plots that show a CCK-8 proliferation assay in HCT-116 and DLD-1 colorectal cancer cell lines expressing either a WT or mutated (-/-) Dicer.
• FIGs. 3 A - 3G are a set of diagrams, images, bar, and dot plots that show RROL’s interaction with chromatin to regulate gene expression.
• FIG. 3A is a diagram that shows Transcriptomic analysis after RROL depletion in MM cell lines, either DROSHA WT (AM01, H929) or KO (AMO1 DR ' KO ) and shows commonly downregulated genes (adj p ⁇ 0.05; lfc ⁇ -l).
• FIG. 3B is a bar plot that illustrates a qRT-PCR analysis of RROL targets in CD138+ cells from 3 MM patients exposed to ASO1 for 24h.
• FIG. 3C is a dot plot and a set of correlation plots showing the correlation between RROL targets (mRNA) and RROL in CD 138+ MM patient cells together and alone for each RROL targets.
• FIG. 3D is a bar plot that illustrates a GLuc/ SEAP dual reporter assay showing reduced activity of ACC1, AN06, CCDC91, EPT1, EXT1, FER, and KIAA1109 promoter activity after RROL knockdown using ASO1.
• FIG. 3E is a set of line plots that illustrate a CCK-8 proliferation assay in 5 MM cells lines after transfection with siRNAs against RROL targets.
• FIG. 3F is a bar plot that illustrates a ChIRP-qPCR analysis showing effective amplification of ACC1 promoter in chromatin purified using 2 RROL antisense probe sets (psi and ps2), compared to chromatin purified using LacZ antisense probes (negative control).
• FIG. 3G is a set of images and a dot plot that illustrates a (left) snapshot obtained by dual RNA-FISH analysis of ACC1 pre- mRNA (green) and RROL (purple) in a representative AM01 cell; (right) box plot showing the distance (nm) of ACC1 pre-RNA spots to the nearest RROL spots or to the nearest random spots.
• FIGs. 4A - 4G are a set of bar and images that illustrate RROL’s promotion of MYC occupancy at the ACC1 promoter.
• FIG. 4A is bar plots that show upstream regulator analysis of transcriptional changes after 3 days of gymnotic exposure to ASO1 in H929 and AM01 cells.
• FIG. 4B a set of bar plots and images that illustrate a ChlP-qPCR analysis of MYC occupancy at ACC1 promoter in AM01, H929 and U266 MYC+ exposed for 24h to AS01 or NC (vehicle).
• FIG. 4A is bar plots that show upstream regulator analysis of transcriptional changes after 3 days of gymnotic exposure to ASO1 in H929 and AM01 cells.
• FIG. 4B a set of bar plots and images that illustrate a ChlP-qPCR analysis of MYC occupancy at ACC1 promoter in AM01, H929 and U266 MYC+ exposed for 24h to AS01 or NC (vehicle).
• FIG. 4C is a bar plot and image that illustrates a qRT-PCR analysis of ACC1 mRNA in P493-6 cells exposed for 2 days to either doxycycline or DMSO to knockdown MYC and for additional 2 days, exposed to either ASO1 or vehicle (NC) to deplete RROL.
• FIG. 4D is a set of images that illustrates a Co-IF/FISH analysis of ACC1 pre-RNA (red), MYC protein (green) and RROL (purple).
• FIG. 4E illustrates a qRT-PCR analysis of RROL (detecting isoform 2) in RIP material precipitated using an anti-MYC antibody (a-MYC) or IgG control.
• FIG. 4F illustrates a Western blot analysis of MYC in RPPD material precipitated with control RNA or RROL transcripts -1 and -2.
• FIG. 4G illustrates an RNA Y3H using MYC as hybrid protein 2 and, as hybrid RNAs, either a negative control RNA (-) or RROL transcripts -1 and -2.
• FIGs. 5A - 51 are a set of schematics, images, and bar plots that illustrate that RROL mediates the assembly of a MYC-WDR82 transcriptional complex, leading to transcriptional and epigenetic activation of ACC1.
• FIG. 5 A schematically shows integrated BioID and Co-IP/MS assays to explore the MYC-protein interacting network in presence or absence of RROL depletion.
• FIG. 5B is an image that illustrates a western blot analysis of WDR82 in RPPD material precipitated with RROL-1 and RROL-2 or with control RNA.
• FIG. 5C is an image that illustrates an RNA Y3H using WDR82 as hybrid protein 2 and, as hybrid RNAs, either a negative control RNA (-) or RROL transcripts -1 and -2.
• FIG. 5D is a bar plot and image that illustrates a ChlP-qPCR analysis of H3K4me3 occupancy at ACC1 promoter in AM01 after silencing of WDR82.
• FIG. 5E is a bar plot and image that illustrates a ChlP-qPCR analysis of MYC occupancy at ACC1 promoter in AM01 after silencing of WDR82.
• FIG. 5F is a bar plot and image that illustrates a qRT-PCR analysis of ACC1 mRNA after transfection of siRNA targeting WDR82 (siWDR82-l or -2) in AM01.
• FIG. 5G is a bar plot and image that illustrates a ChlP-qPCR analysis of WDR82-GFP occupancy at ACC1 promoter in AM01 exposed for 24h to gymnotic ASO1.
• FIG. 5H is a bar plot and an image that illustrates a ChlP-qPCR analysis of H3K4me3 occupancy at ACC1 promoter in AM01 and H929 exposed for 24h to gymnotic ASO1.
• FIG. 51 is a set of images of western blots that shows H3K4me3 levels at the ACC1 promoter site.
• FIGs. 6A - 6E are a set of bar plots, flow cytometry plots, and schematics that show that the RROL/MYC-ACC1 axis regulates de novo lipogenesis.
• FIG. 6A illustrates the incorporation of 14 C-glucose into lipids following either RROL, MYC, or ACC1 depletion / inhibition in MM cell lines or CD138+ MM patient cells.
• FIG. 6B is a set of bar plots that show liquid chromatography - mass spectrometry (LC-MS) based lipid profiling after RROL inhibition in MM cells.
• LC-MS liquid chromatography - mass spectrometry
• FIG. 6C is a bar plot that shows the effect of palmitate on anti- proliferative effects of RROL depletion in MM cells.
• FIG. 6D is a set of flow cytometry plots that show the effect of palmitate on pro-proliferative effects of RROL depletion in MM cells.
• FIG. 6E schematically illustrates the RROL pathway as it affects MM cell growth.
• FIGs. 7A - 7J are a set of dot, bar, and survival plots and images that depict therapeutic inhibitors of RROL that exert potent anti-tumor activity in vitro and in vivo in animal models of human MM.
• FIG. 7A schematically illustrates the multi-step screen to develop RROL therapeutic ASOs.
• FIG. 7B is a set of bar plots that illustrates a CCK-8 proliferation assay in a panel of 11 MM cell lines.
• FIG. 7C is a dot plot that illustrates a subcutaneous in vivo tumor growth of AM01 cells in NOD SCID mice.
• FIG. 7D - FIG. 7E are bar plots that illustrate a qRT-PCR analysis of RROL (FIG.
• FIG. 7D is a bar plot that illustrates a lipid profiling analysis showing modulation of tripalmitin in tumors.
• FIG. 7G is a dot plot and images that illustrate a BLLbased measurement of in vivo tumor growth of MOLP8-luc+ in NSG mice.
• FIG. 7H is a survival plot from experiment in FIG. 7E, black arrows indicate treatments.
• FIG. 71 is a line plot that shows human K light chain ELISA-based measurement of in vivo tumor growth of MM patient cells in NSG mice (PDX-NSG).
• FIG. 7J is a bar plot that shows qRT-PCR analysis of BCL2L11 in tumors retrieved from animals treated with G2-15b*-TO (G) or SB9- 19-TO (SB) or vehicle (NC) as control.
• FIG. 8 A - 8C are a set of bar plots that illustrate the screen data and MIR17HG knockdown with and without antisense oligonucleotides.
• FIG. 8A is a bar plot that shows an analysis of screening data, with the upset plot showing the identification of cell-type unique and shared IncRNA dependencies in MM cells.
• FIG. 8B is a bar plot that shows knockdown of MIR17HG obtained in AM01 engineered to express a dCas9-KRAB fusion protein and anti-MIR17HG gRNAs under the regulation of a conditional promoter.
• FIG. 8C is a bar plot that illustrates a knockdown of MIR17HG obtained in AM01 transfected with two gapmeRs. [0017] FIGs.
• FIG. 9A - 9L are a set of box and bar plots that illustrate RROL expression and correlation with the miR- 17-92 microRNAs.
• FIG. 9A is a box plot that shows RROL expression in newly diagnosed (ND) vs relapsed (R) MM patients.
• FIG. 9B is a box plot that shows RROL expression in ND vs R MM patients from GSE66293.
• FIG. 9C is a Spearman’s correlation that shows the interrelationship between RROL and miR-17-92 in CD138+ cells from 140 MM patients analyzed by RNA-seq and miRNA profiling.
• FIG. 9A is a box plot that shows RROL expression in newly diagnosed (ND) vs relapsed (R) MM patients.
• FIG. 9B is a box plot that shows RROL expression in ND vs R MM patients from GSE66293.
• FIG. 9C is a Spearman’s correlation that shows the inter
• FIG. 9D is a set of bar plots that illustrate qRT-PCR analysis of miR-17-92 microRNAs in AM01 and H929 cells.
• FIG. 9E is a set of bar plots that illustrate a qRT-PCR analysis of miR-17-92 microRNAs in AM01 and H929 either WT or KO for DROSHA.
• FIG. 9F is a set of bar plots that illustrate the knockdown of MIR17HG using 3 different ASOs, or a scramble control (NC), in AMO1 DR KO and H929 DR ' KO .
• FIG. 9G is an RNA-seq coverage plot and junction analysis for RROL in 4 MM samples.
• FIG. 9G is an RNA-seq coverage plot and junction analysis for RROL in 4 MM samples.
• FIG. 9H is a bar plot that shows qRT-PCR analysis of RROL transcript variants 1 and 2 in AM01. Primers amplify the regions indicated in FIG. 9G.
• FIG. 9J is a line plot that shows the effects of RROL depletion in a matrigel-based AM01DR-K0 xenograft in NOD SCID mice.
• FIG. 9K is a bar plot that shows a CCK-8 proliferation assay in HCC-116 cells transfected with ASOs targeting the 5 ’end (5’-ASO) of MIR17HG pre-RNA or a scrambled control (NC).
• FIG. 9L is bar plots that show Ectopic expression of pri-mir- 17-92, lnc-17- 92TV1, and lnc-17-92TV2, was confirmed by qRT-PCR showing: i) upregulation of miR-17 after ectopic expression of pri-mir- 17-92, ii) upregulation of lnc-17-92TVl after its ectopic expression, and iii) upregulation of lnc-17-92TV2 after its ectopic expression.
• FIGs. 10A - 101 are a set of images, heatmaps, and bar plots that illustrate RROL and miR-17-92 miRNA expression and localization in vitro.
• FIG. 10A is a bar plot that shows a sub-cellular qRT-PCR analysis of RROL in AM01 and H929.
• FIG. 10B is a set of images that shows an RNA-FISH analysis of subcellular localization of RROL in AM01.
• FIG. 10C is a heatmap that shows a qRT-PCR analysis of RROL and miR-17-92 miRNAs in AM01 gymnotically exposed to ASO1 or ASO-l-NC.
• FIG. 10A is a bar plot that shows a sub-cellular qRT-PCR analysis of RROL in AM01 and H929.
• FIG. 10B is a set of images that shows an RNA-FISH analysis of subcellular localization of RROL in AM01.
