WO2017223344A1 - Transdifférenciation servant de mécanisme de résistance au traitement pour le cancer de la prostate résistant à la castration - Google Patents

Transdifférenciation servant de mécanisme de résistance au traitement pour le cancer de la prostate résistant à la castration Download PDF

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WO2017223344A1
WO2017223344A1 PCT/US2017/038803 US2017038803W WO2017223344A1 WO 2017223344 A1 WO2017223344 A1 WO 2017223344A1 US 2017038803 W US2017038803 W US 2017038803W WO 2017223344 A1 WO2017223344 A1 WO 2017223344A1
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crpc
expression
genes
prostate cancer
treatment
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Corinne ABATE-SHEN
Michael M. Shen
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Columbia University
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57555—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • markers for determining the development of castration resistant prostate cancer and related treatments and methods are provided herein.
  • ADT androgen-deprivation therapy
  • AR androgen receptor
  • CRPC castration- resistant prostate cancer
  • Treatments for CRPC have focused on inhibition of AR signaling, using anti- androgen agents such as enzalutamide or abiraterone (2). While treatment with these agents improve survival, these gains are often transient as most patients ultimately fail treatment.
  • the present invention relates to a method of detecting castration-resistant prostate cancer (CRPC) and/or super resistant prostate tumors in a subject diagnosed with prostate cancer, comprising:
  • control is selected from the group consisting of a healthy control, an untreated subject, and a composite reference value for human prostate tumors.
  • the method further comprises treating the subject with one or more alternatives to anti-androgen treatment, comprising administering to the subject a therapeutically effective amount of one or any combination of treatments selected from the group consisting of an autologous active cellular immunotherapy (e.g. Sipuleucel-T), a poxvirus based vaccine, a T-cell checkpoint inhibiting monoclonal antibody (anti-CTL4 or anti-PD-1 or anti-PDLl), and cabazitaxe 1, and optionally further comprising monitoring the subject at least monthly.
  • the method further comprises administering to the subject one or more of the following treatments: chemotherapy, radiation therapy, surgery, or any combination thereof, based on the prognosis.
  • the method further comprises treating the subject with one or more alternatives to anti-androgen treatment when CRPC and/or the development of super- resistant prostate tumors is prognosed, wherein the method further comprises administering to the subject an effective amount of one or any combination of the following: androgen synthesis inhibitors (TAK700), androgen receptor (AR) inhibitors (ARN-509, ODM-201, and EZN-4176), AR DNA binding domain inhibitors (EPI-001), selective AR downregulators or SARDs (AZD-3514), including agents that inhibit both androgen synthesis and receptor binding ( ⁇ -001/galeterone).
  • TAK700 androgen synthesis inhibitors
  • AR androgen receptor
  • EPI-001 AR DNA binding domain inhibitors
  • AZD-3514 selective AR downregulators or SARDs
  • the present invention relates to a method of monitoring the treatment of a subject diagnosed with prostate cancer, comprising:
  • the method further comprises treating the subject with one or more alternatives to anti-androgen treatment when CRPC and/or the development of super- resistant prostate tumors is prognosed, comprising administering to the subject a therapeutically effective amount of one or any combination of treatments selected from the group consisting of an autologous active cellular immunotherapy (e.g. Sipuleucel-T), a poxvirus based vaccine, a T-cell checkpoint inhibiting monoclonal antibody (anti-CTL4 or anti-PD-1 or anti-PDLl), and cabazitaxe 1, and further comprising monitoring the subject at least monthly.
  • an autologous active cellular immunotherapy e.g. Sipuleucel-T
  • a poxvirus based vaccine e.g., a poxvirus based vaccine
  • a T-cell checkpoint inhibiting monoclonal antibody anti-CTL4 or anti-PD-1 or anti-PDLl
  • cabazitaxe 1 e.g., cabazitaxe 1, and further comprising
  • the method further comprises detecting one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron- specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), or any combination thereof, wherein the presence any one or more neuroendocrine markers indicates a likelihood of CRPC and/or the development of super-resistant prostate tumors.
  • Syn Synaptophysin
  • CgA chromogranin A
  • NSE neuron- specific enolase
  • DDC 1-DOPA Decarboxylase
  • assaying the gene expression levels is performed using a method comprising at least one of: PCR, RNA sequencing, and microarray.
  • assaying the protein levels is performed using a method comprising at least one of the following methods: quantitative Western blot, immunoblot, quantitative mass spectrometry, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), immunoradiometric assays (IRMA), and immunoenzymatic assays (IEMA) and sandwich assays using monoclonal and polyclonal antibodies.
  • ELISAs enzyme-linked immunosorbent assays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • IEMA immunoenzymatic assays
  • a combination of gene expression and protein expression is performed to arrive at a differential profile, wherein the differential profile indicates CRPC and/or the development of super-resistant prostate tumors, and/or castration resistant prostate cancer with neuroendocrine differentiation (CRPC-NE) or CRPC-NE likelihood.
