WO2016040167A1 - Compositions et méthodes de dépistage et de traitement du cancer du poumon à petites cellules - Google Patents

Compositions et méthodes de dépistage et de traitement du cancer du poumon à petites cellules Download PDF

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WO2016040167A1
WO2016040167A1 PCT/US2015/048602 US2015048602W WO2016040167A1 WO 2016040167 A1 WO2016040167 A1 WO 2016040167A1 US 2015048602 W US2015048602 W US 2015048602W WO 2016040167 A1 WO2016040167 A1 WO 2016040167A1
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nucleic acid
acid molecule
srsfl
expression
sclc
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Brandon Higgs
Jiaqi Huang
Zhan XIAO
Xin Yao
Haihong Zhong
Sarah CONLEY
Yihong Yao
Liyan JIANG
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Brandon Higgs
Jiaqi Huang
Xiao Zhan
Xin Yao
Haihong Zhong
Conley Sarah
Yihong Yao
Jiang Liyan
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • SCLC Small cell lung cancer
  • SRSF1 is linked to the AKT or ERK pathways, two of the most established oncogenic pathways pivotal to tumor growth and survival.
  • SRSF 1 is a key driver oncogene in small cell lung cancer and provides in vivo data to support this conclusion.
  • This novel discovery firmly establishes SRSF1 as a compelling therapeutic target for SCLC, especially for the population with poor outcome as predicted by SRSF1 overexpression.
  • the invention generally features compositions and methods for characterizing and treating small cell lung cancer, as well as for characterizing the prognosis of a patient having small cell lung cancer.
  • the invention provides for a method for reducing the
  • the method comprises contacting the cell with an agent that reduces the expression or activity of an SRSF1 nucleic acid molecule or polypeptide.
  • SCLC small cell lung cancer
  • the invention provides for a method of inducing cell death in a small cell lung cancer cell, where the method comprises contacting the cell with an agent that reduces the expression or activity of an SRSFl nucleic acid molecule or polypeptide.
  • the invention provides for a method for reducing the proliferation or survival of a small cell lung cancer (SCLC) cell, where the method comprises contacting the cell with an inhibitory nucleic acid molecule that reduces the expression of an SRSFl nucleic acid molecule and/or polypeptide.
  • SCLC small cell lung cancer
  • the invention provides for a method of inducing cell death in a small cell lung cancer cell, where the method comprises contacting the cell with an inhibitory nucleic acid molecule that reduces the expression of an SRSFl nucleic acid molecule or polypeptide.
  • the present invention also provides for a method for treating small cell lung cancer
  • SCLC in a subject, where the method comprises administering to the subject an agent that reduces the expression or activity of an SRSFl nucleic acid molecule or polypeptide.
  • the present invention further provides for a method for treating small cell lung cancer (SCLC) in a subject, where the method comprises administering to the subject an inhibitory nucleic acid molecule that reduces the expression of an SRSFl nucleic acid molecule or polypeptide.
  • SCLC small cell lung cancer
  • the invention also provides for a method of treating a subject identified as having SCLC with a poor prognosis, where the method comprises administering to the subject an agent that reduces the expression or activity of an SRSFl nucleic acid molecule or polypeptide, where the subject is identified as having a poor prognosis by detecting SRSFl copy number (CN) gain or increased SRSFl expression in a biological sample of the subject relative to a reference.
  • CN SRSFl copy number
  • the invention also provides for a method of treating a subject identified as having SCLC with a poor prognosis, where the method comprises administering to the subject an inhibitory nucleic acid molecule that reduces the expression of an SRSFl nucleic acid molecule or polypeptide, where the subject is identified as having a poor prognosis by detecting SRSFl copy number (CN) gain or increased SRSFl polynucleotide or polypeptide expression in a biological sample of the subject relative to a reference.
  • CN SRSFl copy number
  • the agent is an inhibitory nucleic acid molecule at least a portion of which is complementary to an SRSFl nucleic acid molecule.
  • the cell is a human cell in vitro or in vivo.
  • the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA.
  • the inhibitory nucleic acid molecule comprises or consists essentially of a nucleic acid molecule having the sequence 5 ' -CCAACAAGATAGAGT AT AA-3'.
  • the cell can be further contacted with cisplatin, topotecan, etoposide, paclitaxel, irinotecan or combinations thereof.
  • the agent is a molecule that suppresses production of SRSF1 polypeptide in vivo.
  • the cell has SRSF1 copy number (CN) gain or increased SRSF1 expression, where the copy number is 3, 4, or more.
  • the agent is an antibody that specifically binds an SRSF1 polypeptide.
  • the subject is human.
  • the combination of the SRSF1 inhibitory nucleic acid molecule and cisplatin, topotecan, etoposide, paclitaxel or irinotecan reduces SCLC proliferation or tumorigenesis more than either the inhibitory nucleic acid molecule or the cisplatin, topotecan, etoposide, paclitaxel or irinotecan administered alone.
