WO2024220373A1 - Assays for monitoring inhibition of rna helicase dhx9 - Google Patents

Assays for monitoring inhibition of rna helicase dhx9 Download PDF

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Publication number
WO2024220373A1
WO2024220373A1 PCT/US2024/024695 US2024024695W WO2024220373A1 WO 2024220373 A1 WO2024220373 A1 WO 2024220373A1 US 2024024695 W US2024024695 W US 2024024695W WO 2024220373 A1 WO2024220373 A1 WO 2024220373A1
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dhx9
cancer
alu
circrna
inhibitor
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PCT/US2024/024695
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French (fr)
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WO2024220373A8 (en
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Jennifer Castro
Scott RIBICH
David Patrick BRENNAN
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Accent Therapeutics, Inc.
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Publication of WO2024220373A1 publication Critical patent/WO2024220373A1/en
Publication of WO2024220373A8 publication Critical patent/WO2024220373A8/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • RNA helicases are an interesting group of proteins serving as potential translational targets in cancer. These proteins belong to superfamily 2, the largest group of eukaryotic RNA helicases, are named after a conserved amino sequence (Asp-Glu-Ala- Asp/His) and have the ability to unwind and restructure RNA molecules with complex secondary structures in an ATPase dependent fashion.
  • DHX9 also known as RNA Helicase A (RHA) or Nuclear DNA Helicase II (NDH II), is a DEAH-box RNA helicase. Due to its regulatory role in processes such as transcription and maintenance of genomic stability, DHX9 has been shown to be a key regulator in a variety of cancer types (Gulliver et al., 2020, Future Science OA (2), FSO650).
  • the present disclosure provides a method of measuring activity of a DHX9 inhibitor in a cell, comprising: contacting a cell with the DHX9 inhibitor; and measuring the level of an Alu-mediated circular RNA (Alu-circRNA) in the cell.
  • the cell is a cancer cell.
  • the cell is a normal cell. In some embodiments, the cell is a non-cancerous cell. In some embodiments, the cell is a peripheral blood mononuclear cell (PBMC). In some embodiments, the contacting is for a sufficient time for the Alu-circRNA to be produced. In some embodiments, the level of Alu-circRNA is correlated to inhibition of a DHX9 activity by the DHX9 inhibitor in the cell. In another aspect, the present disclosure provides a method of measuring activity of a DHX9 inhibitor in a subject, comprising: administering a DHX9 inhibitor to a subject; and measuring the level of an Alu-mediated circular RNA (Alu-circRNA) in a biological sample from the subject.
  • Alu-circRNA Alu-mediated circular RNA
  • the present disclosure provides a method of measuring activity of a DHX9 inhibitor in a subject, comprising: measuring the level of Alu-mediated circular RNA (Alu-circRNA) in a biological sample from a subject, wherein the subject is being treated with a DHX9 inhibitor.
  • the level of Alu-circRNA is correlated to inhibition of DHX9 activity by the DHX9 inhibitor in the subject.
  • the level of Alu- circRNA is correlated to inhibition of DHX9 ATPase activity or resolvase activity.
  • the level of Alu-circRNA is correlated to inhibition of proliferation of MSI cancer cells in the subject.
  • the biological sample is selected from the group consisting of blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue.
  • the biological sample is blood.
  • the method further comprises isolating cells from the blood sample, and measuring the level of the Alu-circRNA in the isolated cells.
  • the biological sample comprises peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the method further comprises isolating PBMCs from a blood sample, and measuring the level of the Alu-circRNA in the PBMCs.
  • the level of Alu-circRNA does not correlate with inhibition of proliferation of PBMCs of the subject.
  • the Alu-circRNA is selected from the group consisting of cirRNA of BRIP1, AKR1A1, BMPR2, CALCOCO2, CLNSIA, DKC1, FAM124A, FBN1, FCHO2, GLS, GON4L, GPR125, NEIL3, PIK3C3, PNN, POMT1, RBM23, SKIL, SLK, SMA, SMG1, SMYD4, TAF2, TFAP4, TMEM41B, USP25, VPS13D, WDR59, and XPR1.
  • the level of the Alu-circRNA is measured by a technique selected from northernblot, reverse transcription-quantitative polymerase chain reaction (RT- qPCR), microarray, droplet digital PCR, next-generation sequence (NGS) RNA sequencing, RNAin situ hypridization (RNA-ISH), in situ hypridization- immunohistochemistry (ISH- IHC), isothermal exponential amplification, and rolling cycle amplification.
  • the method further comprises obtaining the biological sample from the subject.
  • the biological sample is obtained from the subject between 0.5-7 days following administration of the DHX9 inhibitor.
  • the method comprises measuring the level of the Alu-circRNA in biological samples obtained from the subject at more than one time point.
  • the subject has a cancer.
  • the cancer is a microsatellite instability (MSI) cancer.
  • the cancer is selected from the group consisting of colorectal, endometrial, ovarian, gastric, hematopoietic, breast, brain, skin, lung, blood, prostate, head and neck, pancreatic, bladder, bone, soft tissue, kidney, liver, and Ewing’s sarcoma.
  • the method further comprises making a determination on an appropriate therapeutic dose of the DHX9 inhibitor based on a set of data including the level of Alu-circRNA measured.
  • the DHX9 inhibitor is selected from a small molecule, an antisense oligonucleotide, small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA).
  • the DHX9 inhibitor is a small molecule.
  • the DHX9 inhibitor is selected from the inhibitors in Table 1.
  • the present disclosure provides a method of monitoring treatment of a subject with a DHX9 inhibitor, comprising: administering a DHX9 inhibitor to the subject; and obtaining information as to the level of Alu-mediated circular RNA (Alu- circRNA) in a biological sample obtained from the subject after administration of the DHX9 inhibitor.
  • Alu-circRNA Alu-mediated circular RNA
  • the level of Alu-circRNA is correlated to inhibition of DHX9 activity by the DHX9 inhibitor in the subject.
  • the level of Alu- circRNA is correlated to inhibition of DHX9 ATPase activity or resolvase activity.
  • the level of Alu-circRNA is correlated to inhibition of proliferation of MSI cancer cells in the subject.
  • the biological sample is selected from the group consisting of blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue.
  • the biological sample is blood.
  • the method further comprises isolating cells from the blood sample, and measuring the level of the Alu-circRNA in the isolated cells.
  • the biological sample comprises peripheral blood mononuclear cells (PBMCs).
  • the method further comprises isolating PBMCs from a blood sample, and measuring the level of the Alu-circRNA in the PBMCs.
  • the level of Alu-circRNA does not correlate with inhibition of proliferation of PBMCs of the subject.
  • the Alu-circRNA is selected from the group consisting of cirRNA of BRIP1, AKR1A1, BMPR2, CALCOCO2, CLNSIA, DKC1, FAM124A, FBN1, FCHO2, GLS, GON4L, GPR125, NEIL3, PIK3C3, PNN, POMT1, RBM23, SKIL, SLK, SMA, SMG1, SMYD4, TAF2, TFAP4, TMEM41B, USP25, VPS13D, WDR59, and XPR1.
  • the level of the Alu-circRNA is measured by a technique selected from northernblot, reverse transcription-quantitative polymerase chain reaction (RT- qPCR), microarray, droplet digital PCR, next-generation sequence (NGS) RNA sequencing, RNAin situ hypridization (RNA-ISH), in situ hypridization- immunohistochemistry (ISH- IHC), isothermal exponential amplification, and rolling cycle amplification.
  • the method further comprises obtaining the biological sample from the subject.
  • the biological sample is obtained from the subject between 0.5-7 days following administration of the DHX9 inhibitor.
  • the method comprises measuring the level of the Alu-circRNA in biological samples obtained from the subject at more than one time point.
  • the subject has a cancer.
  • the cancer is a microsatellite instability (MSI) cancer.
  • the cancer is selected from the group consisting of colorectal, endometrial, ovarian, gastric, hematopoietic, breast, brain, skin, lung, blood, prostate, head and neck, pancreatic, bladder, bone, soft tissue, kidney, liver, and Ewing’s sarcoma.
  • the method further comprises making a determination on an appropriate therapeutic dose of the DHX9 inhibitor based on a set of data including the level of Alu-circRNA measured.
  • the DHX9 inhibitor is selected from a small molecule, an antisense oligonucleotide, small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA).
  • the DHX9 inhibitor is a small molecule.
  • the DHX9 inhibitor is selected from the inhibitors in Table 1.
  • the subject is a human.
  • FIG. 1 Cells were treated with DHX9 compound 7 for 3 days and probed for multiple Alu-mediated circRNAs, a non-Alu mediated circRNA, as well as each gene’s respective linear RNA form.
  • Alu-mediated circRNAs, BRIP1, AKR1A1 and DKC1 were regulated and induced.
  • FIGs. 2A-2B show that cellular target engagement by DHX9 inhibitors correlates with DHX9 biochemical activity and anti-proliferative activity of DHX9 inhibitors in certain MSI cancers.
  • FIG. 1 Cells were treated with DHX9 compound 7 for 3 days and probed for multiple Alu-mediated circRNAs, a non-Alu mediated circRNA, as well as each gene’s respective linear RNA form.
  • the Alu-mediated circRNAs, BRIP1, AKR1A1 and DKC1 were regulated and induced.
  • FIGs. 2A-2B show that cellular target engagement by DHX9 inhibitor
  • FIG. 2A shows DHX9 ATPase biochemical activity for each DHX9 inhibitor, reported as inflection points (IP) or EC50, plotted against its corresponding circBRIP1 EC50, which is EC50 of circBRIP1 induced in HCT116 CRC-MSI cells measured by cellular DHX9 target engagement assays.
  • FIG. 2B shows anti-proliferative activity of each DHX9 inhibitor in LS411N CRC-MSI cells, measured as IC50, plotted against its corresponding circBRIP1 EC50, measured by cellular DHX9 target engagement assays.
  • FIGs. 3A-3D DHX9 compounds (1 to 4) were dosed in vivo as described in Example 3.
  • FIGs. 4A-4C Mice with human xenograft tumors were dosed with DHX9 inhibitor Compound 1 at 30, 100, 200 and 300 mg/kg orally with a 12 hour schedule as described in Example 4. After 21 days of treatment and 12 hours post last dose of compound tumor volume was recorded, tumors were then harvested and processed for circBRIP1 expression.
  • FIG. 4A shows dose-dependent induction of circBRIP1 in LS411N tumors.
  • FIG. 4B shows individual tumor volumes plotted against each tumor’s corresponding circBRIP1 pharmacodynamic reading in a correlation plot.
  • FIG. 4C shows individual tumor drug exposure of Compound 1 plotted against each tumor’s corresponding circBRIP1 pharmacodynamic reading and fit to four parameter non-linear regression.
  • FIGs. 5A-5B DHX9 compounds were dosed by oral gavage (PO) in vivo as described in Example 5. Whole blood samples were taken at the end of the study 1 hr post last dose, PBMC were isolated and analyzed for mouse linear BRIP1 and/or mouse circBRIP1. Dose dependent induction of circBRIP1 was observed and where applicable, no change in linear BRIP1 was seen.
  • DHX9 compounds were dosed oral gavage (PO) in vivo as described in Example 5.
  • Whole blood samples were taken at the end of the study 24 hr post last dose, PBMC were isolated and analyzed for mouse linear BRIP1 and/or mouse circBRIP1. Dose dependent induction of circBRIP1 was observed , while no linear BRIP1 above baseline was seen.
  • FIG. 7A Colorector cancer cell lines that were either sensitive to DHX9 inhibitor (IC 50 ⁇ 1 ⁇ M or insensitive to DHX9 inhibitor (IC 50 > 1 ⁇ M) were treated with with escalating doses of from 0.001 to 10 ⁇ M Compound 1, as described in Example 6.
  • circBRIP1 EC 50 values were relative to vehicle treated cell.
  • FIG. 7B Several breast, ovarian, lung and colorectal (CRC) cancer cell lines were treated with Compound 1 as described in Example 6. IC50 values for inhibition of proliferative activity by Compound 1 in the cell lines showed no correlation with EC50 values of circBRIP1 induction.
  • FIG. 8A circBRIP1 level was elevated in previously-frozen isolated human PBMCs after treatment with Compound 1 for 24 hours.
  • FIG. 8B Treatment of PBMCs with Compound 1 at concentration of 0.01 ⁇ M to 10 ⁇ M for 10 days did not impact their lif i ( i i i l) FIGs.
  • any and all examples, or exemplary language (e.g. “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed.
  • the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, 0.05%, or 0.01% of the stated value.
  • all numerical values provided herein can be modified by the term about.
  • Alu element refers to a short stretch of DNA (about 300 nucleotides in length), originally characterized by the action of the Alu restriction d l d l ifi d h t i t d l l t (SINE ) Al l t the most abundant transposable elements, and can be divided into five subfamilies of related elements based upon key diagnostic nucleotide positions shared by subfamily members.
  • an Alu element is composed of two non-identical units or arms joined in the middle by an adenosine (A)-rich linker.
  • each Alu repeat also ends with an A-rich tail and is flanked by short direct repeat sequences.
  • biomarker used interchangeably with the term “marker”, is understood to mean a measurable characteristic that reflects in a quantitative or qualitative manner the physiological state of a cell or an organism.
  • biomarkers are characteristics that can be objectively measured and evaluated as indicators of normal processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
  • biomarkers include, for example, polypeptides, peptides, polypeptide fragments, proteins, antibodies, hormones, polynucleotides, RNA or RNA fragments, microRNA (miRNAs), circular RNA (circRNAs), lipids, polysaccharides, and other bodily metabolites.
  • a biomarker is a circRNA, e.g., an Alu-mediated circRNA
  • a biomarker of the present invention is modulated (e.g., increased or decreased level) in a cell or a biological sample from a subject or a group of subjects after having received a treatment as compared to a biological sample from a subject or group of subjects that have not received a treatment (e.g., a control).
  • a biomarker of the present invention is modulated (e.g., increased or decreased level) in a biological sample from a subject or a group of subjects after having received a treatment as compared to a biological sample from the subject or group of subjects before receiving the treatment (e.g., a control).
  • a biomarker may be differentially present at any level, but is generally present at a level that is increased relative to normal or control levels by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, b t l t 100% b t l t 110% b t l t 120% b t l t 130% b t l t 140% b at least 150%, or more; or is generally present at a level that is decreased relative to normal or control levels by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at
  • a biomarker is preferably differentially present at a level that is statistically significant (e.g., a p-value less than 0.05 and/or a q-value of less than 0.10 as determined using either Welch's T-test or Wilcoxon's rank-sum Test).
  • the term “biopsy” or “biopsy tissue” refers to a sample of tissue (e.g., prostate tissue) that is removed from a subject for the purpose of determining if the sample contains cancerous tissue. The biopsy tissue is then examined (e.g., by microscopy) for the presence or absence of cancer.
  • the term “cancer” has the meaning normally accepted in the art. The term can broadly refer to abnormal cell growth.
  • CircRNA refers to noncoding RNA characterized by a covalently closed cyclic structure lacking poly-adenylated tails.
  • CircRNAs are mainly synthesized by the transcription of protein coding genes with RNA polymerase II (Pol II); but unlike linear RNAs, they are not produced by canonical mode of RNA splicing.
  • CircRNA molecules are circularized by joining the 3′ and 5′ ends together with unique back-splicing (see, e.g., Wilusz, Repetitive elements regulate circular RNA biogenesis, Mob Genet Elements, 2015, 5(3):39-45; incorporated herein by reference).
  • CircRNAs are commonly named according to their parental genes or specific functions, e.g., circular RNA derived from the BRIP1 gene is called “circBRIP1.”
  • Several circRNA databases have been constructed to enable organization of discovered and identified circRNAs. A serial number is given to every detected back-spliced junction site. Databases like circBase (circbase.org) and CircNet (circnet. mbc.nctu.edu.tw) provide tissue-specific circRNA expression profiles as well as circRNA-miRNA-gene regulatory networks.
  • Circ2Traits (gyanxet-beta.com/circdb) also allows user to search circRNAs by mutiple diseases. See Zhang et al.
  • Alu-medited circular RNA refers t i RNA h f ti i i t d ith Al l t Al l t i h d i the introns flanking human exons that generate circRNAs; it is estimated that ⁇ 90% of circular RNAs appear to have complementary Alu elements in their flanking introns.
  • RNAs Disruption of base pairing between intronic repeats by mutating several nucleotides in Alu elements have been shown to prevent circularization of certain RNAs (see, e.g., Wilusz, Repetitive elements regulate circular RNA biogenesis, Mob Genet Elements, 2015, 5(3):39- 45; the entitre contents of which are incorporated herein by reference).
  • the formation of the Alu-circRNAs of the instant disclosure are modulated by DHX9.
  • the term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand.
  • an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil.
  • base pairing specific hydrogen bonds
  • a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • control sample refers to any relevant comparative sample, including, for example, a sample from a subject prior to treatment or from an earlier assessment time point, or a cell or a population of cells untreated with any agents.
  • a control sample can be a purified sample and/or nucleic acid (e.g., circRNA) provided with a kit.
  • a control sample can be a sample derived from a subject.
  • a control sample can also be synthetically produced.
  • the level of one or more markers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more markers) in a control sample consists of a group of measurements that may be determined, e.g., based on any appropriate statistical measurement, such as, for example, measures of central tendency including average, median, or modal values. Different from a control is preferably statistically significantly different from a control.
  • control level refers to an accepted or pre-determined level of a marker in a sample.
  • a control level can be a range of values. Marker levels can be compared to a single control value, to a range of control values, to the upper level of normal, or to the lower level of normal as appropriate for the assay.
  • control level is a standardized control level, such as, for example, a level which is predetermined using an average of the levels of expression of one or more markers from a population of cells or subjects prior to receiving a treatment, e.g., prior to administration of a DHX9 inhibitor.
  • control level is a level determined from a sample of a subject collected before the subject receiving a treatment, e.g., prior to administration of a DHX9 inhibitor.
  • control level is a level determined from a sample of a subject collected at an earlier time point.
  • detecting As used herein, “detecting”, “detection”, “determining”, and the like are understood to refer to an assay performed for identification of the presence and/or level of a circRNA (e.g., circBRIP1) and/or an additional one or more specific markers in a sample.
  • the amount of marker detected in the sample can be none or below the level of detection of the assay or method.
  • DHX9 RNA Helicase A (RHA) or Nuclear DNA Helicase II (NDH II) refers to a DEAH-box RNA helicase which shuttles between nucleus and cytoplasm, and can use all four NTPs to power cycles of directional movement from 3’ to 5’.
  • DHX9 can bind to and unwind or resolve dsDNA/RNA, ssDNA/RNA, DNA:RNA hybrids (such as R-loops), circular RNA, and DNA/RNA G quadruplexes.
  • DHX9 has regulatory roles in various RNA and DNA related cellular processes, such as transcription, translation, RNA splicing, editing, RNA transport and processing, microRNA genesis, and maintenance of genomic stability (Pan et al., 2021, Current Protein & Peptide Science (22), 29-40).
  • the NCBI Gene ID for DHX9 is 1660. Human DHX9 nucleotide and amino acid sequences can be found at GenBank Accession No.
  • DHX9 possesses ATPase activity, i.e., DHX9 has an ATPase domain that hydrolyzes ATP i t ADP It h tl b h th t DHX9 l l ti it which represses transposases (see Aktarez et al., DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome, Nature, 2017, 544:112-119; incorporated herein by reference).
  • DNA or "RNA” molecule or sequence (as well as sometimes the term “oligonucleotide”) refers to a molecule comprised generally of the deoxyribonucleotides or ribonucleotides, respectively, of adenine (A), guanine (G), thymine (T) and/or cytosine (C), wherein in “RNA”, T is replaced by uracil (U).
  • adenine A
  • G guanine
  • T thymine
  • C cytosine
  • U uracil
  • disorder disorder
  • disease and “abnormal state” are used inclusively and refer to any deviation from the normal structure or function of any part, organ, or system of the body (or any combination thereof).
  • a specific disease is manifested by characteristic symptoms and signs, including biological, chemical, and physical changes, and is often associated with a variety of other factors including, but not limited to, demographic, environmental, employment, genetic, and medically historical factors. Certain characteristic signs, symptoms, and related factors can be quantitated through a variety of methods to yield important diagnostic information.
  • a sample obtained at an “earlier time point” is a sample that was obtained at a sufficient time in the past such that relevant information could be obtained in the sample from the earlier time point as compared to the later time point.
  • an earlier time point is at least two weeks earlier, at least four weeks earlier, at least six weeks earlier, at least two months earlier, at least three months earlier, at least six months earlier, at least nine months earlier, or at least one year earlier.
  • Multiple subject samples e.g., 3, 4, 5, 6, 7, or more
  • Appropriate intervals for testing for a particular subject can be determined by one of skill in the art based on ordinary considerations.
  • expression is used herein to mean the process by which an RNA or polypeptide is produced from DNA.
  • the process involves the transcription of the gene into RNA if the marker of interest is an RNA, and further involves the translation of an mRNA into a polypeptide if the marker of interest a polypeptide or processing of the initial transcribed RNA into other subtypes of RNA, such as circRNA.
  • expression may refer to the production of RNA, or protein, or both.
  • expression level is used herein to mean the level (e.g., amount) of an RNA l tid t i ll bi l i l l A, “higher level”, “higher level of expression”, “higher level of induction” and the like of a marker refers to the marker’s level in a sample that is greater than the standard error of the assay employed to assess the level, and is preferably at least 25% more, at least 50% more, at least 75% more, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten times the level of the marker in a control sample, and preferably the average level of the marker or markers in several control samples.
  • a subject is “in need of” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment (in some embodiments, a human).
  • the term “inhibit”, “inhibition” or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
  • the term “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
  • level of expression of a gene refers to the level of mRNA, as well as pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s), non-enocoding RNA products (e.g., circRNA) and degradation products, or the level of protein, encoded by the gene in the cell.
  • level of one of more biomarkers means the absolute or relative amount or concentration of the biomarkers in a sample.
  • a “lower level”, “lower level of expression” or “lower level of induction” of a marker refers to the marker’s level in a sample that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the level of the marker in a control sample, and preferably the average level of the marker in several control samples.
  • nucleic acid molecule or “polynucleotides”, refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA, circRNA) and chimeras thereof.
  • the nucleic acid molecule can be obtained by cloning techniques or synthesized.
  • DNA can be double-stranded or single- stranded (coding strand or non-coding strand [antisense]).
  • RNA ribonucleic acid
  • DNA i l d d i th t
  • l i id ribonucleic acid
  • a nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide- nucleic acid bonds (referred to as "peptide nucleic acids” (PNA); Hydig-Hielsen et al., PCT Intl Pub. No.
  • sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy substitutions (containing a 2'-O-methylribofuranosyl moiety; see PCT No. WO 98/02582) and/or 2' halide substitutions.
  • Nitrogenous bases may be conventional bases (A, G, C, T, U), known analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992), or known derivatives of purine or pyrimidine bases (see, Cook, PCT Int'l Pub. No. WO 93/13121) or "abasic" residues in which the backbone includes no nitrogenous base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481).
  • a nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs).
  • the term “obtaining” is understood herein as manufacturing, purchasing, or otherwise coming into possession of a physical entity or a value, e.g., a numerical value.
  • “Obtaining a sample,” as the term is used herein, refers to obtaining possession of a sample, e.g., a tissue sample or cell sample, by “directly acquiring” or “indirectly acquiring” the sample.
  • “Directly acquiring a sample” means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample.
  • Indirectly acquiring a sample refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample).
  • oligonucleotides or “oligos” define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill.
  • An oligonucleotide can be synthesized chemically or derived by cloning according to well-known methods. While they are usually in a single-stranded form, th b i d bl t d d f d t i " l t i " Th contain natural rare or synthetic nucleotides. They can be designed to enhance a chosen criteria like stability for example.
  • Chimeras of deoxyribonucleotides and ribonucleotides may also be within the scope of the present invention.
  • a “patient,” “subject” or “individual” are used interchangeably and refer to either a human or non-human animal.
  • the term includes mammals such as humans. Typically, the animal is a mammal.
  • a subject also refers to for example, non-human primates (e.g., monkey, male or female), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like.
  • the subject is a primate.
  • the subject is a human.
  • peripheral blood mononuclear cells refers to cells isolated from peripheral blood and identified as any blood cell with a round nucleus (i.e., lymphocytes, monocytes, natural killer cells (NK cells) or dendritic cells).
  • PBMCs can be isolated from whole blood as follows, or through equivalent methods: The cell fraction corresponding to red blood cells and granulocytes (neutrophils, basophils and eosinophils) is removed from whole blood by density gradient centrifugation.
  • PBMCs makes up the population of cells that remain in the low density fraction (upper fraction), whilst red blood cells and PMNs have a higher density and are found in the lower fraction.
  • PBMCs include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. In humans, the frequencies of these populations vary across individuals, but typically, lymphocytes are in the range of 70–90 %, monocytes from 10 to 20 %, while dendritic cells are rare, accounting for only 1–2 %. See Kleiveland et al. (Peripheral Blood Mononuclear Cells.
  • a “predetermined threshold value” or “threshold value” of a biomarker refers to the level of the biomarker (e.g., the expression level or quantity in a biological sample) in a corresponding control sample or group of control samples (e.g., an average level or mean level of the biomarker in the group of control samples).
  • the control sample may be from the same subject at a previous time or from different subjects.
  • prophylactic or “therapeutic” treatment refers to administration to the subject of one or more agents or interventions to provide the desired clinical effect. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing at least one sign or symptom of the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or maintain at least one sign or symptom of the existing unwanted condition or side effects therefrom).
  • the unwanted condition e.g., disease or other unwanted state of the host animal
  • a "reference level" of a biomarker means a level of the biomarker that is indicative of therapeutic efficacy of a treatment, e.g., with a DHX9 inhibitor, or lack thereof.
  • a "positive" reference level of a biomarker means a level that is indicative of a treatment, e.g., with a DHX9 inhibitor, having one or more therapeutic effects in a subject.
  • a “negative” reference level of a biomarker means a level that is indicative of a lack of therapeutic effects from a treatment, e.g., with a DHX9 inhibitor.
  • sample or “biological sample” includes a specimen or culture obtained from any source.
  • Biological samples can be obtained from blood (including any blood product, such as whole blood, plasma, serum, or specific types of cells of the blood), interstitial fluid, lymph fluid, urine, saliva, and the like. Biological samples also include tissue samples, such as biopsy tissues or pathological tissues that have previously been fixed (e.g., formaline snap frozen, cytological processing, etc.). In an embodiment, the biological sample is from blood. In another embodiment, the biological sample is a biopsy tissue from a tumor.
  • the term “therapeutic effect” refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance.
  • a therapeutic effect can be understood as a decrease in tumor growth, decrease in tumor growth rate, stabilization or decrease in tumor burden, stabilization or reduction in tumor size, stabilization or decrease in tumor malignancy, increase in tumor apoptosis, and/or a decrease in tumor angiogenesis.
  • “therapeutically effective amount” means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease, e.g., the amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment, e.g., is sufficient to ameliorate at least one sign or symptom of the disease, e.g., to prevent progression of the disease or condition, e.g., prevent tumor growth, decrease tumor size, induce tumor cell apoptosis, reduce tumor angiogenesis, prevent metastasis.
  • the amount is sufficient to avoid or delay onset of the disease.
  • the “therapeutically effective amount” will vary depending on the compound, its therapeutic index, solubility, the disease and its severity and the age, weight, etc., of the patient to be treated, and the like.
  • certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
  • Administration of a therapeutically effective amount of a compound may require the administration of more than one dose of the compound.
  • the term “treat”, “treating” or “treatment” of any disease, condition or disorder refers to the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of the present disclosure to obtaining desired pharmacological and/or physiological effect.
  • the effect can be therapeutic, which includes achieving, partially or substantially, one or more of the following results: partially or totally reducing the extent of the disease, condition or disorder; ameliorating or improving a clinical symptom, complications or indicator associated with the disease, condition or disorder; or delaying, inhibiting or decreasing the likelihood of the progression of the disease, condition or disorder; or eliminating the disease, condition or disorder.
  • the effect can be to prevent the onset of the symptoms or complications of the disease, condition or disorder.
  • CircRNAs are present in small amounts in biological samples, and thought to be not abundant enough to allow for their use as meaningful biomarkers.
  • the methods and assays described herein are based, in part, on the suprising findings that levels of Alu-mediated circular RNAs are detectable in biological samples and that their changes correlate with DHX9 inhibition in vitro and in vivo.
  • assays and methods for detecting, measuring, and monitoring activity of a DHX9 inhibitor in vitro and/or in vivo are provided herein.
  • the present disclosure provides methods for detecting inhibition of DHX9 in a cell, or a population of cells, comprising contacting the cell or population of cells with a DHX9 inhibitor, and detecting or measuring the level of an Alu-mediated circular RNA (Alu-circRNA) in the cell or population of cells.