  • CRPC-NE neuroendocrine differentiation
  • the present invention relates to a method for detecting castration resistant prostate cancer with neuroendocrine differentiation (CRPC-NE) or CRPC- NE likelihood in a patient sample comprising obtaining a sample comprising prostate cancer tumor tissue from the patient and detecting one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron-specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), or any combination thereof, in the sample and diagnosing the patient with CRPC-NE or prognosing the patient as having an increased likelihood of prostate cancer progression (CRPC-NE likelihood) when one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron-specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), or any combination thereof is detected in the sample.
  • CRPC-NE castration resistant prostate cancer with neuroendocrine differentiation
  • DDC 1-DOPA Decarbox
  • anti-androgen treatment is counter-indicated when the patient is diagnosed with CRPC-NE or CRPC-NE likelihood.
  • the method further comprises administering to the subject one or more of chemotherapy, radiation therapy or surgery based on the prognosis. In additional embodiments, the method further comprises administering to the subject one or more of chemotherapy alternatives to anti-androgen treatment, radiation, surgery, or any combination thereof, based on the prognosis.
  • the sample is selected from the group consisting of blood, bronchial lavage fluid, sputum, saliva, urine, amniotic fluid, lymph fluid, tissue or fine needle biopsy samples, peritoneal fluid, and cerebrospinal fluid.
  • the sample also exhibits a decrease in expression or loss of RBI , TP53, PTEN, or a combination thereof; and optionally further exhibits an increase in expression of SOX11 , or any combination thereof.
  • the present invention relates to a method of screening or identifying a test agent for the prevention and/or treatment of prostate cancer comprising: a. contacting or incubating a test agent with a cell comprising a gene listed in Table S4 or Table S8, or corresponding proteins;
  • test agent is therapeutic or preventative for castration- resistant prostate cancer (CRPC) and/or super resistant prostate tumors.
  • CRPC castration- resistant prostate cancer
  • the present invention relates a kit comprising one or more probes for detecting expression levels of one or more genes listed in the gene signatures of Table S4 or Table S8 or corresponding proteins, and optionally, any one or more of TP53, PTEN, SOX11 and/or RBI, and further optionally, wherein any one or more probes are detectably labeled.
  • the kit further comprises at least one sealed container which contains the one or more probes, and optionally, the kit further comprising printed material providing instructions.
  • the kit further comprises any one or combination of antibodies for detecting one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), Neuron-specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), and any combination thereof.
  • Synaptophysin Synaptophysin
  • CgA chromogranin A
  • NSE Neuron-specific enolase
  • DDC 1-DOPA Decarboxylase
  • the present invention relates a kit comprising one or more antibodies for detecting one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron- specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), and any combination thereof, and optionally, any one or more of TP53, PTEN, SOX11 and/or RBI, and further optionally, wherein any one or more of the antibodies or probes are detectably labeled.
  • Synaptophysin Synaptophysin
  • CgA chromogranin A
  • NSE neuron- specific enolase
  • DDC 1-DOPA Decarboxylase
  • the kit further comprises at least one sealed container which contains the one or more probes or antibodies, and optionally, the kit further comprising printed material providing instructions.
  • Figures 1A-F are images, graphs and plots showing that a refined GEM model of CRPC is conserved with human CRPC.
  • Figs. 1A-B show details and percentages of PTEN and TP53 gene alterations in human prostate cancer.
  • Fig. 1A is an oncoprint depicting alterations of PTEN and TP53 in metastatic castration-resistant prostate cancer (CRPC) as reported by the SU2C consortium.
  • Fig. IB is a graph showing the prevalence of alterations in primary prostate tumors (from TCGA (16)) and metastatic CRPC (from SU2C (17)).
  • Fig. 1C are histopathological analyses to determine phenotypic characterization of GEM models.
  • Figs. 1D-F are graphs showing cross-species gene set enrichment analyses (GSEA).
  • MR mouse master regulator
  • PCa treatment-naive androgen-independent versus androgen-dependent primary prostate tumors
  • "NES" stands for normalized enrichment score; GSEA p- values were calculated using 1000 gene permutations.
  • Figures 2A-G are images, graphs, and plots of preclinical analyses of abiraterone showing acceleration of the tumor phenotype in NPp53 CRPC.
  • Fig. 2A is a schematic showing preclinical trial design for the GEM model. Tumors were induced in cohorts of NP or NPp53 mice by delivery of tamoxifen, and mice were subsequently castrated to induce CRPC.
  • Fig. 2B are stains showing the histological phenotype of NP and NPp53 CRPC treated with vehicle or abiraterone. Shown are representative H&E images and immunohistochemical staining for the indicated antibodies.
  • Abiraterone-treated NPp53 CRPC show representative examples of adenocarcinoma and alternative histopathologies, as in Table S3. Scale bars represent 50 microns.
  • FIG. 2C are graphs showing the quantification of cellular proliferation by analysis of Ki67 immunostaining. Data represent the average from 5-8 independent images from 3 independent mice, p-values indicate the difference between bracketed groups and were calculated by a t-test. Note that cellular proliferation for the abiraterone-treated NPp53 CRPC was determined separately for regions of adenocarcinoma and alternative histolopathologies.
