WO2016040167A1

WO2016040167A1 - Compositions and methods for detecting and treating small cell lung cancer

Info

Publication number: WO2016040167A1
Application number: PCT/US2015/048602
Authority: WO
Inventors: Brandon Higgs; Jiaqi Huang; Zhan XIAO; Xin Yao; Haihong Zhong; Sarah CONLEY; Yihong Yao; Liyan JIANG
Original assignee: Brandon Higgs; Jiaqi Huang; Xiao Zhan; Xin Yao; Haihong Zhong; Conley Sarah; Yihong Yao; Jiang Liyan
Priority date: 2014-09-08
Filing date: 2015-09-04
Publication date: 2016-03-17

Abstract

The invention 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.

Description

COMPOSITIONS AND METHODS FOR DETECTING AND TREATING

SMALL CELL LUNG CANCER

BACKGROUND OF THE INVENTION

Small cell lung cancer (SCLC) is an aggressive disease with poor survival. There were an estimated 224,210 new cases of lung cancer diagnosed in 2014 in the United States, and over 159,260 deaths from the disease. The five year survival rate of patients with extensive disease is only four percent, and nearly two-thirds of patients have extensive disease at the time of diagnosis.

Improving survival of lung cancer patients remains difficult despite improved medical therapies. Most lung cancer is detected only in advanced stages when therapy options are limited. There is a growing recognition that lung cancer and other malignancies arise from a variety of pathogenic mechanisms. Methods of characterizing these malignancies at a molecular level is useful for stratifying patients, thereby quickly directing them to effective therapies. Improved methods for predicting the responsiveness of subjects having lung cancer, including small cell lung cancer (SCLC), are urgently required as are new compositions and methods for treating SCLC. The present invention provides the new observation that SRSF1 is linked to the AKT or ERK pathways, two of the most established oncogenic pathways pivotal to tumor growth and survival. The present invention establishes that 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.

SUMMARY OF THE INVENTION

As described below, 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.

In certain embodiments, 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 agent that reduces the expression or activity of an SRSF1 nucleic acid molecule or polypeptide. In other embodiments, 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.

In other embodiments, 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. In certain other embodiments, 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.

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.

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.

In further embodiments of the methods of the invention, the agent is an inhibitory nucleic acid molecule at least a portion of which is complementary to an SRSFl nucleic acid molecule. In certain other embodiments of the methods of the invention, the cell is a human cell in vitro or in vivo. In certain other embodiments of the methods of the invention, the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA. In particular embodiments of the methods of the invention, the inhibitory nucleic acid molecule comprises or consists essentially of a nucleic acid molecule having the sequence 5 ' -CCAACAAGATAGAGT AT AA-3'.

In further embodiments of the methods of the invention, the cell can be further contacted with cisplatin, topotecan, etoposide, paclitaxel, irinotecan or combinations thereof. In other embodiments, the agent is a molecule that suppresses production of SRSF1 polypeptide in vivo. In additional embodiments, the cell has SRSF1 copy number (CN) gain or increased SRSF1 expression, where the copy number is 3, 4, or more. In other embodiments, the agent is an antibody that specifically binds an SRSF1 polypeptide. In additional embodiments, the subject is human.

In certain other embodiments, 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.

In certain other embodiments, 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.

In other embodiments of methods of the invention, the reference is the level of SRSF1 polypeptide or nucleic acid molecule present in a control sample. In particular embodiments, 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.

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.

In particular embodiments of methods of the invention, a biological sample is a tumor sample.

In further embodiments of methods of the invention, 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. In further embodiments, this agent is an inhibitory nucleic acid molecule at least a portion of which is complementary to an SRSFl nucleic acid molecule. In additional embodiments, the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA. In further particular embodiments, the inhibitory nucleic acid molecule comprises or consists essentially of a nucleic acid molecule having the sequence 5 '-CCAACAAGATAGAGTATAA-3 ' . In other

embodiments, the agent is an antibody that specifically binds an SRSFl polypeptide. In additional embodiments, 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. In additional embodiments, 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. In further embodiments, the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA.

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.

1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By "copy number (CN)" 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.

By "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. In one embodiment, the analyte is SRSFl polypeptide, SRSFl DNA or SRSFl mRNA.

By "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.

By "reference" is meant a standard of comparison. In one embodiment, 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. For polypeptides, 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. For nucleic acid molecules, 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.