  • the copy number gain or increase polynucleotide expression is detected by one or more of quantitative PCR, microarray, in situ hybridization, Northern blot, Southern blot, and FISH assay.
  • the reference is the level of SRSF1 polypeptide or nucleic acid molecule present in a control sample.
  • the control sample is derived from a healthy subject. In other particular embodiments, the control sample is derived from the same subject at an earlier point in time.
  • the present invention also provides for a method of identifying a subject as having a poor prognosis, the method comprising detecting SRSF1 copy number (CN) gain or increased SRSF1 expression in a biological sample of the subject relative to a reference, where detection of CN gain and/or increased SRSF1 expression identifies the subject as having a poor prognosis.
  • CN SRSF1 copy number
  • the present invention also provides for a method of identifying a subject as having small cell lung cancer (SCLC) that is responsive to treatment with an agent that reduces the expression or activity of an SRSF1 nucleic acid molecule or polypeptide, where the method comprises detecting SRSF1 copy number (CN) gain or increased SRSF1 expression in a biological sample of the subject relative to a reference, wherein detection CN gain and/or increased SRSF1 expression identifies the subject as responsive to an agent that reduces the expression or activity of an SRSFl nucleic acid molecule or polypeptide.
  • SCLC small cell lung cancer
  • a biological sample is a tumor sample.
  • SCLC is a metastatic neoplasia or a neoplasia having a propensity to metastasize.
  • the present invention further provides for a pharmaceutical composition for the treatment of small cell lung cancer (SCLC), the composition comprising an agent that reduces the expression or activity of an SRSFl nucleic acid molecule or polypeptide and an excipient.
  • this agent is an inhibitory nucleic acid molecule at least a portion of which is complementary to an SRSFl nucleic acid molecule.
  • the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA.
  • the inhibitory nucleic acid molecule comprises or consists essentially of a nucleic acid molecule having the sequence 5 '-CCAACAAGATAGAGTATAA-3 ' .
  • the agent is an antibody that specifically binds an SRSFl polypeptide.
  • the pharmaceutical composition further comprises cisplatin, topotecan, etoposide, paclitaxel, irinotecan or combinations thereof.
  • the present invention also provides for a kit for the treatment of small cell lung cancer (SCLC), the kit comprising an agent that reduces the expression or activity of an SRSFl nucleic acid molecule or polypeptide and instructions for use in the treatment of SCLC.
  • SCLC small cell lung cancer
  • the agent comprised within the kit is an inhibitory nucleic acid molecule at least a portion of which is complementary to an SRSFl nucleic acid molecule.
  • the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA.
  • copy number is meant the number of copies of a gene in the genotype of a cell. Abnormal number of copies of genes arise from alterations in one or more sections of genomic DNA, which is commonly associated with cancer cells. Alterations in gene copy number also result in altered gene dosage of expressed genes.
  • reduces is meant a negative alteration. For example, a reduction of 10%, 25%, 50%, 75%, or 100%.
  • Detect refers to identifying the presence, absence or amount of the analyte to be detected.
  • the analyte is SRSFl polypeptide, SRSFl DNA or SRSFl mRNA.
  • increases is meant a positive alteration. For example, an increase by at least 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 1000%, or more.
  • a reference level is the level of SRSFl expression in a biological sample (e.g., lung cell) obtained from a healthy control subject.
  • a “reference sequence” is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.
  • the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
  • SRSFl Serine/arginine-rich splicing factor 1
  • SRSFl a polypeptide or fragment thereof having at least about 85% or greater amino acid identity to the amino acid sequence provided at NCBI Accession No. NP 001071634 and having SRSFl biological activity.
  • An exemplary SRSFl biological activity is mRNA splicing activity.
  • An exemplary SRSFl amino acid sequence is provided below:
  • Serine/arginine-rich splicing factor 1 nucleic acid molecule is meant a polynucleotide encoding an SRSFl polypeptide.
  • An exemplary SRSFl nucleic acid molecule is provided at NCBI Accession No. NM 001078166.
  • An exemplary SRSFl transcript is provided below:
  • SRSFl inhibitory nucleic acid molecule is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell reduces the expression of SRSFl .
  • an inhibitory nucleic acid molecule comprises at least a portion of a complementary strand of a target nucleic acid molecule.
  • an SRSFl inhibitory nucleic acid molecule inhibits at least about 10%, 25%, 50%, 75%, or even 90-100% of the SRSFl expression in the cell.
  • SRSFl siRNA is meant a double stranded (ds) RNA capable of reducing SRSFl expression in a target cell.
  • dsRNA double stranded RNA capable of reducing SRSFl expression in a target cell.
  • an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end.
  • dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream to reduce the expression of an SRSFl nucleic acid molecule.
  • subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, feline, or murine.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith.
  • treatment of SCLC results in SCLC cell depletion, in reducing or stabilizing the growth or proliferation of a tumor in a subject, in increasing the cell death of a malignant cell, or increasing patient survival. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural. 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 are modified by the term about.