  • Fig. 2D-E are images and graphs reflecting MRI analysis. The abiraterone-treated NPp53 CRPC show examples of Group 1 and Group 2 tumor, as described in the text.
  • Fig. 2D are representative images from 2-D MRI performed immediately prior to the first treatment (pre-treatment) and immediately after the last treatment (post-treatment).
  • Fig. 2E are representative waterfall plots showing the change in tumor volume after treatment; each bar represents a single mouse.
  • Fig. 2F is a plot of principal component analysis (PCA) based on comparison of RNA-sequencing gene expression profiles from NPp53 CRPC treated with vehicle, or abiraterone (Group 1 and Group 2).
  • Fig. 2G is a heatmap of single-sample GSEA (ssGSEA). Single sample gene signatures were defined by comparing each individual abiraterone-treated NPp53 CRPC sample to the average of expression levels in the pooled group of corresponding vehicle-treated samples.
  • PCA principal component analysis
  • ssGSEA single-sample GSEA
  • Cross-species GSEA was performed to compare the single- sample signatures to a human gene signature of treatment-naive androgen-independent versus androgen-dependent tumors of Best et al. The comparison is shown as a heatmap, wherein upper and lower boxes correspond to NESs from the GSEA between the single- sample reference signature and the top 200 over-expressed (top) and under-expressed (bottom) genes from the Best et al. signature (28). See also Figures 8-13 for additional phenotypic and molecular analyses, Tables S2 for data summary, Table S3 for quantitative analysis of the histopatholgy phenotypes, and Dataset 1 for list of differentially expressed genes.
  • Figures 3A-D are graphs and plots showing cross-species computational analyses of adverse treatment response.
  • Fig. 3A is a schematic diagram showing the computational strategy used to predict molecular drivers of adverse treatment response. Illustrated are three possible outcomes: In scenario 1, the "responder” group, drug treatment is predicted to reverse the direction of MR signatures (i.e., activated MRs are repressed (as indicated) and vice versa). In scenario 2, the "non-responder” group, drug treatment is predicted to have minimal effect on MR signatures.
  • Figs. 3B-C are plots showing cross-species GSEA.
  • Fig. 3C is the reference signature, a human MR signature from Beltran et al.
  • NPp53 CRPC "exceptional non-responders”
  • NP CRPC abiraterone-treated "responders”
  • Fig. 3B and Fig. 3C "NES" and p-value were calculated using 1000 gene permutations.
  • the p-value was estimated using a log-rank test to determine the difference in outcomes between patients with higher activity levels of adverse treatment response MR (thin, lower line) versus those with lower/no MR activity (darker, upper line). See also Figures 12 for additional analyses, Table SI for description of human datasets, and Table S4 for summary of MRs identified in Panel Fig. 3C.
  • Figures 4A-E are graphs and plots showing that neuroendocrine differentiation inrP53-deficient CRPC is mediated in part by SOX11.
  • Fig. 4A is a heat map depicting relative expression levels of genes associated with neuroendocrine differentiation in vehicle- or abiraterone-treated mouse CRPC (6) The top right label indicates results for abiraterone (e) which are "exceptional non-responders”.
  • Fig. 4B are graphs showing relative expression level of SOX11 in human prostate cancer showing primary tumors segregated by Gleason grade (from TCGA) or CRPC-Adeno versus CRPC-NE (5).
  • Fig. 4A is a heat map depicting relative expression levels of genes associated with neuroendocrine differentiation in vehicle- or abiraterone-treated mouse CRPC (6) The top right label indicates results for abiraterone (e) which are "exceptional non-responders”.
  • Fig. 4B are graphs showing relative expression level of SOX
  • FIG. 4C is GSEA showing the enrichment of SOX11 target genes, predicted from analyses of a human prostate cancer interactome, in a human signature comparing CRPC-Adeno or CRPC-NE (5). NES and p- value were calculated using 1000 gene permutations.
  • Fig. 4D is a graph of Quantitative realtime PCR results showing expression levels of Soxll and neuron-specific enolase (NSE) in two independent mouse prostate epithelial cell lines from NP or NPp53 tumors (see Fig. 10).
  • NSE Soxll and neuron-specific enolase
  • FIGs. 4E are graphs showing expression levels of Soxll, NSE and Synaptophysin in mouse NPp53 cell lines following knock-down using two independent shRNA for Soxll (shSoxl l#l and shSoxl l#l).
  • shNT is the non-targeting control vector.
  • Figs. 4B, D, and E p-values were calculated using a t-test.
  • Figures 5A-C are graphs showing focal and overt neuroendocrine differentiation arise through transdifferentiation of luminal cells.
  • Fig. 5A shows immunostaining results for Synaptophysin in intact or castrated NP and NPp53 tumors treated with vehicle or abiraterone, as indicated. Shown are regions of focal neuroendocrine differentiation. Scale bars represent 50 microns.
  • Fig. 5B are graphs showing quantification of the percentage of Synaptophysin positive (Syn+) cells in regions of focal and overt neuroendocrine differentiation. Data quantification are provided in Table S5. p-values were calculated by one-way ANOVA. Fig.