  • the cells are maintained in cell culture in vitro.
  • the cell is from a primary cell line.
  • the cell is from an established or immortalized cell line.
  • the cell is an ex vivo cell isolated from a subject.
  • the cell is isolated from a biological sample from a subject, e.g., blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue. In some embodiments, the cell is contacted with the DHX9 in vivo. In some embodiments, the cell is a normal, non-cancerous cell. In some embodiments, the cell is a blood cell. In preferred embodiments, the cell is a peripheral blood mononuclear cell (PBMC). In preferred embodiments, the population of cells comprises or consists of PBMCs. In some embodiments, the cell or population of cells are a cancer cell.
  • PBMC peripheral blood mononuclear cell
  • the cancer is selected from the group consisting of, but not limited to, colorectal, endometrial, ovarian, gastric, hematopoietic, breast, brain, skin, lung, blood, prostate, head and neck, pancreatic, bladder, bone, soft tissue, kidney, liver, and Ewing’s sarcoma.
  • the cancer cell is derived from a cancer that has microsatellite instability (MSI).
  • MSI microsatellite instability
  • the cancer cell is characterized to have genomic instability.
  • the cancer cell is defective in mismatch repair.
  • cancer cell having genomic instability is selected for use in the methods and f th di l
  • the cancer cell is isolated from a tumor from a subject.
  • the cancer cell is isolated from the blood of the a subject having a hematopoeitic cancer.
  • the cells are contacted with one or more amounts or concentrations of a DHX9 inhibitor, and the levels of the Alu-circRNA is measured or determined for each amount or concentration of the inhibitor.
  • the cells are contacted with a DHX9 inhibitor for a period of time sufficient for the inhibitor to inhibit DHX9 expression.
  • the cells are contacted with a DHX9 inhibitor for a period of time sufficient for the inhibitor to inhibit DHX9 activity.
  • the cell or populations of cells is contacted with a DHX9 inhibitor for a period of time sufficient for the Alu-circRNA to be produced, e.g., to detectable levels.
  • the level of the Alu-circRNA is measured or determined between 1 to 24 hours, between 2 to 24 hours, between 4 to 24 hours, between 6 to 24 hours, between 8 to 24 hours, between 10 to 24 hours, between 12 to 24 hours, between 14 to 24 hours, between 16 to 24 hours, between 18 to 24 hours, between 20 to 24 hours, between 22 to 24 hours, between 1 to 20 hours, between 2 to 20 hours, between 4 to 20 hours, between 6 to 20 hours, between 8 to 20 hours, between 10 to 20 hours, between 12 to 20 hours, between 14 to 20 hours, between 16 to 20 hours, between 18 to 20 hours, between 1 to 16 hours, between 2 to 16 hours, between 4 to 16 hours, between 6 to 16 hours, between 8 to 16 hours, between 10 to 16 hours, between 12 to 16 hours, between 14 to 16 hours, between 1 to 12 hours, between 14 to 16 hours, between
  • the level of the Alu-circRNA is measured or determined at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, 102 hours, 108 hours, 114 hours or 120 hours after the cell is contacted with the DHX9 inhibitor.
  • the level of the Alu-circRNA is measured or determined at least 1 day, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, t l t 4 d t l t 45 d t l t 5 d t l t 55 d t l t 6 d t l t 65 days, or at least 7 days after the cell is contacted with the DHX9 inhibitor.
  • the level of the Alu-circRNA is measured or determined about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, or about 7 days after the cell is contacted with the DHX9 inhibitor.
  • the cells are contacted with a DHX9 inhibitor, and the levels of the Alu-circRNA is measured or determined at multiple different times after the cells are first contacted with the inhibitor.
  • the methods further comprise measuring or determining the level of the Alu-circRNA in cells that have not been contacted, or prior to contact, with the DHX9 inhibitor, and this level of the Alu-circRNA is used to establish a control level.
  • the present disclosure generally provides methods for detecting or measuring inhibition of DHX9 in a subject.
  • the present disclosure further generally provides methods for detecting engagement of DHX9 by a DHX9 inhibitor in a subject.
  • the methods comprise detecting or measuring the level of an Alu-circRNA in a biological sample obtained from subject. The subject is scheduled to be administered, is undergoing treatment with, or has recently been administered a DHX9 inhibitor.
  • the sample is obtained from the subject following administration of a DHX9 inhibitor.
  • the method further comprises administering a DHX9 inhibitor to the subject.
  • the present disclosure provides a method for measuring or detecting activity of a DHX9 inhibitor in a subject, comprising administering a DHX9 inhibitor to the subject, and measuring or detecting the level of an Alu-circRNA in a biological sample from the subject.
  • the present disclosure also provides a method for measuring or detecting engagement of DHX9 by a DHX9 inhibitor in a subject, comprising administering a DHX9 inhibitor to the subject, and measuring or detecting the level of an Alu-circRNA in a biological sample from the subject.
  • the present disclosure also provides a method for measuring or detecting activity of a DHX9 inhibitor in a subject, comprising measuring or detecting the level of an Alu-circRNA in a biological sample from the subject, wherein the subject is being treated with (i.e., has been administered) the DHX9 inhibitor.
  • the method further comprises the step of administering the DHX9 inhibitor to a subject.
  • the present disclosure also provides a method for measuring or detecting engagement of DHX9 by a DHX9 inhibitor in a bj t i i i d t ti th l l f Al i RNA i bi l i l sample from the subject, wherein the subject is being treated with (i.e., has been administered) the DHX9 inhibitor.
  • the method further comprises the step of administering the DHX9 inhibitor to a subject.
  • the present disclosure also provides a method for monitoring the treatment of a subject with a DHX9 inhibitor, comprising obtaining information as to the level of Alu- circRNA in a biological sample obtained from the subject after administration of the DHX9 inhibitor.
  • the method further comprises the step of administering the DHX9 inhibitor to the subject.
  • the subject is a mammal, such as a human, a monkey, a cow, a sheep, a goat, a horse, a dog, a cat, a rabbit, a rat, or a mice.
  • the subject is a human.
  • the subject is a healthy, normal subject.
  • the subject has a cancer.
  • the subject is an animal model of a cancer, e.g., a mouse having a human xenograft tumor.
  • the cancer is a microsatellite instability (MSI) cancer.
  • the cancer is microsatellite-instability high (MSI-H). In some embodiments, the cancer is defective in mismatch repair. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, endometrial cancer, ovarian cancer, gastric cancer, hematopoietic cancer, breast cancer, brain cancer, skin cancer, lung cancer, blood cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, bone cancer, soft tissue cancer, kidney cancer, liver cancer, and Ewing’s sarcoma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is colorectal cancer with microsattelite instability (MSI-CRC).
  • MSI-CRC microsattelite instability
  • the biological sample is selected from the group consisting of blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue.
  • the biological sample is blood.
  • the biological sample is cells isolated from blood.
  • the biological sample is peripheral blood mononuclear cells (PBMC).
  • the biological sample comprises peripheral blood mononuclear cells (PBMC).
  • the biological sample consists of peripheral blood mononuclear cells (PBMC).
  • the methods further comprise obtaining the biological sample f th bj t I b di t th th d f th i i l ti t i ll population from the biological sample.
  • the methods may further comprise isolating PBMCs from a blood sample.
  • the cell population can be isolated from the biological sample based on physical properties (e.g., size, density, buoyancy), or based on specific biomarkers.
  • the methods comprise measuring or determining the level of the Alu-circRNA in a whole blood sample.
  • the methods comprise measuring or determining the level of the Alu-circRNA in a certain cell population of the biological sample, e.g., PBMCs in a blood sample.
  • the methods comprise measuring the level of the Alu-circRNA in PBMCs.
  • the DHX9 inhibitor is administered more than once to the subject, i.e., multiple doses of the DHX9 inhibitor are administered to the subject.
  • the biological sample is obtained from the subject following administration of the DHX9 inhibitor.
  • the biological sample is obtained after administration of multiple doses of the DHX9 inhibitor.
  • the level of the Alu-cirRNA is measured in biological samples obtained from the subject at more than one time point.
  • the biological sample is obtained following each administration of the DHX9 inhibitor.
  • biological samples are obtained from the subject at multiple time points after administration of a dose of the DHX9 inhibitor.
  • the biological sample is obtained between 1 to 24 hours, between 2 to 24 hours, between 4 to 24 hours, between 6 to 24 hours, between 8 to 24 hours, between 10 to 24 hours, between 12 to 24 hours, between 14 to 24 hours, between 16 to 24 hours, between 18 to 24 hours, between 20 to 24 hours, between 22 to 24 hours, between 1 to 20 hours, between 2 to 20 hours, between 4 to 20 hours, between 6 to 20 hours, between 8 to 20 hours, between 10 to 20 hours, between 12 to 20 hours, between 14 to 20 hours, between 16 to 20 hours, between 18 to 20 hours, between 1 to 16 hours, between 2 to 16 hours, between 4 to 16 hours, between 6 to 16 hours, between 8 to 16 hours, between 10 to 16 hours, between 12 to 16 hours, between 14 to 16 hours, between 1 to 12 hours, between 2 to 12 hours, between 4 to 12 hours, between 6 to 12 hours, between 8 to 12 hours, between 10 to 12, 1 to 8 hours, between 2 to 8 hours, between 4 to 8 hours, or between 6 to 8 hours after administration of the DHX9
  • the level of the Alu-circRNA is measured or determined at least 0.5 days, at least 1 day, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days , at least 6.5 days, or at least 7 days after administration of the DHX9 inhibitor.
  • the biological sample is obtained about 0.5 days, about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, or about 7 days after administration of the DHX9 inhibitor.
  • the methods further comprise obtaining a biological sample from the subject prior to administration of the DHX9 inhibitor, and the level of the Alu- circRNA in this sample is used to establish a control level.
  • the DHX9 inhibitor is administered to the subject orally, subcutaneously, or intravenously. It will be appreciated that the measurements of the level of the Alu-circRNA obtained from the methods and assays of the disclosure are useful for evaluating the activity or efficacy of a DHX9 inhibitor in vitro and in vivo.
  • the level of the Alu-circRNA determined using the methods of the present disclosure is correlated (e.g., directly correlated) to inhibition of DHX9 activity by the DHX9 inhibitor in the cell or in the subject.
  • the level of the Alu-circRNA is correlated (e.g., directly correlated) to inhibition of the ATPase activity of DHX9.
  • the level of the Alu-circRNA is correlated (e.g., directly correlated) to inhibition of the resolvase activity of DHX9.
  • the level of the Alu-circRNA is correlated to the inhibition of proliferation of a cancer cell, for example an MSI cancer cell, by the DHX9 inhibitor.
  • the level of the Alu-circRNA is correlated to the inhibition of growth of an MSI cancer in a subject by the DHX9 inhibitor. In some embodiments, the level of the Alu-circRNA is not correlated to the inhibition of growth of PBMCs in a subject by the DHX9 inhibitor.
  • the cell can be an isolated cell, a cell in vitro, a cell ex vivo, or a cell in vivo in a subject.
  • the level of Alu-circRNA determined by the methods and assays of the instant disclosure is useful to determine if a DHX9 inhibitor (e.g., a candidate inhibitor) is engaging the DHX9 target, and/or if the DHX9 inhibitor is effective at inhibiting DHX9 activity, in a cell or in a subject.
  • a DHX9 inhibitor e.g., a candidate inhibitor
  • the Alu-circRNA level determined for a cell or cell population after treatment with a candidate DHX9 inhibitor is compared to the Alu-circRNA level determined for a control cell or cell population not treated with a DHX9 inhibitor (e.g., a control level).
  • the candidate DHX9 inhibitor is determined to engage the DHX9 target, and/or to inhibit DHX9 activity, in the cells.
  • the Alu-circRNA level determined for a biological sample obtained from a subject after treatment with a candidate DHX9 inhibitor is compared to the Alu-circRNA level determined for a control sample from a subject not treated with a DHX9 inhibitor (e.g., a control level).
  • the candidate DHX9 inhibitor is determined to engage the DHX9 target, and/or to inhibit DHX9 activity, in the subject. It will be further appreciated that Alu-circRNA levels can be determined and compared among different candidate DHX9 inhibitors to identify a candidate inhibitor that is more or less effective at engaging the DHX9 target and/or inhibiting DHX9 activity in vitro or in vivo.
  • an inhibitor that induces a higher Alu-circRNA level may be considered to be more effective at engaging the DHX9 target and/or inhibiting DHX9 activity than an inhibitor that induces a lower Alu-circRNA level, and an inhibitor that induces a lower Alu-circRNA level may be considered to be less effective at engaging the DHX9 target and/or inhibiting DHX9 activity than an an inhibitor that induces a higher Alu-circRNA level.
  • reference levels can be established for an Alu-cirRNA marker, and levels of the Alu-circRNA induced by a DHX9 inhibitor can be compared to the reference levels to make a determination of DHX9 engagement and/or efficacy of the DHX9 inhibitor.
  • the level of Alu-circRNA determined using the methods and assays of the instant disclosure is useful in determining an appropriate dose, e.g., a th ti ll ff ti d i i ll ff ti d f th DHX9 i hibit Th l l of Alu-circRNA determined using the methods and assays of the instant disclosure is also useful in determining an effective therapeutic treatment regimen for the DHX9 inhibitor.
  • DHX9 Inhibitors The methods provided in the present disclosure are applicable to any DHX9 inhibitors, and are not limited to the exemplary DHX9 inhibitors described herein.
  • the DHX9 inhibitors are previously known in the art, and have been shown to reduce or inhibit the expression or activity of DHX9 in vitro or in vivo.
  • the DHX9 inhibitors are candidate DHX9 inhibitors under discovery and testing for their ability to reduce or inhibit expression or activity of DHX9 in vitro or in vivo.
  • the DHX9 inhibitor is selected from a small molecule, an antisense oligonucleotide, or an RNA interfering agent such as small intering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA).
  • the DHX9 inhibitor is a small molecule.
  • a small molecule inhibitor is typically an organic compound with low molecular weight that has high cell penetration.
  • the small molecule inhibitor of DHX9 is any DHX9 small molecule inhibitor found in the art. Exemplary DHX9 small molecule inhibitors and methods of synthesis thereof can be found at least in international PCT applications PCT/US2023/013303 and PCT/US2023/012929, the entire contents of each of which are incorpoated herein by reference.
  • the DHX9 inhibitor is selected from the compounds listed in Table 1. Table 1. Exemplary DHX9 small molecule inhibitors
  • DMSO dimethylsulfoxide
  • HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate
  • HPLC high pressure liquid chromatography
  • LCMS liquid chromatography mass spectrometry
  • TFA Trifluoroacetic acid
  • KOAc potassium acetate
  • Pd(dppf)Cl 2 [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)
  • K2CO3 potassium carbonate
  • LiOH lithium hydroxide
  • TCFH N′-tetramethylformamidinium hexafluorophosphate
  • HCl hydrochloric acid
  • NMI N-methylimidazole
  • ACN acet
  • LCMS measurement was run on SHIMADZU LCMS-2020 or Agilent 1200 HPLC/6100 SQ System using the follow conditions: Method A: Mobile Phase: A: Water (0.05%TFA) B: Acetonitrile (0.05%TFA); Gradient Phase: 5%B to 100%B within 2.0 min, 100%B with 0.7 min (total runtime: 2.8 min); Flow Rate: 1.5 mL/min; Column: HALO C18, 3.0*30mm, 2.0 ⁇ m; Column Temperature: 40 oC. Detectors: AD2 ELSD, PDA (220 nm and 254 nm), ESI.
  • Method B Mobile Phase: A: Water (0.1%FA) B: Acetonitrile (0.1%FA); Gradient Phase: 5%B to 100%B within 2.0 min, 100%B with 0.7 min (total runtime: 2.8 min); Flow Rate: 1.5 mL/min; Column: HALO C18, 3.0*30mm, 2.0 ⁇ m; Column Temperature: 40 oC. Detectors: AD2 ELSD, PDA (220 nm and 254 nm), ESI.
  • Method C Mobile Phase: A: Water (5mM NH4HCO3) B: Acetonitrile; Gradient Phase: 10%B to 95%B within 2.0 min, 100%B with 0.6 min (total runtime: 2.8 min); Flow Rate: 1.5 mL/min; Column: Poroshell HPH-C18, 3.0*50mm, 4.0 ⁇ m; Column Temperature: 40 oC. Detectors: AD2 ELSD, PDA (220 nm and 254 nm), ESI.
  • Method D Mobile Phase: A: Water (0.01%TFA) B: Acetonitrile (0.01%TFA); Gradient Phase: 5%B to 95%B within 1.4 min, 95%B with 1.6 min (total runtime: 3 min); Flow Rate: 2.0 mL/min; Column: SunFire C18, 4.6*50mm, 3.5 ⁇ m; Column Temperature: 40 oC. Detectors: ADC ELSD, DAD (214 nm and 254 nm), ES-API. Preparation of Intermediate A The compounds claimed herein were prepared following the procedures outlined in the following schemes. Compound names were generated using the software built into ChemDraw.
  • Step 2 To a stirred solution of N-(3-chloro-5-nitrophenyl) methanesulfonamide (41.00 g, 163.57 mmol, 1.00 equiv) and Fe (91.35 g, 1635.75 mmol, 10.00 equiv), NH 4 Cl (87.50 g, 1635.75 mmol, 10.00 equiv) in 500 mL of ethanol and 300 mL of water, this was stirred for 2h at 90 oC. The resulting mixture was filtered, the filter cake was washed with 6 x 50 mL of methanol. The filtrate was concentrated under reduced pressure.
  • Step 2 A solution of methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophene-2- carboxylate (170 g, 634.02 mmol, 1.00 equiv), Pd(dppf)Cl2 (23.20 g, 31.70 mmol, 0.05 equiv), K 2 CO 3 (262.87 g, 1902.06 mmol, 3.00 equiv) and 2-bromo-3-methylpyridine (109.07 g, 634.02 mmol, 1.00 equiv) in 1000 mL of 1,4-dioxane and 200 mL of water, this was stirred for 2 hours at 90 °C under nitrogen atmosphere.
  • Step 2 To a stirred solution of methyl 5-(3,5-difluoropyridin-2-yl)-1-methyl-1H-pyrrole-3- carboxylate (295 g, 1165 mmol, 1 equiv) in tBuOH (3 L) and water (2 L) was added LiOH (246g, 5853 mmol, 5 equiv, in 1L water). The mixture was stirred for 4 hours at 60 °C. The pH value of the solution was adjusted to 3 with 1M of HCl (aq). The product was precipitated from the solution.
  • Step 3 A mixture of 5-(3,5-difluoropyridin-2-yl)-1-methyl-1H-pyrrole-3-carboxylic acid (212 g, 886 mmol, 1 equiv), TCFH (341 g, 1219 mmol, 1.37 equiv), NMI (199 g, 2438 mmol, 2.75 equiv) and N-(3-amino-5-chlorophenyl)methanesulfonamide (214 g, 487 mmol) in ACN (2 L) was stirred for two hours at room temperature. The mixture was quenched with water (2 L). The resulting mixture was extracted with DCM (3 x 3 L) and water.
  • the DHX9 inhibitor is an antisense oligonucleotide (a single- stranded deoxyribonucleotide) complementary to DHX9 mRNA.
  • the DHX9 inhibitor is an RNA intefering agent.
  • the DHX9 inhibitor is selected from the group consisting of small intering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA).
  • short interfering RNA and “siRNA” are used interchangeably to refer to any RNA molecule shorter than about 50, 40, 35, 30, 25, 20, or fewer nucleotides capable of inducing silencing of a gene target based on complementarity.
  • Silencing is typically mediated by an Argonaute protein and is typically initiated by a short RNA molecule trigger. Silencing can be post-transcriptional or transcriptional.
  • the siRNA molecule may be completely or partially complementary to the gene or genes whose expression in reduced, and silencing may be effected with or without cleavage of an mRNA transcript.
  • shRNA short-hairpin RNA
  • shRNAs may be of cellular (endogenous) or artificial (exogenous) origin.
  • Endogenous siRNA includes “piRNA,” which are any of a class of Piwi-interacting RNAs.
  • microRNA miRNA
  • miRNA miRNA
  • the miRNA can be derived from “pre-microRNA” or “pre-miRNA”. “Pre-microRNA” or “pre-miRNA”are used interchangeably to refer to any molecule, the processing of which releases a mature miRNA. Furthermore, as will be appreciated by those skilled in the art, some miRNA can be characterized in terms of their capacities to affect gene regulation in a wide variety of organisms and in a wide variety of tissues. A single gene may be regulated by multiple miRNAs, and miRNA regulation may act synergistically. A single miRNA may also be capable of regulating multiple mRNAs, for example, some estimates have suggested a single human miRNA may regulate up to 100-200 genes.
  • a DHX9 inhibitor It has also been recently described that up to 30% of human genes are targets for miRNA regulation.
  • Method of identifying or verifying a DHX9 inhibitor are known in the art. For example, examplary methods of identifying or verifying a DHX9 inhibitor are described in the Examples of the present disclosure.
  • a candidate molecule that potentially inhibits DHX9 by inhibiting its expression can be verified by determining whether the candidate molecule inhibits or reduces expression of DHX9 mRNA or polypeptide in a cell.
  • Methods for detecting mRNA levels such as northern blotting, in situ hybridization, ribonuclease protection assay (RPA) and RT-qPCR (see, e.g., Karen Reue, mRNA Quantitation Techniques: Considerations for Experimental Design and Application, The Journal of Nutrition, 1998, 128(11): 2038–2044; incorporated herein by reference), and methods for detecting polypeptide levels such as western blotting, ELISA, immunofluorescence staining, immunohistochemistry, and mass spectrometry, are well known in the art.
  • RPA ribonuclease protection assay
  • RT-qPCR see, e.g., Karen Reue, mRNA Quantitation Techniques: Considerations for Experimental Design and Application, The Journal of Nutrition, 1998, 128(11): 2038–2044; incorporated herein by reference
  • methods for detecting polypeptide levels such as western blotting, ELISA, immunofluorescence staining, immunohis
  • a candidate molecule that potentially inhibits DHX9 e.g., by inhibiting DHX9 function or activity of the polypeptide, can be verified by testing whether the candidate molecule inhibits ATPase activity of DHX9 (see, e.g., Example 2 of the present disclosure).
  • Assays and kits for measuring ATPase activity of an ATP-dependent polypeptide such as DHX9 are known in the art and are commercially available.
  • a candidate molecule that potentially inhibits DHX9 e.g., by inhibiting DHX9 function or activity of the polypeptide
  • Circular RNAs Multiple studies have reported that DHX9 binds to or binds near Alu elements more than other RNA-binding proteins (Aktarez et al., 2017; Stagsted et al. 2019, Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles, Life Science Alliance, 2019, 2(3):1-16), incorporated herein by reference). Studies of DHX9 have also shown that DHX9 acts as a nuclear RNA resolvase, and resolves inverted-repeat Alu elements (IAEs) in order to de-repress translation of mRNAs with inverted-repeat Alu elements in their 3’ UTRs (Aktaw et al., 2017).
  • IAEs inverted-repeat Alu elements
  • DHX9 inhibits circRNA production by unwinding and destabilizing RNA structures formed by inverted-repeat Alu elements (IAEs) in flanking regions of circRNAs (Stagsted et al.).
  • Suitable circRNAs for use in the methods and assays of the present disclosure are those circRNAs whose formation is modulated (e.g., reduced or prevented) or controlled by DHX9.
  • a circRNA may be one whose formation is also modulated by mechanisms and/or molecules independent of DHX9, but is nevertheless useful in the methods and assays of the present disclosure as long as inhibition of DHX9 can induce an appreciable increase in formation of the circRNA.
  • the circRNA useful in the methods and assays of the present disclosure is Alu-mediated circRNA. While not wishing to be bound by theory, it is believed that under normal conditions DHX9 binds to inverted-repeat Alu elements in flanking regions of mRNA and inhibits production of the circRNA. When DHX9 is inhibited (e.g., by using an iRNA t k k d DHX9 i ll l l DHX9 i hibit t i hibit it activity), the inhibition of Alu-mediated circRNA production by DHX9 is reduced and the level of the Alu-mediated circRNA is increased.
  • circRNAs for use in the methods and assays of the present disclosure can be identified using methodologies described herein (see, e.g., the methods described in Example 9 of the present disclosure).
  • circRNAs such as those known or predicted in databases (such as circBase (circbase.org) and CircNet (circnet. mbc.nctu.edu.tw)) or available for testing in commercial or customized arrays (e.g., Arraystar Human Circular RNA Array) can be tested for responsiveness to DHX9 inhibition.
  • CircRNAs up-regulated following DHX9 inhibition are useful in the methods of the present disclosure.
  • the Alu-mediated circRNA is selected from the list of Alu- mediated circRNAs listed in Table 2 below. In some embodiments, more than one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Alu-mediated circRNAs, e.g., selected from those listed in Table 2, are detected or measured in the methods of the present disclosure.
  • Table 2 List of Alu-mediated circRNAs (circRNAs) Ge Sym AKR1 BMPR BRIP1 BRIP1 CALC O2 CLNS DKC1 FAM1 FBN1 FCHO GLS (SEQ ID NO: 35)
  • the circRNA is derived from the BRIP1 gene, and called circBRIP1.
  • BRIP1 also known as “BRCA1 Interacting Protein C- terminal helicase 1”
  • BRCA-1 Breast Cancer gene 1
  • Mutations in the BRIP1 gene are known to increase the risk of ovarian and breast cancers.
  • Human BRIP1 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_032043.3, incorporated herein by reference.
  • the circRNA is derived from the AKR1A1 gene.
  • AKR1A1 also known as aldehyde reductase, is involved in the synthesis of ascorbic acid (AsA) as well as the detoxification of aldehydes.
  • Human AKR1A1 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_006066.4, NM_153326.3, NM_001202413.2, or NM_001202414.2, incorporated herein by reference.
  • the circRNA is derived from the BMPR2 gene.
  • the BMPR2 gene also known as “bone morphogenetic protein receptor type 2”, belongs to a family of genes originally identified for its role in regulating the growth and maturation (differentiation) of bone and cartilage.
  • Human BMPR2 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_001204.7, incorporated herein by reference.
  • the circRNA is derived from the CALCOCO2 gene.
  • CALCOCO2 also known as “calcium Binding And Coiled-Coil Domain 2”, is a receptor for ubiquitin-coated bacteria and plays an important role in innate immunity by mediating macroautophagy.
  • Human CALCOCO2 nucleotide and amino acid sequences can be found at GenBank Accession No.
  • the circRNA is derived from the CLNS1A gene.
  • CLNS1A also known as “chloride nucleotide-sensitive channel 1A” functions in multiple regulatory pathways.
  • the encoded protein complexes with numerous cytosolic proteins and performs diverse functions including regulation of small nuclear ribonucleoprotein biosynthesis, platelet activation and cytoskeletal organization.
  • the protein is also found associated with the plasma membrane where it functions as a chloride current regulator.
  • the circRNA is derived from the DKC1 gene.
  • DKC1 also known as “dyskerin pseudouridine synthase 1” plays an active role in telomerase stabilization and maintenance, as well as recognition of snoRNAs containing H/ACA sequences which provides stability during biogenesis and assembly into H/ACA small nucleolar RNA ribonucleoproteins (snoRNPs).
  • NM_001363.5 NM_001142463.3, NM_001288747.2, NR_110021.2, NR_110022.2, or NR_110023.2, incorporated herein by reference.
  • the circRNA is derived from the FAM124A gene.
  • FAM124A is also known as “family with sequence similarity 124 member A”. Human FAM124A nucleotide and amino acid sequences can be found at GenBank Accession No.
  • the circRNA is derived from the FBN1 gene.
  • FBN1 also known as fibrillin 1
  • Fibrillin-1 is an extracellular matrix glycoprotein that serves as a structural component of calcium-binding microfibrils. These microfibrils provide force-bearing structural support in elastic and nonelastic connective tissue throughout the body. Asprosin, secreted by white adipose tissue, has been shown to regulate glucose homeostasis.
  • Human FBN1 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_000138.5, NM_001406716.1, NM_001406717.1, or NM_001406718.1, incorporated herein by reference.
  • the circRNA is derived from the FCHO2 gene.
  • FCHO2 also known as, “FCH and mu domain containing endocytic adaptor 2”, functions in an early step of clathrin-mediated endocytosis.
  • Human FCHO2 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_138782.3 or NM_001146032.2, incorporated herein by reference. 43
  • the circRNA is derived from the GLS gene.
  • GLS also known as glutaminase, an phosphate-activated amidohydrolase that catalyzes the hydrolysis of glutamine to glutamate and ammonia.
  • Human GLS nucleotide and amino acid sequences can be found at GenBank Accession No. NM_014905.5 or NM_001256310.2, incorporated herein by reference.