By "Serine/arginine-rich splicing factor 1 (SRSFl)" is meant 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:

By " 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:

TAAATAGTATTAAAAGATGAGAAACTGTTAGACTGAAGTTCTGTTGTAACATAACCATTATTTCCATCAC

AGTATGAAGACTGCAAACGCAGAAAACAGATTACAGTCTCTTATCCATTTTTTGAAATCCAAAAACTACG AAAACAAAAGATTTTCTGTTGTTGAGCTAATTAAATGTGAACCCTGACCAGAAAAAAAAAAAAAAAAAAA AAAAAAAA

By "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 . Typically, an inhibitory nucleic acid molecule comprises at least a portion of a complementary strand of a target nucleic acid molecule. In one embodiment, 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.

By "SRSFl siRNA" is meant a double stranded (ds) RNA capable of reducing SRSFl expression in a target cell. Optimally, 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.

By "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. For example, 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.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. In one embodiment, 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.

Unless specifically stated or obvious from context, as used herein, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a summary of clinical features of Chinese SCLC patients and the time-to- event analysis schema. In the time-to-event analyses, 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

polymerases and Fanconi anemia pathway genes. 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.

Mutations are shown with red spheres. Figure 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 δ 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.

Mutations are shown with red spheres. 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 4A shows SRSF1 gene expression in copy number gain group and no copy number gain group (line indicated with asterisk) (p=Welch's t-test). Figure 4B shows Kaplan-Meier (KM) curves comparing survival between SRSFl low (n=36) and high mRNA expression (n=13) groups. Similarly, KM curves used to evaluate the difference of survival between different SRSFl CN statuses in discovery set. Figure 4C provides the validation set, and 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

SCLC cell lines. Figure 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 μg/ml) for 48 hours or topotecan (2.5 μg/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 μg/ml) for 48 hours or topotecan (2.5 μg/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. NCI-82, SHP-77 and NIH-H1049 cells 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. 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 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.

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. As reported in more detail below, whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients was conducted. On target in vitro and in vivo studies demonstrate that SRSFl is essential for tumorigenecity of SCLC and plays a key role in DNA repair and chemo-sensitivity. Interestingly, inhibition of SRSFl transcripts decreased the tumorigenicity of SCLC cells.

Inhibition of SRSFl also sensitized SCLC cells to chemotherapeutic agents (e.g., cisplatin, topotecan). These data strongly support SRSFl as a therapeutic target in SCLC and provide a rationale for personalized therapy in SCLC.

Accordingly, 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).

Therapeutic methods

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

pharmaceutical composition, where the pharmaceutical composition comprises an effective amount of the agent and a suitable excipient. In one embodiment, 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) may be administered alone or in combination with a chemotherapeutic agent (e.g., cisplatin, topotecan). While methods of SCLC treatment vary depending on the type of SCLC, the stage of SCLC, and the patient's age, health, and physical condition, more aggressive treatment regimens will be used in patients having a poor prognosis. SCLC patients are identified as having a poor prognosis by detecting SRSFl copy number gain or increased SRSFl expression.

Prognostic methods

The present invention provides assays that are useful for characterizing SCLC in a subject. 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. In one embodiment, 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. In another embodiment, 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.

In one embodiment, 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

immunoquantitative PCR (iqPCR).

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. 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

Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression of a nucleic acid molecule or polypeptide. As reported in detail below, 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. siRNA

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).

Given the sequence of a target gene, 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). The 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat SCLC.

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.

In one embodiment of the invention, 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). Typically, 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. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference. Small hairpin RNAs (shR As) 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. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, 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.

As used herein, the term "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. In some embodiments, 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,

GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1 :5-14 (1990), which is incorporated herein by reference in its entirety. 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. Such 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. The inclusion of 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.

Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this invention, 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. 20, 1988, Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids Research, 18: 299, 1990. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

Essentially 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. Thus 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. For expression within cells, 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). In some embodiments, expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters. Examples of useful promoters in the context of the invention are 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.

Small Molecules

Small molecule agents that directly or indirectly inhibit SRSF1 biological activity or expression.

Delivery of Polynucleotides

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.

Anti-SRSFl Antibodies

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

(including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity (e.g. the antigen binding portion), disulfide-linked Fvs (dsFv), and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies disclosed herein), intrabodies, and epitope-binding fragments of any of the above. In particular, 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. In certain embodiments, 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)). In other embodiments, an anti-SRSFl antibody is an isotype switched variant of a known anti-SRSFl antibody.

Kits

The invention provides kits for the treatment of SCLC. In one embodiment, the kit includes an inhibitory nucleic acid molecule that reduces the expression of an SRSF1 polynucleotide or polypeptide. In some embodiments, 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. If desired 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.

In another embodiment, the invention provides kits for diagnosing SCLC or

characterizing the responsiveness of a subject having SCLC to SRSF1 inhibitory treatment. 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.

In particular embodiments, the instructions include at least one of the following:

description of the therapeutic agent; dosage schedule and administration for treatment of SCLC or symptoms thereof; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. 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.