  • Figure 1 is a summary of clinical features of Chinese SCLC patients and the time-to- event analysis schema.
  • 99 Chinese primary SCLC patients were divided into training and validation cohorts according to the availabilities of matched normal, R Aseq and survival outcome information.
  • the training set includes 22 patients with each patient having tumor and NAT WES data and survival outcome.
  • the test set included 74 patients. WES data from tumor and survival outcome were available for each patient. Among those patients, WES data, RNAseq data, and survival outcome were available for 49 patients.
  • Figures 2A-2D depict factors most frequently mutated in SCLC include DNA
  • Figure 2A is a schematic representation showing amino acid changes in human POLG, POLDl, POLQ proteins.
  • Figure 2B depicts amino acid alterations mapped on a structure of the human POLG catalytic domain. Mutations were mapped onto the structure of human POLG using PDB Id entry 3IKM as template.
  • FIG. 2C depicts relevant amino acid alterations mapped on a structural model of POLDl . Mutations in human POLDl gene were mapped onto structure of the yeast DNA polymerase subunit ⁇ using PDB entry 3IAY (Swan et al, 2009). Orange colored ribbon represents exonuclease domain, blue colored ribbon corresponds to polymerase domain, and the green ribbon represents the N-terminal portion of the protein.
  • Figure 2D is a schematic that depicts mutation prevalence in Fanconi anemia pathway genes.
  • Figure 3 is a heat map depicting exome-wide copy number variations (CNVs) called for 99 SCLC patients.
  • Figures 4A-4E provide five graphs showing that SRSF1 copy number gain and mRNA expression correlates with survival.
  • Figure 4C provides the validation set
  • Figure 4D shows a combination of discovery set and validation set (Figure 4E).
  • "CN" denotes copy number.
  • p* log-rank test
  • p Cox PH regression model
  • HR hazard ratio.
  • Figure 4F shows Kaplan-Meier curves from indications in The Cancer Genome Atlas where at least 3 patients harbored a copy number gain of SRSFl. Plots were generated in OncoLand (OmicSoft Corp; Cary, NC). No amplification line indicated with asterisk.
  • Figures 5A-5C show that SRSFl mediated growth and survival in SRSFl expressing
  • FIG. 5 A is a graph showing the results of TaqMan assays which detected SRSFl DNA CNs in 13 SCLC cell lines.
  • Figure 5B are images of Western blot analysis showing protein expression levels of SRSFl in SCLC cell lines.
  • Figure 5C are graphs showing the effect of siRNA knockdown in SCLC cell lines on proliferation and Caspase-3/7 activities.
  • NCI-H82, SHP-77 and NCI- 1048 cell lines were transfected with non-targeting control or SRSFl -directed siRNAs for 48 hours, then treated with cisplatin (2.5 ⁇ g/ml) for 48 hours or topotecan (2.5 ⁇ g/ml) for 24 hours.
  • Cell growth and Caspase-3/7 activities were assessed and normalized against non-targeting siRNA-transfected cells as 100% control.
  • Figures 6A-6E show that SRSFl was required for tumorigenicity of SCLC.
  • a DMS114 SCLC cell line was transfected with non-targeting control or SRSFl -directed siRNAs for 48 hours, then treated with cisplatin (2.5 ⁇ g/ml) for 48 hours or topotecan (2.5 ⁇ g/ml) for 24 hours.
  • Figure 6A is a graph depicting cell growth assessed and normalized against growth of non- targeting siRNA -transfected cells used as 100% control.
  • Figure 6B is a graph depicting Caspase- 3/7 activities assessed and normalized against Caspase-3/7 activities of non-targeting siRNA- transfected cells used as 100% control.
  • Figure 6C are phase-contrast images of DMS114 cells transfected with non-targeting or SRSFl siRNAs for 48 hours and then seeded in sphere forming media and allowed to grow for 4 days. Phase-contrast images of the sphere formation under each condition were captured and viable cell mass quantitated by CTG assay.
  • Figure 6D is an analysis of reconstitution of SRSFl expression using an siRNA-resistant Flag-tagged SRSFl expression construct carried out in SRSFl siRNA transfected cells. Impact on sphere growth rate was assessed by CTG assay, and successful SRSFl protein re-expression was confirmed by Western blot analysis using either anti-SRSFl antibody or anti-Flag antibody.
  • Figure 6E is an analysis of tumor formation rates in immunocompromised mice implanted with DMS114 cells transfected with non-targeting control siRNA or SRSFl siRNA. Tumor formation rates were monitored and measured as described in the methods herein below.
  • Figures 7A-7D show that SRSFl was required for cell viability and sphere formation in
  • NCI-82, SHP-77 and NIH-H1049 cell lines were transfected with non-targeting and SRSFl siRNAs respectively for 48 hours and then seeded in sphere forming media and allowed to grow for 4 days.