• FIG. 10C is a heatmap that shows a qRT-PCR analysis
• FIG. 10D is a bar plot that shows a qRT-PCR analysis of RROL transcriptional targets in Daudi and Raji cells gymnotically exposed to ASO1 for 24h.
• FIG. 10E is a bar plot that shows a qRT-PCR analysis of murine rrol. accl and fer in 5TGM1 murine MM cells transfected with 3 different ASOs targeting the murine mirl7hg nascent RNA.
• FIG. 10F - FIG. 10G are bar plots that show qRT-PCR analysis of miR-17-92 miRNAs (black bars) and RROL transcriptional targets (white bars) in AM01 transfected with pooled miR-17-92 miRNA inhibitors (FIG. 10F) or mimics (FIG.
• FIG. 10H - FIG. 101 are bar plots that show a ChIRP-qPCR analysis showing the % (FIG. 10H) or CT values (FIG. 101) of positive reactions (amplification) of GAPDH exon2 in chromatin purified using 2 RROL antisense probe sets (psi and ps2) or using LacZ antisense probes (negative control).
• FIGs. 11 A - 11G are a set of photos and a bar plot that show RROL promotes MYC occupancy at the ACC1 promoter.
• FIG. 11A is a western blot of MYC in U266 cells WT or infected with a vector carrying the expression of FLAG or MYC-FLAG.
• FIG. 11B is a schematic illustration of isoforms of RROL.
• FIG. 11C is a qRT-PCR of RROL isoforms -2 and -2 after immunoprecipitation with anti-MYC antibody (a-MYC) or IgG control.
• FIG. 11A is a western blot of MYC in U266 cells WT or infected with a vector carrying the expression of FLAG or MYC-FLAG.
• FIG. 11B is a schematic illustration of isoforms of RROL.
• FIG. 11C is a qRT-PCR of RROL isoforms -2 and -2 after
• FIG. 1 ID is a bar plot that shows qRT-PCR analysis of ACC1 mRNA following treatment with MYC inhibitor 10058-F4 in MM cell lines AM01 and H929.
• FIG. HE is a bar plot that shows incorporation of C 14 -glucose into lipids following treatment with MYC inhibitor 10058-F4 in MM cell lines AM01 and H929.
• FIG. 1 IF is a bar plot that shows a CCK-8 proliferation assay in U266 MYC " and U266 MYC+ after transfection with siRNA targeting ACC1 or a scramble siRNA (NC). Cell viability was measured 48h after transfection and it is represented as % of NC transfected cells.
• FIG. 11G is a photograph of a western blot analysis of MYC in RPPD material precipitated with control RNA or truncated versions of lnc-17- 92TV1; 5% input is used as a reference.
• FIGs. 12A - 12D are a set of images that shows MYC and GFP expression after RROL knockdown or WDR82-GFP fusion protein expression.
• FIG. 12A is an image that illustrates western blot analysis of MYC in FB A-MYC cells with or without doxycycline to induce the FB A-MYC fusion protein and transfected with either NC or ASO1 to knockdown RROL.
• FIG. 12B is an image that illustrates western blot analysis of GFP in AM01 cells WT or infected with a vector carrying the expression of GFP or WDR82-GFP.
• FIG. 12A is an image that illustrates western blot analysis of MYC in FB A-MYC cells with or without doxycycline to induce the FB A-MYC fusion protein and transfected with either NC or ASO1 to knockdown RROL.
• FIG. 12B is an image that illustrates western blot analysis of GFP in AM01 cells WT
• FIG. 12C is an image that shows western blot analysis of WDR82 in RPPD material precipitated with control RNA or truncated versions of lnc-17-92TVl.
• Lamin A/C was used as the protein loading controls (nuclear lysates), and ii. is the quantification of densitometry analysis.
• FIGs. 13A - 13C are a set of bar plots and photographs that show the effect of glucose update and expression of RROL targets after exposure to ASOs that bind RROL.
• FIG. 13A is a bar plot that shows the incorporation of C14-glucose into lipids after transfection of AM01 with miR- 17-92 anti-miRs.
• FIG. IB is a bar plot that shows GEP analysis of miR-17-92’s canonical targets BCL2L11 (BIM) and PTEN in AM01 and H929 exposed to ASO1 for 36h.
• FIG. 13C is a photograph of a western blot analysis of miR-17- 92’s canonical target BIM in AM01 exposed to ASO1 for 36h.
• FIGs. 14A-14C are a set of bar plots that depict a multi-step screen to develop therapeutic ASOs targeting RROL.
• FIG. 14A is a bar plot that shows the results of step 1 identifying ASO-accessible stretches on RROL, 16 sequences (>20-mer) either in “G” or “SB” configuration.
• FIG. 14B is a bar plot that shows the results of step 2 optimizing the G2 and SB9 designs selected from step 1.
• FIG. 14C is a bar plot that shows the results of step 3 where step 2 molecules conjugated with palmitic acid (P), cholesterol (C), or tocopherol (T) were tested.
• P palmitic acid
• C cholesterol
• T tocopherol
• the present disclosure provides antisense oligonucleotide (ASO) that binds the MiR-17-92a-l Cluster Host Gene (MIR17HG) pre-RNA under physiological conditions.
• ASO antisense oligonucleotide
• the MIR17HG gene contains two IneRNAs, lnc-17-92 TV1 and lnc-17-92 TV2 , and six miRNAs, miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a.
• the nucleic acid sequence of the lnc-17-92 TV1 also known as RNA Regulator of Lipogenesis (RROL), Inc- 17- 92 TV1 , and MIR17HG miR ' 17 ' 92 ) transcript is provided at NCBI Accession No. NR_027350, version NR_027350.1.
• Lnc-17-92 TV1 is shown herein to provide a chromatin scaffold mediating a transcription factor complex to drive oncogenic gene translation.
• the nucleic acid sequence of the lnc-17-92 TV2 transcript is provided at NCBI Accession No. NR_027349, version NR_027349.1.
• antisense oligonucleotide refers to a non-naturally occurring polymer of nucleotides (oligomer) capable of binding a target RNA molecule.
• nucleotide unless specifically sated or obvious from context, includes nucleosides that have a ribose sugar (i.e., a ribonucleotide that forms ribonucleic acid, RNA) or a 2’ -deoxyribose sugar (i.e., a deoxyribonucleotide that forms deoxyribonucleic acid, DNA) and a phosphate.
• Nucleotides serve as the monomeric units of nucleic acid polymers or polynucleotides.
• the four nucleobases in DNA are guanine (G), adenine (A), cytosine (C) and thymine (T).
• RNA The four nucleobases in RNA are guanine (G), adenine (A), cytosine (C) and uracil (U).
• G guanine
• A adenine
• C cytosine
• U uracil
• ASOs may be made up of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, or a combination thereof.
• ASOs modulate target RNAs by hybridization through at least partial complementary region(s).
• ASO modulation mechanisms include transcriptional arrest, RNA synthesis disruption (e.g., at various stages including capping, splicing, and/or transport from nucleus to cytoplasm), ribosome attachment, ribonuclease (RNAse) H recruitment, degradation of mRNA (e.g., gapmer ASOs), translational arrest (e.g., ASO binding to a target RNA blocking translation), or steric blocking of target RNA by ASO hybridization.
• RNA synthesis disruption e.g., at various stages including capping, splicing, and/or transport from nucleus to cytoplasm
• RNAse ribonuclease
• degradation of mRNA e.g., gapmer ASOs
• translational arrest e.g., ASO binding to a target RNA blocking translation
• steric blocking of target RNA by ASO hybridization steric blocking of target RNA by ASO hybridization.
• gapmer and “gapmeR” are used herein to refer to short DNA ASO structures with RNA-like wing segments on both sides of a gap, or main region. These linear DNA molecules are designed to hybridize to a target to degrade an RNA through the induction of RNase H cleavage. Binding of a gapmer to the target RNA has a higher affinity due to the modified RNA-like flanking regions, as well as resistance to ASO degradation by nucleases.
• the gapmer gap contains at least one modification that is different from that of modifications in one or both wings. Such modifications include nucleobase, monomeric linkage, and sugar modifications.
• the ASO binds to the 5’ terminal region of the MIR17HG pre-RNA.
• the ASO targets an intronic region proximate to the 5’ terminal end of the MIR17HG pre-RNA.
• the term “intronic region” as used herein refers to regions of an RNA molecule that are removed from a pre-RNA in the course of transcription.
• the ASO targets a region of MIR17HG pre-RNA from which the miRNA primary transcript (pri-miRNA) is spliced (e.g., the pri-mir- 17-92).
• pri-miRNA miRNA primary transcript
• a pre-miRNA is a longer sequence from which a mature miRNA is derived.
• physiological conditions refers to conditions that are normally encountered in mammalian in vivo conditions, for example, an isotonic solution (about 0.9 % normal saline) at body temperature (about 37 °C), at physiological pH (within the range of about 7.3 to about 7.5).
• the minimum percent complementarity of the ASO that is effective may be determined in accordance with standard procedures.
• the ASO has about 85% to about 100% complementarity to the MIR17HG pre-RNA.
• the ASO has 100% complementarity with the MIR17HG pre-RNA sequence that it targets.
• the ASO is at least 99% complementarity, at least 98% complementarity, at least 97% complementarity, at least 96% complementarity, at least 95% complementarity, at last 90% complementarity, at least 85% complementarity to MIR17HG pre-RNA.
• the ASOs have a length ranging from 15 to 30 nucleotides. In some embodiments, ASO is 30 or less nucleotides in length. In some embodiments, the ASO is 25 or less nucleotides in length. In some embodiments, the ASO is 24 or less nucleotides in length. In some embodiments, the ASO is 23 or less nucleotides in length. In some embodiments, the ASO is 20 or less nucleotides in length. In some embodiments, the ASO is 18 or less nucleotides in length. In some embodiments, the ASO is from 15 nucleotides to 24 nucleotides in length.
• the ASO is from 18 nucleotides to 23 nucleotides in length. In some embodiments, the ASO is 18 nucleotides in length (which is referred to herein as an 18-mer). In some embodiments, the ASO is a 20-mer. In some embodiments, the ASO is a 21-mer. In some embodiments, the ASO is a 22-mer. In some embodiments, the ASO is a 23-mer.
• the term “mer” when used herein in reference to a ASO refers to a length of nucleotides. For example, the terms “16-mer” and “16mer” refers to a stretch of 16 nucleotides.
• the ASO is a single stranded RNA, single guide RNA (sgRNA) for the use with CRISPR cas9 gene editing systems.
• sgRNA single guide RNA
• MIR17HG pre-RNA sgRNA #1 AGTGGCGCGAAGGCGCAGGT (SEQ ID NO:
• MIR17HG pre-RNA sgRNA #2 GTGGCGCGAAGGCGCAGGTC (SEQ ID NO:
• MIR17HG pre-RNA sgRNA #3 CCTCGCCCGAGGGCGCGAAG (SEQ ID NO:
• MIR17HG pre-RNA sgRNA #4 GAGGGCGCGAAGTGGCGCGA (SEQ ID NO:
• the ASO is single stranded DNA (ssDNA) molecule.
• ssDNA single stranded DNA
• Nucleic acid sequences of representative ssDNA anti-MIR17HG pre-RNA ASOs are set forth in Table 1.
• ASOs may be further modified, with the addition of a chemical moiety or chemical modification to its nucleobases, nucleotides, or internucleoside linkages.
• ASO modifications enhance stability in vivo, improve specificity, and reduce toxic side effects.
• a representative modification includes modification of nucleotides with 2'-O-methoxyethylribose (MOE) groups.