  • FIG. 5C are images showing histological phenotypes of lineage-marked Synaptophysin+ cells in regions of focal and overt NPp53 CRPC. Shown is representative immunostaining for the indicated antibodies, with arrows indicating key staining. Data quantification are provided in Table S5. Scale bars represent 50 microns.
  • Figure 6 is a schematic depicting the molecular and phenotypic events associated with progression to CRPC including treatment-failure and transdifferentiation to CRPC-NE.
  • Figures 7A-B are graphs showing additional analysis of NP and NPp53 GEM models. Fig.
  • FIGS. 7A are graphs showing Levels of steroid hormones, as determined by mass spectrometry, in serum or dorsolateral prostate from intact or castrated NP or NPp53 mice, as indicated, p-values represent comparisons between bracketed groups and were calculated using a t-test. Boxes without error bars were at or below level of detection.
  • Fig. 7B shows cross species gene set enrichment analyses (GSEA). Comparison of a reference mouse MR signature from NPp53 CRPC versus NP CRPC with a query human MR signature comparing primary tumors having low PTEN/low TP53 versus low PTEN/high TP53 based on the TCGA dataset (low versus high TP53 (TCGA)). "NES" stands for normalized enrichment score; GSEA p-values were calculated using 1000 gene permutations.
  • Figures 8A-D are graphs showing tolerance and efficacy of abiraterone in mouse prostate.
  • Figs. 8A-C are graphs showing optimization of dose and scheduling for abiraterone- acetate treatment performed using non-tumor bearing strain-matched littermates; abiraterone- acetate was delivered by oral gavage.
  • FIG. 8D are graphs showing levels of steroid hormones in serum and prostate tumors following treatment of NPp53 CRPC with abiraterone-acetate (200 mg/kg) for 1 month.
  • Serum and prostate tumors were collected within 24 hours of the last treatment and snap-frozen until processed for mass spectrometry analysis, which was performed in the Steroid Analysis Core of the Fred Hutchinson Cancer Research Center. Shown are the levels of abiraterone and metabolites of steroid biosynthesis, some of which are below the level of detection for the mass spectrometry assay. These data confirm uptake and processing of abiraterone-acetate in mouse CRPC, in addition to the nontumor bearing mice evaluated in Fig. 8A-C, and show that steroids downstream of abiraterone-acetate, namely testosterone and androsterone, are significantly affected by the treatment.
  • FIGS. 9A-C are graphs showing additional analyses of tumor volumes in GEM models before and after treatment.
  • Fig. 9A are images with anterior views from a 3- dimensional volumetric analysis of NP or NPp53 CRPC following treatment with abiraterone.
  • Fig. 9B are growth curve graphs showing the relative tumor volume for individual NPp53 mice after castration and subsequent treatment with abiraterone or vehicle, as indicated.
  • Fig. 9C shows the comparison of tumor volume for individual NPp53 CRPC cases prior to or immediately following treatment with vehicle or abiraterone, as indicated. Mice were randomly assigned to the vehicle or abiraterone treatment groups. Note that there was no significant difference in the tumor volumes of between these groups at the time of enrollment.
  • Figures 10A-D are blots and graphs showing analyses of preclinical treatment of cell lines from NP and NPp53 tumors.
  • Fig. 10A shows western blot analysis using total cell extracts (10 ⁇ g) to detect the indicated proteins.
  • Fig. 10B are graphs using cell culture assays, as follows. To measure the consequences of abiraterone for cell growth, NP or NPp53 cell lines, as indicated, were grown in charcoal- stripped serum in the absence or presence of DHT (20 nM) followed by treatment with abiraterone (10 ⁇ ) or vehicle for 0 to 72 hours.
  • Figs. 10C-D are graphs showing in vivo tumor growth under different conditions. NP or NPp53 cells (1 X 10 6 cells) were implanted into the flank of nude mice, and tumor growth was monitored by caliper measurement until they reached a size of 100-300mm 3 .
  • mice were then randomly enrolled for treatment with abiraterone (200 mg/kg/day) or vehicle 5 times per week for 4 weeks.
  • Fig. IOC shows graphs of tumor growth measurements. * and ** indicate p-values ⁇ 0.05 or 0.01, respectively, calculated by a t-test.
  • Fig. 10D is a graph showing tumor weight at the conclusion of the study, p-values represent comparisons between bracketed groups and were calculated by a t-test.
  • Figure 11 shows images of alternative histopathologies of NPp53 CRPC.
  • Figures 12A-B are additional cross-species computational analyses.
  • Fig. 12A is principal component analysis (PCA) of gene expression profiles from NP CRPC that were treated with vehicle (veh) or abiraterone (abi), as indicated.
  • Fig. 12B shows cross-species GSEA.
  • the reference signature a human MR signature from Best et al. comparing treatment- naive androgen independent versus androgen-dependent prostate tumors, was compared with three independent mouse MR query signatures comparing abiraterone- versus vehicle-treated samples from the "responder", “non-responder", or “exceptional non-responder” groups.