  • Figure 7 A depicts phase-contrast images of sphere formation under each condition captured.
  • Figure 7B is a graph depicting viable cell mass quantitated by CTG assay.
  • Figure 7C is an analysis of reconstitution of SRSFl expression using a siRNA-resistant Flag-tagged SRSFl expression construct carried out in SRSFl siRNA transfected NCI-H82 cells.
  • Figure 7D depicts results of clonogenic assays of DMS-114, NCI-82, SHP-77 and NIH-H1049. Cells were transfected with siRNAs for 48 hours and then seeded in the methylcellulose medium for -7-14 days, colonies with more than 40 cells per colony were counted.
  • Figure 8 is a graph depicting tumor volume of SHP-77 cells transfected with non- targeting control siRNA or SRSFl siRNA implanted into immunocompromised mice. Tumor formation rates were monitored and measured.
  • Figures 9A-9C show mechanism of action for SRSFl in SCLC.
  • Figure 9A is a Western blot showing that SRSFl prevents DNA-damage.
  • DMS114 cells were transfected with control or SRSFl siRNA and then treated with topotecan or Cisplatin for the indicated times.
  • SRSFl, phosphor-H2AX and phosphor-Chk2 were probed with their corresponding antibodies. Equal protein loading across the different samples was demonstrated with the anti -tubulin antibody.
  • Figures 9B and 9C show that SRSFl mediates the activation of AKT and ERK pathways.
  • DMS114 cells transfected with non-targeted or SRSFl -targeted siRNAs were lysed and applied to the phospho-kinase array as detailed in Materials and Methods.
  • the dot blot result was further confirmed by conventional Western blot in both DMS114 and NCI-1048 cells.
  • Figure 10 is a heatmap used as an identity check between matched SCLC tumor and normal specimens. Pearson correlation heatmap was used to compare 300 germline SNP profiles between each of the 25 tumors and matched normals. DETAILED DESCRIPTION OF THE INVENTION
  • the invention features compositions and methods that are useful for characterizing and treating small cell lung cancer (SCLC) in a subject, as well as methods for characterizing the prognosis of a patient with SCLC.
  • SCLC small cell lung cancer
  • the present invention is based, at least in part, on the discovery that SCLC cells characterized by SRSFl copy number gain and/or increased SRSFl mRNA over-expression in tumors is strongly associated with poor survival.
  • whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients was conducted.
  • WES whole exome sequencing
  • transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients was conducted.
  • SRSFl is essential for tumorigenecity of SCLC and plays a key role in DNA repair and chemo-sensitivity.
  • inhibition of SRSFl transcripts decreased the tumorigenicity of SCLC cells.
  • SRSFl Inhibition of SRSFl also sensitized SCLC cells to chemotherapeutic agents (e.g., cisplatin, topotecan).
  • chemotherapeutic agents e.g., cisplatin, topotecan.
  • the present invention provides therapeutic methods for treating SCLC by inhibiting SRSFl expression (e.g., by RNAi), as well as methods for characterizing the prognosis of a patient suffering from SCLC, for example, by detecting SRSFl copy number gain or increased SRSFl expression (e.g., increased SRSFl mRNA or polypeptide expression).
  • the present invention provides methods of treating SCLC, or symptoms thereof, by administering an agent that directly or indirectly inhibits SRSFl biological activity or expression, including but not limited to an siRNA or shRNA that inhibits SRSFl expression, a functional antagonist of SRSFl (such as an inhibitor of SRSFl splicing activity or nuclear import), or an agent that suppresses or inhibits SRSFl production in vivo.
  • an agent that inhibits SRSFl biological activity or expression is provided to a subject having SCLC in a
  • the agent is an SRSFl inhibitory nucleic acid molecule that decreases the expression of an SRSFl nucleic acid molecule or SRSFl polypeptide in a subject.
  • An SRSFl inhibitory nucleic acid molecule e.g., siRNA
  • a chemotherapeutic agent e.g., cisplatin, topotecan.
  • SRSFl can be detected by any suitable method. The methods described herein can be used individually or in combination for a more accurate detection of an SRSFl biomarker.
  • SCLC is characterized by detecting the level of SRSFl expression (e.g., SRSFl polynucleotide or polypeptide level) in a biological sample(e.g., lung tumor) of the subject relative to the expression in a reference (e.g., lung sample from a healthy control subject), where an increase in SRSFl expression is indicative of a poor prognosis.
  • SRSFl expression e.g., SRSFl polynucleotide or polypeptide level
  • the prognosis of a subject with SCLC is characterized by detecting SRSFl copy number in a biological sample (e.g., lung tumor) of the subject, wherein an increase in SRSFl copy number (i.e., copy number gain) relative to a reference is indicative of a poor prognosis.
  • Methods for characterizing SRSFl copy number include, for example, DNA sequencing, TaqMan assays, FISH assays, SNP array, or array comparative genomic hybridization.