• the MIR17HG pre-RNA-binding ASOs may be prepared in a G configuration, in which the ASO contains 5’ terminal nucleotides modified with 2’ -MOE, unmodified DNA (a “DNA gap”), and 3’ terminal nucleotides modified with 2’-M0E.
• MIR17HG pre-RNA-binding ASOs may be prepared in a SB configuration, in which all of the nucleotides in the ASO are modified with 2’MOE. Nucleic acid sequences of representative ASOs are set for in Table 2Error! Reference source not found..
• Gapmer ASOs contain modified nucleotides terminal to a DNA gap.
• the DNA gap is 5, 6, 7, 8, 9, 10, 11, 12, or 15 nucleotides in length.
• the ASO is a 5-8-5 gapmer, where a DNA gap is flanked 5’ by a 5mer of 2’- MOE chemically modified nucleotides, as well as flanked 3’ by a 5mer of 2’ -MOE chemically modified nucleotides.
• Table 2 Nucleic acid sequences of representative MIR17HG pre-RNA-binding ASOs.
• the representative ASOs in Table 2 may be modified, for example, the ASO 2 may be selected as the G configuration ASO for further modification, and the ASO 9 may be selected as the SB configuration ASO for further modification.
• These ASOs may be further modified, as illustrated in Table 3.
• G2 fine tune 15 may be selected as the G configuration ASO (G2-15) as set forth in Table 3
• SB fine tune 19 may be selected as the SB configuration ASO (SB2-19), as set forth in Table 4Error! Reference source not found..
• ASOs may be further modified.
• modifications include conjugation of additional moi eties (e.g., lipids) to one of more ASO nucleotides.
• the ASO may also include one or more chemically modification of one or more nucleobases, nucleotides, or internucleoside linkages.
• Additional moi eties may be conjugated to any one or more nucleotides within an ASO to increase delivery efficiency, specific cellular or tissue targeting, cellular uptake, and/or prolonged circulation time.
• one or more nucleotides within the ASO is conjugated a lipophilic moiety.
• the chemical moiety is conjugated to one or more of the nucleotide on the 5’ or 3’ terminal ends of the ASO (i.e., either 5’ and/or 3’ of the DNA gap in a gapmer).
• every nucleotide of the ASO is conjugated to a chemical moiety i.e., a fully 2’MOE configuration).
• the first 5’ terminal nucleotide is conjugated to a chemical moiety.
• the last 3’ terminal nucleotide is conjugated to a chemical moiety.
• Representative lipophilic moieties include palmitic acid, sterols (e.g., tocopherol, cholesterol), carbohydrates (e.g., N- Acetylgalactosamine; GalNAc), oleyl residues, retinyl residues, cholesteryl residues, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyl oxy hexyl groups, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, myristic acid, 03- (oleoyl)lithocholic acid
• Representative tocopherols include a-tocopherol, P-tocopherol, y-tocopherol, and 6- tocopherol.
• Analogs of a tocopherol include various unsaturated analogs of a-tocotrienol, P- tocotrienol, y-tocotrienol, and 6-tocotrienol.
• one or more nucleotides within the ASO is conjugated with a lipid, such as palmitic acid, tocopherol, or cholesterol.
• a lipid such as palmitic acid, tocopherol, or cholesterol.
• Lipids may be connected to the ASO’s 5’ or 3’ end with a PS (*) or a PO () linkage.
• one or more nucleotides within the ASO is conjugated with a lipid.
• the lipids may be the same or different.
• each of the lipids may be different, e.g., palmitic acid, tocopherol, and cholesterol.
• Additional lipophilic moieties suitable for conjugation to nucleic acids are known in the art. See, e.g., U.S. Patents 8,106,022, 8,404,862, 10,077,443, 10,358,643, 10,441,653, 11,116,843, and 11,260,134 and U.S. Patent Application Publications 2014/0045919, 2016/0289677, 2021/0163934, and 2022/0175817.
• the ASO comprises a chemical modification to one or more nucleobases, sugar moieties, intemucleoside linkages (i.e., the backbone), or combinations thereof.
• Nucleobase modifications include any modification or substitution that is structurally distinguishable from, yet functionally interchangeable with, a nucleobase, including 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines.
• tricyclic pyrimidines include 1,3- diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-l,3- diazaphenoxazine-2-one (G-clamp).
• Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.
• nucleobase modifications are known in the art. See, e.g, U.S. Patents 3,687,808 5,130,302, 5,811,534, 5,830,653, and 6,005,096, U.S. Patent Application Publications 20030158403 and 2003/0175906, and Kroschwitz et al., eds., The Concise Encyclopedia of Polymer Science and Engineering, 1st ed., John Wiley & Sons, 1990.
• Sugar moiety modifications include bicyclic, tricyclic, and non-bicyclic sugar moi eties or sugar surrogates, including furanosyl sugar moi eties (e.g., 2-deoxyfuranosyl sugar moiety modification).
• Representative modified furanosyl sugar moieties include acyclic modifications for example, at the 2’, 4’, and 5’ positions. In some embodiments, the acyclic modification is branched.
• sugar moiety 2’ position examples include 2’ -deoxy-2’ -fluoro (2'-F), 2’-arabino-fluoro (2’-Ara-F), 2’-O-benzyl, 2’-O-methyl-4- pyridine (2’-O-CH2Py(4)), 2’-O-mehtyl (2'-OCH3) (2’-OMe or Me), 2’-O-methoxyethyl (2'- O(CH 2 )2OCH 3 ) (2-0-M0E or MOE), halo, allyl, amino, azido, unlocked nucleic acid (UNA), glycol nucleic acid (GNA), SH, CN, OCN, CF3, OCF3, O — Ci-C 10 alkoxy, O — Ci- C 10 substituted alkoxy, O — C1-C10 alkyl, O — C1-C10 substituted alkyl, S-alkyl, N(Rm)-alkyl, O
• the sugar moiety modification comprises a fluorine modification at the 2’ position (2’ -Fluoro or 2’-F).
• the 2’-F modification provides high RNA binding affinity and resistance to nuclease degradation.
• Preparation methods of 2’-F containing sugar moiety modifications are known in the art. See, e.g., U.S. Patents 5,459,255 and 6,262,241, U.S. Patent Application Publications 20060036087 and 20110269814, and Ludwig, acta Biochim. Biophys. Acad. Sci. Hung. 16(3-4) : 131-3 (1981) and Ludwig and Eckstein J. Org. Chem. 54:631-635 (1989).
• Bicyclic sugar moiety modifications include bridging sugar modifications that form a second ring.
• the second ring comprises a bridge between the 4’ and 2’ positions on a furanose ring.
• the sugar moiety is a ribose.
• Representative 4’ to 2’ bridging sugar moiety modifications include (5)-cEt-BNA, tricyclo- DNA (tcDNA), PMO, 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2', 4'-CH 2 — O-2' (also known as locked nucleic acids or “LNA”), 4'-CH 2 — S-2', 4'-(CH 2 ) 2 — O-2' (also known as ethylene- bridged nucleic acids or “ENA”), 4'-CH(CH 3 ) — O-2' (also known as “constrained ethyl” or “cEt” when in the S configuration), 4'-CH 2 — O — CH 2 -2', 4'-CH 2 — N(R)-2', 4'- CH(CH 2 OCH 3 )— O-2' (“constrained MOE” or “cMOE”), 4'-C(CH 3 )(CH 3 )—
• Internucleoside linkage (i.e., backbone) modifications include any altered 3’ to 5’ phosphodiester linkage, including alkylphosphonates (e.g., methoxypropylphosphonate (MOP)) and phosphorothioates (e.g., phosphorothioate (PS)).
• alkylphosphonates e.g., methoxypropylphosphonate (MOP)
• phosphorothioates e.g., phosphorothioate (PS)
• An ASO comprising multiple modified internucleoside linkages with chiral centers may be stereopure (containing only one stereoisomer), stereorandom (no regularity in stereoisomers), or have a pattern in stereoisomers.
• Preparation methods of phosphorous-containing and non-phosphorous-containing intemucleoside linkages are known in the art. See, e.g., U.S. Patent Application Publication 20220049248 and Hu et al., Signal Transduct. Target. Ther. 5(1)'.101-25 (2020).
• a 2'-deoxyfuranosyl sugar moiety modification involves the addition of a five- member carbon furanosyl ring sugar moiety having two hydrogens at the 2'-position and may be unmodified or further modified at positions other than the 2’ -position.
• a 2’-O- methoxy ethyl sugar moiety modification involves a substitution of the 2’ -OH group of a ribosyl ring with a 2'-O(CH 2 )2-OCH 3 ).
• compositions of the disclosure include a therapeutically effective amount of an ASO and a pharmaceutically acceptable carrier.
• a therapeutically effective amount of an ASO refers to a sufficient amount of an ASO to provide the desired therapeutic effect.
• the effective amount of an ASO for a given patient varies depending one or more factors that may include the age, body weight, type, location, and severity of the cancer and general health of the subject. Ultimately, the attending physician will decide the appropriate dose and dosage regimen. Typically, the ASO will be given in a series of doses, typically a single dose a week for a number of weeks. In some embodiments, the effective amount of the ASO is about 0.03 mg to about 3 kg per dose. In some embodiments, the effective amount of the ASO is about 0.3 mg to about 300 mg per dose. In some embodiments, the effective dose of the ASO is about 30 mg per dose.
• the effective amount of the ASO is about 10 mg/kg subject body weight to about 50 mg/kg subject body weight per dose.
• the ASO is administered once a week for about 30 weeks to about 60 weeks.
• the ASO is administered once a week for about 40 weeks.
• the ASO is administered once a week, twice a week, or every other weekday (e.g., 3 days a week) for about 4 weeks.
• compositions may be provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH.
• Liquid, pharmaceutically acceptable carriers include aqueous or non-aqueous carriers alike.
• pharmaceutically acceptable carrier is art recognized and includes a pharmaceutically acceptable material, composition, or vehicle, suitable for administering ASOs of the present disclosure to mammals.
• the carriers include liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
• Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
• materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide
• liquid carriers include water, saline, phosphate buffered saline, and suitable mixtures thereof.
• the compositions are typically isotonic, /. ⁇ ., they have the same osmotic pressure as blood.
• Sodium chloride, potassium chloride, sodium bicarbonate, sodium carbonate, monobasic sodium phosphate monohydrate, anhydrous sodium phosphate dibasic, potassium phosphate monobasic, dibasic sodium phosphate heptahydrate, and isotonic electrolyte solutions (e.g., Plasma-Lyte®) may be used to achieve the desired isotonicity.
• Hydrochloric acid, and/or sodium hydroxide may be used to adjust the pH of the composition.
• the pH may be in the range of about 7.5 to about 8.5.
• excipients e.g., wetting, dispersing, or emulsifying agents, gelling and viscosity enhancing agents, preservatives and the like as known in the art.
• the compositions include a pharmaceutically acceptable carrier.
• the carrier may be lipid-based, e.g., fatty acids, lipid nanoparticles (LNPs), liposomes, lipid vesicles, or lipoplexes.
• the ASOs are emulsified in a fatty acid carrier.
• Representative fatty acids include ethyl eicosapentaenoate (EPA-E), ethyl octadecatetraenoate (ODTA-E), ethyl nonadecapentaenoate (NDPA-E), ethyl arachidonate (AA-E), ethyl eicosatetraenoate (ETA-E), and ethyl heneicosapentaenoate (HPA-E).
• EPA-E ethyl eicosapentaenoate
• ODTA-E ethyl octadecatetraenoate
• NDPA-E ethyl nonadecapentaenoate
• AA-E ethyl arachidonate
• ETA-E ethyl eicosatetraenoate
• HPA-E ethyl heneicosapentaenoate
• the carrier is an LNP.