  • "NES" and p-value were calculated using 1000 gene permutations.
  • FIGS 13A-B are blots and graphs showing analysis of expression of SOX11 and other NE genes.
  • Fig. 13A are graphs of quantitative real-time PCR showing relative expression levels of Soxl l, Rbl, and Sox2 in individual mouse CRPC tumors treated with vehicle or abiraterone as indicated. The individual mouse ID numbers correspond to the cases shown in Table S3. Gapdh was used as a reference; p values were calculated using a t-test to compare the indicated groups (shown as bracketed) consisting of multiple cases. Data show the mean ⁇ S.D. for triplicates done for each individual tumor.
  • Fig. 13B are additional graphs of quantitative real-time PCR showing relative expression levels of Sox2 and Sox7, using Gapdh as a reference, in the mouse NPp53 cell line 1 (see Fig. 10).
  • Genomic alterations that occur in early localized disease include deletion of 8p21, resulting in haploinsufficiency for the NKX3.1 homeobox gene, as well as the TMPRSS2-ERG chromosomal rearrangement (6).
  • genomic alterations such as those affecting the TP53 tumor suppressor and the PTEN tumor suppressor, occur at low frequencies in primary tumors, but are more prevalent in advanced disease (10).
  • CRPC human castration-resistant prostate cancer
  • CPRC castration-resistant prostate cancer
  • AR target androgen receptor
  • GEM genetically engineered mouse
  • lineage-tracing in vivo demonstrates that neuroendocrine-like cells arise through transdifferentiation of luminal prostate adenocarcinoma cells.
  • the present invention relates to human prostate cancer in which one or a combination of PTEN and TP53 gene alterations are present and indicate a potential to develop non-adenocarcinoma histopathology, which is augmented by abiraterone treatment, and for which abiraterone treatment is counter-indicated, or should be stopped. Consequently, CRPC comprising PTEN and/or TP53 gene alterations may benefit from alternative androgen pathway inhibitors.
  • Novel androgen pathway inhibitors that may be suitable alternatives to abiraterone include androgen synthesis inhibitors (TAK700), androgen receptor inhibitors (ARN-509, ODM-201, and EZN-4176), AR DNA binding domain inhibitors (EPI-001), selective AR downregulators or SARDs (AZD-3514), and agents that inhibit both androgen synthesis and receptor binding ( ⁇ -001/galeterone) (reviewed by Agarwal et al. Ann Oncol. 2014 Sep;25(9): 1700-9; and Leibowitz-Amit R and Joshua AM, Curr Oncol. 2012 Dec; 19(Suppl 3): S22-S31).
  • TAK700 androgen synthesis inhibitors
  • ARN-509, ODM-201, and EZN-4176 AR DNA binding domain inhibitors
  • EPI-001 selective AR downregulators or SARDs
  • ⁇ -001/galeterone selective AR downregulators or SARDs
  • the effects of abiraterone treatment on human prostate cancer in which one or a combination of PTEN and TP53 gene alterations are present may be due to complete lack of dependence on AR signaling in the tumors.
  • most AR-targeted therapies would exhibit negative effects comparable to those of arbiraterone, as shown in the present data.
  • early detection of MR associated with CRPC-NE would allow for identification of patients that would not benefit from AR- targeted therapies.
  • Such patients could be selected for alternative treatment regimen, such as chemotherapy, immunotherapy, or co-targeting therapeutic approaches.
  • Sipuleucel-T an autologous active cellular immunotherapy
  • Additional promising immunotherapeutic agents include poxvirus based vaccines, T-cell checkpoint inhibiting monoclonal antibodies, as well as antibody conjugates.
  • cabazitaxel a new therapeutic option for patients with mCRPC resistant to docetaxel was recently approved by the FDA (Matthew D, Nature Reviews Drug Discovery 2010, 9, 677-678). Eribulin mesylate, a nontaxane halichondrin B analog microtubule inhibitor, has also demonstrated activity in CRPC (Agarwal et al. Ann Oncol. 2014 Sep;25(9): 1700-9).
  • Treating” or “treatment” of a state, disorder or condition includes:
  • the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005). The choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • an “immune response” refers to the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest.
  • a response usually consists of the subject producing antibodies, B cells, helper T cells, suppressor T cells, regulatory T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • a “therapeutically effective amount” means the amount of a compound that, when administered to an animal for treating a state, disorder or condition, is sufficient to effect such treatment.
  • the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, physical condition and responsiveness of the animal to be treated.
  • compositions of the invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of a compound described herein.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of an antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • the present invention provides a pharmaceutical composition or formulation comprising at least one active composition, or a pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent and/or carrier.
  • the excipient, diluent and/or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions of the invention can be formulated for administration in any convenient way for use in human or veterinary medicine.
  • the invention therefore includes within its scope pharmaceutical compositions comprising a product of the present invention that is adapted for use in human or veterinary medicine.
  • the pharmaceutical composition is conveniently administered as an oral formulation.
  • Oral dosage forms are well known in the art and include tablets, caplets, gelcaps, capsules, and medical foods. Tablets, for example, can be made by well-known compression techniques using wet, dry, or fluidized bed granulation methods.