In some embodiments, 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. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory

Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Small cell lung cancer (SCLC) is an aggressive disease with poor survival. Large-scale sequencing studies have revealed potential disease-driving genes in various cancers, although in SCLC, much still remains unknown, particularly in the Asian patient population. Whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients was carried out.

Example 1. Recurrent somatic variants were identified in SCLC Chinese patients.

DNA whole exome sequencing (WES) of 25 normal [normal adjacent tissue (NAT) or blood] and matched tumor, and 74 tumors only (no normal) Chinese SCLC patients identified 34,099 somatic non-silent single nucleotide variants (SNVs) or insertion/deletions (indels). This represented an average of 308 per patient and non-silent/silent ratio of 1.66. The patient clinical summary and cohort analysis flow are shown at Figure 1 and Table 1.

The most frequent transition and transversion changes were G>A and G>T, respectively, consistent with a previous study in SCLC (2). Genes harboring the most recurrent SNVs or indels were TP53 (81%), CSMD3 (43%), RBI (46%), LRPIB (38%) and OBSCN (41%, Table 2) - TP53 and RBI were reported previously (2).

Table 2: Recurrent somatic mutated genes from 99 Chinese SCLC patients

ANK2 17 16 5 1 1 5 12

C1orf173 17 15 3 12 3 14

CACNA1 E 18 16 4 12 5 13

CCDC141 19 17 2 15 2 17

CDH10 17 15 3 12 3 14

FRAS1 20 18 2 16 3 17

TNR 19 17 4 13 4 15

CCDC168 17 17 4 13 4 13

LRP2 15 15 2 13 2 13

LRRC7 14 14 3 1 1 3 1 1

WDFY4 17 16 6 10 6 1 1

TMEM132D 16 14 4 10 4 12

PAPPA2 17 13 1 12 1 16

To better understand the genetic basis of SCLC's chemosensitivity, SNVs and indels in all known DNA repair genes were systematically surveyed (4). Eighty-two percent of patients harbored at least one non-silent mutation in a DNA repair gene other than TP53 (Table 3).

36:c.G259 Nucleotide,

1404204 140420 lT:p.R864 excision. rep

11-1665 Tumor 16 4 44 G T snv ERCC4 L medium air

NM_0001

23:c.A262 Nucleotide,

1.04E+0 7T:p.E876 excision. rep

14 Tumor 13 1.04E+08 8 A T snv ERCC5 V medium air

NM_0001

23:c.C171 Nucleotide,

1.04E+0 2T:p.P571 excision. rep

12-1662 Tumor 13 1.04E+08 8 C T snv ERCC5 L neutral air

NM_0001 Nucleotide,

5074084 507408 24:c.A166 excision. rep

22 Tumor 10 5 45 T c snv ERCC6 G:p.T56A low air

NM_0001

24:c.A115 Nucleotide,

5073231 507323 7T:p.D386 excision. rep

08-3256 Tumor 10 9 19 T A snv ERCC6 V neutral air

NM_0001

24:c.G115 Nucleotide,

5073231 507323 9T:p.E387 excision. rep

11-3377 Tumor 10 7 17 c A snv ERCC6 X 0 air

NM_0001 Nucleotide,

5073257 507325 24:c.C899 excision. rep

08-3699 Tumor 10 7 77 G A snv ERCC6 T:p.A300V low air

Mutations of Fanconi anemia pathway and DNA polymerase genes were most recurrent in SCLC with prevalences of 41% and 30%>, respectively. POD1, POLG and POLQ were most recurrently mutated among the 15 DNA polymerase genes. These mutations cause protein truncations and amino acid changes in the polymerase, exonuclease, and helicase domains (Figures 2A-2C). Within the Fanconi anemia pathway, multiple genes in DNA inter-strand crosslink agents like FANCM (7%) and FANJ (7%) were among the most mutated (Figure 2D).

Somatic copy number variants (CNVs) were identified in both SCLC patient cohorts (Figure 3). Three patients with matched normal were removed due to missing survival outcome (n=22) and a single normal lung sample was used as baseline for the tumor only patient cohort (n=74). 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). Genes with CN losses previously reported in SCLC (Arriola et al, 2008; D'Angelo et al, 2010; Rudin et al, 2012) include RBI (34%), RASSFl (57%), FHIT (54%), KIF2A (16%), and CNTN3 (53%>), as well as a long segment along chromosome 3p22.1 (Table 4)·

Recurrence rates of these genes affected by CNVs were comparable to those reported in Rudin et al, 2012. In addition, recurrent gains of SRSFl were found (51%; 49/96) as well as concordant over-expression of the mRNA for those patients with the gain (p=0.005; two-tailed two-sided Welch's t-test; Figure 4A). Among the 96 Chinese patients, 28% had both CN gain and mRNA over-expression of SRSFl. In a cohort of 25 Caucasian SCLC patients, 32% showed the same result. SRSFl CN status was evaluated by FISH assay (N=34). Using a criterion described herein below for deviations from disomy (8), the positive and negative predictive values were 57% and 69% respectively (Table 5 below).