• an LNP includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and/or crosslinked to one another. Lipid bilayers may include one or more ligands, proteins, or channels.
• Lipid carriers may include one or more cationic/ionizable lipids, one or more polymer conjugated lipids, one or more structural lipids, and/or one or more phospholipids.
• a "cationic lipid” refers to positively charged lipid or a lipid capable of holding a positive charge.
• Cationic lipids include one or more amine group(s) which bear the positive charge, depending on pH.
• a “polymer conjugated lipid” refers to a lipid with a conjugated polymer portion.
• Polymer conjugated lipids include a pegylated lipids, which are lipids conjugated to polyethylene glycol.
• a “structure lipid” refers to a non-cationic lipid that does not have a net charge at physiological pH.
• Exemplary structural lipids include cholesterol, fecosterol, sitosterol, ergosterol, campesterol and the like.
• a “phospholipid” refers to lipids that have a triester of glycerol with two fatty acids and one phosphate ion. Phospholipids in LNPs assemble the lipids into one or more lipid bilayers. LNPs, their method of preparation, formulation, and delivery are disclosed in, e.g., U.S. Patent Application Publication Nos. 2004/0142025, 2007/0042031, and 2020/0237679 and U.S. Patents 9,364,435, 9,518,272, 10,022,435, and 11,191,849.
• Lipoplexes, liposomes, and lipid nanoparticles may include a combination of lipid molecules, e.g., a cationic lipid, a neutral lipid, an anionic lipid, polypeptide-lipid conjugates, and other stabilization components.
• Representative stabilization components include antioxidants, surfactants, and salts.
• Compositions and preparation methods of lipoplexes, liposomes, and lipid nanoparticles are known in the art. See, e.g., U.S. Patents 8,058,069, 8,969,353, 9,682,139, 10,238,754, U.S. Patent Application Publications 2005/0064026 and 2018/0291086, and Lasic, Trends Biotechnol. 76(7):307-21 (1998), Lasic et al., FEBS Lett.
• kits or systems containing one or more ASOs.
• Kits or systems include a package such as a box, carton, tube, or the like, having in close confinement therein one or more containers, such as vials, tubes, ampoules, or bottles, which contain an ASO or a pharmaceutical composition thereof.
• the kits or systems may also include printed instructions for using the ASO and pharmaceutical compositions thereof.
• a kit contains a therapeutically effective amount of an anti- MIR17HG pre-RNA ASO, and printed instructions for using same in the treatment of a MIR17HG pre-RNA-contributed disease in a subject.
• the kit also contains a MYC proto-oncogene, bHLH transcription factor (MYC) inhibitor and printed instructions on the use of the MYC inhibitor, where the MIR17HG pre-RNA ASO and MYC inhibitor or in the same dosage forms or different dosage forms that are disposed in the same or different containers.
• MYC bHLH transcription factor
• the present disclosure is directed to treating a subject having a disease in which aberrant expression and function of the MIR17HG pre-RNA plays a role.
• the method entails administering to a subject in need thereof a pharmaceutical composition that contains a therapeutically effective amount of an ASO.
• the term “disease in which aberrant expression and function of the MIR17HG pre- RNA plays a role” is used herein to refer to a disease which may be improved by the therapeutic targeting of the MIR17HG pre-RNA transcript.
• treatment refers is an approach for obtaining beneficial or desired results, including clinical results.
• results may include one or more of alleviation or amelioration of one or more symptoms of a disease in which MIR17HG pre-RNA plays a role, diminishment of extent of the disease, stabilization of the state of the disease, delay or slowing of the disease, amelioration or palliation of the disease, and remission of the disease (whether partial or total), whether detectable or undetectable.
• subject includes all members of the animal kingdom prone to or suffering from a disease in which aberrant expression and function of the MIR17HG pre-RNA plays a role.
• the subject is a human.
• a subject “having a disease in which aberrant expression and function of MIR17HG pre-RNA plays a role,” “having a neoplasm,” or “in need of’ treatment broadly embraces subjects who have been positively diagnosed, including subjects having active disease who may have been previously treated with one or more rounds of therapy, and subjects who are not currently being treated (e.g., in remission) but who might still be at risk of relapse, and subjects who have not been positively diagnosed but who are predisposed to diseases in which aberrant expression and function of MIR17HG pre-RNA plays a role (e.g., on account of the basis of prior medical history and/or family medical history, or who otherwise present with a one or more risk factors such that a medical professional might reasonably suspect that the subject was predisposed to disease in which aberrant expression and function of MIR17HG pre-RNA plays a role).
• MIR17HG pre-RNA plays a role include, for example, neoplasia (cancer), and non-cancerous diseases such as liver diseases.
• neoplasia as used herein is meant a disease characterized by excess proliferation or reduced apoptosis.
• Illustrative neoplasms for which the disclosure can be used include, but are not limited to pancreatic cancer, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibro
• the disease in which aberrant expression and function of MIR17HG pre-RNA plays a role is multiple myeloma, lymphoma, or colorectal cancer.
• Representative lymphomas include chronic lymphocytic leukemia, cutaneous b-cell lymphoma, cutaneous t-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and waldenstrom macroglobulinemia.
• Representative colorectal cancers include gastrointestinal tract adenocarcinoma, rectal adenocarcinoma, and colon adenocarcinoma).
• MM Multiple myeloma
• the disease in which MIR17HG pre-RNA plays a role is nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver fibrosis, viral hepatitis, or alcoholic liver disease (ALD).
• NAFLD nonalcoholic fatty liver disease
• NASH nonalcoholic steatohepatitis
• liver fibrosis liver fibrosis
• viral hepatitis viral hepatitis
• ALD alcoholic liver disease
• MYC a highly pleiotropic transcription factor that regulates hepatic cell function
• Overexpression of MYC alters a wide range of roles including cell proliferation, growth, metabolism, DNA replication, cell cycle progression, cell adhesion and differentiation. Overexpressed MYC is often seen in patients with liver fibrosis, as described in more detail in Zheng et al., Genes (Basel) 8 123-20 (2017).
• the disease is one in which a transcript of the MIR17HG pre- RNA further interacts with MYC proto-oncogene, bHLH transcription factor (MYC) or MYC-binding partners or is characterized by dysregulated MYC or dysregulated MYC- binding partners.
• MYC bHLH transcription factor
• MYC-binding partners or is characterized by dysregulated MYC or dysregulated MYC- binding partners.
• Representative diseases in which MYC plays a role include multiple myeloma, B-cell lymphomas (e.g., diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and burkitt lymphoma), triple negative breast cancer, pancreatic cancer, liver cancer, and gastric cancer.
• B-cell lymphomas e.g., diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and burkitt lymphoma
• triple negative breast cancer pancreatic cancer, liver cancer
• the therapies of the present disclosure may be used in combination with at least one other active agent in treating diseases and disorders.
• the term “in combination” in this context means that the agents are co-administered, which includes substantially contemporaneous administration, by the same or separate dosage forms, or sequentially, e.g., as part of the same treatment regimen or by way of successive treatment regimens.
• the first of the two therapies is, in some cases, still detectable at effective concentrations at the site of treatment.
• the sequence and time interval may be determined such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise).
• the therapeutics may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they may be administered sufficiently close in time so as to provide the desired therapeutic effect, which may be in a synergistic fashion.
• the terms are not limited to the administration of the active agents at exactly the same time.
• the dosage of the additional therapeutic may be the same or even lower than known or recommended doses. See, Hardman et al., eds., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; Physician's Desk Reference 60th ed., 2006. Anti-cancer agents that may be used in combination with the inventive therapies are known in the art. See, e.g., U.S. Patent 9,101,622 (Section 5.2 thereof).
• an "anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these additional active agents would be provided in a combined amount effective to kill or inhibit proliferation of diseased or cancerous cells. This process may involve contacting the cells with recipient cells and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cells with a single composition or pharmacological formulation that includes both agents, or by contacting the cells with two distinct compositions or formulations, at the same time, wherein one composition includes an ASO and the other includes the second agent(s).
• the therapies of the present disclosure are used in conjunction with chemotherapeutic, radiotherapeutic, immunotherapeutic intervention, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy.
• the therapies of the present disclosure may precede or follow the additional agent (e.g., anti-cancer) treatment by intervals ranging from minutes to weeks.
• additional agent e.g., anti-cancer
• the therapies of the present disclosure and the additional agent may be administered within the same patient visit; in other embodiments, the two agents are administered during different patient visits.
• the therapies of the disclosure and the additional agent are cyclically administered. Cycling therapy involves the administration of one therapeutic for a period of time, followed by the administration of a second therapeutic for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one or both of the additional therapeutics, to avoid or reduce the side effects of one or both of the additional therapeutics, and/or to improve the efficacy of the therapies.
• cycling therapy involves the administration of a first additional therapeutic for a period of time, followed by the administration of a second additional therapeutic for a period of time, optionally, followed by the administration of a third additional therapeutic for a period of time and so forth, and repeating this sequential administration, i.e., the cycle. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the cells of the present disclosure.
• the additional therapeutic is a MYC inhibitor.
• MYC inhibitors include cisplatin, gemcitabine, axitinib, nadroparin, and benzamidine.
• the additional therapeutic is an acetyl-CoA carboxylase-a (ACC1) inhibitor.
• the AAC1 inhibitor is 5-tetradecyl-oxy-2-furoic acid (TOFA).
• the additional therapeutic is an Immunomodulatory imide drug (IMiD).
• IMiDs include thalidomide, lenalidomide, pomalidomide, and iberdomide.
• the additional therapeutic is a proteasome inhibitor.
• Representative proteasome inhibitors include bortezomib, carfilzomib (Kyprolis®), delanzomib, ixazomib, marizomib, and oprozomib.
• myeloma therapeutics that may be suitable for the combination with the inventive therapies described herein include belantamab mafodotin-blmf (Blenrep®), bortezomib (Velcade®), carfilzomib (Kyprolis®), carmustine (BiCNU®), ciltacabtagene autoleucel (Carvykti®), cyclophosphamide, daratumumab (Darzalex®), daratumumab and hyaluronidase-fihj (Darzalex Faspro®), doxorubicin hydrochloride liposome (Doxil®), elotuzumab (Empliciti®), idecabtagene vicleucel (Abecma®), isatuximab-irfc (Sarclisa®), ixazomib citrate (Ninlaro®), len
• Immunotherapy including immune checkpoint inhibitors may be employed to treat a diagnosed cancer.
• Immune checkpoint molecules include, for example, PD1, CTLA4, KIR, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD47, and NKG2A.
• Clinically available examples of immune checkpoint inhibitors include durvalumab (Imfinzi®), atezolizumab (Tecentriq®), and avelumab (Bavencio®).
• Clinically available examples of PD1 inhibitors include nivolumab (Opdivo®), pembrolizumab (Keytruda®), and cemiplimab (Libtayo®). Chemotherapy
• Anti-cancer therapies also include a variety of combination therapies with both chemical and radiation-based treatments.
• Combination chemotherapies include, for example, Abraxane®, altretamine, docetaxel, Herceptin®, methotrexate, Novantrone®, Zoladex®, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, Taxol®, gemcitabien, Navelbine®, farnesyl- protein tansferase inhibitors, transplatinum, 5 -fluorouracil, vincri
• Anti-cancer therapies also include radiation-based, DNA-damaging treatments.
• Combination radiotherapies include what are commonly known as gamma-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells which cause a broad range of damage on DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells and will be determined by the attending physician.
• Radiotherapy may include external or internal radiation therapy.
• External radiation therapy involves a radiation source outside the subject’s body and sending the radiation toward the area of the cancer within the body.
• Internal radiation therapy uses a radioactive substance sealed in needles, seeds, wires, or catheters that are placed directly into or near the cancer.