  • Such oral formulations may be presented for use in a conventional manner with the aid of one or more suitable excipients, diluents, and carriers.
  • Pharmaceutically acceptable excipients assist or make possible the formation of a dosage form for a bioactive material and include diluents, binding agents, lubricants, glidants, disintegrants, coloring agents, and other ingredients.
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • An excipient is pharmaceutically acceptable if, in addition to performing its desired function, it is non-toxic, well tolerated upon ingestion, and does not interfere with absorption of bioactive materials.
  • Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005). The choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are "generally regarded as safe”, e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals, and more particularly in humans.
  • "Patient” or “subject” refers to mammals and includes human and veterinary subjects.
  • the dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the patient's medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like.
  • the initial dose may be larger, followed by smaller maintenance doses.
  • the dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level.
  • oral administration will require a higher dose than if administered intravenously.
  • topical administration will include application several times a day, as needed, for a number of days or weeks in order to provide an effective topical dose.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
  • subject as used in this application means an animal with an immune system such as avians and mammals. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates. Avians include, but are not limited to, fowls, songbirds, and raptors.
  • the invention can be used in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications.
  • patient as used in this application means a human subject. In some embodiments of the present invention, the “patient” is diagnosed with prostate cancer.
  • screen means to test a subject or patient to determine if they have a particular illness or disease, or a particular manifestation of an illness or disease.
  • the term also means to test an agent to determine if it has a particular action or efficacy.
  • identification means to recognize a disease state or a clinical manifestation or severity of a disease state in a subject or patient.
  • the term also is used in relation to test agents and their ability to have a particular action or efficacy.
  • prediction means to tell in advance based upon special knowledge.
  • reference value means an amount or a quantity of a particular protein or nucleic acid in a sample from a healthy control or healthy donor.
  • health control is used interchangeably in this application and are a human subject who is not suffering from prostate cancer or any other cancer-related condition.
  • treat refers to a means to slow down, relieve, ameliorate or alleviate at least one of the symptoms of the disease, or reverse the disease after its onset.
  • prevent refers to acting prior to overt disease onset, to prevent the disease from developing or minimize the extent of the disease or slow its course of development.
  • agent means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, biologies, small organic molecules, antibodies, nucleic acids, peptides, and proteins.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the disease, or results in a desired beneficial change of physiology in the subject.
  • an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, an isolated genomic DNA, or a restriction fragment.
  • an isolated nucleic acid is preferably excised from the chromosome in which it may be found. Isolated nucleic acid molecules can be inserted into plasmids, cosmids, artificial chromosomes, and the like.
  • a recombinant nucleic acid is an isolated nucleic acid.
  • An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
  • An isolated material may be, but need not be, purified.
  • the term "purified” and the like as used herein refers to material that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants.
  • a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell;
  • a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
  • expression profile refers to any description or measurement of one or more of the genes that are expressed by a cell, tissue, or organism under or in response to a particular condition. Expression profiles can identify genes that are up-regulated, down-regulated, or unaffected under particular conditions. Gene expression can be detected at the nucleic acid level or at the protein level. The expression profiling at the nucleic acid level can be accomplished using any available technology to measure gene transcript levels. For example, the method could employ in situ hybridization, Northern hybridization or hybridization to a nucleic acid microarray, such as an oligonucleotide microarray, or a cDNA microarray.
  • the method could employ reverse transcriptase -polymerase chain reaction (RT-PCR) such as fluorescent dye-based quantitative real time PCR (TaqMan® PCR).
  • RT-PCR reverse transcriptase -polymerase chain reaction
  • TaqMan® PCR fluorescent dye-based quantitative real time PCR
  • nucleic acid expression profiles were obtained using Affymetrix GeneChip® oligonucleotide microarrays.
  • the expression profiling at the protein level can be accomplished using any available technology to measure protein levels, e.g., using peptide-specific capture agent arrays.
  • gene signature and “signature genes” will be used interchangeably herein and mean the particular transcripts that have been found to be differentially expressed in some prostate cancer patients, as described herein. For example, those differentially expressed genes listed in Table S4 and/or Table S8. It is noted that differential levels of the corresponding proteins, will also be useful as a marker or signature for prognosing or diagnosing patients as described herein.
  • gene means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription.
  • Transcript or “gene transcript” is a sequence of RNA produced by transcription of a particular gene. Thus, the expression of the gene can be measured via the transcript.
  • antisense DNA is the non-coding strand complementary to the coding strand in double- stranded DNA.
  • genomic DNA means all DNA from a subject including coding and non-coding DNA, and DNA contained in introns and exons.
  • nucleic acid hybridization refers to anti-parallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G.
  • Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters.
  • Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under “low stringency” conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid).
  • vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • Vectors include, but are not limited to, plasmids, phages, and viruses.
  • Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted.
  • a common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites.
  • restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites.
  • a "cassette” refers to a DNA coding sequence or segment of DNA which codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame.
  • foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA.