  • Methods for evaluating increased SRSFl polynucleotide expression include, for example, RNA-seq, quantitative PCR, gene expression microarray, in situ hybridization, Northern blot.
  • SRSFl polypeptide level is measured by immunoassay.
  • Immunoassay typically utilizes an antibody (or other agent that specifically binds the marker) to detect the presence or level of a biomarker in a sample.
  • Antibodies can be produced by methods well known in the art, e.g., by immunizing animals with the biomarkers. Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.
  • the invention contemplates traditional immunoassays including, for example,
  • Immunohistochemistry (IHC), Western blot, sandwich immunoassays including ELISA and other enzyme immunoassays, fluorescence-based immunoassays, and chemiluminescence.
  • Nephelometry is an assay done in liquid phase, in which antibodies are in solution. Binding of the antigen to the antibody results in changes in absorbance,which is measured.
  • Other forms of immunoassay include magnetic immunoassay, radioimmunoassay, and real-time
  • Immunoassays can be carried out on solid substrates (e.g., chips, beads, micro fluidic platforms, membranes) or on any other forms that supports binding of the antibody to the marker and subsequent detection.
  • a single marker may be detected at a time or a multiplex format may be used.
  • Multiplex immunoanalysis may involve planar microarrays (protein chips) and bead-based microarrays (suspension arrays).
  • Patients identified as having increased SRSF1 expression are selected for treatment with an agent that reduces SRSF1 expression or activity (e.g., an SRSF1 inhibitory nucleic acid molecule, such as siRNA), alone or in combination with cisplatin, topotecan, etoposide, paclitaxel, irinotecan or combinations thereof.
  • an agent that reduces SRSF1 expression or activity e.g., an SRSF1 inhibitory nucleic acid molecule, such as siRNA
  • Patients treated with a method of the invention may be monitored by detecting alterations in SRSF1 expression following treatment with, for example, an SRSF1 siRNA and cisplatin or topotecan.
  • Patients showing a reduction in SRSF1 expression, a reduction in tumor volume, or an increase in tumor cell death relative to a reference level are identified as responsive to SRSF1 inhibition.
  • Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression of a nucleic acid molecule or polypeptide.
  • the invention provides methods for inhibiting SRSF1 expression in SCLC cells to reduce their proliferation, survival and/or tumorigenesis. Accordingly, the invention provides single and double stranded inhibitory nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that target SRSF1 and reduce its expression.
  • Exemplary inhibitory acid molecules include siRNA, shRNA, and antisense RNAs.
  • Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down- regulating gene expression (Zamore et al., Cell 101 : 25-33; Elbashir et al., Nature 411 : 494-498, 2001, hereby incorporated by reference).
  • the therapeutic effectiveness of an siRNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38-39.2002).
  • siRNAs may be designed to inactivate that gene. Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically.
  • the nucleic acid sequence of a IL15Ra gene can be used to design small interfering RNAs (siRNAs).
  • siRNAs small interfering RNAs
  • the 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat SCLC.
  • RNAi RNA interference
  • the inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of expression.
  • RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002).
  • the introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of- function phenotypes in mammalian cells.
  • a double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention.
  • the dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA).
  • small hairpin (sh)RNA small hairpin
  • dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired.
  • dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription).
  • Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550- 553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol.
  • Small hairpin RNAs comprise an RNA sequence having a stem-loop structure.
  • a "stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion).
  • the term "hairpin” is also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art.
  • the secondary structure does not require exact base-pairing.
  • the stem can include one or more base mismatches or bulges.
  • the base-pairing can be exact, i.e. not include any mismatches.
  • the multiple stem-loop structures can be linked to one another through a linker, such as, for example, a nucleic acid linker, a miRNA flanking sequence, other molecule, or some combination thereof.
  • small hairpin RNA includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). While there may be some variation in range, a conventional stem-loop shRNA can comprise a stem ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. shRNAs can be expressed from DNA vectors to provide sustained silencing and high yield delivery into almost any cell type.
  • the vector is a viral vector.
  • Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations.
  • Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • a retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP+E-86,
  • the vector can transduce the packaging cells through any means known in the art.
  • a producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein.
  • retroviral vector particles then can be employed, to transduce eukaryotic cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will express a DNA replication protein.
  • Catalytic RNA molecules or ribozymes that include an antisense sequence of the present invention can be used to inhibit expression of a nucleic acid molecule in vivo.
  • ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
  • the design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 Al, each of which is incorporated by reference.
  • the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases.
  • the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8: 183, 1992. Example of hairpin motifs are described by Hampel et al, "RNA Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep.
  • any method for introducing a nucleic acid construct into cells can be employed.
  • Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct.
  • a viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA.
  • Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like.
  • the shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.
  • DNA vectors for example plasmid vectors comprising either an RNA polymerase II or RNA polymerase III promoter can be employed. Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters and in some cases, shRNAs are most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al, 2005, Nat. Genet. 39: 914-921).
  • expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters.
  • RNA polymerase type II promoters examples include tetracycline-inducible promoters (including TRE -tight), IPTG- inducible promoters, tetracycline transactivator systems, and reverse tetracycline transactivator (rtTA) systems.
  • Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types.
  • a certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Patent Application PCT/US2003/030901 (Publication No. WO 2004-029219 A2) and Fewell et al, 2006, Drug Discovery Today 11 : 975-982, for a description of inducible shRNA.
  • Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference). Tools of delivering
  • polynucleotides into cells include nanoparticles, liposomes, or other olignonucleotide- encapsulating vehicles. These vehicles may be additionally charged with cancer-targeted modalities such as antibodies against SCLC-specific targets to facilitate tumor-specific uptake.
  • Subjects having an SCLC responsive to treatment with an anti-SRSFl antibody are identified by characterizing the copy number or expression of SCLC present in a lung cell or tissue. Once selected for treatment, such subjects may be administered virtually any anti-SRSFl antibody known in the art. Suitable anti-SRSFl antibodies include, for example, known anti- SRSFl antibodies, commercially available anti-SRSFl antibodies, or anti-SRSFl antibodies developed using methods well known in the art.
  • Antibodies useful in the invention include immunoglobulins, monoclonal antibodies
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain at least one antigen-binding site.
  • Anti-SRSFl antibodies encompass monoclonal human, humanized or chimeric anti- SRSFl antibodies.
  • Anti-SRSFl antibodies used in compositions and methods of the invention can be naked antibodies, immunoconjugates or fusion proteins.
  • an anti- SRSFl antibody is a human, humanized or chimeric antibody having an IgG isotype, particularly an IgGl, IgG2, IgG3, or IgG4 human isotype or any IgGl, IgG2, IgG3, or IgG4 allele found in the human population.
  • Antibodies of the human IgG class have advantageous functional characteristics, such as a long half-life in serum and the ability to mediate various effector functions (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 1 (1995)).
  • the human IgG class antibody is further classified into the following 4 subclasses: IgGl , IgG2, IgG3 and IgG4.
  • the IgGl subclass has the high ADCC activity and CDC activity in humans (Chemical Immunology, 65, 88 (1997)).
  • an anti-SRSFl antibody is an isotype switched variant of a known anti-SRSFl antibody.
  • kits for the treatment of SCLC includes an inhibitory nucleic acid molecule that reduces the expression of an SRSF1 polynucleotide or polypeptide.
  • the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • an inhibitory nucleic acid molecule of the invention is provided together with instructions for administering the inhibitory nucleic acid molecule to a subject having or at risk of developing SCLC.
  • the invention provides kits for diagnosing SCLC or
  • a diagnostic kit of the invention provides a reagent (e.g., TaqMan primers/ probes for both SRSF1 and housekeeping reference genes) for measuring SRSF1 copy number or expression (e.g., a Taqman probe). If desired, the kit further comprises instructions for measuring SRSF1 copy number or expression and/or instructions for administering an SRSF1 inhibitory therapy to a subject having SCLC.
  • a reagent e.g., TaqMan primers/ probes for both SRSF1 and housekeeping reference genes
  • the kit further comprises instructions for measuring SRSF1 copy number or expression and/or instructions for administering an SRSF1 inhibitory therapy to a subject having SCLC.
  • the instructions include at least one of the following:
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • the kit comprises a sterile container which contains a therapeutic or diagnostic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • a sterile container which contains a therapeutic or diagnostic composition
  • Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • SCLC Small cell lung cancer
  • CNVs Somatic copy number variants
  • Certain genes with recurrent CN gains were previously reported in SCLC (Arriola et al, 2008; D'Angelo et al, 2010; Medina et al, 2009; Rudin et al, 2012), including MYC (8%), KIT (17%)), and SOX4 (19%>), with SOX2 (61%) and multiple other genes located across a segment on chromosome 3q27.1 (2).
  • Example 2 SRSFl CN gain and mRNA over-expression predicted poor survival in Chinese SCLC patients.
  • Kaplan- Meier analyses were conducted between patients with or without CNV alterations in the discovery cohort first as described in the methods herein below. Then this gene list was reduced to those with p ⁇ 0.05 in the validation cohort.
  • SOX2 CN gain was not identified as a correlate with survival.
  • PH Cox proportion hazard
  • Table 6A Kaplan-Meier analysis summary for SRSFl DNA amplification and mRNA expression CNV records events median 0.95LCL 0.95UCL p value
  • Table 6B Cox proportion hazard regression analysis summary for SRSFl DNA amplification and mRNA over-expression
  • SRSFl DNA copy number (CN) was screened in 13 SCLC cell lines using TaqMan assays. Five of 13 (38%) tested SCLC cell lines carried SRSFl CN gain (CN >3): Four cell lines including NCI-H82 had 3 copies, DMSl 14 had 4 copies. These cell lines also expressed high levels of SRSFl mRNA and protein ( Figures 5A and 5B).