  • the circRNA is derived from the GON4L gene.
  • GON4L also known as “GON-4 Like” is a nuclear protein containing two serine phosphosites and a lysine- glutamine cross-link and is thought to be a transcription factor.
  • the circRNA is derived from the GPR125 gene.
  • GPR125 also known as “adhesion G protein-coupled receptor A3” or “ADGRA3”, is a member of the G protein-coupled receptor superfamily. This membrane protein may play a role in tumor angiogenesis through its interaction with the human homolog of the Drosophila disc large tumor suppressor gene.
  • the circRNA is derived from the NEIL3 gene.
  • Neil3 also known as “nei like DNA glycosylase 3”, belongs to a class of DNA glycosylases. These glycosylases initiate the first step in base excision repair by cleaving bases damaged by reactive oxygen species and introducing a DNA strand break via the associated lyase reactionHuman NEIL3 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_018248.3, incorporated herein by reference.
  • the circRNA is derived from the PIK3C3 gene.
  • PIK3C3 also known as “phosphatidylinositol 3-kinase catalytic subunit type 3”, belongs to the phosphoinositide 3-kinase (PI3K) family.
  • PIK3C3 can phosphorylate phosphatidylinositol (PtdIns) to generate phosphatidylinositol 3-phosphate (PI3P), a phospholipid central to autophagy. Inhibition of PIK3C3 successfully inhibits autophagy.
  • Human PIK3C3 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_002647.4 or NM_001308020.2, incorporated herein by reference. 44
  • the circRNA is derived from the PNN gene.
  • PNN also known as “pinin, desmosome associated protein”
  • Pnn/DRS/memA is a potential tumor suppressor involved in the regulation of cell adhesion and cell migration.
  • Human PNN nucleotide and amino acid sequences can be found at GenBank Accession No. NM_002687.4, incorporated herein by reference.
  • the circRNA is derived from the POMT1 gene.
  • POMT1 also known as “protein O-mannosyltransferase 1” is a key enzyme in the glycosylation of ⁇ - dystroglycan.
  • RNA binding motif protein 23 is a member of the U2AF-like family of RNA binding proteins. This protein interacts with some steroid nuclear receptors, localizes to the promoter of a steroid- responsive gene, and increases transcription of steroid-responsive transcriptional reporters in a hormone-dependent manner.
  • Human RBM23 nucleotide and amino acid sequences can be found at GenBank Accession No.
  • the circRNA is derived from the SKIL gene.
  • SKIL also known as “SKI like proto-oncogene”, is a component of the SMAD pathway, which regulates cell growth and differentiation through transforming growth factor-beta (TGFB).
  • the encoded protein binds to the promoter region of TGFB-responsive genes and recruits a nuclear repressor complex.
  • TGFB signaling causes SMAD3 to enter the nucleus and degrade this protein, allowing these genes to be activated.
  • Human SKIL nucleotide and amino acid sequences can be found at GenBank Accession No. NM_005414.5, NM_001145097.2, NM_001145098.3, or NM_001248008.1, incorporated herein by reference.
  • the circRNA is derived from the SLK gene. Human SLK nucleotide and amino acid sequences can be found at GenBank Accession No. NM_014720.4 or NM_001304743.2, incorporated herein by reference. 45
  • the circRNA is derived from the SMA gene.
  • SMA also known as “survival of motor neuron 1”
  • This duplicated region contains at least four genes and repetitive elements which make it prone to rearrangements and deletions.
  • the protein encoded by this gene localizes to both the cytoplasm and the nucleus. Within the nucleus, the protein localizes to subnuclear bodies called gems which are found near coiled bodies containing high concentrations of small ribonucleoproteins (snRNPs).
  • This protein forms heteromeric complexes with proteins such as SIP1 and GEMIN4, and also interacts with several proteins known to be involved in the biogenesis of snRNPs, such as hnRNP U protein and the small nucleolar RNA binding protein.
  • Human SMA nucleotide and amino acid sequences can be found at GenBank Accession No. NM_001297715.1, NM_022874.2, or NM_000344.4, incorporated herein by reference.
  • the circRNA is derived from the SMG1 gene.
  • SMG1 also known as “SMG1 nonsense mediated mRNA decay associated PI3K related kinase”
  • NMD nonsense-mediated mRNA decay
  • the protein has kinase activity and is thought to function in NMD by phosphorylating the regulator of nonsense transcripts 1 protein.
  • Human SMG1 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_015092.5, incorporated herein by reference.
  • the circRNA is derived from the SMYD4 gene.
  • SMYD4 also known as “SET and MYND domain containing 4”
  • is a potential tumor suppressor Human SMYD4 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_052928.4, incorporated herein by reference.
  • the circRNA is derived from the TAF2 gene.
  • TAF2 TATA-box binding protein associated factor 2 encodes one of the larger subunits of transcription factor IID (TFIID) that is stably associated with the TFIID complex. It contributes to interactions at and downstream of the transcription initiation site, interactions that help determine transcription complex response to activators.
  • Human TAF2 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_003184.4, incorporated herein by reference.
  • the circRNA is derived from the TFAP4 gene.
  • TFAP4 also known as “transcription factor AP-4”, is a transcription factor of the basic helix-loop-helix- zipper (bHLH-ZIP) family contain a basic domain, which is used for DNA binding, and HLH 46
  • Transcription factor AP4 activates both viral and cellular genes by binding to the symmetrical DNA sequence CAGCTG.
  • Human TFAP4 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_003223.3, incorporated herein by reference.
  • the circRNA is derived from the TMEM41B gene.
  • TMEM41B also known as “transmembrane protein 41B”, is an integral endoplasmic reticulum (ER) membrane protein distantly that plays roles in autophagosome biogenesis.
  • Human TMEM41B nucleotide and amino acid sequences can be found at GenBank Accession No. NM_015012.4, incorporated herein by reference.
  • the circRNA is derived from the USP25 gene.
  • USP25 also known as “ubiquitin specific peptidase 25” is a deubiquitinating enzyme.
  • Human USP25 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_013396.6, NM_001283041.3, NM_001283042.3, NM_001352560.2, NM_001352561.2, NM_001388299.1, NM_001388300.1, NM_001388301.1 or, NM_001388302.1, incorporated herein by reference.
  • the circRNA is derived from the VPS13D gene.
  • VPS13D also known as “vacuolar protein sorting 13 homolog D”
  • VPS13D is a protein belonging to the vacuolar- protein-sorting-13 gene family.
  • vacuolar-protein-sorting-13 proteins are involved in trafficking of membrane proteins between the trans-Golgi network and the prevacuolar compartment.
  • Human VPS13D nucleotide and amino acid sequences can be found at GenBank Accession No. NM_015378.4 or NM_018156.4, incorporated herein by reference.
  • the circRNA is derived from the WDR59 gene.
  • WDR59 also known as “WD repeat domain 59”
  • WDR59 is involved in cellular response to amino acid starvation and positive regulation of TOR signaling.
  • the circRNA is derived from the XPR1 gene.
  • XPR1 also known as “xenotropic and polytropic retrovirus receptor 1”, is a receptor for the xenotropic and polytropic classes of murine leukemia viruses.
  • the encoded protein is involved in phosphate homeostasis by mediating phosphate export from the cell.Human XPR1 nucleotide and amino 47
  • CircRNA can be detected and measured using methods commonly known in the art.
  • circRNA is detected and/or measured by employing a technique selected from the group consisting of northernblot, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), microarray, droplet digital PCR, next-generation sequence (NGS) RNA sequencing, RNAin situ hypridization (RNA-ISH), in situ hypridization- immunohistochemistry (ISH-IHC), isothermal exponential amplification and rolling cycle amplification.
  • RT-qPCR reverse transcription-quantitative polymerase chain reaction
  • NGS next-generation sequence
  • RNA-ISH RNAin situ hypridization
  • ISH-IHC in situ hypridization- immunohistochemistry
  • circRNA can be detected using quantitative real-time PCR, e.g.,Taqman PCR, e.g., Taqman Multiplex PCR.
  • circRNA can be detected using SYBR Green PCR.
  • circRNA is detected by RT-qPCR.
  • the circRNA is a circBRIP1, and the circBRIP1 is detected by RT-qPCR using the primers as described in any of Examples 1-9 of the present disclosure.
  • the forward and reverse primers used to detect circBRIP are TCTGTGTGCCAGACTGTGAG (SEQ ID NO: 9) and ACACCAAGTTCTGACGAAAAGG (SEQ ID NO: 10), respectively.
  • the forward and reverse primers used to detect circBRIP are GTGAGCCAAGGAATTTTGTGTTT (SEQ ID NO: 19) and GGTCTGAACTTTGCCATTAATATCTG (SEQ ID NO: 20), respectively.
  • the forward and reverse primers used to detect circBRIP are TCAAAATAGAAGCCAGTGGATGAG (SEQ ID NO: 22) and AATGGCATGCACAAGAACATG (SEQ ID NO: 23), respectively.
  • Levels of circRNA markers can be detected based on the absolute level or a normalized or relative level. Detection of absolute circRNA levels may be preferable when monitoring the treatment of a cell or subject. For example, the level of one or more circRNA markers (e.g., 48
  • circBRIP1 can be monitored in a cell or a subject receiving a DHX9 inhibitor, e.g., at regular intervals, such as daily or weekly intervals.
  • a modulation in the level of one or more circRNA markers can be monitored over time to observe trends in changes in marker levels.
  • Expression levels of the markers of the invention, e.g., circBRIP1, in the subject may be higher than the expression level of those markers in a prior expression level (e.g., prior to treatment, or from an earlier time point), thus indicating engagement of the DHX9 target by the inhibitor, inhibition of DHX9 by the inhibitor, or responsiveness or potential therapeutic effects of the treatment regimen for the subject.
  • determinations may be based on the normalized level of the marker.
  • Levels are normalized by correcting the absolute level of a marker by comparing its level to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or GAPDH gene. This normalization allows the comparison of the level in one sample to another sample (e.g., comparison between samples from cells or subjects receiving different amount of the same inhibitor, or comparison between samples from cells or subjects receiving different inhibitors).
  • HCT116 cells were obtained from ATCC (CCL-247) and were grown and assayed in McCoy’s 5a growth media supplemented with 10%FBS. The cells were plated at pre-determined cell densities in 384-well cell culture plates and incubated overnight in 37 o C at 5% CO2. In a separate plate, reference and test compounds were prepared in DMSO stock solution and serially diluted 3-fold for 10 points. Compound 7 (0.1% 49
  • qPCR was carried out using the below custom primer sequences obtained at Integrated DNA Technologies (IDT): Table 3: DNA Oligo Primers DNA Oligo Primers Circular (Circ) Forward (F) SEQ ID a separate plate for each gene. Data analysis was performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ⁇ Ct for circRNA or linRNA is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity is calculated by the following formula, whereas: 50
  • CirRNAs of BRIP1, A Alu-mediated, and cirRNA of SETD3 is non-Alu-mediated.
  • the Alu-mediated circRNAs namely BRIP1, AKR1A1 and DKC1, were induced, while no induction of SETD3 cirRNA was observed (see FIG. 1).
  • Example 2 Correlations of circBRIP1 with anti-tumor activity of DHX9 inhibitors in vitro This study was conducted to evaluate the circular format of BRIP1 in response to treatment with a panel of DHX9 inhibitors in vitro.
  • Methods DHX9 Inhibitor compounds A panel of DHX9 inhibitor compounds were used in this Example. The panel includes Compounds 1, 2, 3, 4 as shown in Table 1 and compounds which have been described in International Application No. PCT/US2023/012929, the entire contents of which are expressly incorporated by reference herein.
  • ATPase Assay DHX9 ATPase assay was performed in small-volume, nonbinding, 384- well white plates at a final volume of 10 ⁇ L/well.
  • test compounds (10 mM solution in DMSO; 100 nL/well) were serially diluted on Bravo (Agilent, Santa Clara, CA) and dispensed into wells of columns 3–22 of the plates using an Echo 555 acoustic dispenser (Labcyte, Sunnyvale, CA). 100nL of Aurintricarboxylic acid was dispensed into low control wells and 100 nL of DMSO was dispensed into high control wells.
  • Bravo Alent, Santa Clara, CA
  • Echo 555 acoustic dispenser Labcyte, Sunnyvale, CA
  • a Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific, Waltham, MA) was used to add a solution of DHX9 (1.25 nM, 5 ⁇ L/well) in assay buffer (40 mM HEPES [pH 7.5], 0.01% Tween 20, 0.01%BSA, 1 mM DTT, 5 mM MgCl2, 0.004U/ml RNAseOUT).
  • the reaction was initiated by the addition of 5 ⁇ L of substrate solution (30 nM double-stranded RNA substrate, 10 ⁇ M Ultra Pure ATP in assay buffer) into the wells. The plates were incubated at room temperature for 1 h. After the indicated 51
  • the percentage inhibition was calculated based on the high control (DMSO) as 0% inhibition, and low control (10 ⁇ M Aurintricarboxylic acid) control as 100% inhibition and used for the calculation of IC 50 and IP (inflection point) values by fitting the dose–response curves to a four-parameter logistic model. Assay results reported IP values instead of IC50 values since several compounds did not reach 100% inhibition at higher compound concentrations.
  • DMSO high control
  • low control 10 ⁇ M Aurintricarboxylic acid
  • IP inflection point
  • LS411N cells (CRL- 2159), obtained from ATCC, were grown in RPMI-1640 media supplemented with 10% FBS. The cells were plated at pre-determined cell densities in 384-well solid white cell culture plates and incubated overnight in 37 o C at 5% CO 2 . In a separate plate, reference and a panel of test compounds were prepared in DMSO stock solution and serially diluted 3-fold for 10 points. Compound (0.1% final DMSO concentration) was transferred to the cell plate in designated wells, and incubated 37 o C at 5% CO2 for 120 hours. CellTiter-Glo reagent was prepared fresh according to manufacturer’s (Promega) directions and added to each well.
  • the plate was then shaken at 300rpm for 10 minutes at RT, and then read on an Envision plate reader using a Luminescence protocol.
  • Data analysis was performed by normalizing the raw data (raw luminescence units or RSample) to an average of the positive control values for wells containing culture media only (100% cell death or RLC) and the negative control values for 0.1% DMSO (0% cell death or RHC).
  • An IC 50 was calculated using a 4-parameter logistic nonlinear regression model in Scigilian Analyzer or GraphPad Prism, constraining the top parameter to 100 and the bottom parameter to 0.
  • IC50 Fit formula (A+((B-A)/(1+((x/C) ⁇ D))) ... whereas A:Bottom; B:Top; C:IC50; D:Slope circBRIP1 Cellular Target Engagement Taqman Multiplex Assay: Evaluation of the circular format of BRIP1 mRNA in HCT116 cells by Quantitative Polymerase Chain Reaction (qPCR) using the Taqman Multiplex assay.
  • HCT116 cells were obtained from ATCC (CCL-247) and were grown and assayed in McCoy’s 5a growth media supplemented with 10%FBS. The cells were plated at pre-determined cell densities in 384-well cell culture plates and incubated overnight in 37 oC at 5% CO2.
  • ⁇ Ct for circBRIP1 or linBRIP1 is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity is calculated by the following formula, whereas: • ⁇ Cts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ⁇ Ctc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. 2 ⁇ Cts
  • EC50 was then calculated using a 4-parameter logistic nonlinear regression model in Scigilian Analyzer or GraphPad Prism, floating both the top and bottom parameters.
  • IC50 Fit formula (A+((B-A)/(1+((x/C) ⁇ D)))) ... whereas A:Bottom; B:Top; C:IC50; D:Slope circBRIP1 Cellular Target Engagement SYBR Green Assay: The circular format of BRIP1 mRNA in HCT116 cells was additionally evaluated by Quantitative Polymerase Chain Reaction (qPCR) using the SYBR Green assay.
  • HCT116 cells were obtained from ATCC (CCL-247) and were grown and assayed in McCoy’s 5a growth media supplemented with 10%FBS.
  • ⁇ Ct for circRNA is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity is calculated by the following formula, whereas: • ⁇ Cts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ⁇ Ctc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. 54
  • Example 3 Evaluation of circBRIP1 in human xenograft tumor This study was conducted to evaluate the circular and linear format of human BRIP1 mRNA in LS411N xenograft tumors by qPCR. Methods Experiments were performed in female BALB/c nude mice (GenPharmatech Co.). Animals were allowed to acclimate for 7 days before the study.
  • mice were kept in laminar flow rooms at constant temperature and humidity with 3-5 mice in each cage. Animals were housed in polycarbonate cage which is the size of 300 x 180 x 150 mm 3 and in an environmentally monitored, well-ventilated room maintained at a temperature of (23 ⁇ 3°C) and a relative humidity of 40% 70%. Fluorescent lighting provided illumination approximately 12 hours per day.
  • LS411N (ATCC; CRL-2159) tumor cell lines were maintained in vitro as monolayer in RPMI 1640 medium supplemented with 10% heat inactivated FBS, at 37°C in an atmosphere of 5% CO2 in air.
  • the tumor cells were sub-cultured, not exceeding 4-5 passages, and cells growing in an exponential growth phase were harvested and counted for tumor inoculation.
  • Each mouse was inoculated subcutaneously on the right flank with LS411N tumor cells (2 ⁇ 10 6 ) in 0.1 mL of RPMI-1640 with Matrigel (1:1) for model development. 55
  • DHX9 compound was dissolved in in 0.96 mL Solutol while vortexing and sonicating for 1 hour to obtain a suspension. Then 2.4 mL of 1% MC (with 100mg/mL PVP VA64) was added and vortexed for 5 min to obtain homogeneous suspension. The mixture was kept stirring at room temperature for 5 min, and then 1.44 mL of water was added while vortexing to make the final volume of 4.8 mL. The mixture was kept stirring at RT for 30 min and homogenized for 3 min. The resulting solution was used for in vivo studies. Treatment was started when the mean tumor size reached approximately 100-150 mm 3 , at which time the mice were randomized into treatment groups.
  • DHX9 compound Compounds 1, 2, 3 and 4, as shown in Table 1
  • daily BID (12 hour schedule) by oral gavage at a final dosing volume of 10 mL/kg.
  • Tumor samples 2-3mm 3 in size were transferred into a clean tube and 300 ⁇ L of fresh lysis buffer containing 1% 2- 0.12mercaptoethanol was added.
  • the samples were then homogenized using a TissueLyser at a frequency of 30/s for 5 min. The homogenate samples are then centrifuged at 1200 rpm for 15 min.
  • RNA is then purified using the PureLink RNA Mini Kit according to the manufacturer’s (Invitrogen) protocol, and cDNA is reverse transcribed using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s (Invitrogen) protocol.
  • qPCR is carried out using TaqMan Gene Expression Assay with VIC Dye for Linear BRIP1 (Assay ID: Hs00908156_m1), TaqMan GAPDH primer limited assay with JUN dye/QSY probe, and the below custom TaqMan Gene Expression Assay with FAM Dye for circBRIP1: • Forward Primer: GTGAGCCAAGGAATTTTGTGTTT (SEQ ID NO: 19) • Reverse Primer: GGTCTGAACTTTGCCATTAATATCTG (SEQ ID NO: 20) • Probe: CCATCTTACAAGGCCTTT (SEQ ID NO: 21) Note: All target genes can be combined in one well per sample. 56
  • ⁇ Ct for circBRIP1 or linBRIP1 is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample.
  • the inhibition activity is calculated by the following formula, whereas: • ⁇ Cts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ⁇ Ctc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control.
  • mice were allowed to acclimate for 7 days before the study. The general health of the animals were evaluated by a veterinarian, and complete health checks were performed prior to the study. General procedures for animal care and housing were in accordance with the standard, Commission on Life Sciences, National Research Council, Standard Operating Procedures (SOPs) of Pharmaron, Inc. The mice were kept in laminar flow rooms at constant temperature and humidity with 3-5 mice in each cage. Animals were housed in polycarbonate cage which is the size of 300 x 180 x 150 mm 3 and in an environmentally monitored, well- ventilated room maintained at a temperature of (23 ⁇ 3°C) and a relative humidity of 40% 70%. Fluorescent lighting provided illumination approximately 12 hours per day. Animals had free access to irradiation sterilized dry granule food during the entire study period except for time 57
  • the LS411N (ATCC; CRL-2159) tumor cell lines were maintained in vitro as monolayer in RPMI 1640 medium supplemented with 10% heat inactivated FBS, at 37°C in an atmosphere of 5% CO2 in air.
  • the tumor cells were sub-cultured, not exceeding 4-5 passages, and cells growing in an exponential growth phase were harvested and counted for tumor inoculation.
  • Each mouse was inoculated subcutaneously on the right flank with LS411N tumor cells (2 ⁇ 10 6 ) in 0.1 mL of RPMI-1640 with Matrigel (1:1) for model development.
  • DHX9 compound Depending on the target concentration, 14.4 – 144 mg DHX9 compound was dissolved in in 0.96 mL Solutol while vortexing and sonicating for 1 hour to obtain a suspension. Then 2.4 mL of 1% MC (with 100mg/mL PVP VA64) was added and vortexed for 5 min to obtain homogeneous suspension. The mixture was kept stirring at room temperature for 5 min, and then 1.44 mL of water was added while vortexing to make the final volume of 4.8 mL. The mixture was kept stirring at RT for 30 min and homogenized for 3 min. The resulting solution was used for in vivo studies.
  • 1% MC with 100mg/mL PVP VA64
  • mice were randomized into treatment groups. Animals were then treated with vehicle (20% Solutol/ 50% 1% MC (4000 cp) with 100 mg/mL PVP VA64/ 30% water) or indicated mg/kg of DHX9 compound 1 daily BID (12 hour schedule) by oral gavage at a final dosing volume of 10 mL/kg.
  • vehicle 20% Solutol/ 50% 1% MC (4000 cp) with 100 mg/mL PVP VA64/ 30% water
  • mg/kg of DHX9 compound 1 daily BID (12 hour schedule) by oral gavage at a final dosing volume of 10 mL/kg.
  • the measurement of tumor size was conducted with a caliper and recorded twice per week.
  • Intra-tumoral Pharmacokinetics Bioanalytical Assay Tumors were collected at 1 h and 12 h post the last dose and cut into 3 pieces. One piece of the tumor was weighted and snap frozen for drug exposure analysis. On the assay day, 3 volumes of H2O were added to the tumor tissue (3 mL to each gram of tissue), and homogenate at 4 degrees Celsius. The homogenate (30 ⁇ L) was mixed with 15 ⁇ L blank solution, then extracted and protein precipitation with 200 ⁇ L of acetonitrile containing IS (internal standard, dexamethasone). After vortexed for 30 s, the samples were centrifuged at 4 degrees Celsius, 3900 rpm for 15 minutes. The supernatant was 58
  • HPLC Shimadzu system (DGU-20A5R, LC-30AD, SIL-30AC, Rack Changer II, CTO-30A, and CBM-20A).
  • concentration in the homogenate was quantified using standards prepared in control tumor tissues with concentrations range 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL. The determined concentration in the tumor homogenate was then back calculated to that in tumor tissue by multiplying with a dilution factor of 4.
  • Intra-tumoral Human circBRIP1 Analysis Tumor samples 2-3mm 3 in size were transferred into a clean tube and 300 ⁇ L of fresh lysis buffer containing 1% 2-mercaptoethanol was added. The samples were then homogenized using a TissueLyser at a frequency of 30/s for 5 min.
  • Target gene Ct to the GAPDH housekeeping gene Ct for each sample.
  • mice were housed in polycarbonate cage which is the size of 300 x 180 x 150 mm 3 and in an environmentally monitored, well-ventilated room maintained at a temperature of (23 ⁇ 3°C) and a relative humidity of 40% 70%. Fluorescent lighting provided illumination approximately 12 hours per day. Animals had free access to irradiation sterilized dry granule food during the entire study period except for time periods specified by the protocol, as well as sterile drinking water in a bottle was available ad libitum during the quarantine and study periods.
  • the LS411N (ATCC; CRL-2159) tumor cell lines were maintained in vitro as monolayer in RPMI 1640 medium supplemented with 10% heat inactivated FBS, at 37°C in an atmosphere of 5% CO2 in air.
  • the tumor cells were sub-cultured, not exceeding 4-5 passages, and cells growing in an exponential growth phase were harvested and counted for tumor inoculation.
  • Each mouse was inoculated subcutaneously on the right flank with LS411N tumor cells (2 ⁇ 106) in 0.1 mL of RPMI-1640 with Matrigel (1:1) for model development.
  • DHX9 compound (Compound 2, 3, or 4, as shown in Table 1) was dissolved in in 0.96 mL Solutol while vortexing and sonicating for 1 hour to obtain a suspension. Then 2.4 mL of 1% MC (with 100mg/mL PVP VA64) was added and vortexed for 5 min to obtain homogeneous suspension. The mixture was kept stirring at room temperature for 5 min, and then 1.44 mL of water was added while vortexing to make the final volume of 4.8 mL. The mixture was kept stirring at RT for 30 min and homogenized for 3 min. The resulting solution was used for in vivo studies.
  • 1% MC with 100mg/mL PVP VA64
  • mice were randomized into treatment groups. Animals were then treated with vehicle (20% Solutol/ 50% 1% MC (4000 cp) with 100 mg/mL PVP VA64/ 30% water) or indicated mg/kg of DHX9 compound daily BID (12 hour schedule) by oral gavage at a final dosing volume of 10 mL/kg.
  • the layer of lymphocytes was transferred into a new 1.5 mL tube containing 0.5 mL of 1 ⁇ PBS, and centrifuged at 1500 rpm for 5 min.
  • the samples were then 61
  • PBMC samples were transferred into a clean tube and 300 ⁇ L of fresh lysis buffer containing 1% 2-mercaptoethanol. The samples were then homogenized using a TissueLyser at a frequency of 30/s for 5 min. The homogenate samples were then centrifuged at 1200 rpm for 15 min. The supernatant was transferred to a clean tube, and one volume of 70% ethanol to each volume of homogenate supernatant was added, and the samples were then vortexed.
  • ⁇ Ct for circBRIP1 or linBRIP1 was calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity was calculated by the following formula, whereas: • ⁇ Cts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ⁇ Ctc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. ) ⁇ 62
  • CRC cell lines including MSI-H CRC cells (sensitive to DHX9 inhibitor) and MSS CRC cells (insensitive to DHX9 inhibitor) were treated with with escalating doses of from 0.001 to 10 ⁇ M Compound 1, including HCT116 (CRC MSI; Sensitive), HT-29 (CRC MSS; Insensitive), DLD-1 (CRC MSI; Sensitive), HCT-15 (CRC MSI; Sensitive), LS411N (CRC MSI; Sensitive), NCI-H747 (CRC MSS; Insensitive), LS174T (CRC MSI; Sensitive), LOVO (CRC MSI; Sensitive), SW48 (CRC MSI; Insensitive), and SNU-407 (CRC MSI; Insensitive) Reference and test compounds were prepared in DMSO stock solution and serially diluted 3-fold for 10 points.
  • ⁇ Ct for circBRIP1 is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample.
  • Example 7 Evaluation of circBRIP1 in frozen human PBMCs As proof of concept, circBRIP1 levels were examined in peripheral blood mononuclear cells (PMBCs) using frozen PBMCs from donors after treatment with a DHX9 inhibitor.
  • PMBCs peripheral blood mononuclear cells
  • Frozen PBMCs were thawed in a 37 o C water bath, washed, and reconstituted in media. The same number of cells were added to each well of 12-well plates and let rest for 2 hours before being subjected to testing conditions. Compound 1 was dissolved in DMSO at 10 mM as a stock solution, and diluted to a desired concentration. Frozen PBMCs from three donors or LS411N cells (positive control) were treated with 10 ⁇ M of Compound 1 or control vehicle for 24 hours. The circBRIP1 level was determined using methods essentially as decribed above. As shown in FIG. 8A, treatment with Compound 1 significantly elevated the circBRIP1 level in PBMCs as compared to vehicle-treated cells.
  • PBMCs Human PBMCs were thawed from a frozen cryotube in a 37 0 C water bath and transferred to a flask with medium X-Vivo 15 supplemented with 5% Fetal Bovine Serum (FBS), 1% PenStrep, and 1% Glutamax. Flasks were incubated at 37C for 30 minutes. PBMCs were harvested from flask and counted. PBMCs were diluted in medium and added to 96 well plates at 100,000 PBMCs per well. A stimulus mixture containing IL-15 at 1ng/well, IL-21 at 5ng/well, and CD40L at 5ng/well was added to each well.