  • a segment or sequence of DNA having inserted or added DNA can also be called a "DNA construct” or "gene construct.”
  • a common type of vector is a "plasmid", which generally is a self-contained molecule of double- stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • a plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA.
  • Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme.
  • Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA.
  • Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms.
  • a large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
  • Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
  • Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.
  • host cell means any cell of any organism that is selected, modified, transformed, grown, used or manipulated in any way, for the production of a substance by the cell, for example, the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays, as described herein.
  • a "polynucleotide” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a nucleic acid, such as DNA and RNA, and means any chain of two or more nucleotides.
  • a nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide.
  • PNA protein nucleic acids
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
  • nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • the nucleic acids may also be modified by many means known in the art.
  • Non-limiting examples of such modifications include methylation, "caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, and carbamates) and with charged linkages (e.g., phosphorothioates, and phosphorodithioates).
  • Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, and poly-L- lysine), intercalators (e.g.
  • polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Modifications of the ribose-phosphate backbone may be done to facilitate the addition of labels, or to increase the stability and half-life of such molecules in physiological environments. Nucleic acid analogs can find use in the methods of the invention as well as mixtures of naturally occurring nucleic acids and analogs. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, and bio tin.
  • polypeptide as used herein means a compound of two or more amino acids linked by a peptide bond.
  • Polypeptide is used herein interchangeably with the term “protein.”
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system, i.e. , the degree of precision required for a particular purpose, such as a pharmaceutical formulation.
  • “about” can mean within 1 or more than 1 standard deviations, per the practice in the art.
  • “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • the term "about” meaning within an acceptable error range for the particular value should be assumed.
  • the present invention relates to a method of detecting castration-resistant prostate cancer (CRPC) and/or super resistant prostate tumors in a subject diagnosed with prostate cancer, comprising:
  • control is selected from the group consisting of a healthy control, an untreated subject, and a composite reference value for human prostate tumors.
  • the present invention relates to a method of monitoring the treatment of a subject diagnosed with prostate cancer, comprising:
  • test expression profile a. assaying gene expression of one or more genes from Table S4 or Table S8 or corresponding protein levels, in a sample from the subject to obtain a test expression profile, b. comparing the test expression profile of the genes or corresponding proteins with a reference expression profile, wherein the reference expression profile comprises the expression level of the same genes or corresponding proteins obtained from the subject prior to treatment;
  • the method further comprises treating the subject with one or more alternatives to anti-androgen treatment when CRPC and/or the development of super- resistant prostate tumors is prognosed, comprising administering to the subject a therapeutically effective amount of one or any combination of treatments selected from the group consisting of an autologous active cellular immunotherapy (e.g. Sipuleucel-T), a poxvirus based vaccine, a T-cell checkpoint inhibiting monoclonal antibody (anti-CTL4 or anti-PD-1 or anti-PDLl), and cabazitaxe 1, and further comprising monitoring the subject at least monthly.
  • an autologous active cellular immunotherapy e.g. Sipuleucel-T
  • a poxvirus based vaccine e.g., a poxvirus based vaccine
  • a T-cell checkpoint inhibiting monoclonal antibody anti-CTL4 or anti-PD-1 or anti-PDLl
  • cabazitaxe 1 e.g., cabazitaxe 1, and further comprising
  • the method further comprises detecting one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron- specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), or any combination thereof, wherein the presence any one or more neuroendocrine markers indicates a likelihood of CRPC and/or the development of super-resistant prostate tumors.
  • Syn Synaptophysin
  • CgA chromogranin A
  • NSE neuron- specific enolase
  • DDC 1-DOPA Decarboxylase
  • assaying the gene expression levels is performed using a method comprising at least one of: PCR, RNA sequencing, and microarray.
  • assaying the protein levels is performed using a method comprising at least one of the following methods: quantitative Western blot, immunoblot, quantitative mass spectrometry, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), immunoradiometric assays (IRMA), and immunoenzymatic assays (IEMA) and sandwich assays using monoclonal and polyclonal antibodies.
  • ELISAs enzyme-linked immunosorbent assays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • IEMA immunoenzymatic assays
  • a combination of gene expression and protein expression is performed to arrive at a differential profile, wherein the differential profile indicates CRPC and/or the development of super-resistant prostate tumors, and/or castration resistant prostate cancer with neuroendocrine differentiation (CRPC-NE) or CRPC-NE likelihood.
  • CRPC-NE neuroendocrine differentiation
  • the present invention relates to a method for detecting castration resistant prostate cancer with neuroendocrine differentiation (CRPC-NE) or CRPC- NE likelihood in a patient sample comprising obtaining a sample comprising prostate cancer tumor tissue from the patient and detecting one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron-specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), or any combination thereof, in the sample and diagnosing the patient with CRPC-NE or prognosing the patient as having an increased likelihood of prostate cancer progression (CRPC-NE likelihood) when one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron-specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), or any combination thereof is detected in the sample.
  • CRPC-NE castration resistant prostate cancer with neuroendocrine differentiation
  • DDC 1-DOPA Decarbox
  • traditional anti-androgen treatment is counter-indicated when the patient is diagnosed with CRPC-NE or CRPC-NE likelihood.