Example 2. SRSFl CN gain and mRNA over-expression predicted poor survival in Chinese SCLC patients.

For patients with both survival and whole exome sequencing (WES) data (n=96), genes within CN gain or lost regions were correlated with patient survival. The patient cohorts were separated into a discovery set including patients with tumors and matched normal (n=22) and a validation set including patients with tumors only (n=74). 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. Interestingly, SOX2 CN gain was not identified as a correlate with survival. For the remaining genes, patients with both RNA sequencing data (RNASeq) and survival data were interrogated (n=49). SRSFl was the only gene correlating between both CN gain and mRNA over-expression (Figure 4A), as well as between over-expression and survival, using a Cox proportion hazard (PH) regression model adjusting for age, gender, tumor stage, and chemotherapy status (p=0.034; HR=3.0; log-rank test: p*=0.074; Figure 4B). Summaries of Kaplan-Meier analyses as well as Cox proportion hazard analyses are presented in Tables 6A and 6B below.

Patients with SRSFl mRNA over-expression or CN gain demonstrated significantly worse survival. The discovery (log-rank test p=0.062), validation (log-rank test p=0.03), and combined patient cohort (Cox PH p=0.021 ; HR=2.1; log-rank test p*=0.005) survival analyses are provided in Figures 4C-4E. The combined patient cohort exhibited a more substantial difference between patients with SRSFl CN gain and those without (p=0.005) than either individual cohort. SRSFl CNV status evaluated by TaqMan assays (N=67) were also used to validate sequencing results. The positive predictive value and negative predictive value were 33% and 95% respectively. The adjusted survival difference showed that patients with a CN gain in SRSFl had significantly worse survival (p=0.029; HR=3; log- rank test: p*=0.043). CN gains in SRSFl from The Cancer Genome Atlas (TCGA) were interrogated for correlation with survival (Figure 4F). Among the cancer indications in TCGA with at least 3 patients harboring a CN gain in SRSFl (BRCA, KIRP, SARC, SKCM, and UCEC), UCEC was the only indication with a correlation between patients harboring CN gain of SRSFl and poor survival (log-rank test p=0.003), though the number of patients harboring a CN gain was highly unbalanced compared to those without (n=8 vs. n=437, respectively).

Table 6A: Kaplan-Meier analysis summary for SRSFl DNA amplification and mRNA expression CNV records events median 0.95LCL 0.95UCL p value

SRSF1 CN gain No 16 7 36.5 22.7 NA 0.062 (matched samples) Yes 6 3 17 6 NA

SRSF1 CN gain (tumor No 31 14 39.6 23.2 NA 0.03 only) Yes 43 29 17 1 1 31.7

Table 6B: Cox proportion hazard regression analysis summary for SRSFl DNA amplification and mRNA over-expression

Example 3. SRSFl mediated growth and survival in SRSFl high-expressing SCLC.

The following studies address whether SRSFl acts as a tumor driver in SCLC.

Previous studies have shown SRSFl as a potential candidate oncogene in breast cancer and non-SCLC (NSCLC). However, these observations were based on over-expression of SRSFl in non-transformed cells to demonstrate the transforming potential of the substantially overexpressed SRSFl protein. 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. However, a combination of cisplatin and SRSFl siRNA significantly enhanced the overall growth inhibition effect. A similar effect was observed with topotecan (Figure 6A).

Caspase 3/7 assays were performed on similarly treated cells (Figure 6B). SRSFl siRNA alone induced modest, but significant caspase-3 activation, similar to cisplatin treatment alone. However, the combination of the two produced a substantially higher caspase induction. A similar trend was observed using topotecan. Comparable results were also obtained in other SCLC models (Figure 5C).

The effect of 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). Example 4. SRSFl was required for in vivo tumorigenicity of SCLC.

A tumor formation study was carried out using siRNA-transfected DMS 114 and SHP- 77 cells. Equal numbers of viable transfected cells were injected in immunocompromised mice, and tumor growth was monitored for up to three weeks. DMS 114 and SHP-77 cells transfected with SRSFl siRNA showed significantly reduced tumor volumes compared to those transfected with control siRNAs (Figure 6E and Figure 8). Thus, SRSFl expression was shown to be important for in vivo tumorigenicity of SCLC cells.