• Cell lines were grown at 37°C, 5% CO2.
• MM-CLs AM01, NCI-H929, SK-MM-1, U266, JJN3 and KMS-12-BM were purchased from DSMZ (Braunschweig, Germany).
• MM. IS, MM.1R and RPMI-8226 were purchased from ATCC (Manassas, VA, USA).
• ABZB CL is AM01 bortezomib -resistant and ACFZ Cl is AM01 carfilzomib-resistant (Morelli et al., Blood 132: 1050-1063 (2016)).
• LR7 CL is U266 melphalan-resistant (Morelli et al., Blood 132: 1050-1063 (2016)). These cells were cultured in RPMI-1640 medium (Gibco® Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Lonza Group Ltd., Basel, Switzerland) and 1% penicillin/streptomycin (Gibco®, Life Technologies), b) B-cell lymphoma cell lines (BCL0CL): Maver-1, Jeko-1 (mantle cell lymphoma), Sultan, P3HR1, Daudi and Raji (Burkitt lymphoma) (purchased from ATCC) were cultured in RPMI-1640 medium (Gibco® Life Technologies) supplemented with 10% fetal bovine serum (Lonza Group Ltd.) and 1% penicillin/streptomycin (Gibco®, Life Technologies), c) Non-malignant cell lines: HK-2 (human kidney cells, cortex/proximal
• Flp-In T-REx cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) (Gibco®, Life Technologies) supplemented with 10% fetal bovine serum (Lonza Group Ltd.) and 1% penicillin/streptomycin (Gibco®, Life Technologies), e) P493-6 were cultured in RPML1640 medium (Gibco® Life Technologies) supplemented with 10% fetal bovine serum (Lonza Group Ltd.) and 1% penicillin/streptomycin (Gibco®, Life Technologies), f) 5TGM1 murine MM cells were cultured in IMDM (Iscove modified Dulbecco medium) (Gibco®, Life Technologies) supplemented with 10% fetal bovine serum (Lonza Group Ltd.) and 1% penicillin/streptomycin (Gibco®, Life Technologies), g) Colorectal cancer cell lines HCT116 and DLD-1, parental and
• CD 138+ cells were isolated from the BM aspirates of MM patients by Ficoll-Hypaque (Lonza Group, Basel, Switzerland) density gradient sedimentation; followed by antibody-mediated positive selection using anti-CD138 magnetic activated cell separation microbeads (Miltenyi Biotech, Gladbach, Germany). Purity of immunoselected cells was assessed by flow-cytometry analysis using a phycoerythrin-conjugated CD138 monoclonal antibody by standard procedures.
• CD 138+ cells were cultured physically separated from HS-5 cells by means of Falcon Cell Culture Inserts (Corning, New York, NY, USA), according to manufacturer’s instructions, as previously described (Morelli et al., Blood 132: 1050-1063 (2016)).
• Peripheral blood mononuclear cells Following informed consent approved by the Dana-Farber Cancer Institute Institutional Review Board, CD138+ cells were isolated from the BM aspirates of MM patients by Ficoll-Hypaque (Lonza Group, Basel, Switzerland) density gradient sedimentation; followed by antibody-mediated positive selection using anti- CD138 magnetic activated cell separation microbeads (Miltenyi Biotech, Gladbach, Germany). Purity of immunoselected cells was assessed by flow-cytometry analysis using a phycoerythrin-conjugated CD138 monoclonal antibody by standard procedures.
• CD 138+ cells were cultured physically separated from HS-5 cells by means of Falcon Cell Culture Inserts (Corning, New York, NY, USA), according to manufacturer’s instructions, as previously described (Morelli et al., Blood 132: 1050-1063 (2016)).
• RNA-seq as primary dataset, previously published RNAseq data from CD 138+ MM cells from 360 MM patients from IFM/DFCI 20019 clinical trial (NCT01191060) were used (Samur et al., Leukemia 32:2626-2635 (2016)). This dataset was used to assess expression of IncRNAs in newly diagnosed MM patients. Unstranded paired-end RNA sequencing were quantified using quasi-mapping with Salmon. Reference transcripts for GRCh38 transcripts were downloaded from Gencode v24. After QC controls TPM values for genes generated from isoform level TPMs with tximport.
• Microarray-based gene expression analysis RROL expression level was evaluated in a publicly available dataset (GSE66293) (Lionetti et al., Oncotarget 6:24205-17 (2015)) including 129 newly diagnosed and 12 relapsed MM cases that were profiled by GeneChip Human Gene 1.0 ST array (Affymetrix, Santa Clara, CA, USA) (Todoerti et al., Clin. Cancer. Res. 79:3247-58 (2013)). Normalized and re-annotated expression levels were obtained as described (Todoerti et al., Clin. Cancer. Res.
• miRNA profiling miRNA expression data for IFM cohort were generated using Affymetrix GeneChip® miRNA Array 4.0 platform. Affy and oligo packages from Bioconductor was used to normalize the miRNA expression data. [00101] Correlation analysis: Spearman correlation was used to evaluate correlation between IncRNA, mRNAs and miRNAs.
• survival analysis survival analysis was performed using survival package in R, and log rank test was used to compare groups.
• dCAS9-KRAB cell lines expressing the dCas9-KRAB fusion protein were generated as previously described Morelli et al., Methods Mol. Biol. 23 8: 189-204 (2021). Briefly, cells were infected with a lentivirus expressing the dCas9- BFP-KRAB transgene (Addgene, Plasmid #46911) and sorted for clones stably expressing high BFP. Infection was performed at low MOI ( ⁇ 0.4).
• sgRNA for ENO1 gRNA_EN01 : CCGGCGAGATCTCCGTGCTC (SEQ ID NO: 66) or a non-targeting negative control (gRNA NC: GATGTGGTCATTCGTCATGA (SEQ ID NO: 67).
• sgRNAs were cloned into pU6-sgRNA EFlAlpha-puro-T2A-BFP (Plasmid #60955).
• CRISPRi viability screens were used designed using the Broad Institute web portal (now called CRISPick: portals.broadinstitute.org/gppx/crispick/public).
• CRISPick portals.broadinstitute.org/gppx/crispick/public.
• target IncRNAs were selected based on median TPM>0.5 in the IFM/DFCI cohort.
• target IncRNAs were selected based on primary screen results (i.e., targeted by significantly depleted or enriched gRNAs, FDR ⁇ 0.25); plus additional IncRNAs identified through a de- novo assembly of RNA-seq data and manually selected IncRNAs selected based on their impact on the clinical outcome of MM patients enrolled in the IFM/DFCI clinical study.
• gRNA pool library production Primary CRISPRi library consisting of 7,500 gRNAs or secondary CRISPRi library consisting of 3,750 gRNAs were co-transfected with packaging plasmids (psPAX2, Addgene #12260; pMD2.G, Addgene #12259) into HEK293T cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) following the manufacture’s protocol.
• Library DNA (4pg), psPAX2 DNA (4pg) and VSV-G DNA (2pg) was mixed and transfected into HEK293T cells in a T75 flask (x10).
• DMEM media supplemented with 10% of FBS
• lentiviral media was harvested, concentrated using Lenti-XTM Concentrator (Takara, cat. no. 631232) and stored at -80°C.
• Virus titer determination 1 ⁇ 10 6 cells (each cell line) were plated per well of a 6- well plate. Cells were infected with different amounts of lentivirus overnight in the presence of 8pg/ml of polybrene. The titering of lentiviral particles was performed by flow-cytometry following protocol from Cellecta, section 5.3 and 5.4.
• Genomic DNA was isolated using Blood & Cell Culture DNA Maxi/Midi Kit (Qiagen #13362,13343) following the manufacturer’s protocol.
• Cellecta (Mountain View, CA) performed PCR amplification of the gRNA cassette for Illumina sequencing of gRNA representation. Protocols for PCR and Illumina sequencing are available online.
• gRNA constructs were co-transfected with packaging plasmids (psPAX2, Addgene #12260; pMD2.G, Addgene #12259) into HEK293T cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) following the manufacture’s protocol.
• Virus was harvested 48 hours later, concentrated, and stored at -80°C.
• MM cell lines stably expressing dCas9-KRAB fusion protein were infected with a lentivirus driving expression of individual sgRNAs. Infected cells were selected using puromycin. Expression of sgRNAs was obtained by doxycycline (0.5pg/mL, every other day).
• Antisense oligonucleotides synthetic miRNA mimics and inhibitors, siRNAs. Long Non-Coding LNA gapmerRs, SEQ ID NOs: 2-14, were custom-designed and purchased from Exiqon (Vedbaek, Denmark).
• Synthetic mimics and inhibitors for miR-17a, miR-18a, miR-19a, miR-20a, miR-19b-l and miR-92al were purchased from Ambion (Applied Biosystems, CA, US).
• Silencer selected siRNAs were purchased from Ambion (Applied Biosystems, CA, US).
• Design of t-ASOs is described in Table 2 - Table 4.
• Gymnosis Gymonotic experiments were performed as previously described (Taiana et al., Methods Mol. Biol. 2375: 157-166 (2021)). Briefly: cells were seeded at plating density to reach confluence on the final day of the experiments. Cell number at plating ranged from 0,5 to 2,5 x 10 3 in 96-well plates, from 2,5 to 10 x 10 4 in 12-well plates, from 1 to 3 x 10 5 in 6-well plates. For ChIP and Co-IP experiments, cell number at plating was 1 x 10 6 in T75 flask (10mL final volume).
• AM01 were transduced with Lenti ORF clone of Human WD repeat domain 82 (WDR82), mGFP tagged (RC216325L4) (Origene Technologies, Rockville, Maryland, MD).
• WDR82 Human WD repeat domain 82
• mGFP tagged RC216325L4
• pLX_311-Cas9 Additional Biotech, Inc.
• CRISPR/CAS9 gene knockout To generate DROSHA KO cells, AM01 and H929 stably expressing Cas9 were transduced with transEDIT CRISPR single gRNA lentiviral expression vectors targeting DROSHA (CMV promoter, ZsGreen, TEVH-1203933) (transOMIC technologies Inc., Huntsville, AL, USA). ZsGreen+ cells were sorted (BD FACSARIA III; BD Biosciences, Qume Drive San Jose, CA, USA) 5 days after infection and cultured.
• CMV promoter, ZsGreen, TEVH-1203933 transOMIC technologies Inc., Huntsville, AL, USA
• Cell viability assay Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies) and 7-AminoactinoMYCin (7-AAD) flow cytometry assays (BD biosciences), according to manufacturer’s instructions. Flow cytometry analysis was performed either by FACS CANTO II (BD biosciences) or by Attune NxT Flow cytometer (Thermo Fisher Scientific).
• Apoptosis was investigated by Annexin V/7-AAD flow cytometry assay (BD biosciences) and by electronic microscopy. Flow cytometry analysis was performed either by FACS CANTO II (BD biosciences) or by Attune NxT Flow cytometer (Thermo Fisher Scientific).
• RNA extraction, reverse transcription (RT) and quantitative real-time amplification (qRT-PCR) were performed as previously described (Morelli et al., Blood 732: 1050-1063 (2016)). Briefly, total RNA was extracted from cells with TRIzol® Reagent (Thermo Fisher Scientific), according to manufacturer’s instructions. Nuclear and cytosolic subcellular RNA purification was performed using RNA Subcellular Isolation Kit (cat. no. 25501) (Active Motif, Carlsbad, CA), according to manufacturer’s instructions. The integrity of total RNA was verified by nanodrop (Celbio Nanodrop Spectrophotometer nd- 1000).