  • FBS Fetal Bovine Serum
  • PBS Fetal Bovine Serum
  • PenStrep 1% PenStrep
  • Glutamax Glutamax
  • Test compounds were prepared in DMSO stock solution and serially diluted. Compound (0.1% final DMSO concentration) was added to each well. DMSO was used as a negative control (high control, HC) and staurosporine was used a positive control (low control, LC). Plates were incubated at 37C for 0, 2, 4, 6, 8, 10 days. Every other day, medium was replenished with stimulus and diluted compounds. At each CTG time point, plate was removed from incubator and Cell-Titer Glo 2.0 reagent was added to each well according to manufacturer’s (Promega) protocol and detected on an Envision.
  • Example 8 Evaluation of circBRIP1 in human PBMCs This study was conducted to evaluate the circular format of BRIP1 mRNA in freshl human PBMC cells by qPCR.
  • Human whole blood was obtained from Research Blood Components. Blood of each donor was aliquoted into vacutainer tubes containing Acid Citrate Dextrose (ACD). Test compounds were prepared in DMSO stock solution and serially diluted. Compound (0.1% final DMSO concentration) was added to vacutainer tubes containing human whole blood. Tubes were placed in tube rotator and incubated at 37C for 24 hours. PBMCs were isolated from human whole blood after compound treatment by density gradient centrifugation using Ficoll-Paque Plus.
  • qPCR was carried out using TaqMan Gene Expression Assay with VIC Dye for Linear BRIP1 (Assay ID: Hs00908156_m1), TaqMan GAPDH primer limited assay with JUN dye/QSY probe, and the below custom TaqMan Gene Expression Assay with FAM Dye for circBRIP1: • Forward Primer: GTGAGCCAAGGAATTTTGTGTTT (SEQ ID NO: 19) • Reverse Primer: GGTCTGAACTTTGCCATTAATATCTG (SEQ ID NO: 20) • Probe: CCATCTTACAAGGCCTTT (SEQ ID NO: 21) Note: All target genes can be combined in one well per sample.
  • ⁇ Ct for circBRIP1 or linBRIP1 was calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity was calculated by the following formula, whereas: • ⁇ Cts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ⁇ Ctc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. 66
  • Test compounds are prepared in DMSO stock solution and serially diluted. Compound (0.1% final DMSO concentration) is added to vacutainer tubes containing human whole blood. Tubes are placed in tube rotator and incubated at 37C for 24 hours. Total cells are isolated from human whole blood after compound treatment by density gradient centrifugation using Ficoll-Paque Plus. RNA is extracted from cells using RNeasy Plus Kits according to the manufacturer’s (Qiagen) protocol. cDNA is reverse transcribed using RT 2 First Strand Kit according to the manufacturer’s (Qiagen) protocol.
  • qPCR is carried out using TaqMan Gene Expression Assay with VIC Dye for Linear BRIP1 (Assay ID: Hs00908156_m1), TaqMan GAPDH primer limited assay with JUN dye/QSY probe, and the below custom TaqMan Gene Expression Assay with FAM Dye for circBRIP1: • Forward Primer: GTGAGCCAAGGAATTTTGTGTTT (SEQ ID NO: 19) • Reverse Primer: GGTCTGAACTTTGCCATTAATATCTG (SEQ ID NO: 20) • Probe: CCATCTTACAAGGCCTTT (SEQ ID NO: 21) Note: All target genes can be combined in one well per sample. 67
  • ⁇ Ct for circBRIP1 or linBRIP1 is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample.
  • the inhibition activity is calculated by the following formula, whereas: • ⁇ Cts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ⁇ Ctc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control.
  • Example 10 Identification of additional Alu-circRNAs mediated by DHX9 This study was conducted to identify additional Alu-mediated circRNAs that can be markers for evaluating DHX9 inhibition. Methods Screening for circRNA upregulated after knockdown of DHX9: HCT116 CRC-MSI cells were treated with DHX9 siRNA for a total of 3 days.
  • RNA from each sample was quantified using the NanoDrop ND-1000.
  • the sample preparation and microarray hybridization were performed based on the Arraystar Human Circular RNA Array’s standard protocols. Briefly, total RNAs were digested with Rnase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8x15K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. 68
  • Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software limma package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering. Differentially expressed circRNAs between two samples (DHX9 siRNA-treated and scrambled control siRNA treated) were identified through Fold Change filtering. Hierarchical Clustering was performed to show the distinguishable circRNAs expression pattern among samples.
  • Identifying Alu-mediated circRNA among circRNAs upregulated by knockdown of DHX9 Up-regulated circRNA that were found to be differentially regulated by DHX9 knockdown were categorized as Alu mediated by methods as described by Stagsted et al. 2019 (Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles, Life Science Alliance, 2019, 2(3):1-16), the entire contents of which are incorporated herein by reference). Stagsted et al. states that Alu-mediated circRNA biogenesis occurs by aberrant backsplicing of RNA transcripts as stimulated by bringing Inverted Alu Elements (IAE) into close proximity with each other.
  • IAE Inverted Alu Elements
  • circRNA can also occur when long flanking introns backsplice on each other, which is termed “AUG circRNA” and relies on IAE-independent (i.e., Alu-independent) mode of biogenesis, whereas “other circRNA” relies on IAE-dependent biogenesis.
  • HITS-CLIP CrossLinking ImmunoPrecipitation
  • CircRNAs up-regulated by DHX9 knockdown identified by the Arraystar Human Circular RNA Array was compared to a list of circRNAs sensitive to DHX9 expression provided in Supplemental Table 4 of Stagsted et al. 2019. CircRNAs annotated as “AUG circRNA” by Statsted et al. were considered to not be sensitive to DHX9 activity, and thus not dependent on IAE for biogenesis. CircRNAs annotated as “Ambiguous circRNA” were also irrelevant for this analysis, as they are divergent across multiple host genes Results 69

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Abstract

The present disclosure provides methods of measuring the activity of a DHX9 inhibitor in cells or in a subject, the methods comprising measuring the level of an Alu-mediated circular RNA induced by the DHX9 inhibitor.

Description

ASSAYS FOR MONITORING INHIBITION OF RNA HELICASE DHX9 REFERENCES TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Applicatino No. 63/459632, filed on April 15, 2023, the entire content of which is incorporated herein by reference SEQUENCE LISTING The instant application contains a Sequence Listing XML which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 15, 2024, is named 130090-01520.xml and is 51,303 bytes in size. BACKGROUND OF THE INVENTION Genomic instability is a characteristic of most cancers. In hereditary cancers, genomic instability results from mutations in DNA repair genes and drives cancer development. Interfering with players that maintain genome stability or manage replication stress, such as RNA helicases, is an evolving an approach to specifically target cancer with genomic instability. DEAD/H box RNA helicases are an interesting group of proteins serving as potential translational targets in cancer. These proteins belong to superfamily 2, the largest group of eukaryotic RNA helicases, are named after a conserved amino sequence (Asp-Glu-Ala- Asp/His) and have the ability to unwind and restructure RNA molecules with complex secondary structures in an ATPase dependent fashion. They remodel complex RNA structures like hairpins and mRNP complexes and have been reported to play a pivotal role in virtually all steps of mRNA processing and translation. Interestingly, cancer cells seem to rely heavily on RNA helicases to meet not only the increased general protein synthesis demand, but also for translation of specific pro-oncogenic mRNAs to enhance survival. DHX9, also known as RNA Helicase A (RHA) or Nuclear DNA Helicase II (NDH II), is a DEAH-box RNA helicase. Due to its regulatory role in processes such as transcription and maintenance of genomic stability, DHX9 has been shown to be a key regulator in a variety of cancer types (Gulliver et al., 2020, Future Science OA (2), FSO650). Specifically, microsatellite instable cancers, such as Microsatellite Instable (MSI) colorectal cancer, and tumors with defective Mismatch Repair (MMR) exhibit a strong dependence on With the development of DHX9 inhibitors as potential therapeutic agents for treating certain cancers, there is a need in the art for non-invasive biomarkers for their clinical applications. SUMMARY OF THE INVENTION In one aspect, the present disclosure provides a method of measuring activity of a DHX9 inhibitor in a cell, comprising: contacting a cell with the DHX9 inhibitor; and measuring the level of an Alu-mediated circular RNA (Alu-circRNA) in the cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a normal cell. In some embodiments, the cell is a non-cancerous cell. In some embodiments, the cell is a peripheral blood mononuclear cell (PBMC). In some embodiments, the contacting is for a sufficient time for the Alu-circRNA to be produced. In some embodiments, the level of Alu-circRNA is correlated to inhibition of a DHX9 activity by the DHX9 inhibitor in the cell. In another aspect, the present disclosure provides a method of measuring activity of a DHX9 inhibitor in a subject, comprising: administering a DHX9 inhibitor to a subject; and measuring the level of an Alu-mediated circular RNA (Alu-circRNA) in a biological sample from the subject. In another aspect, the present disclosure provides a method of measuring activity of a DHX9 inhibitor in a subject, comprising: measuring the level of Alu-mediated circular RNA (Alu-circRNA) in a biological sample from a subject, wherein the subject is being treated with a DHX9 inhibitor. In some embodiments, the level of Alu-circRNA is correlated to inhibition of DHX9 activity by the DHX9 inhibitor in the subject. In some embodiments, the level of Alu- circRNA is correlated to inhibition of DHX9 ATPase activity or resolvase activity. In some embodiments, the level of Alu-circRNA is correlated to inhibition of proliferation of MSI cancer cells in the subject. In some embodiments, the biological sample is selected from the group consisting of blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue. In some embodiments, the biological sample is blood. In some embodiments, the method further comprises isolating cells from the blood sample, and measuring the level of the Alu-circRNA in the isolated cells. In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the method further comprises isolating PBMCs from a blood sample, and measuring the level of the Alu-circRNA in the PBMCs. In some embodiments, the level of Alu-circRNA does not correlate with inhibition of proliferation of PBMCs of the subject. In some embodiments, the Alu-circRNA is selected from the group consisting of cirRNA of BRIP1, AKR1A1, BMPR2, CALCOCO2, CLNSIA, DKC1, FAM124A, FBN1, FCHO2, GLS, GON4L, GPR125, NEIL3, PIK3C3, PNN, POMT1, RBM23, SKIL, SLK, SMA, SMG1, SMYD4, TAF2, TFAP4, TMEM41B, USP25, VPS13D, WDR59, and XPR1. In some embodiments, the level of the Alu-circRNA is measured by a technique selected from northernblot, reverse transcription-quantitative polymerase chain reaction (RT- qPCR), microarray, droplet digital PCR, next-generation sequence (NGS) RNA sequencing, RNAin situ hypridization (RNA-ISH), in situ hypridization- immunohistochemistry (ISH- IHC), isothermal exponential amplification, and rolling cycle amplification. In some embodiments, the method further comprises obtaining the biological sample from the subject. In some embodiments, the biological sample is obtained from the subject between 0.5-7 days following administration of the DHX9 inhibitor. In some embodiments, the method comprises measuring the level of the Alu-circRNA in biological samples obtained from the subject at more than one time point. In some embodiments, the subject has a cancer. In some embodiments, the cancer is a microsatellite instability (MSI) cancer. In some embodiments, the cancer is selected from the group consisting of colorectal, endometrial, ovarian, gastric, hematopoietic, breast, brain, skin, lung, blood, prostate, head and neck, pancreatic, bladder, bone, soft tissue, kidney, liver, and Ewing’s sarcoma. In some embodiments, the method further comprises making a determination on an appropriate therapeutic dose of the DHX9 inhibitor based on a set of data including the level of Alu-circRNA measured. In some embodiments, the DHX9 inhibitor is selected from a small molecule, an antisense oligonucleotide, small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA). In some embodiments, the DHX9 inhibitor is a small molecule. In some embodiments, the DHX9 inhibitor is selected from the inhibitors in Table 1. I b di t th bj t i h In yet another aspect, the present disclosure provides a method of monitoring treatment of a subject with a DHX9 inhibitor, comprising: administering a DHX9 inhibitor to the subject; and obtaining information as to the level of Alu-mediated circular RNA (Alu- circRNA) in a biological sample obtained from the subject after administration of the DHX9 inhibitor. In some embodiments, the level of Alu-circRNA is correlated to inhibition of DHX9 activity by the DHX9 inhibitor in the subject. In some embodiments, the level of Alu- circRNA is correlated to inhibition of DHX9 ATPase activity or resolvase activity. In some embodiments, the level of Alu-circRNA is correlated to inhibition of proliferation of MSI cancer cells in the subject. In some embodiments, the biological sample is selected from the group consisting of blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue. In some embodiments, the biological sample is blood. In some embodiments, the method further comprises isolating cells from the blood sample, and measuring the level of the Alu-circRNA in the isolated cells. In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the method further comprises isolating PBMCs from a blood sample, and measuring the level of the Alu-circRNA in the PBMCs. In some embodiments, the level of Alu-circRNA does not correlate with inhibition of proliferation of PBMCs of the subject. In some embodiments, the Alu-circRNA is selected from the group consisting of cirRNA of BRIP1, AKR1A1, BMPR2, CALCOCO2, CLNSIA, DKC1, FAM124A, FBN1, FCHO2, GLS, GON4L, GPR125, NEIL3, PIK3C3, PNN, POMT1, RBM23, SKIL, SLK, SMA, SMG1, SMYD4, TAF2, TFAP4, TMEM41B, USP25, VPS13D, WDR59, and XPR1. In some embodiments, the level of the Alu-circRNA is measured by a technique selected from northernblot, reverse transcription-quantitative polymerase chain reaction (RT- qPCR), microarray, droplet digital PCR, next-generation sequence (NGS) RNA sequencing, RNAin situ hypridization (RNA-ISH), in situ hypridization- immunohistochemistry (ISH- IHC), isothermal exponential amplification, and rolling cycle amplification. In some embodiments, the method further comprises obtaining the biological sample from the subject. In some embodiments, the biological sample is obtained from the subject between 0.5-7 days following administration of the DHX9 inhibitor. In some embodiments, the method comprises measuring the level of the Alu-circRNA in biological samples obtained from the subject at more than one time point. In some embodiments, the subject has a cancer. In some embodiments, the cancer is a microsatellite instability (MSI) cancer. In some embodiments, the cancer is selected from the group consisting of colorectal, endometrial, ovarian, gastric, hematopoietic, breast, brain, skin, lung, blood, prostate, head and neck, pancreatic, bladder, bone, soft tissue, kidney, liver, and Ewing’s sarcoma. In some embodiments, the method further comprises making a determination on an appropriate therapeutic dose of the DHX9 inhibitor based on a set of data including the level of Alu-circRNA measured. In some embodiments, the DHX9 inhibitor is selected from a small molecule, an antisense oligonucleotide, small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA). In some embodiments, the DHX9 inhibitor is a small molecule. In some embodiments, the DHX9 inhibitor is selected from the inhibitors in Table 1. In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: Cells were treated with DHX9 compound 7 for 3 days and probed for multiple Alu-mediated circRNAs, a non-Alu mediated circRNA, as well as each gene’s respective linear RNA form. Upon DHX9 inhibition, the Alu-mediated circRNAs, BRIP1, AKR1A1 and DKC1, were regulated and induced. FIGs. 2A-2B show that cellular target engagement by DHX9 inhibitors correlates with DHX9 biochemical activity and anti-proliferative activity of DHX9 inhibitors in certain MSI cancers. FIG. 2A shows DHX9 ATPase biochemical activity for each DHX9 inhibitor, reported as inflection points (IP) or EC50, plotted against its corresponding circBRIP1 EC50, which is EC50 of circBRIP1 induced in HCT116 CRC-MSI cells measured by cellular DHX9 target engagement assays. FIG. 2B shows anti-proliferative activity of each DHX9 inhibitor in LS411N CRC-MSI cells, measured as IC50, plotted against its corresponding circBRIP1 EC50, measured by cellular DHX9 target engagement assays. FIGs. 3A-3D: DHX9 compounds (1 to 4) were dosed in vivo as described in Example 3. Tumor samples were taken at the end of the study 1 hr post last dose and analyzed for human linear BRIP1 and/or human circBRIP1. Dose dependent induction of circBRIP1 was observed and where applicable, no change in linear BRIP1 was seen. FIGs. 4A-4C: Mice with human xenograft tumors were dosed with DHX9 inhibitor Compound 1 at 30, 100, 200 and 300 mg/kg orally with a 12 hour schedule as described in Example 4. After 21 days of treatment and 12 hours post last dose of compound tumor volume was recorded, tumors were then harvested and processed for circBRIP1 expression. FIG. 4A shows dose-dependent induction of circBRIP1 in LS411N tumors. FIG. 4B shows individual tumor volumes plotted against each tumor’s corresponding circBRIP1 pharmacodynamic reading in a correlation plot. FIG. 4C shows individual tumor drug exposure of Compound 1 plotted against each tumor’s corresponding circBRIP1 pharmacodynamic reading and fit to four parameter non-linear regression. FIGs. 5A-5B: DHX9 compounds were dosed by oral gavage (PO) in vivo as described in Example 5. Whole blood samples were taken at the end of the study 1 hr post last dose, PBMC were isolated and analyzed for mouse linear BRIP1 and/or mouse circBRIP1. Dose dependent induction of circBRIP1 was observed and where applicable, no change in linear BRIP1 was seen. FIG. 6: DHX9 compounds were dosed oral gavage (PO) in vivo as described in Example 5. Whole blood samples were taken at the end of the study 24 hr post last dose, PBMC were isolated and analyzed for mouse linear BRIP1 and/or mouse circBRIP1. Dose dependent induction of circBRIP1 was observed , while no linear BRIP1 above baseline was seen. FIG. 7A: Colorector cancer cell lines that were either sensitive to DHX9 inhibitor (IC50 < 1 µM or insensitive to DHX9 inhibitor (IC50 > 1 µM) were treated with with escalating doses of from 0.001 to 10 µM Compound 1, as described in Example 6. circBRIP1 EC50 values were relative to vehicle treated cell. Similar potency of circBRIP1 induction was seen in both sensitive and insenstive cells. FIG. 7B: Several breast, ovarian, lung and colorectal (CRC) cancer cell lines were treated with Compound 1 as described in Example 6. IC50 values for inhibition of proliferative activity by Compound 1 in the cell lines showed no correlation with EC50 values of circBRIP1 induction. FIG. 8A: circBRIP1 level was elevated in previously-frozen isolated human PBMCs after treatment with Compound 1 for 24 hours. FIG. 8B: Treatment of PBMCs with Compound 1 at concentration of 0.01 µM to 10 µM for 10 days did not impact their lif i ( i i i l) FIGs. 9A-9D: Four fresh human blood samples were obtained and processed as described in Example 8. Results show an induced level of human circBRIP1 in a dose response when treated with DHX9 compound 1 at varying levels per donor. Linear BRIP1 was not significantly changed. DETAILED DESCRIPTION OF THE INVENTION I. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. The term "such as" is used herein to mean, and is used interchangeably, with the phrase "such as but not limited to." As used herein, the term “a,” “an,” “the” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g. “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about. As used herein, the term “Alu element” refers to a short stretch of DNA (about 300 nucleotides in length), originally characterized by the action of the Alu restriction d l d l ifi d h t i t d l l t (SINE ) Al l t the most abundant transposable elements, and can be divided into five subfamilies of related elements based upon key diagnostic nucleotide positions shared by subfamily members. Typically, an Alu element is composed of two non-identical units or arms joined in the middle by an adenosine (A)-rich linker. Typically, each Alu repeat also ends with an A-rich tail and is flanked by short direct repeat sequences. Many Alu elements have been annotated and characterized in the art (see, e.g., Dagan et al. Alu Gene: a database of Alu elements incorporated within protein‐coding genes , Nucleic Acids Research, 32(1), 2004, Pages D489–D492; Hormozdiari et al., Alu repeat discovery and characterization within human genomes Genome Res., 2011, 21: 840-849; the entire contents of each of which are incorpated herein by reference) As used herein, the term “biomarker”, used interchangeably with the term “marker”, is understood to mean a measurable characteristic that reflects in a quantitative or qualitative manner the physiological state of a cell or an organism. Said another way, biomarkers are characteristics that can be objectively measured and evaluated as indicators of normal processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Examples of biomarkers include, for example, polypeptides, peptides, polypeptide fragments, proteins, antibodies, hormones, polynucleotides, RNA or RNA fragments, microRNA (miRNAs), circular RNA (circRNAs), lipids, polysaccharides, and other bodily metabolites. In a preferred embodiment, a biomarker is a circRNA, e.g., an Alu-mediated circRNA Preferably, a biomarker of the present invention is modulated (e.g., increased or decreased level) in a cell or a biological sample from a subject or a group of subjects after having received a treatment as compared to a biological sample from a subject or group of subjects that have not received a treatment (e.g., a control). In some embodiments a biomarker of the present invention is modulated (e.g., increased or decreased level) in a biological sample from a subject or a group of subjects after having received a treatment as compared to a biological sample from the subject or group of subjects before receiving the treatment (e.g., a control). A biomarker may be differentially present at any level, but is generally present at a level that is increased relative to normal or control levels by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, b t l t 100% b t l t 110% b t l t 120% b t l t 130% b t l t 140% b at least 150%, or more; or is generally present at a level that is decreased relative to normal or control levels by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by 100% (i.e., absent). A biomarker is preferably differentially present at a level that is statistically significant (e.g., a p-value less than 0.05 and/or a q-value of less than 0.10 as determined using either Welch's T-test or Wilcoxon's rank-sum Test). As used herein, the term “biopsy” or “biopsy tissue” refers to a sample of tissue (e.g., prostate tissue) that is removed from a subject for the purpose of determining if the sample contains cancerous tissue. The biopsy tissue is then examined (e.g., by microscopy) for the presence or absence of cancer. As used herein, the term “cancer” has the meaning normally accepted in the art. The term can broadly refer to abnormal cell growth. As used herein, the term “circular RNA,” abbreviated as “circRNA,” refers to noncoding RNA characterized by a covalently closed cyclic structure lacking poly-adenylated tails. CircRNAs are mainly synthesized by the transcription of protein coding genes with RNA polymerase II (Pol II); but unlike linear RNAs, they are not produced by canonical mode of RNA splicing. CircRNA molecules are circularized by joining the 3′ and 5′ ends together with unique back-splicing (see, e.g., Wilusz, Repetitive elements regulate circular RNA biogenesis, Mob Genet Elements, 2015, 5(3):39-45; incorporated herein by reference). CircRNAs are commonly named according to their parental genes or specific functions, e.g., circular RNA derived from the BRIP1 gene is called “circBRIP1.” Several circRNA databases have been constructed to enable organization of discovered and identified circRNAs. A serial number is given to every detected back-spliced junction site. Databases like circBase (circbase.org) and CircNet (circnet. mbc.nctu.edu.tw) provide tissue-specific circRNA expression profiles as well as circRNA-miRNA-gene regulatory networks. Circ2Traits (gyanxet-beta.com/circdb) also allows user to search circRNAs by mutiple diseases. See Zhang et al. (Circular RNAs: Promissing Biomarkers for Human Diseases, EBioMedicine 34 (2018) 267–274), the entire contents of which are incorporated herein by reference. The term “Alu-medited circular RNA”, abbreviated as “Alu-circRNA” herein, refers t i RNA h f ti i i t d ith Al l t Al l t i h d i the introns flanking human exons that generate circRNAs; it is estimated that ~90% of circular RNAs appear to have complementary Alu elements in their flanking introns. Disruption of base pairing between intronic repeats by mutating several nucleotides in Alu elements have been shown to prevent circularization of certain RNAs (see, e.g., Wilusz, Repetitive elements regulate circular RNA biogenesis, Mob Genet Elements, 2015, 5(3):39- 45; the entitre contents of which are incorporated herein by reference). In some embodiments, the formation of the Alu-circRNAs of the instant disclosure are modulated by DHX9. As used herein, the term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. The term “control sample,” as used herein, refers to any relevant comparative sample, including, for example, a sample from a subject prior to treatment or from an earlier assessment time point, or a cell or a population of cells untreated with any agents. A control sample can be a purified sample and/or nucleic acid (e.g., circRNA) provided with a kit. A control sample can be a sample derived from a subject. A control sample can also be synthetically produced. The level of one or more markers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more markers) in a control sample consists of a group of measurements that may be determined, e.g., based on any appropriate statistical measurement, such as, for example, measures of central tendency including average, median, or modal values. Different from a control is preferably statistically significantly different from a control. The term “control level” refers to an accepted or pre-determined level of a marker in a sample. A control level can be a range of values. Marker levels can be compared to a single control value, to a range of control values, to the upper level of normal, or to the lower level of normal as appropriate for the assay. In some embodiments, the control level is a standardized control level, such as, for example, a level which is predetermined using an average of the levels of expression of one or more markers from a population of cells or subjects prior to receiving a treatment, e.g., prior to administration of a DHX9 inhibitor. In some embodiments, the control level is a level determined from a sample of a subject collected before the subject receiving a treatment, e.g., prior to administration of a DHX9 inhibitor. In some embodiments, the control level is a level determined from a sample of a subject collected at an earlier time point. As used herein, “detecting”, “detection”, “determining”, and the like are understood to refer to an assay performed for identification of the presence and/or level of a circRNA (e.g., circBRIP1) and/or an additional one or more specific markers in a sample. The amount of marker detected in the sample can be none or below the level of detection of the assay or method. As used herein, the term “DHX9”, RNA Helicase A (RHA) or Nuclear DNA Helicase II (NDH II) refers to a DEAH-box RNA helicase which shuttles between nucleus and cytoplasm, and can use all four NTPs to power cycles of directional movement from 3’ to 5’. Functionally, DHX9 can bind to and unwind or resolve dsDNA/RNA, ssDNA/RNA, DNA:RNA hybrids (such as R-loops), circular RNA, and DNA/RNA G quadruplexes. As such, DHX9 has regulatory roles in various RNA and DNA related cellular processes, such as transcription, translation, RNA splicing, editing, RNA transport and processing, microRNA genesis, and maintenance of genomic stability (Pan et al., 2021, Current Protein & Peptide Science (22), 29-40). The NCBI Gene ID for DHX9 is 1660. Human DHX9 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_001357.5 (transcript variant 1) and NR_033302.2 (transcript variant 2). (The GenBank number is incorporated herein by reference in the version available on the filing date of the application to which this application claims priority). DHX9 possesses ATPase activity, i.e., DHX9 has an ATPase domain that hydrolyzes ATP i t ADP It h tl b h th t DHX9 l l ti it which represses transposases (see Aktaş et al., DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome, Nature, 2017, 544:112-119; incorporated herein by reference). As used herein, the term "DNA" or "RNA" molecule or sequence (as well as sometimes the term "oligonucleotide") refers to a molecule comprised generally of the deoxyribonucleotides or ribonucleotides, respectively, of adenine (A), guanine (G), thymine (T) and/or cytosine (C), wherein in "RNA", T is replaced by uracil (U). The terms “disorder”, “disease”, and “abnormal state” are used inclusively and refer to any deviation from the normal structure or function of any part, organ, or system of the body (or any combination thereof). A specific disease is manifested by characteristic symptoms and signs, including biological, chemical, and physical changes, and is often associated with a variety of other factors including, but not limited to, demographic, environmental, employment, genetic, and medically historical factors. Certain characteristic signs, symptoms, and related factors can be quantitated through a variety of methods to yield important diagnostic information. As used herein, a sample obtained at an “earlier time point” is a sample that was obtained at a sufficient time in the past such that relevant information could be obtained in the sample from the earlier time point as compared to the later time point. In certain embodiments, an earlier time point is at least two weeks earlier, at least four weeks earlier, at least six weeks earlier, at least two months earlier, at least three months earlier, at least six months earlier, at least nine months earlier, or at least one year earlier. Multiple subject samples (e.g., 3, 4, 5, 6, 7, or more) can be obtained at regular or irregular intervals over time and analyzed for trends in changes in marker levels. Appropriate intervals for testing for a particular subject can be determined by one of skill in the art based on ordinary considerations. The term “expression” is used herein to mean the process by which an RNA or polypeptide is produced from DNA. The process involves the transcription of the gene into RNA if the marker of interest is an RNA, and further involves the translation of an mRNA into a polypeptide if the marker of interest a polypeptide or processing of the initial transcribed RNA into other subtypes of RNA, such as circRNA. Depending on the context in which used, “expression” may refer to the production of RNA, or protein, or both. The term “expression level” is used herein to mean the level (e.g., amount) of an RNA l tid t i ll bi l i l l A, “higher level”, “higher level of expression”, "higher level of induction” and the like of a marker refers to the marker’s level in a sample that is greater than the standard error of the assay employed to assess the level, and is preferably at least 25% more, at least 50% more, at least 75% more, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten times the level of the marker in a control sample, and preferably the average level of the marker or markers in several control samples. As used herein, a subject is “in need of” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment (in some embodiments, a human). As used herein, the term “inhibit”, “inhibition” or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process. As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment. The terms “level of expression of a gene”, “gene expression level”, “level of a marker”, and the like refer to the level of mRNA, as well as pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s), non-enocoding RNA products (e.g., circRNA) and degradation products, or the level of protein, encoded by the gene in the cell. The “level” of one of more biomarkers means the absolute or relative amount or concentration of the biomarkers in a sample. A “lower level”, “lower level of expression” or “lower level of induction” of a marker refers to the marker’s level in a sample that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the level of the marker in a control sample, and preferably the average level of the marker in several control samples. As used herein, "nucleic acid molecule" or "polynucleotides", refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA, circRNA) and chimeras thereof. The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single- stranded (coding strand or non-coding strand [antisense]). Conventional ribonucleic acid (RNA) d d ib l i id (DNA) i l d d i th t " l i id" d polynucleotides as are analogs thereof. A nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide- nucleic acid bonds (referred to as "peptide nucleic acids" (PNA); Hydig-Hielsen et al., PCT Intl Pub. No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages or combinations thereof. Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy substitutions (containing a 2'-O-methylribofuranosyl moiety; see PCT No. WO 98/02582) and/or 2' halide substitutions. Nitrogenous bases may be conventional bases (A, G, C, T, U), known analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992), or known derivatives of purine or pyrimidine bases (see, Cook, PCT Int'l Pub. No. WO 93/13121) or "abasic" residues in which the backbone includes no nitrogenous base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481). A nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs). An "isolated nucleic acid molecule", as is generally understood and used herein, refers to a polymer of nucleotides, and includes, but should not limited to DNA and RNA. The "isolated" nucleic acid molecule is purified from its natural in vivo state, obtained by cloning or chemically synthesized. As used herein, the term “obtaining” is understood herein as manufacturing, purchasing, or otherwise coming into possession of a physical entity or a value, e.g., a numerical value. "Obtaining a sample," as the term is used herein, refers to obtaining possession of a sample, e.g., a tissue sample or cell sample, by "directly acquiring" or "indirectly acquiring" the sample. "Directly acquiring a sample" means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample. "Indirectly acquiring a sample" refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample). As used herein, "oligonucleotides" or "oligos" define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill. An oligonucleotide can be synthesized chemically or derived by cloning according to well-known methods. While they are usually in a single-stranded form, th b i d bl t d d f d t i " l t i " Th contain natural rare or synthetic nucleotides. They can be designed to enhance a chosen criteria like stability for example. Chimeras of deoxyribonucleotides and ribonucleotides may also be within the scope of the present invention. As used herein, a “patient,” “subject” or “individual” are used interchangeably and refer to either a human or non-human animal. The term includes mammals such as humans. Typically, the animal is a mammal. A subject also refers to for example, non-human primates (e.g., monkey, male or female), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a primate. In some embodiments, the subject is a human. As used herein, the term “peripheral blood mononuclear cells,” abbreviated as “PBMCs”, refers to cells isolated from peripheral blood and identified as any blood cell with a round nucleus (i.e., lymphocytes, monocytes, natural killer cells (NK cells) or dendritic cells). PBMCs can be isolated from whole blood as follows, or through equivalent methods: The cell fraction corresponding to red blood cells and granulocytes (neutrophils, basophils and eosinophils) is removed from whole blood by density gradient centrifugation. A gradient medium with a density of ~1.077 g/ml separates whole blood into two fractions; PBMCs makes up the population of cells that remain in the low density fraction (upper fraction), whilst red blood cells and PMNs have a higher density and are found in the lower fraction. PBMCs include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. In humans, the frequencies of these populations vary across individuals, but typically, lymphocytes are in the range of 70–90 %, monocytes from 10 to 20 %, while dendritic cells are rare, accounting for only 1–2 %. See Kleiveland et al. (Peripheral Blood Mononuclear Cells. In: Verhoeckx K, Cotter P, López-Expósito I, et al., editors. The Impact of Food Bioactives on Health: in vitro and ex vivo models [Internet]. Cham (CH): Springer; 2015. Chapter 15), incorporated herein by reference.. The phrase “pharmaceutically acceptable” indicates that the substance, composition or dosage form must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith. As used herein, a “predetermined threshold value” or “threshold value” of a biomarker refers to the level of the biomarker (e.g., the expression level or quantity in a biological sample) in a corresponding control sample or group of control samples (e.g., an average level or mean level of the biomarker in the group of control samples). The d t i d th h ld l b d t i d i t tl ith t of marker levels in a biological sample. The control sample may be from the same subject at a previous time or from different subjects. As used herein, “prophylactic” or “therapeutic” treatment refers to administration to the subject of one or more agents or interventions to provide the desired clinical effect. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing at least one sign or symptom of the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or maintain at least one sign or symptom of the existing unwanted condition or side effects therefrom). As used herein, a "reference level" of a biomarker means a level of the biomarker that is indicative of therapeutic efficacy of a treatment, e.g., with a DHX9 inhibitor, or lack thereof. A "positive" reference level of a biomarker means a level that is indicative of a treatment, e.g., with a DHX9 inhibitor, having one or more therapeutic effects in a subject. A "negative" reference level of a biomarker means a level that is indicative of a lack of therapeutic effects from a treatment, e.g., with a DHX9 inhibitor. As used herein, “sample” or “biological sample” includes a specimen or culture obtained from any source. Biological samples can be obtained from blood (including any blood product, such as whole blood, plasma, serum, or specific types of cells of the blood), interstitial fluid, lymph fluid, urine, saliva, and the like. Biological samples also include tissue samples, such as biopsy tissues or pathological tissues that have previously been fixed (e.g., formaline snap frozen, cytological processing, etc.). In an embodiment, the biological sample is from blood. In another embodiment, the biological sample is a biopsy tissue from a tumor. The term “therapeutic effect” refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or in the enhancement of desirable physical or mental development and conditions in an animal or human. A therapeutic effect can be understood as a decrease in tumor growth, decrease in tumor growth rate, stabilization or decrease in tumor burden, stabilization or reduction in tumor size, stabilization or decrease in tumor malignancy, increase in tumor apoptosis, and/or a decrease in tumor angiogenesis. As used herein, “therapeutically effective amount” means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease, e.g., the amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment, e.g., is sufficient to ameliorate at least one sign or symptom of the disease, e.g., to prevent progression of the disease or condition, e.g., prevent tumor growth, decrease tumor size, induce tumor cell apoptosis, reduce tumor angiogenesis, prevent metastasis. When administered for preventing a disease, the amount is sufficient to avoid or delay onset of the disease. The “therapeutically effective amount” will vary depending on the compound, its therapeutic index, solubility, the disease and its severity and the age, weight, etc., of the patient to be treated, and the like. For example, certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment. Administration of a therapeutically effective amount of a compound may require the administration of more than one dose of the compound. As used herein, the term “treat”, “treating” or “treatment” of any disease, condition or disorder, refers to the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of the present disclosure to obtaining desired pharmacological and/or physiological effect. The effect can be therapeutic, which includes achieving, partially or substantially, one or more of the following results: partially or totally reducing the extent of the disease, condition or disorder; ameliorating or improving a clinical symptom, complications or indicator associated with the disease, condition or disorder; or delaying, inhibiting or decreasing the likelihood of the progression of the disease, condition or disorder; or eliminating the disease, condition or disorder. In certain embodiments, the effect can be to prevent the onset of the symptoms or complications of the disease, condition or disorder. Reference will now be made in detail to exemplary embodiments of the invention. While the invention will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the invention to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. II. Methods and Assays CircRNAs are present in small amounts in biological samples, and thought to be not abundant enough to allow for their use as meaningful biomarkers. The methods and assays described herein are based, in part, on the suprising findings that levels of Alu-mediated circular RNAs are detectable in biological samples and that their changes correlate with DHX9 inhibition in vitro and in vivo. Thus, provided herein are assays and methods for detecting, measuring, and monitoring activity of a DHX9 inhibitor in vitro and/or in vivo. In some aspects, the present disclosure provides methods for detecting inhibition of DHX9 in a cell, or a population of cells, comprising contacting the cell or population of cells with a DHX9 inhibitor, and detecting or measuring the level of an Alu-mediated circular RNA (Alu-circRNA) in the cell or population of cells. In some embodiments, the cells are maintained in cell culture in vitro. In some embodiments, the cell is from a primary cell line. In some embodiments, the cell is from an established or immortalized cell line. In some embodiments, the cell is an ex vivo cell isolated from a subject. In some embodiments, the cell is isolated from a biological sample from a subject, e.g., blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue. In some embodiments, the cell is contacted with the DHX9 in vivo. In some embodiments, the cell is a normal, non-cancerous cell. In some embodiments, the cell is a blood cell. In preferred embodiments, the cell is a peripheral blood mononuclear cell (PBMC). In preferred embodiments, the population of cells comprises or consists of PBMCs. In some embodiments, the cell or population of cells are a cancer cell. In some embodiments, the cancer is selected from the group consisting of, but not limited to, colorectal, endometrial, ovarian, gastric, hematopoietic, breast, brain, skin, lung, blood, prostate, head and neck, pancreatic, bladder, bone, soft tissue, kidney, liver, and Ewing’s sarcoma. In some embodiments, the cancer cell is derived from a cancer that has microsatellite instability (MSI). In some embodiments, the cancer cell is characterized to have genomic instability. In some embodiments, the cancer cell is defective in mismatch repair. In some embodiments, cancer cell having genomic instability is selected for use in the methods and f th di l In some embodiments, the cancer cell is isolated from a tumor from a subject. In some embodiments, the cancer cell is isolated from the blood of the a subject having a hematopoeitic cancer. In some embodiments, the cells are contacted with one or more amounts or concentrations of a DHX9 inhibitor, and the levels of the Alu-circRNA is measured or determined for each amount or concentration of the inhibitor. In some embodiments, the cells are contacted with a DHX9 inhibitor for a period of time sufficient for the inhibitor to inhibit DHX9 expression. In some embodiments, the cells are contacted with a DHX9 inhibitor for a period of time sufficient for the inhibitor to inhibit DHX9 activity. In some embodiments, the cell or populations of cells is contacted with a DHX9 inhibitor for a period of time sufficient for the Alu-circRNA to be produced, e.g., to detectable levels. In some embodiments, the level of the Alu-circRNA is measured or determined between 1 to 24 hours, between 2 to 24 hours, between 4 to 24 hours, between 6 to 24 hours, between 8 to 24 hours, between 10 to 24 hours, between 12 to 24 hours, between 14 to 24 hours, between 16 to 24 hours, between 18 to 24 hours, between 20 to 24 hours, between 22 to 24 hours, between 1 to 20 hours, between 2 to 20 hours, between 4 to 20 hours, between 6 to 20 hours, between 8 to 20 hours, between 10 to 20 hours, between 12 to 20 hours, between 14 to 20 hours, between 16 to 20 hours, between 18 to 20 hours, between 1 to 16 hours, between 2 to 16 hours, between 4 to 16 hours, between 6 to 16 hours, between 8 to 16 hours, between 10 to 16 hours, between 12 to 16 hours, between 14 to 16 hours, between 1 to 12 hours, between 2 to 12 hours, between 4 to 12 hours, between 6 to 12 hours, between 8 to 12 hours, between 10 to 12, 1 to 8 hours, between 2 to 8 hours, between 4 to 8 hours, or between 6 to 8 hours after the cell is contacted with the DHX9 inhibitor. In some embodiments, the level of the Alu-circRNA is measured or determined at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, 102 hours, 108 hours, 114 hours or 120 hours after the cell is contacted with the DHX9 inhibitor. In some embodiments, the level of the Alu-circRNA is measured or determined at least 1 day, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, t l t 4 d t l t 45 d t l t 5 d t l t 55 d t l t 6 d t l t 65 days, or at least 7 days after the cell is contacted with the DHX9 inhibitor. In some embodiments, the level of the Alu-circRNA is measured or determined about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, or about 7 days after the cell is contacted with the DHX9 inhibitor. In some embodiments, the cells are contacted with a DHX9 inhibitor, and the levels of the Alu-circRNA is measured or determined at multiple different times after the cells are first contacted with the inhibitor. In some embodiments, the methods further comprise measuring or determining the level of the Alu-circRNA in cells that have not been contacted, or prior to contact, with the DHX9 inhibitor, and this level of the Alu-circRNA is used to establish a control level. In other aspects, the present disclosure generally provides methods for detecting or measuring inhibition of DHX9 in a subject. The present disclosure further generally provides methods for detecting engagement of DHX9 by a DHX9 inhibitor in a subject. The methods comprise detecting or measuring the level of an Alu-circRNA in a biological sample obtained from subject. The subject is scheduled to be administered, is undergoing treatment with, or has recently been administered a DHX9 inhibitor. In some embodiments, the sample is obtained from the subject following administration of a DHX9 inhibitor. In some embodiments, the method further comprises administering a DHX9 inhibitor to the subject. The present disclosure provides a method for measuring or detecting activity of a DHX9 inhibitor in a subject, comprising administering a DHX9 inhibitor to the subject, and measuring or detecting the level of an Alu-circRNA in a biological sample from the subject. The present disclosure also provides a method for measuring or detecting engagement of DHX9 by a DHX9 inhibitor in a subject, comprising administering a DHX9 inhibitor to the subject, and measuring or detecting the level of an Alu-circRNA in a biological sample from the subject. The present disclosure also provides a method for measuring or detecting activity of a DHX9 inhibitor in a subject, comprising measuring or detecting the level of an Alu-circRNA in a biological sample from the subject, wherein the subject is being treated with (i.e., has been administered) the DHX9 inhibitor. In some embodiments, the method further comprises the step of administering the DHX9 inhibitor to a subject. The present disclosure also provides a method for measuring or detecting engagement of DHX9 by a DHX9 inhibitor in a bj t i i i d t ti th l l f Al i RNA i bi l i l sample from the subject, wherein the subject is being treated with (i.e., has been administered) the DHX9 inhibitor. In some embodiments, the method further comprises the step of administering the DHX9 inhibitor to a subject. The present disclosure also provides a method for monitoring the treatment of a subject with a DHX9 inhibitor, comprising obtaining information as to the level of Alu- circRNA in a biological sample obtained from the subject after administration of the DHX9 inhibitor. In some embodiments, the method further comprises the step of administering the DHX9 inhibitor to the subject. In certain embodiments, the subject is a mammal, such as a human, a monkey, a cow, a sheep, a goat, a horse, a dog, a cat, a rabbit, a rat, or a mice. In some embodiments, the subject is a human. In some embodiments, the subject is a healthy, normal subject. In some embodiments, the subject has a cancer. In some embodiments, the subject is an animal model of a cancer, e.g., a mouse having a human xenograft tumor. In some embodiments, the cancer is a microsatellite instability (MSI) cancer. In some embodiments, the cancer is microsatellite-instability high (MSI-H). In some embodiments, the cancer is defective in mismatch repair. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, endometrial cancer, ovarian cancer, gastric cancer, hematopoietic cancer, breast cancer, brain cancer, skin cancer, lung cancer, blood cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, bone cancer, soft tissue cancer, kidney cancer, liver cancer, and Ewing’s sarcoma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is colorectal cancer with microsattelite instability (MSI-CRC). In certain embodiments, the biological sample is selected from the group consisting of blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue. In some embodiments, the biological sample is blood. In some embodiments, the biological sample is cells isolated from blood. In some embodiments, the biological sample is peripheral blood mononuclear cells (PBMC). In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMC). In some embodiments, the biological sample consists of peripheral blood mononuclear cells (PBMC). In some embodiments, the methods further comprise obtaining the biological sample f th bj t I b di t th th d f th i i l ti t i ll population from the biological sample. In some embodiments, the methods may further comprise isolating PBMCs from a blood sample. The cell population can be isolated from the biological sample based on physical properties (e.g., size, density, buoyancy), or based on specific biomarkers. In some embodiments, the methods comprise measuring or determining the level of the Alu-circRNA in a whole blood sample. In some embodiments, the methods comprise measuring or determining the level of the Alu-circRNA in a certain cell population of the biological sample, e.g., PBMCs in a blood sample. In some embodiments, the methods comprise measuring the level of the Alu-circRNA in PBMCs. In some embodiments, the DHX9 inhibitor is administered more than once to the subject, i.e., multiple doses of the DHX9 inhibitor are administered to the subject. In some embodiments, the biological sample is obtained from the subject following administration of the DHX9 inhibitor. In some embodiments, the biological sample is obtained after administration of multiple doses of the DHX9 inhibitor. In some embodiments, the level of the Alu-cirRNA is measured in biological samples obtained from the subject at more than one time point. In some embodiments, the biological sample is obtained following each administration of the DHX9 inhibitor. In some embodiments, biological samples are obtained from the subject at multiple time points after administration of a dose of the DHX9 inhibitor. In some embodiments, the biological sample is obtained between 1 to 24 hours, between 2 to 24 hours, between 4 to 24 hours, between 6 to 24 hours, between 8 to 24 hours, between 10 to 24 hours, between 12 to 24 hours, between 14 to 24 hours, between 16 to 24 hours, between 18 to 24 hours, between 20 to 24 hours, between 22 to 24 hours, between 1 to 20 hours, between 2 to 20 hours, between 4 to 20 hours, between 6 to 20 hours, between 8 to 20 hours, between 10 to 20 hours, between 12 to 20 hours, between 14 to 20 hours, between 16 to 20 hours, between 18 to 20 hours, between 1 to 16 hours, between 2 to 16 hours, between 4 to 16 hours, between 6 to 16 hours, between 8 to 16 hours, between 10 to 16 hours, between 12 to 16 hours, between 14 to 16 hours, between 1 to 12 hours, between 2 to 12 hours, between 4 to 12 hours, between 6 to 12 hours, between 8 to 12 hours, between 10 to 12, 1 to 8 hours, between 2 to 8 hours, between 4 to 8 hours, or between 6 to 8 hours after administration of the DHX9 inhibitor.In some embodiments, the biological sample is obtained at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 h 11 h 12 h 13 h 14 h 15 h 16 h 17 h 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, 102 hours, 108 hours, 114 hours or 120 hours after administration of the DHX9 inhibitor. In some embodiments, the level of the Alu-circRNA is measured or determined at least 0.5 days, at least 1 day, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days , at least 6.5 days, or at least 7 days after administration of the DHX9 inhibitor. In some embodiments, the biological sample is obtained about 0.5 days, about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, or about 7 days after administration of the DHX9 inhibitor. In some embodiments, the methods further comprise obtaining a biological sample from the subject prior to administration of the DHX9 inhibitor, and the level of the Alu- circRNA in this sample is used to establish a control level. In certain embodiments, the DHX9 inhibitor is administered to the subject orally, subcutaneously, or intravenously. It will be appreciated that the measurements of the level of the Alu-circRNA obtained from the methods and assays of the disclosure are useful for evaluating the activity or efficacy of a DHX9 inhibitor in vitro and in vivo. For example, in some embodiments, the level of the Alu-circRNA determined using the methods of the present disclosure is correlated (e.g., directly correlated) to inhibition of DHX9 activity by the DHX9 inhibitor in the cell or in the subject. In some embodiments, the level of the Alu-circRNA is correlated (e.g., directly correlated) to inhibition of the ATPase activity of DHX9. In some embodiments, the level of the Alu-circRNA is correlated (e.g., directly correlated) to inhibition of the resolvase activity of DHX9. In some embodiments, the level of the Alu-circRNA is correlated to the inhibition of proliferation of a cancer cell, for example an MSI cancer cell, by the DHX9 inhibitor. In some embodiments, the level of the Alu-circRNA is correlated to the inhibition of growth of an MSI cancer in a subject by the DHX9 inhibitor. In some embodiments, the level of the Alu-circRNA is not correlated to the inhibition of growth of PBMCs in a subject by the DHX9 inhibitor. In various embodiments, the cell can be an isolated cell, a cell in vitro, a cell ex vivo, or a cell in vivo in a subject. It will further be appreciated that the level of Alu-circRNA determined by the methods and assays of the instant disclosure is useful to determine if a DHX9 inhibitor (e.g., a candidate inhibitor) is engaging the DHX9 target, and/or if the DHX9 inhibitor is effective at inhibiting DHX9 activity, in a cell or in a subject. In a non-limiting example, the Alu-circRNA level determined for a cell or cell population after treatment with a candidate DHX9 inhibitor is compared to the Alu-circRNA level determined for a control cell or cell population not treated with a DHX9 inhibitor (e.g., a control level). If the level of the Alu-circRNA in the treated cells is increased as compared to the level of the Alu-circRNA in the control cells, the candidate DHX9 inhibitor is determined to engage the DHX9 target, and/or to inhibit DHX9 activity, in the cells. Similarly, the Alu-circRNA level determined for a biological sample obtained from a subject after treatment with a candidate DHX9 inhibitor is compared to the Alu-circRNA level determined for a control sample from a subject not treated with a DHX9 inhibitor (e.g., a control level). If the level of the Alu-circRNA in the sample obtained after treatment with the candidate DHX9 inhibitor is increased as compared to the level of the Alu-circRNA in the control sample, the candidate DHX9 inhibitor is determined to engage the DHX9 target, and/or to inhibit DHX9 activity, in the subject. It will be further appreciated that Alu-circRNA levels can be determined and compared among different candidate DHX9 inhibitors to identify a candidate inhibitor that is more or less effective at engaging the DHX9 target and/or inhibiting DHX9 activity in vitro or in vivo. In some embodiments, if the same dosing regimen is utilized for two or more inhibitors, an inhibitor that induces a higher Alu-circRNA level may be considered to be more effective at engaging the DHX9 target and/or inhibiting DHX9 activity than an inhibitor that induces a lower Alu-circRNA level, and an inhibitor that induces a lower Alu-circRNA level may be considered to be less effective at engaging the DHX9 target and/or inhibiting DHX9 activity than an an inhibitor that induces a higher Alu-circRNA level. In some embodiments, reference levels can be established for an Alu-cirRNA marker, and levels of the Alu-circRNA induced by a DHX9 inhibitor can be compared to the reference levels to make a determination of DHX9 engagement and/or efficacy of the DHX9 inhibitor. In certain embodiments, the level of Alu-circRNA determined using the methods and assays of the instant disclosure is useful in determining an appropriate dose, e.g., a th ti ll ff ti d i i ll ff ti d f th DHX9 i hibit Th l l of Alu-circRNA determined using the methods and assays of the instant disclosure is also useful in determining an effective therapeutic treatment regimen for the DHX9 inhibitor. DHX9 Inhibitors The methods provided in the present disclosure are applicable to any DHX9 inhibitors, and are not limited to the exemplary DHX9 inhibitors described herein. In some embodiments the DHX9 inhibitors are previously known in the art, and have been shown to reduce or inhibit the expression or activity of DHX9 in vitro or in vivo. In some embodiments the DHX9 inhibitors are candidate DHX9 inhibitors under discovery and testing for their ability to reduce or inhibit expression or activity of DHX9 in vitro or in vivo. In some embodiments, the DHX9 inhibitor is selected from a small molecule, an antisense oligonucleotide, or an RNA interfering agent such as small intering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA). In some embodiments, the DHX9 inhibitor is a small molecule. A small molecule inhibitor is typically an organic compound with low molecular weight that has high cell penetration. In some embodiments, the small molecule inhibitor of DHX9 is any DHX9 small molecule inhibitor found in the art. Exemplary DHX9 small molecule inhibitors and methods of synthesis thereof can be found at least in international PCT applications PCT/US2023/013303 and PCT/US2023/012929, the entire contents of each of which are incorpoated herein by reference. In some embodiments, the DHX9 inhibitor is selected from the compounds listed in Table 1. Table 1. Exemplary DHX9 small molecule inhibitors
Figure imgf000026_0001
O
Figure imgf000027_0001
Methods for preparing the compounds listed in Table 1 are provided below. Abbreviations: DMSO = dimethylsulfoxide HATU = 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HPLC = high pressure liquid chromatography LCMS = liquid chromatography mass spectrometry TFA = Trifluoroacetic acid AcOK = KOAc = potassium acetate Pd(dppf)Cl2 = [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) K2CO3 = potassium carbonate LiOH = lithium hydroxide TCFH = N′-tetramethylformamidinium hexafluorophosphate K3PO4 = potassium phosphate tBuOH = tert-butanol HCl = hydrochloric acid NMI = N-methylimidazole ACN = acetonitrile DCM = dichloromethane DMF = N,N-dimethylformamide EA = ethyl acetate PE = petroleum ether H2O = water EtOH = ethanol General Methods: 1. 1H NMR spectra were recorded on any of the following: NMR10 Bruker AVANCE Ⅲ HD 300MHz NMR16 Bruker AVANCE Ⅲ HD 300MHz NMR19 Bruker AVANCE Ⅲ HD 400MHz NMR24 Bruker AVANCE NEO 400MHz NMR30 Bruker AVANCE NEO 400MHz Bruker AVβ 400 2. LCMS measurement was run on SHIMADZU LCMS-2020 or Agilent 1200 HPLC/6100 SQ System using the follow conditions: Method A: Mobile Phase: A: Water (0.05%TFA) B: Acetonitrile (0.05%TFA); Gradient Phase: 5%B to 100%B within 2.0 min, 100%B with 0.7 min (total runtime: 2.8 min); Flow Rate: 1.5 mL/min; Column: HALO C18, 3.0*30mm, 2.0µm; Column Temperature: 40 ºC. Detectors: AD2 ELSD, PDA (220 nm and 254 nm), ESI. Method B: Mobile Phase: A: Water (0.1%FA) B: Acetonitrile (0.1%FA); Gradient Phase: 5%B to 100%B within 2.0 min, 100%B with 0.7 min (total runtime: 2.8 min); Flow Rate: 1.5 mL/min; Column: HALO C18, 3.0*30mm, 2.0µm; Column Temperature: 40 ºC. Detectors: AD2 ELSD, PDA (220 nm and 254 nm), ESI. Method C: Mobile Phase: A: Water (5mM NH4HCO3) B: Acetonitrile; Gradient Phase: 10%B to 95%B within 2.0 min, 100%B with 0.6 min (total runtime: 2.8 min); Flow Rate: 1.5 mL/min; Column: Poroshell HPH-C18, 3.0*50mm, 4.0µm; Column Temperature: 40 ºC. Detectors: AD2 ELSD, PDA (220 nm and 254 nm), ESI. Method D: Mobile Phase: A: Water (0.01%TFA) B: Acetonitrile (0.01%TFA); Gradient Phase: 5%B to 95%B within 1.4 min, 95%B with 1.6 min (total runtime: 3 min); Flow Rate: 2.0 mL/min; Column: SunFire C18, 4.6*50mm, 3.5µm; Column Temperature: 40 ºC. Detectors: ADC ELSD, DAD (214 nm and 254 nm), ES-API. Preparation of Intermediate A The compounds claimed herein were prepared following the procedures outlined in the following schemes. Compound names were generated using the software built into ChemDraw. To the extent that there are discrepancies between the name of a compound and its depicted structure, the depicted chemical structure is to be taken as the appropriate compound. Intermediate A: N-(3-amino-5-chlorophenyl)methanesulfonamide
Figure imgf000029_0001
Step 1: To a stirred mixture of 1-chloro-3-fluoro-5-nitrobenzene (45 g, 256.35 mmol, 1.00 equiv) and methane sulfonamide (24.38 g, 256.35 mmol, 1.00 equiv), Cs2CO3 (250.57 g, 769.05 mmol, 3.00 equiv) in 500 mL of DMSO, the resulted solution was stirred for 2h at 60 ºC. The mixture was cooled then quenched with 1000 mL of water and extracted with 3 x 1000 mL of ethyl acetate and dried over anhydrous Na2SO4 and concentrated and the residue was purified onto silica gel column eluted with 50% of ethyl acetate in petroleum ether to afford N-(3-chloro-5-nitrophenyl) methane sulfonamide (41 g, 63.81%) as a light yellow solid. LCMS (ESI) [M + H]+: 249.9 Step 2: To a stirred solution of N-(3-chloro-5-nitrophenyl) methanesulfonamide (41.00 g, 163.57 mmol, 1.00 equiv) and Fe (91.35 g, 1635.75 mmol, 10.00 equiv), NH4Cl (87.50 g, 1635.75 mmol, 10.00 equiv) in 500 mL of ethanol and 300 mL of water, this was stirred for 2h at 90 ºC. The resulting mixture was filtered, the filter cake was washed with 6 x 50 mL of methanol. The filtrate was concentrated under reduced pressure. The residue was purified onto silica gel column eluted with 50% of ethyl acetate in petroleum ether to afford N-(3- amino-5-chlorophenyl) methanesulfonamide (25.3 g, 70.1%) as a light yellow solid. LCMS (ESI) [M + H]+: 220.01 1H NMR (300 MHz, DMSO-d6) δ 9.64 (s, 1H), 6.41 – 6.31 (m, 3H), 5.54 (s, 2H), 2.98 (s, 3H) Intermediate E: N-(3-amino-5-chlorophenyl)propane-1-sulfonamide
Figure imgf000030_0001
A solution of 3-bromo-5-chloroaniline (1 g, 4.843 mmol, 1 equiv), propane-1-sulfonamide (1.19 g, 9.686 mmol, 2 equiv), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (2.07 g, 14.529 mmol, 3 equiv), CuI (2.77 g, 14.529 mmol, 3 equiv) and K2CO3 (2.01 g, 14.529 mmol, 3 equiv) in ACN (10 mL) was stirred for 2 hours at 110°C under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE / EA (1:1) to afford N-(3-amino-5- chlorophenyl)propane-1-sulfonamide (540 mg, 45% yield) as a yellow solid. LCMS (ESI) [M + H]+: 249 Compound 1: N-(3-chloro-5-methanesulfonamidophenyl)-4-(3-methylpyridin-2- yl)thiophene-2-carboxamide
Figure imgf000031_0001
Step 1: A solution of methyl 4-bromothiophene-2-carboxylate (150 g, 678.52 mmol, 1.00 equiv), Pd(dppf)Cl2 (24.82 g, 33.93 mmol, 0.05 equiv), AcOK (199.77 g, 2035.55 mmol, 3.00 equiv) and bis(pinacolato)diboron (206.76 g, 814.22 mmol, 1.2 equiv) in 1000 mL of dioxane. This was stirred for 2 hours at 80 ºC under nitrogen atmosphere. The resulting mixture was cooled and concentrated. The residue was purified onto silica gel column eluted with 10% of ethyl acetate in petroleum ether to afford methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl) thiophene-2-carboxylate (170 g, 92.5%) as a yellow solid. Step 2: A solution of methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophene-2- carboxylate (170 g, 634.02 mmol, 1.00 equiv), Pd(dppf)Cl2 (23.20 g, 31.70 mmol, 0.05 equiv), K2CO3 (262.87 g, 1902.06 mmol, 3.00 equiv) and 2-bromo-3-methylpyridine (109.07 g, 634.02 mmol, 1.00 equiv) in 1000 mL of 1,4-dioxane and 200 mL of water, this was stirred for 2 hours at 90 °C under nitrogen atmosphere. The resulting mixture was cooled and concentrated. The residue was purified onto silica gel column chromatography, eluted with 50% of ethyl acetate in petroleum ether to afford methyl 4-(3-methylpyridin-2-yl)thiophene - 2-carboxylate (115 g, 77%) as a yellow solid. LCMS (ESI) [M + H]+: 234 Step 3: To a stirred solution of methyl 4-(3-methylpyridin-2-yl)thiophene-2-carboxylate (115 g, 492.95 mmol, 1.00 equiv), LiOH (118.06 g, 4929.49 mmol, 10.00 equiv) in 250 mL of ethanol and 250 mL of water. Then the mixture was stirred for 2 hours at room temperature. The mixture was adjusted pH to 5 with citric acid. The precipitated solids were collected by filtration and washed with water. The resulted solid was dried under vacuum to afford 4-(3- methylpyridin-2-yl)thiophene-2-carboxylic acid (60 g, 54.9%) as a yellow solid. LCMS (ESI) [M + H]+: 220 Step 4: To a stirred solution of 4-(3-methylpyridin-2-yl)thiophene-2-carboxylic acid (60 g, 273.65 mmol, 1.00 equiv), TCFH (115.17 g, 410.47 mmol, 1.50 equiv) and NMI (67.40 g, 820.94 mmol, 3.00 equiv) in 1000 mL of acetonitrile, to this was added N-(3-amino-5- chlorophenyl)methanesulfonamide (50 g, 226.58 mmol, 0.83 equiv).Then the mixture was stirred for 2 hours at room temperature. The mixture was concentrated and purified by reverse flash chromatography eluting with 56% acetonitrile in water (0.05% FA) to afford N- (3-chloro-5-methanesulfonamidophenyl)-4-(3-methylpyridin-2-yl)thiophene-2-carboxamide (49.6634 g, 42.8%) as a white solid. LCMS (ESI) [M + H]+: 422. 1H NMR (300 MHz, Methanol-d4) δ 8.49 – 8.41 (m, 1H), 8.21 (d, J = 1.4 Hz, 1H), 7.99 (d, J = 1.4 Hz, 1H), 7.85 – 7.75 (m, 1H), 7.69 (t, J = 1.9 Hz, 1H), 7.60 (t, J = 1.9 Hz, 1H), 7.35 (dd, J = 7.7, 4.9 Hz, 1H), 7.05 (t, J = 2.0 Hz, 1H), 3.06 (s, 3H), 2.50 (s, 3H). Compound 2: N-(3-chloro-5-methanesulfonamidophenyl)-5-(3,5-difluoropyridin-2-yl)-1- methyl-1H-pyrrole-3-carboxamide
Figure imgf000032_0001
Step 1: To a mixture of methyl 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H- pyrrole-3-carboxylate (116 g, 440 mmol, 1 equiv), 2-bromo-3,5-difluoropyridine (85 g, 440 mmol, 1 equiv), Pd(dppf)Cl2 (18 g, 22 mmol, 0.05 equiv) and K3PO4 (200 g, 880 mmol, 2 equiv) was added dioxane (1 L) and H2O (100 mL). The resulting mixture was stirred for 2 hours at 80 °C under N2 atmosphere The reaction mixture was extracted with ethyl acetate The organic phase was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 30% of ethyl acetate in petroleum ether to afford methyl 5-(3,5-difluoropyridin-2-yl)-1-methyl-1H-pyrrole-3-carboxylate (98g, 88.6% yield) as a white solid. Step 2: To a stirred solution of methyl 5-(3,5-difluoropyridin-2-yl)-1-methyl-1H-pyrrole-3- carboxylate (295 g, 1165 mmol, 1 equiv) in tBuOH (3 L) and water (2 L) was added LiOH (246g, 5853 mmol, 5 equiv, in 1L water). The mixture was stirred for 4 hours at 60 °C. The pH value of the solution was adjusted to 3 with 1M of HCl (aq). The product was precipitated from the solution. The mixture was filtered and the filter cake was washed with water (3 x 200 mL) to afford 5-(3,5-difluoropyridin-2-yl)-1-methyl-1H-pyrrole-3-carboxylic acid (212 g, 76.0% yield) as a white solid. LCMS [M+H]+: 239. Step 3: A mixture of 5-(3,5-difluoropyridin-2-yl)-1-methyl-1H-pyrrole-3-carboxylic acid (212 g, 886 mmol, 1 equiv), TCFH (341 g, 1219 mmol, 1.37 equiv), NMI (199 g, 2438 mmol, 2.75 equiv) and N-(3-amino-5-chlorophenyl)methanesulfonamide (214 g, 487 mmol) in ACN (2 L) was stirred for two hours at room temperature. The mixture was quenched with water (2 L). The resulting mixture was extracted with DCM (3 x 3 L) and water. The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM / ACN (5:1) to afford N-(3-chloro-5- methanesulfonamidophenyl)-5-(3,5-difluoropyridin-2-yl)-1-methyl-1H-pyrrole-3- carboxamide (155g, 39.5% yield) as a white solid. LCMS [M+H]+: 441. 1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 9.90 (s, 1H), 8.59 (d, J = 2.4 Hz, 1H), 8.08 (ddd, J = 11.1, 8.9, 2.5 Hz, 1H), 7.79 – 7.69 (m, 2H), 7.64 (t, J = 1.9 Hz, 1H), 7.21 (dd, J = 3.6, 1.9 Hz, 1H), 6.91 (t, J = 2.0 Hz, 1H), 3.89 (s, 3H), 3.06 (s, 3H).