Example 5. SRSFl knockdown triggered DNA-damage response and cell-cycle arrest

To explore additional mechanisms of tumor suppression by SRSFl knockdown, a potential role for DNA-damage induction was assessed by analysis of DNA-repair factors.

Clear inductions of p-H2AX and Chk2 were consistently observed upon SRSFl abrogation in DMS 114 and SHP-77 cells (Figure 6F and Figure 9A). Increased phosphorylation was also observed with a combination of SRSFl siRNA transfection and treatment by cisplatin or topotecan (Figure 6F and Figure 9A). Finally, the impact of SRSF 1 loss on kinase signaling pathways in SCLC cells through phospho-kinase array profiling was investigated (Figure 9B). Control siRNA- transfected DMSl 14 displayed strong phospho-AKT and ERK signals, which were abrogated by SRSFl siRNA. Western blot confirmed this in both DMSl 14 and NCI-H1048 cells (Figure 9C).

This study represents the first comprehensive genetic landscape survey in Chinese SCLC patients (with detailed clinical history) revealing key genetic alterations. Of particular importance is the prevalence of SRSFl copy number gain (51% in Chinese and 32% in Caucasian SCLC patients) and mRNA over-expression, and its role as a prognostic marker for poor patient survival - reported for the first time in SCLC. Furthermore, SRSFl is a key cancer driver, as demonstrated by the profound tumor-suppressive effect of specific SRSFl knockdown in SRSFl -amplified or overexpressed SCLC models. 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

mechanisms driving oncogenic phenotypes, though these were not detected with reliability using RNASeq here - a challenge with this technology that currently persists in splice variant detection. Without being bound by theory, it is likely that 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. Furthermore, the present invention establishes that 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. In comparison, 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.

SCLC patient summary

Ninety-nine Chinese patients with primary SCLC who had surgical treatment in

Shanghai Chest Hospital or Jiangsu Cancer Hospital, Nanjing were recruited. Of the 99 patients, 25 had matched normal adjacent tissue, while 74 patients only had tumor specimens. The tumoral purity was assessed >70% for all patient tumor specimens. Patients with histopathologically diagnosed incident SCLC were recruited prospectively onto an ongoing study at the Jiangsu Cancer Hospital or Shanghai Chest Hospital from July 2004 to July 20013. All patients were genetically unrelated, ethnic Han Chinese. The follow-up was conducted by telephone calls after the first visit to the hospital. All clinical features of the patients are summarized in Figure 1 and Table 1.

The study protocol and informed consent from all studies in this study were approved by the Ethics Committee of Shanghai Chest Hospital and Nanjing Medical University.

Informed consent in writing was obtained from each patient and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Ethics Committee of Renji Hospital. No donor organs were obtained from executed prisoners or other institutionalized persons.

DNA sequence read mapping and variant calling

DNA whole exome sequence (WES) and RNA sequencing data (RNASeq) data were generated by using the Illumina standard library preparation and sequencing. 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.

All sequence data was QCd for read counts, quality values, kmer usage, GC-content, and all other relevant parameters within FastQC (vO.10.1). The DNA sequence was aligned to the human genome (UCSC hgl9; Feb 2009 release; Genome Reference Consortium

GRCh37) using GATK (v2.3.4) and both indel realignment and PCR duplicate removal was conducted using GATK (v2.3.4) and Picard (vl.85), respectively. For the 25tumor-normal matched specimens, two different pipelines were employed and common SNVs between the two were retained: 1) SAMtools (v0.1.18) mpileup was used (Qphred>30 and mapping quality>30 with minimum coverage >20) to call SNVs relative to the human reference genome for all 25 tumors. MuTect (vl .1.4) was used to identify somatic single nucleotide variants (SNVs) with default settings. GATK SomaticIndelDetector (v2.3.4) with default setting and SAMtools (v0.1.18) mpileup were used for small insertion/deletions (indels) identification. The SNVs and indels which were in common between MuTect /GATK and Samtools were retained. SNV and indel were further filtered by 1000 genomes minor allele frequency (MAF) in all races of <1% or unknown MAF. The retained SNVs/INDELs were further filtered by dbSNP129 and dbSNP135. All dbSNPs with Cosmic ID were noted for further study.

For the 74 DNA tumor specimens without a matched normal specimen, 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. SNV and indel filtering using 1000 genomes minor allele frequency (MAF) in all races of <1% or unknown MAF was employed. The retained SNVs/INDELs were further filtered by dbSNP 129 and dbSNP135. All dbSNPs with Cosmic ID were noted for further study.