• oligo-dT-primed cDNA was obtained through the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) and then used as a template to quantify:
• [00118] a) human RROL (Hs03295901), ACACA or ACC1 (Hs01046047_ml), AN06 (Hs03805835_ml), EXT1 (Hs00609156_ml), FER (Hs00245497_ml), MALAT1 (Hs00273907_sl) AND PVT1 (Hs00413039_ml). Normalization was performed with human GAPDH (Hs03929097_gl) or ACTB (Hs03023943_gl) or 18S (Hs03003631_gl).
• Single-tube TaqMan miRNA assay was used to detect and quantify miR-17 (002308), miR-18a (002422), miR-19a (000395), miR-20a (000580), miR-19b (000396) and miR-92a-l (000431), according to the manufacturer’s instructions, by the use of ViiA7 RT reader (Thermo Fisher Scientific). Mature miRNAs expression was normalized on RNU44 (Thermo Fisher Scientific, assay Id: Hs03929097_gl).
• RROL isoforms were also detected by SYBR Green qRT-PCR using the following primers: RROL-1 (Fw, 5’-CCTGCAACTTCCTGGAGAAC (SEQ ID NO: 68); Rev, 5’- GTCTCAAGTGGGCATGATGA (SEQ ID NO: 69)), RROL-2 (Fw, 5’- GACCCTCTTTTAAGTTGGGTG (SEQ ID NO: 70; Rev, 5’-
• nitrocellulose membranes were blocked and probed over-night with primary antibodies at 4 °C, then the membranes were washed 3 times in PBS- Tween and then incubated with a secondary antibody conjugated with horseradish peroxidase for 2 hours at room temperature. Chemiluminescence was detected using Western Blotting Luminol Reagent (sc-2048, Santa Cruz, Dallas, TX, USA).
• Anti-MYC [D84C1] (#5605), anti-WDR82 [D2I3B] (#99715), anti-H3K4me3 [C42D8] (#9751) and anti -Lamin A/C (#2032) antibodies were purchased from Cell Signaling Biotechnology (Danvers, MA).
• Anti-Drosha antibody [EPR12794] (abl83732) was purchased from Abeam (Cambridge, UK).
• Anti-MYC [9E10] (sc-40), GAPDH (sc-25778) and ⁇ -actin (ab96682) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
• Monoclonal ANTI-FLAG® M2 antibody (F3165) was purchased from Millipore Sigma (Bedford, MA). Secondary antibodies: Anti-rabbit IgG, HRP-linked Antibody (#7074) and Anti-mouse IgG, HRP-linked Antibody (#7076) were purchased from Cell Signaling Biotechnology (Danvers, MA).
• RNA FISH RNA FISH.
• RNA-FISH experiments were conducted according to established protocols (Raj et al., Nat. Methods 5:877-9 (2008); Shaffer et al., PLoS One S:e75120-9 (2013)).
• Cells were plated on coverslips coated with poly-L-lysine and allowed to attach for at least 1 hour. The media was then removed, the cells were washed once with IX PBS, and then fixed and permeabilized in ice cold 95% methanol/5% acetic acid at 4°C for 10 minutes.
• RNA FISH probes Stellaris
• Hybridization buffer 50% Stellaris RNA FISH Hybridization Buffer, Biosearch Technologies, SMF-HB1-10; 10% Deionized Formamide
• Co-immunofluorescence with RNA FISH (Co-IF/FISH).
• Co-IF/FISH experiments were conducted in a similar fashion to the dual RNA-FISH experiment with the following modifications. After adhering the cells to coverslips, the cells were fixed with 4% PF A (VWR, BT140770) in RNase-free PBS for 10 minutes at room temperature. After washing the cells 3X for 5 minutes with PBS, the cells were permeabilized with ice cold 95% methanol/5% acetic acid at 4°C for 10 minutes.
• RNA FISH Microarray-based gene expression profiling after RROL depletion. Microarray-based analysis of gene expression changes after treatment with ASO1 was performed as previously described (Morelli et al., Blood 132: 1050-1063 (2016)).
• RNA-seq analysis of AMO1 DR-KO after RROL depletion Total RNA was extracted as described above and submitted to NovaSeq RNAseq analysis followed by VIPER NGS Analysis pipeline (Cornwell et al., BMC Bioinformatics 79: 135-14 (2016)). List of differentially expressed genes (DEGs) were applied to the GSEA or IPA software to reveal biological pathways modulated by RROL.
• DEGs differentially expressed genes
• Luciferase reporter assay Promoter reporter clones for human ACC1 (NM_198834), AN06 (NM_001025356), CCDC91 (NM_018318), EPT1 (NM_033505), EXT1 (NM_000127), FER (NM_001308028) and ZYGI 1 A (NM_001004339) were cloned into GLuc-ONTM Promoter Reporter Vector (GeneCopoeia, Rockville, MD). Luciferase reporter assay was performed according to manufacturer’s instructions.
• ChIRP ChIRP.
• RROL and LacZ antisense DNA probes were designed using the online probe designer at singlemoleculefish.com. Oligonucleotides were biotinylated at the 3' end with an 18-carbon spacer arm. AM01 cells were collected and subjected to ChIRP using the EZ- Magna ChIRP RNA Interactome Kit (Millipore Sigma, Bedford, MA), according to manufacturer’s instructions and established protocols (Chu et al., J. Vis. Exp. 67:3912-6 (2012)).
• Lipid profiling Lipids were extracted from MM cells, dried, and stored under argon until analysis. Lipid species were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer (6500+ QTRAP system; AB SCIEX) by Lipometrix, at KU Leuven, Belgium.
• LC-ESI/MS/MS liquid chromatography electrospray ionization tandem mass spectrometry
• Nexera X2 UHPLC system Shiadzu
• hybrid triple quadrupole/linear ion trap mass spectrometer 6500+ QTRAP system; AB SCIEX
• Lipid extraction lipid extraction was performed with 1 N HC1:CH 3 OH 1 :8 (v/v), 900 ⁇ l CHCl 3 and 200 pg/ml of the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT; Sigma Aldrich).
• BHT 2,6-di-tert-butyl-4-methylphenol
• a mixture of deuterium labeled lipids SPLASH® LIPIDOMIX® Mass Spec Standard #330707, Avanti Polar Lipids was spiked into the extract mix.
• the organic fraction was evaporated using a Savant Speedvac spd111v (Thermo Fisher Scientific) at room temperature and the remaining lipid pellet was stored at - 20°C under argon.
• Sphingomyelins, ceramides, dihydroceramides, hexosylceramides and lactosylceramides were measured in positive ion mode with a precursor scan of 184.1, 264.4, 266.4, 264.4 and 264.4 respectively.
• Triacylglycerides and diacylglycerides were measured in positive ion mode with a neutral loss scan for one of the fatty acyl moieties.
• Lipid species signals were corrected for isotopic contributions (calculated with Python Molmass 2019.1.1) and were quantified based on internal standard signals and adheres to the guidelines of the Lipidomics Standards Initiative (LSI) (level 2 type quantification as defined by the LSI).
• LSI Lipidomics Standards Initiative
• ChlP-qPCR was performed as previously described (Fulciniti et al., Cell Rep. 25:3693-3705 (2016)). Briefly, 1 ⁇ 10 7 cells (AM01, H929 and U266 MYC+ , with corresponding treatments) were cross-linked with 1% formaldehyde for 10 minutes at 37°C. The cross-linked chromatin was then extracted, diluted with lysis buffer, and sheared by sonication. The chromatin was divided into equal samples for immunoprecipitation with specific antibodies. The immunoprecipitates were pelleted by centrifugation and incubated at 68°C to reverse the protein-DNA cross-linking.
• the DNA was extracted from the elute by the Qiaquick PCR purification kit (QIAGEN).
• Antibodies used were as follows: endogenous MYC (Cell Signaling Technology, #13987), MYC-DDK (Santa Cruz Biotechnology, 9E10- x), GFP (Abeam, #ab290), H3K4me3 (#ab8580), Normal Rabbit IgG (Cell Signaling Technology, #2729), Normal Mouse IgG (Santa Cruz Biotechnology, sc-2025).
• a parallel sample of input DNA from the same cells was used as control. ChIP and input DNA were analyzed using SYBR Green real-time PCR analysis (Applied Biosystems).
• ACC1 Fw TTTCTCTCTTGCAGAGTGAGGTGTGG (SEQ ID NO: 72) and ACC1 Rv: TACAAAGGCACGGAGAGAGCAAGT (SEQ ID NO: 73).
• RNA-Protein Pull-Down RROL transcripts were cloned into a pBlueScript vector and sequence verified. In vitro transcription and biotynilation was performed using AmpliScribeTM T7-FlashTM Biotin-RNA Transcription Kit (Lucigen, cat. no. #ASB71110), according to manufacturer’s instructions. Cell nuclear lysates (from 1 ⁇ 10 7 AM01 cells) were incubated with biotinylated RNA and streptavidin beads for RNA pull-down incubation, using PierceTM Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, cat. no. #20164), according to manufacturer’s instructions. RNA-associated proteins were eluted and analyzed by western blotting.
• RNA Yeast 3 Hybrid Saccharomyces cerevisiae strain YLW3 was transformed with RNA plasmids, using standard protocols. They were tested for viability by spotting on SC plates depleted of uracil (SC-U). Protein plasmids were transformed into the yeast strain Y8800 and grown in SC-plates depleted of tryptophan (SC-W). Yeast strains YLW3 containing the examined RNA plasmid were mated with the Y8800 yeast strains containing the protein plasmid. Mating was performed according to the manufacturer’s protocol in YPD media.
• Diploids carrying both plasmids were selected in SC media depleted of tryptophan and uracil (SD-WU), and dimerization was tested by growth in (SC-WUH) media, also depleted of histidine.
• SC-WUH tryptophan and uracil
• RNA immunoprecipitation (RIP) experiments were performed using the Magna RIP RNA-binding Protein Immunoprecipitation Kit (Millipore Sigma, cat. no. 17- 701), according to manufacturer’s instructions.
• the anti-MYC antibody [Y69] used for RIP was purchased from Abeam (ab32072). Normal Rabbit IgG was purchased from Cell Signaling Technology (cat. no. #2729).
• the primers used for detecting RROL are listed above.
• Co-IP Co-immunoprecipitation
• Protein lysates were obtained from 1 ⁇ 10 7 cells (AM01, H929 and U266 MYC+ , with corresponding treatments). Coimmunoprecipitation was performed using PierceTM Co-Immunoprecipitation Kit (Thermo Fisher Scientific, cat. no. 26149), according to manufacturer’s instructions.
• IP antibodies used were as follows: anti- MYC antibody [Y69] was purchased from Abeam (ab32072), Anti-FLAG® M2 antibody was purchased from Millipore Sigma (F3165), Normal Rabbit IgG was purchased from Cell Signaling Technology (2729).
• BioID Proximity-dependent biotin identification
• BioID was performed as described by Kalkat et al., Mol. Cell 72:836-848 (2016). Briefly, FBA-MYC cells were grown to 60% confluence into T75 flasks prior to transfection with ASO1 (50nM, using Lipofectamine 2000 as described above) and treatment with 1 mg/mL doxycycline (Millipore Sigma), 1 ⁇ M MG132 (Millipore Sigma) and 50 mM biotin (Bio Basic) for 24 hours. Experiments with FBA-MYC cells, exposed to doxycycline, included 16 biological replicates (8 with RROL depletion and 8 without RROL depletion).
• Negative controls used for the analysis included 6 biological replicates of FBA-MYC cells not exposed to doxycycline. Cells were harvested by scraping and washed three times with 50mL of PBS prior to flash freezing. Cell pellets were lysed in 1 mL of modified RIPA buffer (1% NP-40, 50 mM Tris- HC1 pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1 : 100 protease inhibitor cocktail (Thermo Fisher Scientific), 0.5% sodium deoxycholate), with 250U of benzonase (Millipore).