Compound 3: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-isopropoxypyrimidin-2- yl)-5-methylthiophene-2-carboxamide
Figure imgf000034_0001
Step 1: A mixture of 2-bromopyrimidin-5-ol (15.0 g, 85.7 mmol, 1 equiv), K2CO3 (35.48 g, 257.1 mmol, 3 equiv) and 2-bromopropane (13.71 g, 111.5 mmol, 1.3 equiv) in DMF (150 mL) was stirred for 5 hours at room temperature. The reaction mixture was diluted with water (1 L). The resulting solution was extracted with EA (3 x 800 mL) and the organic layers were combined. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography on silica gel column eluting with EA/PE (20%) to afford 2-bromo-5-isopropoxypyrimidine (15.0 g, 81% yield) as a white solid. LCMS (ESI) [M + H]+: 217 Step 2: A mixture of 2-bromo-5-isopropoxypyrimidine (14.0 g, 64.5 mmol, 1 equiv), Pd(dppf)Cl2 (3.3 g, 4.5 mmol, 0.07 equiv), K3PO4 (41.1 g, 193.5 mmol, 3 equiv) and methyl 5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) thiophene-2-carboxylate (23.66 g, 83.8 mmol, 1.3 equiv) in 1,4-dioxane (150 mL) and H2O (15 mL) was stirred for 2 hours at 80 °C under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE / EA (32%) to afford methyl 4-(5-isopropoxypyrimidin-2-yl)-5-methylthiophene-2-carboxylate (12.70 g, 67% yield) as a white solid. LCMS (ESI) [M + H]+: 293 Step 3: To a stirred solution of methyl 4-(5-isopropoxypyrimidin-2-yl)-5-methylthiophene-2- carboxylate (12.7 g, 43.4 mmol, 1 equiv) in EtOH (150 mL) and H2O (150 mL) was added LiOH (10.4 g, 434.4 mmol, 10 equiv). Then the mixture was stirred for 4 hours at room temperature. The mixture was acidified to pH 5 with HCl (aq.). The precipitated solids were collected by filtration and washed with water. The resulting solid was dried under vacuum to afford 4-(5-isopropoxypyrimidin-2-yl)-5- methylthiophene-2-carboxylic acid (10.4 g, 86% yield) as a white solid. LCMS (ESI) [M + H]+: 279 Step 4: To a stirred solution of 4-(5-isopropoxypyrimidin-2-yl)-5-methylthiophene-2- carboxylic acid (10.4 g, 37.4 mmol, 1 equiv), TCFH (20.97 g, 74.7 mmol, 2 equiv) and NMI (12.27 g, 149.5 mmol, 4 equiv) in ACN (180 mL) was added N-(3-amino-5- chlorophenyl)methanesulfonamide (8.25 g, 37.4 mmol, 1 equiv). Then the mixture was stirred for 2 hours at room temperature. The mixture was concentrated and purified by reverse phase flash chromatography eluting with ACN/H2O (56%, 0.1%FA) to afford N-(3- chloro-5-(methylsulfonamido)phenyl)-4-(5-isopropoxypyrimidin-2-yl)-5-methylthiophene-2- carboxamide (10.0113 g, 55.7% yield) as a white solid. LCMS (ESI) [M + H]+: 481.1H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 10.06 (s, 1H), 8.64 (d, J = 2.7 Hz, 3H), 7.69 (dt, J = 6.5, 1.9 Hz, 2H), 6.95 (t, J = 2.0 Hz, 1H), 4.88 (p, J = 6.0 Hz, 1H), 3.08 (s, 3H), 2.83 (s, 3H), 1.34 (d, J = 6.0 Hz, 6H). Compound 4: 4-(5-(tert-butoxy)pyrimidin-2-yl)-N-(3-chloro-5- (methylsulfonamido)phenyl)-5-methylthiophene-2-carboxamide
Figure imgf000035_0001
4-(5-(tert-butoxy)pyrimidin-2-yl)-N-(3-chloro-5-(methylsulfonamido)phenyl)-5- methylthiophene-2-carboxamide was prepared using a procedure similar to that described for Compounds 2 and 3. LCMS (ESI) [M + H]+: 495.1H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 10.07 (s, 1H), 8.66 (d, J = 13.9 Hz, 3H), 7.69 (dt, J = 8.3, 1.8 Hz, 2H), 6.95 (t, J = 1.9 Hz, 1H), 3.08 (s, 3H), 2.86 (s, 3H), 1.38 (s, 9H). Compound 7: 4-bromo-N-(3-methanesulfonamidophenyl)thiophene-2-carboxamide
Figure imgf000036_0001
To a solution of 4-bromothiophene-2-carboxylic acid (100 mg, 482 µmol), HATU (274 mg, 722 µmol) and N,N-diisopropylethylamine (185 mg, 1.44 mmol) in N,N-dimethylformamide (1.5 mL) was added N-(3-aminophenyl)methanesulfonamide (89.7 mg, 482 µmol). The mixture was stirred at room temperature overnight. The residue was purified by preparative HPLC to afford 4-bromo-N-(3-methanesulfonamidophenyl)thiophene-2-carboxamide (100 mg, 266 µmol). 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 9.84 (s, 1H), 8.10 (d, J = 1.4 Hz, 1H), 8.02 (d, J = 1.4 Hz, 1H), 7.65 (t, J = 2.0 Hz, 1H), 7.51 (dt, J = 8.0, 1.3 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.00 – 6.85 (m, 1H), 3.01 (s, 3H). LCMS (ESI-MS): [M+1] = 377.00 In other embodiments, the DHX9 inhibitor is an antisense oligonucleotide (a single- stranded deoxyribonucleotide) complementary to DHX9 mRNA. In some embodiments, the DHX9 inhibitor is an RNA intefering agent. In some embodiments, the DHX9 inhibitor is selected from the group consisting of small intering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA). As used herein, the terms “short interfering RNA” and “siRNA” are used interchangeably to refer to any RNA molecule shorter than about 50, 40, 35, 30, 25, 20, or fewer nucleotides capable of inducing silencing of a gene target based on complementarity. Silencing is typically mediated by an Argonaute protein and is typically initiated by a short RNA molecule trigger. Silencing can be post-transcriptional or transcriptional. The siRNA molecule may be completely or partially complementary to the gene or genes whose expression in reduced, and silencing may be effected with or without cleavage of an mRNA transcript. As used herein, the terms “short-hairpin RNA” and “shRNA” are used interchangeably to refer to any single-stranded RNA molecule having two regions capable of base-pairing with one another separated by a region that does not participate in base pairing, the processing of which releases one or more siRNAs. shRNAs may be of cellular (endogenous) or artificial (exogenous) origin. Endogenous siRNA includes “piRNA,” which are any of a class of Piwi-interacting RNAs. As used herein, the term “microRNA,” “mature microRNA” and “miRNA” are used interchangeably to refer to a non-coding RNA molecule that has a length in the range of 19 to 30 nucleotides that participates in gene regulation. In some embodiments, the miRNA can be derived from “pre-microRNA” or “pre-miRNA”. “Pre-microRNA” or “pre-miRNA”are used interchangeably to refer to any molecule, the processing of which releases a mature miRNA. Furthermore, as will be appreciated by those skilled in the art, some miRNA can be characterized in terms of their capacities to affect gene regulation in a wide variety of organisms and in a wide variety of tissues. A single gene may be regulated by multiple miRNAs, and miRNA regulation may act synergistically. A single miRNA may also be capable of regulating multiple mRNAs, for example, some estimates have suggested a single human miRNA may regulate up to 100-200 genes. It has also been recently described that up to 30% of human genes are targets for miRNA regulation. Method of identifying or verifying a DHX9 inhibitor are known in the art. For example, examplary methods of identifying or verifying a DHX9 inhibitor are described in the Examples of the present disclosure. In a non-limiting example, a candidate molecule that potentially inhibits DHX9 by inhibiting its expression can be verified by determining whether the candidate molecule inhibits or reduces expression of DHX9 mRNA or polypeptide in a cell. Methods for detecting mRNA levels such as northern blotting, in situ hybridization, ribonuclease protection assay (RPA) and RT-qPCR (see, e.g., Karen Reue, mRNA Quantitation Techniques: Considerations for Experimental Design and Application, The Journal of Nutrition, 1998, 128(11): 2038–2044; incorporated herein by reference), and methods for detecting polypeptide levels such as western blotting, ELISA, immunofluorescence staining, immunohistochemistry, and mass spectrometry, are well known in the art. Alternatively, a candidate molecule that potentially inhibits DHX9, e.g., by inhibiting DHX9 function or activity of the polypeptide, can be verified by testing whether the candidate molecule inhibits ATPase activity of DHX9 (see, e.g., Example 2 of the present disclosure). Assays and kits for measuring ATPase activity of an ATP-dependent polypeptide such as DHX9 are known in the art and are commercially available. In some embodiments, a candidate molecule that potentially inhibits DHX9, e.g., by inhibiting DHX9 function or activity of the polypeptide, can be verified by testing whether the candidate molecule inhibits or reduces a level of a product or a process that is dependent on DHX9 activity, such as RNA processing (see, e.g., Aktaş et al., DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome, Nature, 2017, 544:112-119; the entire contents of which are incorporated herein by reference). Circular RNAs (circRNAs) Multiple studies have reported that DHX9 binds to or binds near Alu elements more than other RNA-binding proteins (Aktaş et al., 2017; Stagsted et al. 2019, Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles, Life Science Alliance, 2019, 2(3):1-16), incorporated herein by reference). Studies of DHX9 have also shown that DHX9 acts as a nuclear RNA resolvase, and resolves inverted-repeat Alu elements (IAEs) in order to de-repress translation of mRNAs with inverted-repeat Alu elements in their 3’ UTRs (Aktaş et al., 2017). It has been further reported that DHX9 inhibits circRNA production by unwinding and destabilizing RNA structures formed by inverted-repeat Alu elements (IAEs) in flanking regions of circRNAs (Stagsted et al.). Suitable circRNAs for use in the methods and assays of the present disclosure are those circRNAs whose formation is modulated (e.g., reduced or prevented) or controlled by DHX9. In some embodiments a circRNA may be one whose formation is also modulated by mechanisms and/or molecules independent of DHX9, but is nevertheless useful in the methods and assays of the present disclosure as long as inhibition of DHX9 can induce an appreciable increase in formation of the circRNA. In some embodiments, the circRNA useful in the methods and assays of the present disclosure is Alu-mediated circRNA. While not wishing to be bound by theory, it is believed that under normal conditions DHX9 binds to inverted-repeat Alu elements in flanking regions of mRNA and inhibits production of the circRNA. When DHX9 is inhibited (e.g., by using an iRNA t k k d DHX9 i ll l l DHX9 i hibit t i hibit it activity), the inhibition of Alu-mediated circRNA production by DHX9 is reduced and the level of the Alu-mediated circRNA is increased. Accordingly, circRNAs for use in the methods and assays of the present disclosure can be identified using methodologies described herein (see, e.g., the methods described in Example 9 of the present disclosure). For example, circRNAs, such as those known or predicted in databases (such as circBase (circbase.org) and CircNet (circnet. mbc.nctu.edu.tw)) or available for testing in commercial or customized arrays (e.g., Arraystar Human Circular RNA Array) can be tested for responsiveness to DHX9 inhibition. CircRNAs up-regulated following DHX9 inhibition are useful in the methods of the present disclosure. Such circRNAs can be compared to database or publications that annotate Alu elements (e.g., Stagsted et al. 2019) to arrive at Alu-mediated circRNAs that are DHX9- mediated. It is predicted that any circRNA identified as containing Alu elements (e.g., utilizing the foregoing databases) would be useful in the the methods and assays of the present disclosure. In some embodiments, the Alu-mediated circRNA is selected from the list of Alu- mediated circRNAs listed in Table 2 below. In some embodiments, more than one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Alu-mediated circRNAs, e.g., selected from those listed in Table 2, are detected or measured in the methods of the present disclosure.
Table 2: List of Alu-mediated circRNAs (circRNAs) Ge Sym AKR1 BMPR BRIP1 BRIP1 CALC O2 CLNS DKC1 FAM1 FBN1 FCHO GLS
Figure imgf000040_0001
(SEQ ID NO: 35)
39 ME148112605v.1
Docket No.: 130090-01520 GON4 GPR1 NEIL3 NEIL3 PIK3C PNN POMT RBM2 SKIL SLK SMA SMG1
Figure imgf000041_0001
(SEQ ID NO: 47) 40 ME148112605v.1
Docket No.: 130090-01520 SMYD TAF2 TFAP TMEM B USP25 VPS13 WDR5 XPR1 XPR1
Figure imgf000042_0001
(SEQ ID NO: 56) 41 ME148112605v.1
In some embodiments, the circRNA is derived from the BRIP1 gene, and called circBRIP1. BRIP1, also known as “BRCA1 Interacting Protein C- terminal helicase 1”, is a tumor suppressor gene interacting with another known DNA repair gene, BRCA-1 (Breast Cancer gene 1), involved in repair by homologous recombination. Mutations in the BRIP1 gene are known to increase the risk of ovarian and breast cancers. Human BRIP1 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_032043.3, incorporated herein by reference. In some embodiments, the circRNA is derived from the AKR1A1 gene. AKR1A1, also known as aldehyde reductase, is involved in the synthesis of ascorbic acid (AsA) as well as the detoxification of aldehydes. Human AKR1A1 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_006066.4, NM_153326.3, NM_001202413.2, or NM_001202414.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the BMPR2 gene. The BMPR2 gene, also known as “bone morphogenetic protein receptor type 2”, belongs to a family of genes originally identified for its role in regulating the growth and maturation (differentiation) of bone and cartilage. Recently, researchers have found that this gene family plays a broader role in regulating the growth and differentiation of numerous types of cells. Human BMPR2 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_001204.7, incorporated herein by reference. In some embodiments, the circRNA is derived from the CALCOCO2 gene. CALCOCO2, also known as “calcium Binding And Coiled-Coil Domain 2”, is a receptor for ubiquitin-coated bacteria and plays an important role in innate immunity by mediating macroautophagy. Human CALCOCO2 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_005831.5, NM_001261390.2, or NM_001261391.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the CLNS1A gene. CLNS1A, also known as “chloride nucleotide-sensitive channel 1A” functions in multiple regulatory pathways. The encoded protein complexes with numerous cytosolic proteins and performs diverse functions including regulation of small nuclear ribonucleoprotein biosynthesis, platelet activation and cytoskeletal organization. The protein is also found associated with the plasma membrane where it functions as a chloride current regulator. Human CLNS1Anucleotide and amino acid
42
sequences can be found at GenBank Accession No. NM_001293.3, NM_001311199.2, NM_001311200.2, NM_001311201.2, or NM_001311202.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the DKC1 gene. DKC1, also known as “dyskerin pseudouridine synthase 1” plays an active role in telomerase stabilization and maintenance, as well as recognition of snoRNAs containing H/ACA sequences which provides stability during biogenesis and assembly into H/ACA small nucleolar RNA ribonucleoproteins (snoRNPs). This gene is highly conserved and widely expressed, and may play additional roles in nucleo-cytoplasmic shuttling, DNA damage response, and cell adhesion. Human DKC1nucleotide and amino acid sequences can be found at GenBank Accession No. NM_001363.5, NM_001142463.3, NM_001288747.2, NR_110021.2, NR_110022.2, or NR_110023.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the FAM124A gene. FAM124A, is also known as “family with sequence similarity 124 member A”. Human FAM124A nucleotide and amino acid sequences can be found at GenBank Accession No. NM_145019.4, NM_001242312.2, or NM_001330522.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the FBN1 gene. FBN1, also known as fibrillin 1, is proteolytically processed to generate two proteins including the extracellular matrix component fibrillin-1 and the protein hormone asprosin. Fibrillin-1 is an extracellular matrix glycoprotein that serves as a structural component of calcium-binding microfibrils. These microfibrils provide force-bearing structural support in elastic and nonelastic connective tissue throughout the body. Asprosin, secreted by white adipose tissue, has been shown to regulate glucose homeostasis. Human FBN1 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_000138.5, NM_001406716.1, NM_001406717.1, or NM_001406718.1, incorporated herein by reference. In some embodiments, the circRNA is derived from the FCHO2 gene. FCHO2, also known as, “FCH and mu domain containing endocytic adaptor 2”, functions in an early step of clathrin-mediated endocytosis. Human FCHO2 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_138782.3 or NM_001146032.2, incorporated herein by reference. 43
In some embodiments, the circRNA is derived from the GLS gene. GLS, also known as glutaminase, an phosphate-activated amidohydrolase that catalyzes the hydrolysis of glutamine to glutamate and ammonia. Human GLS nucleotide and amino acid sequences can be found at GenBank Accession No. NM_014905.5 or NM_001256310.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the GON4L gene. GON4L, also known as “GON-4 Like”, is a nuclear protein containing two serine phosphosites and a lysine- glutamine cross-link and is thought to be a transcription factor. Human GON4L nucleotide and amino acid sequences can be found at GenBank Accession No. NM_001282856.2, NM_032292.6, NM_001282860.2, NM_001282861.2, or NM_001282858.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the GPR125 gene. GPR125, also known as “adhesion G protein-coupled receptor A3” or “ADGRA3”, is a member of the G protein-coupled receptor superfamily. This membrane protein may play a role in tumor angiogenesis through its interaction with the human homolog of the Drosophila disc large tumor suppressor gene. Human GPR125 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_145290.4, incorporated herein by reference. In some embodiments, the circRNA is derived from the NEIL3 gene. Neil3, also known as “nei like DNA glycosylase 3”, belongs to a class of DNA glycosylases. These glycosylases initiate the first step in base excision repair by cleaving bases damaged by reactive oxygen species and introducing a DNA strand break via the associated lyase reactionHuman NEIL3 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_018248.3, incorporated herein by reference. In some embodiments, the circRNA is derived from the PIK3C3 gene. PIK3C3, also known as “phosphatidylinositol 3-kinase catalytic subunit type 3”, belongs to the phosphoinositide 3-kinase (PI3K) family. PIK3C3 can phosphorylate phosphatidylinositol (PtdIns) to generate phosphatidylinositol 3-phosphate (PI3P), a phospholipid central to autophagy. Inhibition of PIK3C3 successfully inhibits autophagy. Human PIK3C3 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_002647.4 or NM_001308020.2, incorporated herein by reference. 44
In some embodiments, the circRNA is derived from the PNN gene. PNN, also known as “pinin, desmosome associated protein”, is a nuclear and cell adhesion-related protein. Studies have suggested that Pnn/DRS/memA is a potential tumor suppressor involved in the regulation of cell adhesion and cell migration. Human PNN nucleotide and amino acid sequences can be found at GenBank Accession No. NM_002687.4, incorporated herein by reference. In some embodiments, the circRNA is derived from the POMT1 gene. POMT1, also known as “protein O-mannosyltransferase 1”, is a key enzyme in the glycosylation of α- dystroglycan. Human POMT1 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_007171.4, NM_001077365.2, or NM_001077366.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the RBM23 gene. RMB23, also known as “RNA binding motif protein 23”, is a member of the U2AF-like family of RNA binding proteins. This protein interacts with some steroid nuclear receptors, localizes to the promoter of a steroid- responsive gene, and increases transcription of steroid-responsive transcriptional reporters in a hormone-dependent manner. Human RBM23 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_018107.5, NM_001077351.2, NM_001077352.2, NM_001308044.2, NM_001352762.2, NM_001352763.2, NM_001352764.2, NM_001352765.2 or NM_001352766.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the SKIL gene. SKIL, also known as “SKI like proto-oncogene”, is a component of the SMAD pathway, which regulates cell growth and differentiation through transforming growth factor-beta (TGFB). In the absence of ligand, the encoded protein binds to the promoter region of TGFB-responsive genes and recruits a nuclear repressor complex. TGFB signaling causes SMAD3 to enter the nucleus and degrade this protein, allowing these genes to be activated. Human SKIL nucleotide and amino acid sequences can be found at GenBank Accession No. NM_005414.5, NM_001145097.2, NM_001145098.3, or NM_001248008.1, incorporated herein by reference. In some embodiments, the circRNA is derived from the SLK gene. Human SLK nucleotide and amino acid sequences can be found at GenBank Accession No. NM_014720.4 or NM_001304743.2, incorporated herein by reference. 45
In some embodiments, the circRNA is derived from the SMA gene. SMA, also known as “survival of motor neuron 1”, is part of a 500 kb inverted duplication on chromosome 5q13. This duplicated region contains at least four genes and repetitive elements which make it prone to rearrangements and deletions. The protein encoded by this gene localizes to both the cytoplasm and the nucleus. Within the nucleus, the protein localizes to subnuclear bodies called gems which are found near coiled bodies containing high concentrations of small ribonucleoproteins (snRNPs). This protein forms heteromeric complexes with proteins such as SIP1 and GEMIN4, and also interacts with several proteins known to be involved in the biogenesis of snRNPs, such as hnRNP U protein and the small nucleolar RNA binding protein. Human SMA nucleotide and amino acid sequences can be found at GenBank Accession No. NM_001297715.1, NM_022874.2, or NM_000344.4, incorporated herein by reference. In some embodiments, the circRNA is derived from the SMG1 gene. SMG1, also known as “SMG1 nonsense mediated mRNA decay associated PI3K related kinase”, is involved in nonsense-mediated mRNA decay (NMD) as part of the mRNA surveillance complex. The protein has kinase activity and is thought to function in NMD by phosphorylating the regulator of nonsense transcripts 1 protein. Human SMG1 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_015092.5, incorporated herein by reference. In some embodiments, the circRNA is derived from the SMYD4 gene. SMYD4, also known as “SET and MYND domain containing 4”, is a potential tumor suppressor Human SMYD4 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_052928.4, incorporated herein by reference. In some embodiments, the circRNA is derived from the TAF2 gene. TAF2 (TATA-box binding protein associated factor 2) encodes one of the larger subunits of transcription factor IID (TFIID) that is stably associated with the TFIID complex. It contributes to interactions at and downstream of the transcription initiation site, interactions that help determine transcription complex response to activators. Human TAF2 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_003184.4, incorporated herein by reference. In some embodiments, the circRNA is derived from the TFAP4 gene. TFAP4, also known as “transcription factor AP-4”, is a transcription factor of the basic helix-loop-helix- zipper (bHLH-ZIP) family contain a basic domain, which is used for DNA binding, and HLH 46
and ZIP domains, which are used for oligomerization. Transcription factor AP4 activates both viral and cellular genes by binding to the symmetrical DNA sequence CAGCTG. Human TFAP4 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_003223.3, incorporated herein by reference. In some embodiments, the circRNA is derived from the TMEM41B gene. TMEM41B, also known as “transmembrane protein 41B”, is an integral endoplasmic reticulum (ER) membrane protein distantly that plays roles in autophagosome biogenesis. Human TMEM41B nucleotide and amino acid sequences can be found at GenBank Accession No. NM_015012.4, incorporated herein by reference. In some embodiments, the circRNA is derived from the USP25 gene. USP25, also known as “ubiquitin specific peptidase 25”, is a deubiquitinating enzyme. Human USP25 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_013396.6, NM_001283041.3, NM_001283042.3, NM_001352560.2, NM_001352561.2, NM_001388299.1, NM_001388300.1, NM_001388301.1 or, NM_001388302.1, incorporated herein by reference. In some embodiments, the circRNA is derived from the VPS13D gene. VPS13D, also known as “vacuolar protein sorting 13 homolog D”, is a protein belonging to the vacuolar- protein-sorting-13 gene family. In yeast, vacuolar-protein-sorting-13 proteins are involved in trafficking of membrane proteins between the trans-Golgi network and the prevacuolar compartment. Human VPS13D nucleotide and amino acid sequences can be found at GenBank Accession No. NM_015378.4 or NM_018156.4, incorporated herein by reference. In some embodiments, the circRNA is derived from the WDR59 gene. WDR59, also known as “WD repeat domain 59”, is involved in cellular response to amino acid starvation and positive regulation of TOR signaling. Human WDR59 nucleotide and amino acid sequences can be found at GenBank Accession No. NM_030581.4, NM_001324171.2, or NM_001324172.2, incorporated herein by reference. In some embodiments, the circRNA is derived from the XPR1 gene. XPR1, also known as “xenotropic and polytropic retrovirus receptor 1”, is a receptor for the xenotropic and polytropic classes of murine leukemia viruses. The encoded protein is involved in phosphate homeostasis by mediating phosphate export from the cell.Human XPR1 nucleotide and amino 47
acid sequences can be found at GenBank Accession No. NM_004736.4, NM_001135669.2, NM_001328662.2, or NR_137330.2, incorporated herein by reference. Methods of Detecting circRNAs CircRNA can be detected and measured using methods commonly known in the art. For example, in some embodiments, circRNA is detected and/or measured by employing a technique selected from the group consisting of northernblot, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), microarray, droplet digital PCR, next-generation sequence (NGS) RNA sequencing, RNAin situ hypridization (RNA-ISH), in situ hypridization- immunohistochemistry (ISH-IHC), isothermal exponential amplification and rolling cycle amplification. In some embodiments, circRNA can be detected using quantitative real-time PCR, e.g.,Taqman PCR, e.g., Taqman Multiplex PCR. In some embodiments, circRNA can be detected using SYBR Green PCR. Additional methods of detecting circRNA are known in the art, and can be found at, for example, in Mi et al., Circular RNA detection methods: A minireview, Talanta, 2022, 238(2): 123066, the entire contents of which are incorporated herein by reference. In some embodiments, circRNA is detected by RT-qPCR. In some embodiments, the circRNA is a circBRIP1, and the circBRIP1 is detected by RT-qPCR using the primers as described in any of Examples 1-9 of the present disclosure. In some embodiments, the forward and reverse primers used to detect circBRIP are TCTGTGTGCCAGACTGTGAG (SEQ ID NO: 9) and ACACCAAGTTCTGACGAAAAGG (SEQ ID NO: 10), respectively. In some embodiments, the forward and reverse primers used to detect circBRIP are GTGAGCCAAGGAATTTTGTGTTT (SEQ ID NO: 19) and GGTCTGAACTTTGCCATTAATATCTG (SEQ ID NO: 20), respectively. In some embodiments, the forward and reverse primers used to detect circBRIP are TCAAAATAGAAGCCAGTGGATGAG (SEQ ID NO: 22) and AATGGCATGCACAAGAACATG (SEQ ID NO: 23), respectively. Levels of circRNA markers can be detected based on the absolute level or a normalized or relative level. Detection of absolute circRNA levels may be preferable when monitoring the treatment of a cell or subject. For example, the level of one or more circRNA markers (e.g., 48