Patient identity QC

To verify the identity and matching between the tumor and normal paired WES samples, a selection of 300 heterozygous single nucleotide polymorphisms (SNPs) with MAFs>0.3 and <0.7 were selected from the 1000 genomes database. Then all DNA samples were clustered to observe any major discrepancies in subject or specimen labeling (Figure 10).

DNA polymerases structure modeling

Amino acid change mutations were mapped onto corresponding structures using mutagenesis wizard implemented in PyMOL (Schrodinger, LLC). For POLG coordinates of human mitochondrial DNA polymerase holoenzyme from Protein Data Bank (PDB) (Nucl.

Acids Res. (2000) 28 (1): 235-242) entry 3IKM ((2009) Cell, 139: 312-324) were used. The

Q52E mutation could not be mapped since that part of the protein was absent in the structure.

For DNA polymerase delta subunit the PDB entry 3IAY of yeast that shares 48/65% sequence identity/similarity over 908 amino acids was used.

RNA sequence read mapping and differential expression analysis

For RNASeq data, the average read count per mate was 50 million. 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.

Somatic copy number variation (CNV) analysis

For copy number variation (CNV) analysis, the DNA sequence was aligned to the human genome (UCSC hgl9; Feb 2009 release; Genome Reference Consortium GRCh37) using Bowtie2 (v2.0.0-beta7) and indel realignment was conducted using GATK (v2.3.4). A mapping summary for all specimens is provided at Figure 3. R package ExomeCNV was used to identify CNVs based on the read depth of coverage derived from the WES alignments. For the 22 matched specimens, the standard ExomeCNV pipeline was employed.

For the 74 tumor specimens without matched normal tissue, 1 normal FFPE lung tissue specimen was used as baseline with each of the 74 tumor specimens using ExomeCNV. The overview of segmentation log ratios for matched or unmatched results are shown in Figure 9.

Time-to-event analyses

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. Those genes with a trend of significance (log-rank p<0.1) in the discovery cohort (n=22) and with 10% CNV calls among the cohort were evaluated in the validation cohort (n=74; 1,707 genes). Since the discovery cohort was approximately 1/3 the size of the validation cohort and thus less powered, a modest log-rank test threshold was used. Among those 1,707 genes, 215 had p-values<0.05 from the log-rank test and CNV calls in more than 10% of the patients in the cohort. The gene expression of SRSFl in SCLC patients were divided into two groups according to SRSFl gene expression level (>75% percentile of overall expression and <=75% percentile of overall expression). Similar Kaplan-Meier analysis and Cox proportion hazard (PH) regression model were performed to compare the survival curves of different gene expression groups and the difference assessed with p-values for the grouping difference and the hazard ratio adjusting for age, gender, tumor stages and chemotherapy treatment status before sampling. KM analysis of correlation between SRSFl CN status and survival for the samples was performed with matched tumor and normal pairs and the samples without adjacent normal tissue pairs respectively. Both KM analysis and Cox PH analysis were performed for combined samples with and without normal pairs for correlation between SRSF 1 CN gain status and survival and SRSFl gene expression status and survival (see Example 2 for more details). R package survival was used to perform the above analysis. Model summaries are provided in Figure 3 and Tables 5A and 5B.

Taqman validation for SRSFl CNV status as prognostic marker for survival

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).

TaqMan gene expression assay

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. The average Ct values of reference gene assays (ACTB, Hs99999903_ml ; GAPDH, Hs99999905_ml ; UBC Hs00824723_ml) within a sample were utilized for calculation of ACt (ACT = the average CT of target gene - the average CT of the reference gene).

Cell culture, antibodies, and function assays All SCLC cell lines were grown in RPMI1640 medium supplemented with 10% fetal bovine serum. SRSF1 (SF2/ASF) antibody (96) was supplied by Santa Cruz Biotechnology. Phospho-Histone H2A.X (Serl39) (20E3) and Phospho-Chk2 (Thr68) (C13C1) were supplied by Cell Signaling Technology. Cell proliferation was determined by CellTiter-Glo Luminescent Cell Viability Assay (Promega). Caspase-Glo 3/7 Assay Systems (Promega) were used to analyze cell apoptosis. siRNA transfection

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 ΙΟΟηΜ. 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.

Colony formation assays

For clonogenic assay, 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.

Sphere forming assays

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 μg/mL insulin, 1% KnockOut Serum Replacement (Life Technologies). Cells were treated with Cisplatin (0.001 μg - 10 μg/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.