• modified RIPA buffer 1% NP-40, 50 mM Tris- HC1 pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1 : 100 protease inhibitor cocktail (Thermo Fisher Scientific), 0.5% sodium deoxycholate), with 250U of benzonase (
• Lysate was rotated for 1 h at 4°C, sonicated 3x30 s, then centrifuged at 27000 g for 30 min at 4°C.
• Biotinylated proteins were isolated by affinity purification with 30 mg of washed streptavidin-Sepharose beads (GE) with rotation for 2 h at 4°C. Beads were then washed 7x1 mL 50 mM ammonium bicarbonate (pH 8.0) prior to tryptic digestion.
• Mass Spectrometry Analysis of Co-IP and BioID samples was performed at the Taplin Mass Spectrometry Facility (Harvard Medical School, Boston, MA), according to established protocols.
• AMO1 DR ' KO xenograft model AMO DR ' KO were gymnotically exposed to ASO1 (2,5uM) or ASO-NC (2,5uM) for 2 days before subcutaneous injection into SCID NOD mice. The day of injection (day 0), cell viability was assessed by Annexin V / 7-AAD flow cytometry assay, confirming no detectable pro-apoptotic activity of ASO-1 at this time point (not shown).
• tumor cells injection cells were resuspended in PBS1X supplemented with ASO1 (5uM) or ASO-NC (5uM); and then mixed with equivalent volume of Matrigel (Corning, #354230) reaching a final oligo concentration of 2,5uM. 5 ⁇ 10 6 cells were subcutaneously injected per mice (5 mice per group). Tumor sizes were measured by electronic caliper.
• AM01 xenograft model 5 ⁇ 10 6 AM01 cells were subcutaneously injected in NOD SCID mice. As tumor became palpable ( ⁇ 50mm), mice were randomized to receive G2-15b*- TO or SB9-19-TO or vehicle (-) as control (3 groups, 5 mice/group). Treatments were administered via I.P. injection, every other day per 2 weeks, at 10mg/kg. Tumor sizes were measured by electronic caliper. In an independent experiment used for qRT-PCR analysis of RROL and ACC1, mice were enrolled to receive treatment after tumors reached the volume of ⁇ 200mm and treated at day 1-3-5. Tumors were then collected at day 6.
• MOLP8-luc+ xenograft model 1X10 6 MOLP8-1UC+ cells were injected via tail vein in 28 NSG mice. 3 mice (marked by a X) were then excluded for failed injection. The day after, 11 mice were assigned to the control group, 8 mice for treatment with G2-19b*-TO and 6 mice for treatment with SB9-19-TO. Treatments were administered via I.P. injection, every other day per 2 weeks, at 10mg/kg. At the end of the treatment cycle (day 15), BLI was measured as indication of tumor growth.
• %TGI Tumor growth inhibition
• Example 2 A genome-wide CRISPRi viability screen identifies MIR17HG as a leading dependency in MM
• RNA-seq data from 360 newly diagnosed MM patients was analyzed and 913 IncRNA transcripts were identified as expressed in primary MM cells, as illustrated in FIG. 1 A left panel labeled I, and in a panel of 70 MM cell lines.
• 3 MM cell lines H929, KMS-11 and KMS-12- BM
• TSS 913 transcription start sites
• 576 negative control sgRNAs see FIG. 1A middle panel labeled II.
• MAGeCK Genome-wide CRISPR-Cas9 Knockout
• RRA rank aggregation
• sgRNAs were further tested in secondary screens using a pooled library targeting 224 IncRNA TSS, the TSS of known protein coding oncogenes (MYC, IRF4) (Chesi et al., Cancer Cell 73: 167-80 (2008); Shaffer et al., Nature 757:226-31 (2008)) or tumor suppressors (TP53) (Jovanovic et al., Front. Oncol. 5:665-7 (2019)) as positive controls, and 2245 non-targeting sgRNAs as negative control, as illustrated in FIG. 1 A, right panel labeled III.
• MYC protein coding oncogenes
• IRF4 tumor suppressors
• TP53 tumor suppressors
• MM cell lines H929, KMS11, KMS12BM and AM01
• sgRNAs targeting IRF4 and MYC were significantly depleted in three (MYC) or all (IRF4) cell lines
• sgRNAs targeting TP53 were significantly enriched in both TP53 wild-type cell lines (AM01 and H929) ( Tessoulin et al., J. Hematol. Oncol. 77: 137 (2018)).
• FIG. IB highlights the top IncRNA dependency MIR17HG, along with protein coding genes IRF4 and MYC that were used as positive controls.
• FIG. 1C illustrates the CCK-8 proliferation assay of the MM cell lines AM01, H929, KMS11 and KMS12BM which stably expressed the KRAB-dCAS9 fusion protein and transduced with lentivectors to conditionally express anti-MIR17HG sgRNAs.
• a CCK-8 assay was performed at indicated time points after exposure to doxycycline (0.5 pg/mL). Cell proliferation was calculated compared to parental cells infected with the empty sgRNA vector and exposed to doxycycline under some conditions. Focusing on depleted sgRNAs, IncRNA dependencies in MM cells that were either cell type specific (54%) or shared by two or more cell lines (46%) (see, FIG. 8A) were identified.
• MIR17HG as the leading IncRNA dependency in the screen, with RRA scores equal or superior to those obtained by targeting MYC or IRF4 in all cell lines tested (FIG.8B).
• MM cell lines expressing dCAS9-KRAB fusion protein were transduced with the top four sgRNAs targeting MIR17HG under the regulation of a tetracycline-inducible promoter and observed reduced cell growth compared to cells infected with non-targeting sgRNAs after continued exposure to doxycycline, as illustrated in FIG. 1C and FIG. 8B.
• MIR17HG expression was investigated by qRT-PCR five days after induction of gRNAs with doxycycline. The results shown in FIG. 8B as the average RNA expression levels after normalization with ACTB and ⁇ Ct calculations.
• LNA gapmeR ASOs targeting the MIR17HG nascent RNA (pre-RNA) for RNase H-mediated degradation (Lai et al., Mol. Cell 77: 1032-1043 (2020); Lee and Mendell, 2020) were used to transfect 11 MM cell lines including those resistant to conventional anti -MM agents (AMO1-ABZB resistant to bortezomib; AMO1-ACFZ resistant to carfilzomib; MM.1R resistant to dexamethasone); and confirmed significant impact on MM cell viability independent of the genetic and molecular background, as illustrated in FIG. ID and FIG. 8C.
• LNA locked nucleic acid
• FIG. ID and FIG. IE Data present mean ⁇ s.d.in FIG. ID and FIG. IE. */? ⁇ 0.05 by Student’s t test.
• FIG. 8C shows MIR17HG expression by qRT-PCR 24h after transfection. The results shown are an of average RNA expression levels after normalization with ACTB and ⁇ Ct calculations. One (1) of three independent experiments is shown.
• Example 3 MIR17HG RROL (RROL) mediates dependency in microRNA-independent manner.
• MIR17HG miR ' 17 ' 92 miR-17/-18a/-19a/-20a/-19b/-92al
• MIR17HG also produces as yet poorly characterized IncRNA transcript lnc-17-92 TV1 (also known as MIR17HG RROL ) (He et al., Nature 735:828-33 (2005); Ota et al., Cancer Res. 67:3087-95 (2004)), as illustrated in FIG. 2A.
• RROL expression was higher during disease progression in 2 independent datasets from MM patients enrolled in the IFM/DFCI clinical trial (NCT01191060) analyzed at diagnosis and/or relapse (see, FIG. 9A - FIG. 9B); and that higher expression of RROL was associated with shorter event-free (EFS) and overall (OS) survival in 3 large cohorts of newly diagnosed MM patients (FIG. 2B).
• EFS event-free
• OS overall survival in 3 large cohorts of newly diagnosed MM patients
• FIG. 9D shows cells infected with a lentiviral vector containing pri-mir-17-92 (Lenti-pri-mir) or a lentiviral vector containing GFP.
• the results shown are average miRNA expression levels after normalization with RNU44 and ⁇ Ct calculations.
• ASOs were designed to target MIR17HG at the 5’-end (5’-ASOs), a sequence not included in the ectopic pri-mir-17-92.
• two DROSHA knockout (DR-KO) MM cell lines (AMO1 DR ' KO and H929 DR ' KO ) were established, which are unable to produce miR-17-92s (FIG. 9E).
• RNA-seq (FIG. 2G) and qRT-PCR (FIG. 2H) indicated preferential expression of RROL TV1, which is the isoform further investigated and hereafter referred to as RROL (also known as Inc- 17-92).
• RROL also known as Inc- 17-92
• ASO1 abrogated the ability of AMO1 DR ' KO to establish tumors into NOD SCID mice, as detected by tumor growth of AMO1 DR ' KO with (ASO-1) or without (NC) RROL depletion (FIG. 2E) and prolonged animal survival (FIG. 2F).
• the easy- to-transfect colorectal cancer cell line HCT-116 which is driven by MIR17HG 34 , was used to find that ectopic expression of lnc-17-92 TV1 significantly rescued the anti-proliferative activity of ASOs targeting MIR17HG pre-RNA more effectively than ectopic lnc-17-92TV2 or pri-mir-17-92 (FIG.
• Ectopic expression of pri-mir-17-92, lnc-17-92TVl, and lnc-17-92TV2 was confirmed by qRT-PCR showing: i) upregulation of miR-17 after ectopic expression of pri-mir-17-92; ii) upregulation of lnc-17-92TVl after its ectopic expression; iii) upregulation of lnc-17-92TV2 after its ectopic expression.
• Example 4 RROL interacts with chromatin to regulate gene expression.
• IncRNAs The functional role of IncRNAs depends on their subcellular localization Ulitsky et al., Cell 15 :26-46 (2013).
• MALAT1 and GAPDH were used as internal controls for nuclear- and cytosolic- enriched RNAs, respectively.
• the results are average fold enrichment of nuclear vs cytosolic fraction after 2-ACt calculations. This finding was confirmed by RNA FISH, as illustrated in FIG. 10B. On this basis, next the transcriptional network regulated by RROL in MM was explored.
• FIG. 10C shows average RROL or miRNA expression levels after normalization with GAPDH or RNU44, respectively, and ⁇ Ct calculations.
• RROL was also depleted in DR-WT (AM01 and H929) and DR-KO (AM01DR-K0) MM cell lines using early exposure to gymnotic ASO1 to avoid modulation of RROL’ s canonical targets in DROSHA WT cells (FIG. 13B and FIG. 13C).
• FIG. 10E are the average mRNA expression levels after normalization with murine gapdh and ⁇ Ct calculations. Conversely, the expression of these genes was not affected by modulation of individual members of miR- 17-92 by synthetic mimics or inhibitors (FIG. 10F - FIG. 10G). The results shown are average miRNA or mRNA expression levels after normalization with RNU44 or GAPDH, respectively, and ⁇ Ct calculations. Moreover, significant positive correlation (Spearman r > 0.3; p ⁇ 0.001) between RROL and its target genes in at least 1 out of 2 large RNA-seq MM patient datasets (IFM/DFCI and MMRF/CoMMpass) were observed, as illustrated in FIG. 3C; supporting clinical relevance of these regulatory axis.
• FIG. 10E are the average mRNA expression levels after normalization with murine gapdh and ⁇ Ct calculations. Conversely, the expression of these genes was not affected by modulation of individual members of miR- 17-92 by synthetic mimics or inhibitors (FI
• a luciferase reporter assay performed in 293T DR ' KO cells in presence or absence of RROL depletion, demonstrates the regulatory control of RROL over these genes, except AN06, occurs at the promoter level (FIG. 3D).