circBRIP1) can be monitored in a cell or a subject receiving a DHX9 inhibitor, e.g., at regular intervals, such as daily or weekly intervals. A modulation in the level of one or more circRNA markers can be monitored over time to observe trends in changes in marker levels. Expression levels of the markers of the invention, e.g., circBRIP1, in the subject may be higher than the expression level of those markers in a prior expression level (e.g., prior to treatment, or from an earlier time point), thus indicating engagement of the DHX9 target by the inhibitor, inhibition of DHX9 by the inhibitor, or responsiveness or potential therapeutic effects of the treatment regimen for the subject. As an alternative to making determinations based on the absolute expression level of the circRNA marker, determinations may be based on the normalized level of the marker. Levels are normalized by correcting the absolute level of a marker by comparing its level to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or GAPDH gene. This normalization allows the comparison of the level in one sample to another sample (e.g., comparison between samples from cells or subjects receiving different amount of the same inhibitor, or comparison between samples from cells or subjects receiving different inhibitors). EXAMPLES Example 1: Probing circRNAs after DHX9 inhibition in vitro This experiment was conducted to evaluate circular and linear formats of different mRNA in HCT116 cells after treatment with DHX9 inhibitors by Quantitative Polymerase Chain Reaction (qPCR). Methods HCT116 cells were obtained from ATCC (CCL-247) and were grown and assayed in McCoy’s 5a growth media supplemented with 10%FBS. The cells were plated at pre-determined cell densities in 384-well cell culture plates and incubated overnight in 37oC at 5% CO2. In a separate plate, reference and test compounds were prepared in DMSO stock solution and serially diluted 3-fold for 10 points. Compound 7 (0.1% 49
final DMSO concentration) was transferred to the cell plate in designated wells, and incubated 37oC at 5% CO2 for 72 hours. RNA was extracted from the cells using the SYBR Green Fast Advanced Cell-to-CT kit according to manufacturer’s (Invitrogen) protocol, and cDNA was reverse transcribed using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s (Invitrogen) protocol. Using RT2 SYBR Green ROX qPCR Mastermix (Invitrogen) and thermal cycler conditions for the QuantStudio Thermocycler, qPCR was carried out using the below custom primer sequences obtained at Integrated DNA Technologies (IDT): Table 3: DNA Oligo Primers DNA Oligo Primers Circular (Circ) Forward (F) SEQ ID a
Figure imgf000051_0001
separate plate for each gene. Data analysis was performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ΔCt for circRNA or linRNA is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity is calculated by the following formula, whereas: 50
• ΔCts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ΔCtc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. 2^^^^ %^^^^^^^ = 100% × 2^^^^ Results CirRNAs of BRIP1, A
Figure imgf000052_0001
Alu-mediated, and cirRNA of SETD3 is non-Alu-mediated. Upon DHX9 inhibition by Compound 7, the Alu-mediated circRNAs, namely BRIP1, AKR1A1 and DKC1, were induced, while no induction of SETD3 cirRNA was observed (see FIG. 1). Example 2: Correlations of circBRIP1 with anti-tumor activity of DHX9 inhibitors in vitro This study was conducted to evaluate the circular format of BRIP1 in response to treatment with a panel of DHX9 inhibitors in vitro. Methods DHX9 Inhibitor compounds: A panel of DHX9 inhibitor compounds were used in this Example. The panel includes Compounds 1, 2, 3, 4 as shown in Table 1 and compounds which have been described in International Application No. PCT/US2023/012929, the entire contents of which are expressly incorporated by reference herein. ATPase Assay: DHX9 ATPase assay was performed in small-volume, nonbinding, 384- well white plates at a final volume of 10 µL/well. A panel of test compounds (10 mM solution in DMSO; 100 nL/well) were serially diluted on Bravo (Agilent, Santa Clara, CA) and dispensed into wells of columns 3–22 of the plates using an Echo 555 acoustic dispenser (Labcyte, Sunnyvale, CA). 100nL of Aurintricarboxylic acid was dispensed into low control wells and 100 nL of DMSO was dispensed into high control wells. Then, a Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific, Waltham, MA) was used to add a solution of DHX9 (1.25 nM, 5 µL/well) in assay buffer (40 mM HEPES [pH 7.5], 0.01% Tween 20, 0.01%BSA, 1 mM DTT, 5 mM MgCl2, 0.004U/ml RNAseOUT). The reaction was initiated by the addition of 5 µL of substrate solution (30 nM double-stranded RNA substrate, 10 µM Ultra Pure ATP in assay buffer) into the wells. The plates were incubated at room temperature for 1 h. After the indicated 51
incubation times, 10 µL ADP-Glo reagent was added to the reactions and the plate was incubated at room temperature for 40 min. Then, 20 µL of kinase detection reagent was added and after an incubation time of 40 min, luminescence was recorded on Envision plate reader (Perkin-Elmer, Billerica, MA). All data was analyzed by Accent Therapeutics internal data analysis software developed by Scigilian (Montreal, Canada). The percentage inhibition was calculated based on the high control (DMSO) as 0% inhibition, and low control (10µM Aurintricarboxylic acid) control as 100% inhibition and used for the calculation of IC50 and IP (inflection point) values by fitting the dose–response curves to a four-parameter logistic model. Assay results reported IP values instead of IC50 values since several compounds did not reach 100% inhibition at higher compound concentrations. Cellular Proliferation Assay: The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. LS411N cells (CRL- 2159), obtained from ATCC, were grown in RPMI-1640 media supplemented with 10% FBS. The cells were plated at pre-determined cell densities in 384-well solid white cell culture plates and incubated overnight in 37oC at 5% CO2. In a separate plate, reference and a panel of test compounds were prepared in DMSO stock solution and serially diluted 3-fold for 10 points. Compound (0.1% final DMSO concentration) was transferred to the cell plate in designated wells, and incubated 37oC at 5% CO2 for 120 hours. CellTiter-Glo reagent was prepared fresh according to manufacturer’s (Promega) directions and added to each well. The plate was then shaken at 300rpm for 10 minutes at RT, and then read on an Envision plate reader using a Luminescence protocol. Data analysis was performed by normalizing the raw data (raw luminescence units or RSample) to an average of the positive control values for wells containing culture media only (100% cell death or RLC) and the negative control values for 0.1% DMSO (0% cell death or RHC). An IC50 was calculated using a 4-parameter logistic nonlinear regression model in Scigilian Analyzer or GraphPad Prism, constraining the top parameter to 100 and the bottom parameter to 0.
Figure imgf000053_0001
52 IC50 Fit formula = (A+((B-A)/(1+((x/C)^D)))) … whereas A:Bottom; B:Top; C:IC50; D:Slope circBRIP1 Cellular Target Engagement Taqman Multiplex Assay: Evaluation of the circular format of BRIP1 mRNA in HCT116 cells by Quantitative Polymerase Chain Reaction (qPCR) using the Taqman Multiplex assay. HCT116 cells were obtained from ATCC (CCL-247) and were grown and assayed in McCoy’s 5a growth media supplemented with 10%FBS. The cells were plated at pre-determined cell densities in 384-well cell culture plates and incubated overnight in 37 ºC at 5% CO2. In a separate plate, reference and a panel of test compounds were prepared in DMSO stock solution and serially diluted 3-fold for 10 points. Compound (0.1% final DMSO concentration) was transferred to the cell plate in designated wells, and incubated 37 ºC at 5% CO2 for 72 hours. RNA was extracted from the cells using the TaqMan Gene Expression Cells-to-CT™ kit according to manufacturer’s (Invitrogen) protocol, and cDNA was reverse transcribed using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s (Invitrogen) protocol. Using TaqMan Multiplex Mastermix and thermal cycler conditions for the QuantStudio Thermocycler, qPCR was carried out using TaqMan GAPDH primer limited assay with JUN dye/QSY probe, and the below custom TaqMan Gene Expression Assay with FAM Dye for circBRIP1: • Forward Primer: GTGAGCCAAGGAATTTTGTGTTT (SEQ ID NO: 19) • Reverse Primer: GGTCTGAACTTTGCCATTAATATCTG (SEQ ID NO: 20) • Probe: CCATCTTACAAGGCCTTT (SEQ ID NO: 21) Note: All target genes can be combined in one well per sample. Data analysis was performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ΔCt for circBRIP1 or linBRIP1 is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity is calculated by the following formula, whereas: • ΔCts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ΔCtc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. 2ΔCts
Figure imgf000054_0001
An EC50 was then calculated using a 4-parameter logistic nonlinear regression model in Scigilian Analyzer or GraphPad Prism, floating both the top and bottom parameters. IC50 Fit formula = (A+((B-A)/(1+((x/C)^D)))) … whereas A:Bottom; B:Top; C:IC50; D:Slope circBRIP1 Cellular Target Engagement SYBR Green Assay: The circular format of BRIP1 mRNA in HCT116 cells was additionally evaluated by Quantitative Polymerase Chain Reaction (qPCR) using the SYBR Green assay. HCT116 cells were obtained from ATCC (CCL-247) and were grown and assayed in McCoy’s 5a growth media supplemented with 10%FBS. The cells were plated at pre-determined cell densities in 384-well cell culture plates and incubated overnight in 37oC at 5% CO2. In a separate plate, reference and test compounds were prepared in DMSO stock solution and serially diluted 3-fold for 10 points. Compound 7 (0.1% final DMSO concentration) was transferred to the cell plate in designated wells, and incubated 37oC at 5% CO2 for 72 hours. RNA was extracted from the cells using the SYBR Green Fast Advanced Cell-to-CT kit according to manufacturer’s (Invitrogen) protocol, and cDNA was reverse transcribed using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s (Invitrogen) protocol. Using RT2 SYBR Green ROX qPCR Mastermix (Invitrogen) and thermal cycler conditions for the QuantStudio Thermocycler, qPCR was carried out using the below custom primer sequences: • BRIP1-Circular-Forward (TCT GTG TGC CAG ACT GTG AG) (SEQ ID NO: 9) • BRIP1-Circular-Reverse (ACA CCA AGT TCT GAC GAA AAG G) (SEQ ID NO: 10) • GAPDH-Forward (GTCTCCTCTGACTTCAACAGCG) (SEQ ID NO: 17) • GAPDH-Reverse (ACCACCCTGTTGCTGTAGCCAA) (SEQ ID NO: 18) Note: Forward and Reverse primers are combined per gene, and each sample has to be run on a separate plate for each gene. Data analysis was performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ΔCt for circRNA is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity is calculated by the following formula, whereas: • ΔCts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ΔCtc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. 54
2^^^^ %^^^^^^^ = 100% × Results The results of this study, hat cellular target engagement
Figure imgf000056_0001
by the DHX9 inhibitors and the resulting induction of circBRIP1 correlates with the inhibition of DHX9 ATPase activity and anti-proliferative activity of DHX9 inhibitors in certain MSI cancers. Example 3: Evaluation of circBRIP1 in human xenograft tumor This study was conducted to evaluate the circular and linear format of human BRIP1 mRNA in LS411N xenograft tumors by qPCR. Methods Experiments were performed in female BALB/c nude mice (GenPharmatech Co.). Animals were allowed to acclimate for 7 days before the study. The general health of the animals were evaluated by a veterinarian, and complete health checks were performed prior to the study. General procedures for animal care and housing were in accordance with the standard, Commission on Life Sciences, National Research Council, Standard Operating Procedures (SOPs) of Pharmaron, Inc. The mice were kept in laminar flow rooms at constant temperature and humidity with 3-5 mice in each cage. Animals were housed in polycarbonate cage which is the size of 300 x 180 x 150 mm3 and in an environmentally monitored, well-ventilated room maintained at a temperature of (23 ± 3°C) and a relative humidity of 40% 70%. Fluorescent lighting provided illumination approximately 12 hours per day. Animals had free access to irradiation sterilized dry granule food during the entire study period except for time periods specified by the protocol, as well as sterile drinking water in a bottle was available ad libitum during the quarantine and study periods. The LS411N (ATCC; CRL-2159) tumor cell lines were maintained in vitro as monolayer in RPMI 1640 medium supplemented with 10% heat inactivated FBS, at 37°C in an atmosphere of 5% CO2 in air. The tumor cells were sub-cultured, not exceeding 4-5 passages, and cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Each mouse was inoculated subcutaneously on the right flank with LS411N tumor cells (2 × 106) in 0.1 mL of RPMI-1640 with Matrigel (1:1) for model development. 55
Depending on the target concentration, DHX9 compound was dissolved in in 0.96 mL Solutol while vortexing and sonicating for 1 hour to obtain a suspension. Then 2.4 mL of 1% MC (with 100mg/mL PVP VA64) was added and vortexed for 5 min to obtain homogeneous suspension. The mixture was kept stirring at room temperature for 5 min, and then 1.44 mL of water was added while vortexing to make the final volume of 4.8 mL. The mixture was kept stirring at RT for 30 min and homogenized for 3 min. The resulting solution was used for in vivo studies. Treatment was started when the mean tumor size reached approximately 100-150 mm3, at which time the mice were randomized into treatment groups. Animals were then treated with vehicle (20% Solutol/ 50% 1% MC (4000 cp) with 100 mg/mL PVP VA64/ 30% water) or indicated mg/kg of DHX9 compound (Compounds 1, 2, 3 and 4, as shown in Table 1) daily BID (12 hour schedule) by oral gavage at a final dosing volume of 10 mL/kg. Tumor samples 2-3mm3 in size were transferred into a clean tube and 300 µL of fresh lysis buffer containing 1% 2- 0.12mercaptoethanol was added. The samples were then homogenized using a TissueLyser at a frequency of 30/s for 5 min. The homogenate samples are then centrifuged at 1200 rpm for 15 min. The supernatant was transferred to a clean tube, and one volume of 70% ethanol to each volume of homogenate supernatant is added, and the samples are then vortexed. RNA is then purified using the PureLink RNA Mini Kit according to the manufacturer’s (Invitrogen) protocol, and cDNA is reverse transcribed using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s (Invitrogen) protocol. Using TaqMan Multiplex Mastermix and thermal cycler conditions for the QuantStudio Thermocycler, qPCR is carried out using TaqMan Gene Expression Assay with VIC Dye for Linear BRIP1 (Assay ID: Hs00908156_m1), TaqMan GAPDH primer limited assay with JUN dye/QSY probe, and the below custom TaqMan Gene Expression Assay with FAM Dye for circBRIP1: • Forward Primer: GTGAGCCAAGGAATTTTGTGTTT (SEQ ID NO: 19) • Reverse Primer: GGTCTGAACTTTGCCATTAATATCTG (SEQ ID NO: 20) • Probe: CCATCTTACAAGGCCTTT (SEQ ID NO: 21) Note: All target genes can be combined in one well per sample. 56
Data analysis was performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ΔCt for circBRIP1 or linBRIP1 is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity is calculated by the following formula, whereas: • ΔCts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ΔCtc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. 2)^^^^ %^^^^^^^ = 100% × )^^^^ Results In tumors of mice tr
Figure imgf000058_0001
mpounds, a dose dependent induction of circBRIP1 was observed and no change in linear BRIP1 was seen (see FIGs. 3A- 3D). Example 4: Correlations of intra-tumoral circBRIP1 with anti-tumor activity of DHX9 inhibitors in vivo This study was conducted to evaluate pharmacodynamics (PD) and pharmacokinetics (PK) of intra-tumoral circular BRIP1 with efficacy of a DHX9 inhibitor (Compound 1, as shown in Table 1). Methods In vivo Efficacy Study: Experiments were performed in female BALB/c nude mice (GenPharmatech Co.). Animals were allowed to acclimate for 7 days before the study. The general health of the animals were evaluated by a veterinarian, and complete health checks were performed prior to the study. General procedures for animal care and housing were in accordance with the standard, Commission on Life Sciences, National Research Council, Standard Operating Procedures (SOPs) of Pharmaron, Inc. The mice were kept in laminar flow rooms at constant temperature and humidity with 3-5 mice in each cage. Animals were housed in polycarbonate cage which is the size of 300 x 180 x 150 mm3 and in an environmentally monitored, well- ventilated room maintained at a temperature of (23 ± 3°C) and a relative humidity of 40% 70%. Fluorescent lighting provided illumination approximately 12 hours per day. Animals had free access to irradiation sterilized dry granule food during the entire study period except for time 57
periods specified by the protocol, as well as sterile drinking water in a bottle was available ad libitum during the quarantine and study periods. The LS411N (ATCC; CRL-2159) tumor cell lines were maintained in vitro as monolayer in RPMI 1640 medium supplemented with 10% heat inactivated FBS, at 37°C in an atmosphere of 5% CO2 in air. The tumor cells were sub-cultured, not exceeding 4-5 passages, and cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Each mouse was inoculated subcutaneously on the right flank with LS411N tumor cells (2 × 106) in 0.1 mL of RPMI-1640 with Matrigel (1:1) for model development. Depending on the target concentration, 14.4 – 144 mg DHX9 compound was dissolved in in 0.96 mL Solutol while vortexing and sonicating for 1 hour to obtain a suspension. Then 2.4 mL of 1% MC (with 100mg/mL PVP VA64) was added and vortexed for 5 min to obtain homogeneous suspension. The mixture was kept stirring at room temperature for 5 min, and then 1.44 mL of water was added while vortexing to make the final volume of 4.8 mL. The mixture was kept stirring at RT for 30 min and homogenized for 3 min. The resulting solution was used for in vivo studies. Treatment was started when the mean tumor size reached approximately 100-150 mm3, at which time the mice were randomized into treatment groups. Animals were then treated with vehicle (20% Solutol/ 50% 1% MC (4000 cp) with 100 mg/mL PVP VA64/ 30% water) or indicated mg/kg of DHX9 compound 1 daily BID (12 hour schedule) by oral gavage at a final dosing volume of 10 mL/kg. The measurement of tumor size was conducted with a caliper and recorded twice per week. The tumor volume (TV) (mm3) was estimated using the formula: TV = a × b2/2, where “a” and “b” are long and short diameters of a tumor, respectively. Intra-tumoral Pharmacokinetics Bioanalytical Assay: Tumors were collected at 1 h and 12 h post the last dose and cut into 3 pieces. One piece of the tumor was weighted and snap frozen for drug exposure analysis. On the assay day, 3 volumes of H2O were added to the tumor tissue (3 mL to each gram of tissue), and homogenate at 4 degrees Celsius. The homogenate (30 µL) was mixed with 15 µL blank solution, then extracted and protein precipitation with 200 μL of acetonitrile containing IS (internal standard, dexamethasone). After vortexed for 30 s, the samples were centrifuged at 4 degrees Celsius, 3900 rpm for 15 minutes. The supernatant was 58
diluted 3 times with water and 2 µL of diluted supernatant was injected into the LC/MS/MS system for quantitative analysis (AB API 5500). The Q1 to Q3 mass transition (m/z, Da) were 393.11 to 373.10 for dexamethasone and 421.98 to 202.30 for test compound. HPLC separation was achieved on an Agilent poroshell EC-C184 µm (50 × 2.1 mm) column with gradient from 5% Acetonitrile in Water (0.1% Formic acid) to 95% Acetonitrile in Water (0.1% Formic acid) in 2.5 minutes. HPLC: Shimadzu system (DGU-20A5R, LC-30AD, SIL-30AC, Rack Changer II, CTO-30A, and CBM-20A). The concentration in the homogenate was quantified using standards prepared in control tumor tissues with concentrations range 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL. The determined concentration in the tumor homogenate was then back calculated to that in tumor tissue by multiplying with a dilution factor of 4. Intra-tumoral Human circBRIP1 Analysis: Tumor samples 2-3mm3 in size were transferred into a clean tube and 300 µL of fresh lysis buffer containing 1% 2-mercaptoethanol was added. The samples were then homogenized using a TissueLyser at a frequency of 30/s for 5 min. The homogenate samples are then centrifuged at 1200 rpm for 15 min. The supernatant was transferred to a clean tube, and one volume of 70% ethanol to each volume of homogenate supernatant was added, and the samples were then vortexed. RNA was then purified using the PureLink RNA Mini Kit according to the manufacturer’s (Invitrogen) protocol, and cDNA is reverse transcribed using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s (Invitrogen) protocol. Using SYBR Green Master Mix (Invitrogen) and thermal cycler conditions for the QuantStudio Thermocycler, qPCR was carried out using the below custom primer sequences: • BRIP1-Circular-Forward (TCT GTG TGC CAG ACT GTG AG) (SEQ ID NO: 9) • BRIP1-Circular-Reverse (ACA CCA AGT TCT GAC GAA AAG G) (SEQ ID NO: 10) • GAPDH-Forward (GTCTCCTCTGACTTCAACAGCG) (SEQ ID NO: 17) • GAPDH-Reverse (ACCACCCTGTTGCTGTAGCCAA) (SEQ ID NO: 18) Note: Forward and Reverse primers are combined per gene, and each sample has to be run on a separate plate for each gene. Data analysis was performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ΔCt for circBRIP1 or linBRIP1 is calculated by normalizing the 59
target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity is calculated by the following formula, whereas: • ΔCts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ΔCtc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. )^^^^ %^^^^^^^ = 100% × 2 2)^^^^ Results Compound 1 was trea
Figure imgf000061_0001
using BID oral application when the tumor volume was an average of 133 mm3 (n=8/group). After 21 days of treatment and 12 hours post last dose of compound tumor volume was recorded, tumors were then harvested and processed for circBRIP1 expression. Dose-dependent intra-tumoral circBRIP1 induction was observed, and was statistically significant compared to vehicle control based on two way ordinary ANOVA test, with indicated doses resulting in p-values <0.0001 (see FIG. 4A). Individual tumor volumes were plotted against each tumor’s corresponding circBRIP1 pharmacoynamic reading in a correlation plot (see FIG. 4B). Individual tumor drug exposure of Compound 1 were plotted against each tumor’s corresponding circBRIP1 pharmacodynamic reading and fit to four parameter non-linear regression (see FIG. 4C). These results show that both tumor volume and tumor drug exposure correlate to circBRIP1 induction. Example 5: Evaluation of circBRIP1 in peripheral blood mononuclear cells (PBMC) of mice This study was conducted to evaluate pharmacodynamics (PD) of the circular format of mouse BRIP1 mRNA in mouse PBMC cells by qPCR. Methods Experiments were performed in female BALB/c nude mice (GenPharmatech Co.). Animals were allowed to acclimate for 7 days before the study. The general health of the animals were evaluated by a veterinarian, and complete health checks were performed prior to the study. General procedures for animal care and housing were in accordance with the standard, Commission on Life Sciences, National Research Council, Standard Operating Procedures (SOPs) of Pharmaron, Inc. The mice were kept in laminar flow rooms at constant temperature 60
and humidity with 3-5 mice in each cage. Animals were housed in polycarbonate cage which is the size of 300 x 180 x 150 mm3 and in an environmentally monitored, well-ventilated room maintained at a temperature of (23 ± 3°C) and a relative humidity of 40% 70%. Fluorescent lighting provided illumination approximately 12 hours per day. Animals had free access to irradiation sterilized dry granule food during the entire study period except for time periods specified by the protocol, as well as sterile drinking water in a bottle was available ad libitum during the quarantine and study periods. The LS411N (ATCC; CRL-2159) tumor cell lines were maintained in vitro as monolayer in RPMI 1640 medium supplemented with 10% heat inactivated FBS, at 37°C in an atmosphere of 5% CO2 in air. The tumor cells were sub-cultured, not exceeding 4-5 passages, and cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Each mouse was inoculated subcutaneously on the right flank with LS411N tumor cells (2 × 106) in 0.1 mL of RPMI-1640 with Matrigel (1:1) for model development. Depending on the target concentration, DHX9 compound (Compound 2, 3, or 4, as shown in Table 1) was dissolved in in 0.96 mL Solutol while vortexing and sonicating for 1 hour to obtain a suspension. Then 2.4 mL of 1% MC (with 100mg/mL PVP VA64) was added and vortexed for 5 min to obtain homogeneous suspension. The mixture was kept stirring at room temperature for 5 min, and then 1.44 mL of water was added while vortexing to make the final volume of 4.8 mL. The mixture was kept stirring at RT for 30 min and homogenized for 3 min. The resulting solution was used for in vivo studies. Treatment was started when the mean tumor size reached approximately 100-150 mm3, at which time the mice were randomized into treatment groups. Animals were then treated with vehicle (20% Solutol/ 50% 1% MC (4000 cp) with 100 mg/mL PVP VA64/ 30% water) or indicated mg/kg of DHX9 compound daily BID (12 hour schedule) by oral gavage at a final dosing volume of 10 mL/kg. Whole blood was collected into tubes coated with EDTA-K2. 1×PBS (volume ratio=1:1) to blood sample was added and mixed gently. The blood sample was transferred into a new tube containing Ficoll-PaqueTM PREMIUM 1.084 (volume ratio=1:5), and centrifuged at 400 g for 20 min at room temperature. The layer of lymphocytes was transferred into a new 1.5 mL tube containing 0.5 mL of 1×PBS, and centrifuged at 1500 rpm for 5 min. The samples were then 61