SRSFl rescue assays

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. After 48 hr, cells were harvested and then seeded in ultralow attachment plates (Corning) in sphere forming media: DMEM/F12 with 0.4% BSA, 10 ng/mL bFGF, 20 ng/mL EGF, 5 μg/mL insulin, 1% KnockOut Serum Replacement (Life Technologies). Cells were also harvested and lysed with Novex Tris- Glycine SDS Sample Buffer (Life Technologies) for Western blotting. Viability of spheres was quantitated after 4 days by CellTiter-Glo Assay (Promega). Images were taken with EVOS FL Auto Cell Imaging System

Xenograft studies in mice

All animal procedures were conducted in accordance with all appropriate regulatory standards under protocols approved by the Medimmune Institutional Animal Care and Use Committee. Immunocompromised athymic nude (nu/nu) female mice were purchased from Harlon Laboratories at 3-4 weeks of age. SHP-77 and DMS-114 cells were transfected with either control siRNA or SRSF1 siRNA at a final concentration of ΙΟΟηΜ. Two days after transfection, ten million viable cells in 50% matrigel were inoculated subcutaneously (SC) into right flank of each mouse. The length and width of each tumor was measured with an electronic caliper 2 times per week. Tumor volume (mm³) was calculated based on the following formula: [ length (mm) x width (mm)² ] ÷ 2.

References

1. Sher, T., Dy, G. K. & Adjei, A. A. Small cell lung cancer. Mayo Clin. Proc. 83, 355-367 (2008).

2. Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, et al, Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer. Nat Genet. 2012 Oct; 44(10): 1 11 1-6.

3. Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, et al., Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nat Genet. 2012 Oct;44 (10): 1104-10.

4. Wood RD, Mitchell M, Lindahl T. Human DNA repair genes, 2005. Mutat Res. 2005 Sep 4;577(l-2):275-83.

5. Arriola El, Canadas I, Arumi M, Rojo F, Rovira A, et al. Genetic changes in small cell lung carcinoma. Clin. Transl. Oncol. 10, 189-197 (2008). 6. D'Angelo, S.P. & Pietanza, M.C. The molecular pathogenesis of small cell lung cancer. Cancer Biol. Ther. 10, 1-10 (2010).

7. Medina PP, Castillo SD, Blanco S, Sanz-Garcia M, Largo C, et al. The SRYHMG box gene, SOX4, is a target of gene amplification at chromosome 6p in lung cancer. Hum. Mol.

Genet. 18, 1343-1352 (2009).

8. Cappuzzo F, Hirsch FR, Rossi E, Bartolini S, Ceresoli GL, Bemis L, Haney J, Witta S, Danenberg K, Domenichini I, Ludovini V, Magrini E, Gregorc V, Doglioni C, Sidoni A, Tonato M, Franklin WA, Crino L, Bunn PA Jr, Varella-Garcia M. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst. 2005 May 4;97(9):643-55.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions.

Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method for reducing the proliferation or survival of a small cell lung cancer (SCLC) cell, the method comprising contacting the cell with an agent that reduces the expression or activity of an SRSF1 nucleic acid molecule or polypeptide.

2. A method of inducing cell death in a small cell lung cancer cell, the method comprising contacting the cell with an agent that reduces the expression or activity of an SRSF1 nucleic acid molecule or polypeptide.

3. The method of claim 1 or 2, wherein the agent is an inhibitory nucleic acid molecule at least a portion of which is complementary to an SRSF 1 nucleic acid molecule.

4. A method for reducing the proliferation or survival of a small cell lung cancer (SCLC) cell, the method comprising contacting the cell with an inhibitory nucleic acid molecule that reduces the expression of an SRSF1 nucleic acid molecule and/or polypeptide.

5. A method of inducing cell death in a small cell lung cancer cell, the method comprising contacting the cell with an inhibitory nucleic acid molecule that reduces the expression of an SRSF1 nucleic acid molecule or polypeptide.

6. The method of any of claims 1-5, wherein the cell is a human cell in vitro or in vivo.

7. The method of any of claims 4-6, wherein the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA.

8. The method of claim 7, wherein the inhibitory nucleic acid molecule comprises or consists essentially of a nucleic acid molecule having the sequence 5'- CCAACAAGATAGAGTATAA-3 ' .

9. The method of any one of claims 1-8, further comprising contacting the cell with cisplatin, topotecan, etoposide, paclitaxel, irinotecan or combinations thereof.

10. The method of claim 1 or 2, wherein the agent is a molecule that suppresses production of SRSF1 polypeptide in vivo.

11. The method of any one of claims 1-1 1, wherein the cell has SRSF1 copy number (CN) gain or increased SRSF1 expression.

12. The method of claim 1 1, wherein the copy number is 3, 4, or more.

13. A method for treating small cell lung cancer (SCLC) in a subject, the method comprising administering to the subject an agent that reduces the expression or activity of an SRSF1 nucleic acid molecule or polypeptide.