  • SRSFl siRNA was transfected into DMSl 14 cells, and the growth effect of SRSFl ablation in two dimensional cell culture either alone or in conjunction with a sub-lethal dose of cisplatin or topotecan - two of the most frequently used standard of care compounds in SCLC, were evaluated (Figure 6A).
  • SRSFl siRNA but not the control siRNA decreased the SRSFl protein level. This caused a 35% decrease in the proliferation rate of DMS 1 14.
  • Treatment with a low dose of cisplatin only induced a modest decrease in cell growth.
  • a combination of cisplatin and SRSFl siRNA significantly enhanced the overall growth inhibition effect. A similar effect was observed with topotecan (Figure 6A).
  • SRSFl knockdown was investigated on SCLC cells when grown as 3D spheroids.
  • Cells transfected with non-targeting or SRSFl siRNA produced large and well- organized spheroids or did not form well-organized structures ( Figure 6C, 7 A, and 7B). The results were confirmed by colony formation assays ( Figure 7D).
  • the effect of SRSFl siRNA was mediated by specific target loss as demonstrated by a reconstitution study.
  • An siRNA- resistant Flag-tagged expression construct was able to efficiently rescue spheroid growth in the presence of the SRSFl siRNA in DMS 114 cells ( Figure 6D) and NCI-H82 cells ( Figure 7C).
  • SRSFl was required for in vivo tumorigenicity of SCLC.
  • SRSFl expression was shown to be important for in vivo tumorigenicity of SCLC cells.
  • SRSFl was identified as a candidate oncogene in breast cancer (9) and NSCLC (10) based on SRSFl over-expression in non-transformed cells to demonstrate the transforming potential of the substantially over- expressed SRSFl protein.
  • RSF1 promotes tumorigenesis primarily through its canonical RNA splicing function on various oncogenic or tumor-suppressor effector molecules (11).
  • Das et al previously summarized various spliced products of SRSFl and isoform
  • SRSFl relies on certain non- canonical pathways to sustain the tumorigenicity of SCLC cells. SRSFl loss induced p- H2AX signal, suggesting that SRSF 1 may help maintain the genomic integrity of SCLC to safeguard against DNA-damage and cell death. Furthermore, SRSFl mediates the activation of both PI3K/AKT and MEK/ERK pathways.
  • the present invention provides the new observation that SRSFl is linked to the AKT or ERK pathways, two of the most established oncogenic pathways pivotal to tumor growth and survival.
  • SRSF 1 is a key driver oncogene in small cell lung cancer and provides for the first time, in vivo data to supports this conclusion.
  • previous studies related to breast cancer and NSCLC have utilized a platform where SRSF 1 is inherently overexpressed, and thus are flawed with respect to confirming the oncogenic activity of SRSF 1.
  • This novel discovery firmly establishes SRSF 1 as a compelling therapeutic target for SCLC, especially for the population with poor outcome as predicted by SRSFl overexpression. The results described herein were obtained using the following materials and methods.
  • RNA whole exome sequence WES
  • RNASeq RNA sequencing data
  • Paired end FASTQ files of 90 mer sequence reads for both sequence data types were provided to Medlmmune.
  • RNASeq data has been deposited into GEO under accession GSE60052 while WES data was deposited into dBGaP under access 12059.
  • GATK SomaticIndelDetector (v2.3.4) with default setting and SAMtools (v0.1.18) mpileup were used for small insertion/deletions (indels) identification.
  • SAMtools vO.1.18 mpileup was used (Qphred>30 and mapping quality>30 with minimum coverage >20) to call SNVs relative to the human reference genome.
  • the retained SNVs/INDELs were further filtered by dbSNP 129 and dbSNP135. All dbSNPs with Cosmic ID were noted for further study.
  • RNA reads were mapped to the human genome (UCSC hgl9; Feb 2009 release; Genome Reference Consortium GRCh37) using TopHat2 (V2.0.9; Kim et al, 2013) and the human reference gtf annotation file (GRCh37.68). Transcript counts were calculated and normalized using htseq- count and DESeq (vl .12.1). The DESeq negative binomial distribution was used to calculate the p-value and fold changes between 49 lung tumor and 7 normal lung samples using adjusted p ⁇ 0.05 and fold change>2 as a threshold.
  • CNV copy number variation
  • Time-to-event analyses were used to correlate the copy number (CN) gain status of SRSFl and SRSFl gene expression with overall survival of SCLC patients respectively.
  • Two different analyses were conducted: a Kaplan-Meier (KM) analysis was used to evaluate the difference of survival curves for SRSFl CN gain group and no CN gain group.
  • a multivariate Cox proportion hazard (PH) regression model was used to evaluate the difference of survival for SRSFl CN gain group and no CN gain group adjusting for age, gender, tumor stages and chemotherapy treatment status before sampling.