washed twice with 1.0 ml of 1×PBS, and red blood cells were lysed with 1× red blood cell lysing buffer. Resulting isolated PBMC were then suspended in 1×PBS for further analysis. PBMC samples were transferred into a clean tube and 300 µL of fresh lysis buffer containing 1% 2-mercaptoethanol. The samples were then homogenized using a TissueLyser at a frequency of 30/s for 5 min. The homogenate samples were then centrifuged at 1200 rpm for 15 min. The supernatant was transferred to a clean tube, and one volume of 70% ethanol to each volume of homogenate supernatant was added, and the samples were then vortexed. RNA was purified using the PureLink RNA Mini Kit according to the manufacturer’s (Invitrogen) protocol, and cDNA was reverse transcribed using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s (Invitrogen) protocol. Using TaqMan Multiplex Mastermix (Thermo Fisher) and thermal cycler conditions for the QuantStudio Thermocycler, qPCR was carried out using TaqMan Gene Expression Assay with FAM Dye for Mouse linear BRIP1 (Assay ID: Mm01297848_m1), TaqMan mouse GAPDH primer limited assay with VIC dye (Assay ID: Mm99999915_g1), and the below custom TaqMan Gene Expression Assay with FAM Dye for mouse circBRIP1: • Forward Primer: TCAAAATAGAAGCCAGTGGATGAG (SEQ ID NO: 22) • Reverse Primer: AATGGCATGCACAAGAACATG (SEQ ID NO: 23) • Probe: CCTGAGGCACCACCT (SEQ ID NO: 24) Note: each sample needed to be run in 2 different plates, one with mouse linear BRIP1 and GAPDH, and the other plate with mouse circBRIP1 and GAPDH. Data analysis was performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ΔCt for circBRIP1 or linBRIP1 was calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity was calculated by the following formula, whereas: • ΔCts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ΔCtc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. )^^^^
Figure imgf000063_0001
62
Results At 1 hr (FIGs 5A-B) and 24 hours (FIG. 6) after the last dose of DHX9 inhibitor, dose dependent induction of circBRIP1 was observed, and no induction in linear BRIP1 above baseline was seen. Example 6: Comparison of circBRIP1 induction by DHX9 inhibitor and sensitivity to DHX9 inhibitor in cancer cell lines This study was conducted to determine inhibition of DHX9 in a range of cancer cell lines and whether circBRIP1 correlates with sensitivity to DHX9 inhibition. Methods Cells were plated at pre-determined cell densities in 384-well cell culture plates and incubated overnight in 37oC at 5% CO2. A number of colorectal cancer (CRC) cell lines, including MSI-H CRC cells (sensitive to DHX9 inhibitor) and MSS CRC cells (insensitive to DHX9 inhibitor) were treated with with escalating doses of from 0.001 to 10 µM Compound 1, including HCT116 (CRC MSI; Sensitive), HT-29 (CRC MSS; Insensitive), DLD-1 (CRC MSI; Sensitive), HCT-15 (CRC MSI; Sensitive), LS411N (CRC MSI; Sensitive), NCI-H747 (CRC MSS; Insensitive), LS174T (CRC MSI; Sensitive), LOVO (CRC MSI; Sensitive), SW48 (CRC MSI; Insensitive), and SNU-407 (CRC MSI; Insensitive) Reference and test compounds were prepared in DMSO stock solution and serially diluted 3-fold for 10 points. Compound 1 (0.1% final DMSO concentration) was transferred to the cell plate in designated wells, and incubated 37oC at 5% CO2 for 72 hours. Compound 1 was prepared to a 10 mM stock solution, and 10 tested doses were created by titration by diluting the compound 1 fold each step. RNA was extracted from the cells using the TaqMan Gene Expression Cells-to-CT™ kit according to manufacturer’s (Invitrogen) protocol, and cDNA was reverse transcribed using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s (Invitrogen) protocol. Using TaqMan Multiplex Mastermix and thermal cycle conditions for the Quant Studio Thermocycler, qPCR was carried out as described above to quantify the level of circBRIP1. 63
Data analysis was performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ΔCt for circBRIP1 is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity (% Vehicle) is calculated by the following formulas, whereas: − ΔCts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample − ΔCtc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control − mRNA level = 2-ΔCtc − % Vehicle = 100 x [mRNA (compound treated) / mRNA (DMSO)] An EC50 was then calculated using a 4-parameter logistic nonlinear regression model in Scigilian Analyze or GraphPad Prism, floating both the top and bottom parameters. − EC50 Fit formula = Y=A + (B-A)/(1+10^((LogC-X)*D)) whereas A:Bottom; B:Top; C:EC50; D:Slope Results Induction of circBRIP1 was observed in both the DHX9-dependent and independent CRC cell lines with similar potency (see FIG. 7A). Thus, induction of circBRIP1 can serve as a clear and robust DHX9-specific target engagement biomarker across DHX9 sensitive and insensitive cells. A similar experiment was carried out using assays as described above, testing levels of circBRIP1 and inhibition of proliferation for breast, ovarian, lung, and colorectal cancer (CRC) cell lines after treatment with DHX9 inhibitor (Compound 1). Consistent with the observations made in CRC cell lines, circBRIP1 levels were also upregulated in breast, ovarian, and lung cell lines, and the levels of circBRIP1 did not correlate with anti-proliferation effects of the DHX9 inhibitor. In summary, induction of circBRIP1 can be used as a biomarker for DHX9 inhibition across DHX9 sensitive and insensitive cells. Example 7: Evaluation of circBRIP1 in frozen human PBMCs As proof of concept, circBRIP1 levels were examined in peripheral blood mononuclear cells (PMBCs) using frozen PBMCs from donors after treatment with a DHX9 inhibitor. 64
Frozen PBMCs were thawed in a 37oC water bath, washed, and reconstituted in media. The same number of cells were added to each well of 12-well plates and let rest for 2 hours before being subjected to testing conditions. Compound 1 was dissolved in DMSO at 10 mM as a stock solution, and diluted to a desired concentration. Frozen PBMCs from three donors or LS411N cells (positive control) were treated with 10 µM of Compound 1 or control vehicle for 24 hours. The circBRIP1 level was determined using methods essentially as decribed above. As shown in FIG. 8A, treatment with Compound 1 significantly elevated the circBRIP1 level in PBMCs as compared to vehicle-treated cells. Next, the proliferation of PMBCs was examined following treatment with DHX9 inhibitor using the following assay. Human PBMCs were thawed from a frozen cryotube in a 370C water bath and transferred to a flask with medium X-Vivo 15 supplemented with 5% Fetal Bovine Serum (FBS), 1% PenStrep, and 1% Glutamax. Flasks were incubated at 37C for 30 minutes. PBMCs were harvested from flask and counted. PBMCs were diluted in medium and added to 96 well plates at 100,000 PBMCs per well. A stimulus mixture containing IL-15 at 1ng/well, IL-21 at 5ng/well, and CD40L at 5ng/well was added to each well. Test compounds were prepared in DMSO stock solution and serially diluted. Compound (0.1% final DMSO concentration) was added to each well. DMSO was used as a negative control (high control, HC) and staurosporine was used a positive control (low control, LC). Plates were incubated at 37C for 0, 2, 4, 6, 8, 10 days. Every other day, medium was replenished with stimulus and diluted compounds. At each CTG time point, plate was removed from incubator and Cell-Titer Glo 2.0 reagent was added to each well according to manufacturer’s (Promega) protocol and detected on an Envision. The inhibition activity was calculated following the formula below: • % Inhibition = 100 x (ReadoutHC - ReadoutSample) / (ReadoutHC -ReadoutLC) PBMCs were exposed to Compound 1 at concentrations ranging from 0.01 µM to 10 µM for 10 days. PMBCs were treated with staustoporine as positive control for inhibition. As shown in FIG. 8B, staustoporine inhibited proliferation of PBMCs. Surprisingly, Compound 1 did not inhibit proliferation of PBMCs through the duration tested. In summary, treatment of PBMCs elevated circBRIP1 level in the cells, but did not impact their proliferation. 65
Example 8: Evaluation of circBRIP1 in human PBMCs This study was conducted to evaluate the circular format of BRIP1 mRNA in freshl human PBMC cells by qPCR. Methods Human whole blood was obtained from Research Blood Components. Blood of each donor was aliquoted into vacutainer tubes containing Acid Citrate Dextrose (ACD). Test compounds were prepared in DMSO stock solution and serially diluted. Compound (0.1% final DMSO concentration) was added to vacutainer tubes containing human whole blood. Tubes were placed in tube rotator and incubated at 37C for 24 hours. PBMCs were isolated from human whole blood after compound treatment by density gradient centrifugation using Ficoll-Paque Plus. RNA was extracted from PBMCs using RNeasy Plus Kits according to the manufacturer’s (Qiagen) protocol. cDNA was reverse transcribed using RT2 First Strand Kit according to the manufacturer’s (Qiagen) protocol. Using TaqMan Multiplex Mastermix and thermal cycler conditions for the QuantStudio Thermocycler, qPCR was carried out using TaqMan Gene Expression Assay with VIC Dye for Linear BRIP1 (Assay ID: Hs00908156_m1), TaqMan GAPDH primer limited assay with JUN dye/QSY probe, and the below custom TaqMan Gene Expression Assay with FAM Dye for circBRIP1: • Forward Primer: GTGAGCCAAGGAATTTTGTGTTT (SEQ ID NO: 19) • Reverse Primer: GGTCTGAACTTTGCCATTAATATCTG (SEQ ID NO: 20) • Probe: CCATCTTACAAGGCCTTT (SEQ ID NO: 21) Note: All target genes can be combined in one well per sample. Data analysis was performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ΔCt for circBRIP1 or linBRIP1 was calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity was calculated by the following formula, whereas: • ΔCts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ΔCtc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. 66
2^^^^ %^^^^^^^ = 100% × Results
Figure imgf000068_0001
In all donor’s blood, treatment with DHX9 inhibitor compound 1 at varying concentrations induced the presence of human circBRIP1 in a dose-responsive manner. Linear BRIP1 was not significantly changed (see FIGs. 9A-9D). This result indicates that circBRIP1 in PBMCs may be used to monitor DHX9 target engagement and inhibition in vivo. Example 9: Evaluation of circBRIP1 in human whole blood This study is conducted to evaluate the circular format of BRIP1 mRNA in fresh human whole blood by qPCR. Methods Human whole blood IS obtained from Research Blood Components. Blood of each donor IS aliquoted into vacutainer tubes containing Acid Citrate Dextrose (ACD). Test compounds are prepared in DMSO stock solution and serially diluted. Compound (0.1% final DMSO concentration) is added to vacutainer tubes containing human whole blood. Tubes are placed in tube rotator and incubated at 37C for 24 hours. Total cells are isolated from human whole blood after compound treatment by density gradient centrifugation using Ficoll-Paque Plus. RNA is extracted from cells using RNeasy Plus Kits according to the manufacturer’s (Qiagen) protocol. cDNA is reverse transcribed using RT2 First Strand Kit according to the manufacturer’s (Qiagen) protocol. Using TaqMan Multiplex Mastermix and thermal cycler conditions for the QuantStudio Thermocycler, qPCR is carried out using TaqMan Gene Expression Assay with VIC Dye for Linear BRIP1 (Assay ID: Hs00908156_m1), TaqMan GAPDH primer limited assay with JUN dye/QSY probe, and the below custom TaqMan Gene Expression Assay with FAM Dye for circBRIP1: • Forward Primer: GTGAGCCAAGGAATTTTGTGTTT (SEQ ID NO: 19) • Reverse Primer: GGTCTGAACTTTGCCATTAATATCTG (SEQ ID NO: 20) • Probe: CCATCTTACAAGGCCTTT (SEQ ID NO: 21) Note: All target genes can be combined in one well per sample. 67
Data analysis is performed by using the QuantStudio Thermocycler software to determine threshold Ct values. ΔCt for circBRIP1 or linBRIP1 is calculated by normalizing the target gene Ct to the GAPDH housekeeping gene Ct for each sample. The inhibition activity is calculated by the following formula, whereas: • ΔCts: Mean of Ct(target gene) – Mean of Ct(GAPDH) for each sample • ΔCtc: Mean of Ct(target gene) – Mean of Ct(GAPDH) for DMSO control. 2^^^^ %^^^^^^^ = 100% × 2^^^^ Results It is expected that trea
Figure imgf000069_0001
men w n or compound 1 at varying concentrations will induce the presence of human circBRIP1 in a dose-responsive manner, with linear BRIP1 not significantly changed, suggesting that circBRIP1 in whole blood may be used to monitor DHX9 target engagement and inhibition in vivo. Example 10: Identification of additional Alu-circRNAs mediated by DHX9 This study was conducted to identify additional Alu-mediated circRNAs that can be markers for evaluating DHX9 inhibition. Methods Screening for circRNA upregulated after knockdown of DHX9: HCT116 CRC-MSI cells were treated with DHX9 siRNA for a total of 3 days. Cells were harvested, and lysed with 1 ml of the TRIzol Reagent per 5-10 x 106 cells. Total RNA from each sample was quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on the Arraystar Human Circular RNA Array’s standard protocols. Briefly, total RNAs were digested with Rnase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8x15K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. 68
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software limma package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering. Differentially expressed circRNAs between two samples (DHX9 siRNA-treated and scrambled control siRNA treated) were identified through Fold Change filtering. Hierarchical Clustering was performed to show the distinguishable circRNAs expression pattern among samples. Identifying Alu-mediated circRNA among circRNAs upregulated by knockdown of DHX9: Up-regulated circRNA that were found to be differentially regulated by DHX9 knockdown were categorized as Alu mediated by methods as described by Stagsted et al. 2019 (Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles, Life Science Alliance, 2019, 2(3):1-16), the entire contents of which are incorporated herein by reference). Stagsted et al. states that Alu-mediated circRNA biogenesis occurs by aberrant backsplicing of RNA transcripts as stimulated by bringing Inverted Alu Elements (IAE) into close proximity with each other. Through a different mechanism, circRNA can also occur when long flanking introns backsplice on each other, which is termed “AUG circRNA” and relies on IAE-independent (i.e., Alu-independent) mode of biogenesis, whereas “other circRNA” relies on IAE-dependent biogenesis. Stagsted et al. shows through High-Throughput Sequencing of RNA isolated by CrossLinking ImmunoPrecipitation (HITS-CLIP) that DHX9 occupancy shows a clear selection for binding at proximal flanking regions of Alu-dependent circRNAs, as well as a clear preference for non-AUG over AUG circRNA. CircRNAs up-regulated by DHX9 knockdown identified by the Arraystar Human Circular RNA Array was compared to a list of circRNAs sensitive to DHX9 expression provided in Supplemental Table 4 of Stagsted et al. 2019. CircRNAs annotated as “AUG circRNA” by Statsted et al. were considered to not be sensitive to DHX9 activity, and thus not dependent on IAE for biogenesis. CircRNAs annotated as “Ambiguous circRNA” were also irrelevant for this analysis, as they are divergent across multiple host genes Results 69
The analysis as described above yielded the circRNAs listed in Table 2 as Alu-mediated circRNAs useful in evaluating DHX9 inhibition in the methods and assays of the present disclosure. Larger panels of circRNA other than those included in the Arraystar Human Circular RNA Array are tested using this same or similar approach to identify additional Alu-mediated circRNAs useful for the methods and assays provided herei 70
EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 71

Claims

We claim: 1. A method of measuring activity of a DHX9 inhibitor in a cell, comprising: contacting a cell with the DHX9 inhibitor; and measuring the level of an Alu-mediated circular RNA (Alu-circRNA) in the cell.
2. The method of claim 1, wherein the cell is a cancer cell.
3. The method of claim 1, wherein the cell is a non-cancerous cell.
4. The method of claim 1, wherein the cell is a peripheral blood mononuclear cell (PBMC).
5. The method of any one of claims 1-3, wherein the contacting is for a sufficient time for the Alu-circRNA to be produced.
6. The method of claim 5, wherein the level of Alu-circRNA is correlated to inhibition of a DHX9 activity by the DHX9 inhibitor in the cell.
7. A method of measuring activity of a DHX9 inhibitor in a subject, comprising: administering a DHX9 inhibitor to a subject; and measuring the level of an Alu-mediated circular RNA (Alu-circRNA) in a biological sample from the subject.
8. A method of measuring activity of a DHX9 inhibitor in a subject, comprising: measuring the level of Alu-mediated circular RNA (Alu-circRNA) in a biological sample from a subject, wherein the subject is being treated with a DHX9 inhibitor.
9. The method of claim 7 or 8, wherein the level of Alu-circRNA is correlated to inhibition of DHX9 activity by the DHX9 inhibitor in the subject. 72
10. The method of claim 9, wherein the level of Alu-circRNA is correlated to inhibition of DHX9 ATPase activity or resolvase activity.
11. The method of claim 7 or 8, wherein the level of Alu-circRNA is correlated to inhibition of proliferation of MSI cancer cells in the subject.
12. The method of any one of claims 7-11, wherein the biological sample is selected from the group consisting of blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue.
13. The method of claim 12 wherein the biological sample is blood.
14. The method of claim 13 wherein the method further comprises isolating cells from the blood sample, and measuring the level of the Alu-circRNA in the isolated cells.
15. The method of any one of claims 7-11, wherein the biological sample comprises peripheral blood mononuclear cells (PBMCs).
16. The method of claim 15, wherein the method further comprises isolating PBMCs from a blood sample, and measuring the level of the Alu-circRNA in the PBMCs.
17. The method of claim 15, wherein the level of Alu-circRNA does not correlate with inhibition of proliferation of PBMCs of the subject.
18. The method of claim any one of claims 1-17, wherein the Alu-circRNA is selected from the group consisting of cirRNA of BRIP1, AKR1A1, BMPR2, CALCOCO2, CLNSIA, DKC1, FAM124A, FBN1, FCHO2, GLS, GON4L, GPR125, NEIL3, PIK3C3, PNN, POMT1, RBM23, SKIL, SLK, SMA, SMG1, SMYD4, TAF2, TFAP4, TMEM41B, USP25, VPS13D, WDR59, and XPR1. 73
19. The method of any one of claims 1-18, wherein the level of the Alu-circRNA is measured by using one or more techniques selected from northernblot, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), microarray, droplet digital PCR, next-generation sequence (NGS) RNA sequencing, RNAin situ hypridization (RNA-ISH), in situ hypridization- immunohistochemistry (ISH-IHC), isothermal exponential amplification, and rolling cycle amplification.
20. The method of any one of claims 7-19, further comprising obtaining the biological sample from the subject.
21. The method of any one of claims 7-20, wherein the biological sample is obtained from the subject between 0.5-7 days following administration of the DHX9 inhibitor.
22. The method of any one of claims 7-21, wherein the method comprises measuring the level of the Alu-circRNA in biological samples obtained from the subject at more than one time point.
23. The method of any one of claims 7-22, wherein the subject has a cancer.
24. The method of claim 23, wherein the cancer is a microsatellite instability (MSI) cancer.
25. The method of claim 23 or 24, wherein the cancer is selected from the group consisting of colorectal cancer, endometrial cancer, ovarian cancer, gastric cancer, hematopoietic cancer, breast cancer, brain cancer, skin cancer, lung cancer, blood cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, bone cancer, soft tissue cancer, kidney cancer, liver cancer, and Ewing’s sarcoma.
26. The method of any one of claims 7-25, further comprising making a determination on an appropriate therapeutic dose of the DHX9 inhibitor based on a set of data including the level of Alu-circRNA measured. 74
27. The method of any one of claims 1-26, wherein the DHX9 inhibitor is selected from a small molecule, an antisense oligonucleotide, small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA).
28. The method of any one of claims 1-26, wherein the DHX9 inhibitor is a small molecule.
29. The method of any one of claims 1-26, wherein the DHX9 inhibitor is selected from the DHX9 inhibitors in Table 1.
30. The method of any one of claims 7-29, wherein the subject is a human.
31. A method of monitoring treatment of a subject with a DHX9 inhibitor, comprising: administering a DHX9 inhibitor to the subject; and obtaining information as to the level of Alu-mediated circular RNA (Alu- circRNA) in a biological sample obtained from the subject after administration of the DHX9 inhibitor.
32. The method of claim 31, wherein the level of Alu-circRNA is correlated to inhibition of DHX9 activity by the DHX9 inhibitor in the subject.
33. The method of claim 32, wherein the level of Alu-circRNA is correlated to inhibition of DHX9 ATPase activity or resolvase activity.
34. The method of claim 31, wherein the level of Alu-circRNA is correlated to inhibition of proliferation of MSI cancer cells in the subject.
35. The method of any one of claims 31-34, wherein the biological sample is selected from the group consisting of blood, interstitial fluid, lymph fluid, organ tissue, biopsy tissue, and skin tissue. 75
36. The method of claim 35, wherein the biological sample is blood.
37. The method of claim 36, wherein the method further comprises isolating cells from the whole blood sample, and measuring the level of the Alu-circRNA in the isolated cells.
38. The method of any one of claims 31-34, wherein the biological sample comprises peripheral blood mononuclear cells (PBMCs).
39. The method of claim 38, wherein the method further comprises isolating PBMCs from a blood sample, and measuring the level of the Alu-circRNA in the PBMCs.
40. The method of claim 38 or 39, wherein the level of Alu-circRNA does not correlate with inhibition of proliferation of PBMCs cells of the subject.
41. The method of claim any one of claims 31-40, wherein the Alu-circRNA is selected from the group consisting of cirRNA of BRIP1, AKR1A1, BMPR2, CALCOCO2, CLNSIA, DKC1, FAM124A, FBN1, FCHO2, GLS, GON4L, GPR125, NEIL3, PIK3C3, PNN, POMT1, RBM23, SKIL, SLK, SMA, SMG1, SMYD4, TAF2, TFAP4, TMEM41B, USP25, VPS13D, WDR59, and XPR1.
42. The method of any one of claims 31-41, wherein the level of the Alu-circRNA is measured by using one or more techniques selected from northernblot, reverse transcription- quantitative polymerase chain reaction (RT-qPCR), microarray, droplet digital PCR, next- generation sequence (NGS) RNA sequencing, RNAin situ hypridization (RNA-ISH), in situ hypridization- immunohistochemistry (ISH-IHC), isothermal exponential amplification, and rolling cycle amplification.
43. The method of any one of claims 31-42, further comprising obtaining the biological sample from the subject. 76
44. The method of any one of claims 31-43, wherein the biological sample is obtained from the subject between 0.5-7 days following administration of the DHX9 inhibitor.
45. The method of any one of claims 31-44, wherein the method comprises measuring the level of the Alu-circRNA in biological samples obtained from the subject at more than one time point.
46. The method of any one of claims 31-45, wherein the subject has a cancer.
47. The method of claim 46, wherein the cancer is a microsatellite instability (MSI) cancer.
48. The method of claim 46 or 47, wherein the cancer is selected from the group consisting of colorectal cancer, endometrial cancer, ovarian cancer, gastric cancer, hematopoietic cancer, breast cancer, brain cancer, skin cancer, lung cancer, blood cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, bone cancer, soft tissue cancer, kidney cancer, liver cancer, and Ewing’s sarcoma.
49. The method of any one of claims 31-48, further comprising making a determination on an appropriate therapeutic dose of the DHX9 inhibitor based on a set of data including the level of Alu-circRNA measured.
50. The method of any one of claims 31-49, wherein the DHX9 inhibitor is selected from a small molecule, an antisense oligonucleotide, small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA).
51. The method of any one of claims 31-49, wherein the DHX9 inhibitor is a small molecule.
52. The method of any one of claims 31-49, wherein the DHX9 inhibitor is selected from the inhibitors in Table 1. 77
53. The method of any one of claims 31-49, wherein the subject is a human. 78
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993013121A1 (en) 1991-12-24 1993-07-08 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
WO1995032305A1 (en) 1994-05-19 1995-11-30 Dako A/S Pna probes for detection of neisseria gonorrhoeae and chlamydia trachomatis
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
WO1998002582A2 (en) 1996-07-16 1998-01-22 Gen-Probe Incorporated Methods for detecting and amplifying nucleic acid sequences using modified oligonucleotides having increased target specific t¿m?
EP4137585A1 (en) * 2021-08-20 2023-02-22 Institut D'Investigacions Biomèdiques August Pi I Sunyer - IDIBAPS Cancer informative biomarker signature

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
WO1993013121A1 (en) 1991-12-24 1993-07-08 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
WO1995032305A1 (en) 1994-05-19 1995-11-30 Dako A/S Pna probes for detection of neisseria gonorrhoeae and chlamydia trachomatis
WO1998002582A2 (en) 1996-07-16 1998-01-22 Gen-Probe Incorporated Methods for detecting and amplifying nucleic acid sequences using modified oligonucleotides having increased target specific t¿m?
EP4137585A1 (en) * 2021-08-20 2023-02-22 Institut D'Investigacions Biomèdiques August Pi I Sunyer - IDIBAPS Cancer informative biomarker signature

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NM_001328662.2
"The Biochemistry of the Nucleic Acids", 1992, pages: 5 - 36
AKTA ET AL.: "DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome", NATURE, vol. 544, 2017, pages 112 - 119
DAGAN ET AL.: "Alu Gene: a database of Alu elements incorporated within protein-coding genes", NUCLEIC ACIDS RESEARCH, vol. 32, no. 1, 2004, pages D489 - D492
GULLIVER CHLOE ET AL: "The enigmatic helicase DHX9 and its association with the hallmarks of cancer", FUTURE SCIENCE OA, vol. 7, no. 2, 1 February 2021 (2021-02-01), XP093035283, DOI: 10.2144/fsoa-2020-0140 *
GULLIVER ET AL., FUTURE SCIENCE OA, no. 2, 2020, pages FS0650
HORMOZDIARI ET AL.: "Alu repeat discovery and characterization within human genomes", GENOME RES, vol. 21, 2011, pages 840 - 849
JENNIFER CASTRO ET AL: "Abstract 1136: Targeting DHX9 inhibition as a novel therapeutic modality in microsatellite instable colorectal cancer", CANCER RESEASRCH; AACR ANNUAL MEETING 2023; APRIL 14-19, 2023; ORLANDO, FL, USA; PART 1, UNIVERSITY OF CHICAGO PRESS, vol. 83, no. 7_Supplement, 4 April 2023 (2023-04-04), pages 1136, XP009555249, ISSN: 0099-7374, DOI: 10.1158/1538-7445.AM2023-1136 *
KAREN REUE: "mRNA Quantitation Techniques: Considerations for Experimental Design and Application", THE JOURNAL OF NUTRITION, vol. 128, no. 11, 1998, pages 2038 - 2044
KLEIVELAND ET AL.: "Peripheral Blood Mononuclear Cells", 2015, SPRINGER, article "The Impact of Food Bioactives on Health: in vitro and ex vivo models [Internet"
MI ET AL.: "Circular RNA detection methods: A minireview", TALANTA, vol. 238, no. 2, 2022, pages 123066
PAN ET AL., CURRENT PROTEIN & PEPTIDE SCIENCE, no. 22, 2021, pages 29 - 40
STAGSTED ET AL.: "Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles", LIFE SCIENCE ALLIANCE, vol. 2, no. 3, 2019, pages 1 - 16
WILUSZ: "Repetitive elements regulate circular RNA biogenesis", MOB GENET ELEMENTS, vol. 5, no. 3, 2015, pages 39 - 45, XP055324358, DOI: 10.1080/2159256X.2015.1045682
ZHANG ET AL.: "Circular RNAs: Promissing Biomarkers for Human Diseases", EBIOMEDICINE, vol. 34, 2018, pages 267 - 274

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