14. The method of claim 13, wherein the agent is an inhibitory nucleic acid molecule at least a portion of which is complementary to an SRSF 1 nucleic acid molecule.

15. A method for treating small cell lung cancer (SCLC) in a subject, the method comprising administering to the subject an inhibitory nucleic acid molecule that reduces the expression of an SRSF1 nucleic acid molecule or polypeptide.

16. The method of claim 14 or 15, wherein the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA.

17. The method of claim 16, wherein the inhibitory nucleic acid molecule comprises or consists essentially of a nucleic acid molecule having the sequence 5'- CCAACAAGATAGAGTATAA-3 ' .

18. The method of claim 13, wherein the agent is an antibody that specifically binds an SRSF1 polypeptide.

19. The method of any of claims 13-18, further comprising contacting the cell with cisplatin, topotecan, etoposide, paclitaxel, irinotecan or combinations thereof.

20. The method of any one of claims 13-19, wherein the subject is human.

21. A method of treating a subject identified as having SCLC with a poor prognosis, the method comprising administering to the subject an agent that reduces the expression or activity of an SRSFl nucleic acid molecule or polypeptide, wherein 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.

22. A method of treating a subject identified as having SCLC with a poor prognosis, the method comprising administering to the subject an inhibitory nucleic acid molecule that reduces the expression of an SRSFl nucleic acid molecule or polypeptide, wherein 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.

23. The method of claim 21 or 22, wherein the method further comprises administering cisplatin, topotecan, etoposide, paclitaxel, irinotecan or combinations thereof.

24. The method of claim 23, wherein the combination of the SRSFl 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.

25. The method of any one of claims 21-24, wherein 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.

26. The method of any one of claims 21-25, wherein the biological sample is a tumor sample.

27. The method of any one of claims 21-26, wherein the SCLC is a metastatic neoplasia or a neoplasia having a propensity to metastasize.

28. The method of any one of claims 21-27, wherein the CN is 3, 4, or more.

29. The method of any one of claims 21-28, wherein the subject is human.

30. The method of any one of claims 21-29, wherein the reference is the level of SRSFl polypeptide or nucleic acid molecule present in a control sample.

31. The method of any one of claims 21-30, wherein the control sample is derived from a healthy subject.

32. The method of any one of claims 21-30, wherein the control sample is derived from the same subject at an earlier point in time.

33. A method of identifying a subject as having a poor prognosis, the method comprising detecting SRSFl copy number (CN) gain or increased SRSFl expression in a biological sample of the subject relative to a reference, wherein detection of CN gain and/or increased SRSFl expression identifies the subject as having a poor prognosis.

34. 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 SRSFl nucleic acid molecule or polypeptide, the method comprising detecting SRSFl copy number (CN) gain or increased SRSFl expression in a biological sample of the subject relative to a reference, wherein detection CN gain and/or increased SRSFl expression identifies the subject as responsive to an agent that reduces the expression or activity of an SRSFl nucleic acid molecule or polypeptide.

35. The method of claim 33 or 34, wherein the biological sample is a tumor sample.

36. 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.

37. The pharmaceutical composition of claim 36, wherein the agent is an inhibitory nucleic acid molecule at least a portion of which is complementary to an SRSF 1 nucleic acid molecule.

38. The pharmaceutical composition of claim 37, wherein the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA.

39. The pharmaceutical composition of claim 38, wherein the inhibitory nucleic acid molecule comprises or consists essentially of a nucleic acid molecule having the sequence 5'- CCAACAAGATAGAGTATAA-3 '

40. The pharmaceutical composition of claim 36, wherein the agent is an antibody that specifically binds an SRSF1 polypeptide.

41. The pharmaceutical composition of claim 36, further comprising cisplatin, topotecan, etoposide, paclitaxel, irinotecan or combinations thereof.

42. A kit for the treatment of small cell lung cancer (SCLC), the kit comprising an agent that reduces the expression or activity of an SRSF1 nucleic acid molecule or polypeptide and instructions for use in the treatment of SCLC.

43. The kit of claim 42, wherein the agent is an inhibitory nucleic acid molecule at least a portion of which is complementary to an SRSF 1 nucleic acid molecule.

44. The kit of claim 42, wherein the inhibitory nucleic acid molecule is an antisense molecule, shRNA, or siRNA.

45. The kit of claim 42, wherein the inhibitory nucleic acid molecule comprises or consists essentially of a nucleic acid molecule having the sequence 5'-

CCAACAAGATAGAGTATAA-3 ' .

46. The kit of claim 42, wherein the agent is an antibody that specifically binds an SRSF1 polypeptide.

47. The kit of claim 42, further comprising cisplatin, topotecan, etoposide, paclitaxel, irinotecan or combinations thereof.