  • Taqman assay for evaluate SRSFl CN variation was performed on residual DNA samples from 67 patients.
  • Genomic DNAs (gDNA) from cultured cells were prepared using QIAamp DNA Micro Kit.
  • Copy number assay of SRSFl (Hs00944074_cn) and reference assay RNase P (VIC) were ordered from AB I/Life Technologies. Assays were performed based on ABI reference with four replicates for each samples. The assays were run on ABI 7900HT (SDS v2.X) and the data files were analyzed using the CopyCaller Software.
  • Reference probe RNAse-P was used to determine the SRSFl CN gain status: copy number > 2 were considered as a gain status. Similar Kaplan-Meier analysis and Cox PH analysis were performed to evaluate the correlation between SRSFl CN gain status and survival (see Time- to-event analyses section for details).
  • Single-stranded cDNA was generated from total RNA using the Superscript® III First-Strand Synthesis SuperMix. Samples of cDNA were pre-amplified using TaqMan Pre- Amp Master Mix. Samples and assays were loaded into 48 x 48 dynamic array chips, and the chip was loaded on the BioMark RT-PCR System. All gene expression assays were ordered from ABI/Life Technologies. The assay ID of SRSFl is Hs00199471_ml.
  • siRNA reverse transfections were carried out using Lipofectamine RNAiMAX (Life Technologies). siRNAs targeting SRSF1 were ordered as "HP custom siRNA" from Qiagen. The sequence 5 ' -CCAACAAGATAGAGTATAA-3 ' (SRSFl siRNA) was used. AllStars Neg. Control siRNA (Qiagen) was used as negative control for transfection. Both control siRNA and SRSFl siRNAs were transfected at a final concentration of ⁇ . Culture medium was replaced with fresh medium at 48 hours after transfection, and cell lysates were prepared at 72 hours after transfection for Western blotting.
  • SCLC cell lines were transfected with SRSFl siRNAs for 48 hours and then seeded in a 1% methylcellulose H4100 medium (StemCell Technologies) consisting of RPMI1640 medium with 10% FBS at 2,000 cells/mL. After 5 days, colonies with more than 40 cells per colony were counted.
  • a 1% methylcellulose H4100 medium StemCell Technologies
  • SCLC cell lines were transfected with SRSFl siRNAs for 48 hours and then seeded in ultralow attachment plates (Corning) in sphere forming media: DMEM/F12 with 0.4% BSA, lOng/mL bFGF, 20 ng/mL EGF, 5 ⁇ g/mL insulin, 1% KnockOut Serum Replacement (Life Technologies).
  • Cells were treated with Cisplatin (0.001 ⁇ g - 10 ⁇ g/ml) for 4 days, after which viability of spheres was quantitated by CellTiter-Glo Assay (Promega). Images were taken with EVOS FL Auto Cell Imaging System.
  • SCLC cell lines were cotransfected with 800 ng myc/flag-tagged SRSFl vector (Origene) encoding the open reading frame of either the wildtype gene (NM_006924.4 with 25 nM of either non-targeting siRNA or SRSF1 siRNA-2 using Lipofectamine RNAiMAX (Life Technologies).
  • SRSF1 siRNA targeted the 3'UTR of SRSF1, and therefore did not affect expression of the SRSF1 ORF vector.

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Abstract

L'invention concerne des compositions et des méthodes pour caractériser et traiter le cancer du poumon à petites cellules, ainsi que pour caractériser le pronostic d'un patient atteint d'un cancer du poumon à petites cellules.
PCT/US2015/048602 2014-09-08 2015-09-04 Compositions et méthodes de dépistage et de traitement du cancer du poumon à petites cellules WO2016040167A1 (fr)

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EP3392345A1 (fr) * 2017-04-22 2018-10-24 Mologen AG Biomarqueur pour la thérapie du cancer pulmonaire à petites cellules
US10801027B2 (en) 2016-06-01 2020-10-13 University Of Sheffield Inhibitors of SRSF1 to treat neurodegenerative disorders
EP3949986A4 (fr) * 2019-03-26 2023-12-27 FUJIFILM Corporation Composition pharmaceutique pour inhiber la production d'une protéine du virus de l'hépatite b, et méthode permettant de cribler une telle composition

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10801027B2 (en) 2016-06-01 2020-10-13 University Of Sheffield Inhibitors of SRSF1 to treat neurodegenerative disorders
EP3392345A1 (fr) * 2017-04-22 2018-10-24 Mologen AG Biomarqueur pour la thérapie du cancer pulmonaire à petites cellules
WO2018193137A1 (fr) * 2017-04-22 2018-10-25 Mologen Ag Biomarqueur pour le traitement du cancer du poumon à petites cellules
EP3949986A4 (fr) * 2019-03-26 2023-12-27 FUJIFILM Corporation Composition pharmaceutique pour inhiber la production d'une protéine du virus de l'hépatite b, et méthode permettant de cribler une telle composition

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