• the reporter vectors were co-transfected into 293T cells with either ASO1 or control ASO. Cells were harvested for luciferase activity assay 48h after transfection. Results are shown as % of normalized Glue activity in ASO1 transfected cells compared to control.
• RNAi-based LOF screen in 5 MM cell lines identified ACC1 (Acetyl-CoA Carboxylase Alpha) as the RROL target gene with most significant impact on MM cell proliferation and survival (FIG. 3E). These results provided the rationale to further explore the role of RROL-ACC1 axis in MM pathobiology. As shown in FIG. 3E, 2 siRNAs were used for each target, plus a scramble siRNA (NC) as control. Cell viability was measured at the indicated time points. It is represented as % of NC transfected cells.
• NC scramble siRNA
• RROL interaction at the promoter region of the top target ACC1 was confirmed by chromatin isolation by RNA precipitation (ChIRP) assay followed by qRT-PCR analysis (FIG. 3F and FIG. 10H - FIG. 101).
• ChIRP RNA precipitation
• Example 5 RROL promotes MYC occupancy at the ACC1 promoter.
• ACC1 expression levels in cells exposed to NC were set as an internal reference.
• FISH/IF immunofluorescence analysis of MYC protein
• RNA immunoprecipitation (RIP) assay was performed with MYC antibody, which showed a specific enrichment of RROL isoform 2 (RROL-2) in the MYC-bound RNA, as illustrated in FIG. 4E and FIG. 1 IB - FIG. 11C.
• LncRNA PVT1 was used as positive control for the known role as MYC interactor (FIG. 4E).
• RPPD RNA-Protein pull-down
• RNA yeast-3 -hybrid (Y3H) assay was adapted to confirm the RROL-MYC interaction in an in vivo cellular model (Hook et al., RNA 77:227-33 (2005)).
• a direct RROL-MYC interaction activates a reporter gene allowing for yeast colony growth; and, as shown in FIG. 4G, we detected yeast colony growth in the presence of RROL-2 as hybrid RNA.
• Example 6 RROL mediates the assembly of a MYC-WDR82 transcriptional complex, leading to transcriptional and epigenetic activation of ACC1.
• FIG. 1 ID shows ACC1 mRNA following treatment with MYC inhibitor 10058-F4 in MM cell lines AM01 and H929.
• Raw Ct values were normalized to GAPDH mRNA and expressed as ⁇ Ct values calculated using the comparative cross threshold method.
• ACC1 expression levels in cells treated with DMSO (NC) were set as an internal reference. Therefore, the functional interplay between MYC and MIR17HG was investigated.
• MYC activity has been shown to be modulated through the interaction with transcriptional and epigenetic co-regulators (Gouw et al.. Cell Metab. 30:556-572 (2019)).
• BioID proximity-dependent biotin identification
• Co-IP/MS mass-spectrometry analysis
• RNA-protein interaction between RROL and WDR82 was further confirmed by both RPPD (FIG. 5B) and RNA Y3H (FIG. 5C) assays. Black arrows in FIG. 5C indicate yeast colony growth.
• An analysis using truncated versions of lnc-17-92TVl further indicated the 3’-end regions, which do not include miR-17-92, as particularly relevant for the interaction with MYC in MM cells (FIG. 11G).
• WDR82 is a regulatory component of the SET1 methyltransferase complex catalyzing the histone H3 ‘Lys-4’ trimethylation (H3K4me3) at the transcription start sites of active loci (Lee and Skalnik, Mol. Cell. Biol. 25:609-18 (2008)), a sine qua non condition for MYC binding to chromatin and transactivation Amente et al., Am. J. Cancer Res. 7:413-418 (2011). Consistently, depletion of WDR82 resulted in a global reduction of H3K4me3 (FIG. 5D and FIG. 12D) and a reduced occupancy of MYC at ACC1 promoter (FIG.
• FIG. 5E shows a western blot analysis of WDR82 and H3K4me3 (represented as % of input chromatin). Lamin A/C was used as protein loading controls (nuclear lysates).
• FIG. 5E shows a western blot analysis of WDR82 and MYC from paired samples (represented as % of input chromatin), a-tubulin was used as protein loading controls.
• Data in FIG. 5F are shown as raw Ct values that were normalized to GAPDH mRNA and expressed as ⁇ Ct values calculated using the comparative cross threshold method. ACC1 expression levels in ells transfected with NC were set as an internal reference.
• FIG. 5G illustrates a ChlP-qPCR analysis of WDR82-GFP occupancy at ACC1 promoter in AM01 exposed for 24h to gymnotic ASO1 as a western blot analysis of WDR82-GFP from paired samples (represented as % of input chromatin), a-tubulin was used as protein loading controls. Furthermore, using MM cells expressing an ectopic WDR82-GFP fusion protein (FIG. 12B), it was demonstrated that RROL expression is essential for WDR82 occupancy at the ACC1 promoter (FIG. 5H), without globally impacting the H3K4 methylation status (FIG. 51).
• FIG. 12B MM cells expressing an ectopic WDR82-GFP fusion protein
• 5H illustrates a ChlP-qPCR analysis of H3K4me3 occupancy at ACC1 promoter in AM01 and H929 exposed for 24h to gymnotic ASO1 as a shown a western blot analysis of H3K4me3 from paired samples (represented as % of input chromatin).
• Lamin A/C was used as protein loading controls (nuclear lysates). *p ⁇ 0.05, Student t test.
• RROL depletion resulted in reduced levels of H3K4me3 at the ACC1 promoter site, without detectable impact on the total H3K4me3 (FIG. 51).
• Example 7 The RROL/MYC-ACC1 axis regulates de novo lipogenesis.
• ACC1 catalyzes the carboxylation of acetyl-CoA into malonyl-CoA, the rate limiting step during de novo lipogenesis (DNL) (Beloribi-Djefaflia et al., Oncogenesis 5:el89-10 (2016)), a metabolic pathway aberrantly activated in cancer cells (Rschreibig and Schulze., Nat. Rev. Cancer 76:732-749 (2016)).
• RROL depletion similarly to either MYC or ACC1 inhibition, significantly reduced the incorporation of C 14 -radiolabeled glucose into the lipid pool - indicative of a reduced DNL (Zadra et al., Proc. Natl. Acad. Sci.
• NS indicates p > 0.05 after Student t test.
• Liquid Chromatography - Mass Spectrometry (LC-MS) based lipid profiling after RROL inhibition in MM cells confirmed depletion of several saturated (SFA) and monounsaturated (MUFA) phospholipid species (FIG. 6B) produced via DNL.
• SFA saturated
• MUFA monounsaturated
• lipid profiling analysis showing modulation of major membrane phospholipid classes [phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholin (PC) and phosphatidylinositol (PI)], with saturated (SFA) or monounsaturated (MUFA) acyl chains, after treatment of AM01 and H929 with ASO1 is shown in FIG. 6B. (Rysman et al., Cancer Res. 70:8117-26 (2010); Zaidi et al., Prog. Lipid Res. 52:585-9 (2013)).
• PG phosphatidylglycerol
• PE phosphatidylethanolamine
• PS phosphatidylserine
• PC phosphatidylcholin
• PI phosphatidylinositol
• FIG. 6D shows an annexin V / 7-AAD flow cytometry assay in AM01 exposed for 6 days to ASO1 at indicated concentrations, with supplementation of either BSA (control) or BSA-PA (20 ⁇ M).
• Example 8 Therapeutic inhibitors of RROL exert potent anti -tumor or activity in vitro and in vivo in animal models of human MM,
• More than 80 fully phosphorothioated (PS), 2’-O-methoxyethyl (2’-M0E)- modified, lipid-conjugated ASOs were screened to explore the therapeutic potential of RROL and to develop inhibitors. These ASOs were screened for their potential to either trigger RNase H-mediated degradation of RROL (gapmeRs) or exert function via an RNase Id- independent mechanism (steric blockers) (Puttaraju et al., Nat. Med. 27:526-535 (2021)) (FIG. 7A and FIG. 14A - FIG. 14C).
• FIG. 7B shows the MM cell lines AM01, ABZB, ACFZ, H929, MM.
• FIG. 14A - FIG. 14C The testing results of the multi-step screen to develop therapeutic ASOs targeting RROL is shown in FIG. 14A - FIG. 14C.
• the results of step 1 are shown in FIG. 14A, which identified ASO-accessible stretches on RROL, 16 sequences (>20-mer) either in “G” or “SB” configuration for a total of 32 ASOs were tested; based on KD activity in AM01 cells assessed by qRT-PCR, the sequences G2 (21-mer) and SB9 (22-mer) were selected for further investigation.
• the results of step 2 are shown in FIG.
• step 3 shows the testing of the two selected step 2 molecules conjugated with palmitic acid (P) or cholesterol (C) or tocopherol (T). Based on KD activity in AM01 cells assessed by qRT-PCR.
• the TO-conjugated molecules were selected as the leading compounds.
• the G2-15-T ASO was further optimized by replacing the 10-mer “core” DNA gap with an 8-mer “core” DNA gap (G2-15b-T).
• a subcutaneous AM01 xenograft model in immunocompromised NOD SCID mice was used to assess the in vivo anti -turn or activity of both compounds.
• Analysis of tumors retrieved from mice following this treatment confirmed reduced expression of RROL (FIG. 7D) and its targets (FIG. 7E), target BIM (aka /A7.2/./7) (FIG.
• Raw Ct values were normalized to ACTB mRNA and expressed as ⁇ Ct values calculated using the comparative cross threshold method. Expression levels in NC were set as an internal reference.
• FIG. 7G a scatter plot showing the analysis of bioluminescence intensity. Black bars indicate median value. Bioluminescence was measured at the end of treatment cycle (day 15). On the right of FIG. 7G, image acquisition is shown. Mice removed from the study due to failed I V. injection of tumor cells are covered by a black rectangle. Importantly, both inhibitors significantly prolonged the animal survival (FIG. 7H).
• RROL is a leading dependency in MM.
• RROL host gene, MIR17HG is often amplified and/or overexpressed in human cancer with driver role.
• RROL is a regulator of gene expression via chromatin occupancy and interaction with transcription factors and epigenetic modulators, such as MYC and WDR82.
• DNL has emerged as an essential pathway for the onset and progression of MYC-driven cancers, that are susceptible to pharmacologic inhibition of ACC1 (Gouw et al., Cell Metab 30:556-572 (2019)).
• the roles of ACC1 and DNL in tumorigenesis seem particularly relevant in MM, where tumor cells need to adapt their metabolic pathways to meet the high bioenergetic and biosynthetic demand posed by the malignant cell growth coupled with unceasing production of monoclonal immunoglobulin (El Arfani et al., Int J Mol Sci 19: 1200-19 (2016); Masarwi et al., JBMR Plus 3:el0173-10 (2019)).
• the in vivo working example utilized two of the inventive ASOs that target RROL via different mechanisms of action (i.e., RNase H-dependent or -independent).
• RNase H-dependent or -independent RNA medicine
• the use of ASOs to therapeutically antagonize disease-driver genes is becoming increasing possible (Dhuri et al., J Clin Med 9:2004-24 (2020); Puttaraju et al., Nat Med 27:526-535 (2021)), including in MM therapy (Mondala et al., Cell Stem Cell 28:623-636 (2021); Morelli et al., Blood 132: 1050-1063 (2016); (Mondala et al., 2021; Morelli et al., 2018
• this working example establishes RROL as a IncRNA that facilitates MYC-WDR82 protein complex formation and its chromatin binding, impacting lipid metabolism and ultimately tumor cell growth.

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• 2022
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