  • alternative treatments should be considered for treatment. Examples of such alternative treatments, that are expected to be less likely to lead to development of CRPC or super-resistant prostate tumors include at least the following: a therapeutically effective amount of treatment selected from the group consisting of an autologous active cellular immunotherapy (e.g.
  • Sipuleucel- T a poxvirus based vaccine, a T-cell checkpoint inhibiting monoclonal antibody (anti-CTL4 or anti-PD-1 or anti-PDLl), and cabazitaxe 1, and further comprising monitoring the subject at least monthly.
  • the present invention also provides methods for stratifying subjects prior to treatment and/or monitoring subjects for their responses to treatment, e.g, administration of agents, both oral and topical, life style alterations such as diet and exercise, and non-traditional treatment such as acupuncture. This is useful in both patient care as well as clinical trials.
  • the methods comprise obtaining the expression of at least one gene in at least one gene signature, or the corresponding protein level, in a subject prior to any treatment.
  • the methods comprise obtaining the expression of at least one gene in at least one gene signature (or the corresponding protein) in a normal subject that serves as a reference expression value.
  • the measurement of expression of the same gene or genes (or corresponding proteins) is measured, and differential expression or protein level, when compared to the reference (e.g. prior to treatment levels, control levels, or NES) would indicate that the subject is susceptible or likely to develop CRPC or to develop super-resistant tumors.
  • the expression of at least one gene in at least one gene signature, or corresponding protein level would be measured before and after treatment. In an alternative embodiment, more than one gene from each signature, or corresponding protein would be measured.
  • a combination of genes and other markers such as one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), Neuron-specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), and any combination thereof, can be detected and form a diagnostic or signature group for determine whether the subject is susceptible or likely to develop CRPC or to develop super-resistant tumors, or the likelihood of CRPC-NE.
  • Synaptophysin Syn
  • CgA chromogranin A
  • NSE Neuron-specific enolase
  • DDC 1-DOPA Decarboxylase
  • the present invention also provides a method for determining target genes or proteins for drug development.
  • the invention also contemplates that the protein products of any of the genes in the gene signatures found for example in Table S4, Table S8, or in dataset land/or described in any of the Tables or Figures herein may have diagnostic value, as well as to serve as potential therapeutic targets for patient monitoring, stratification, or drug development.
  • a sample of biological tissue or bodily fluid from a subject with prostate cancer is obtained.
  • the sample is tested for protein levels (for protein corollaries of any of the genes listed in Tables S4 or S8, for example, or any of the Tables or Figures herein.
  • the protein sample can be obtained from any biological tissue.
  • Preferred biological tissues include, but are not limited to, epidermal, whole blood, and plasma.
  • the protein sample can be obtained from any biological fluid.
  • Preferred fluids include, but are not limited to, plasma, saliva, and urine. Protein can be isolated and/or purified from the sample using any method known in the art, including but not limited to immunoaffinity chromatography.
  • preferred methods for detecting and measuring increase levels of the proteins in a protein sample include quantitative Western blot, immunoblot, quantitative mass spectrometry, enzyme -linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), immunoradiometric assays (IRMA), and immunoenzymatic assays (IEMA) and sandwich assays using monoclonal and polyclonal antibodies.
  • ELISAs enzyme -linked immunosorbent assays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • IEMA immunoenzymatic assays
  • Antibodies are a preferred method of detecting and measuring target or desired proteins in a sample. Such antibodies are available commercially or can be made by conventional methods known in the art. Such antibodies can be monoclonal or polyclonal and fragments thereof, and immunologic binding equivalents thereof.
  • the term "antibody” means both a homologous molecular entity as well as a mixture, such as a serum product made up of several homologous molecular entities.
  • such antibodies will immunoprecipitate the desired proteins from a solution as well as react with desired/target proteins on a Western blot, immunoblot, ELISA, and other assays listed above.
  • Antibodies for use in these assays can be labeled covalently or non-covalently with an agent that provides a detectable signal. Any label and conjugation method known in the art can be used. Labels, include but are not limited to, enzymes, fluorescent agents, radiolabels, substrates, inhibitors, cofactors, magnetic particles, and chemiluminescent agents. A number of fluorescent materials are known and can be utilized as detectable labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • Any desired targets or binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • the enzymes which can be used in these procedures are known and can be utilized.
  • the enzymes can be are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • sample refers to a sample of biological fluid, tissue, or cells, in a healthy and/or pathological state obtained from a subject.
  • samples include, but are not limited to, blood, bronchial lavage fluid, sputum, saliva, urine, amniotic fluid, lymph fluid, tissue or fine needle biopsy samples, peritoneal fluid, cerebrospinal fluid, and includes supernatant from cell lysates, lysed cells, cellular extracts, and nuclear extracts.
  • the whole blood sample is further processed into serum or plasma samples.
  • the sample includes blood spotting tests.
  • kits form for use by a health care provider and/or a diagnostic laboratory.
  • kits may include probes for one or more of the genes from one or more signatures, as described herein, reagents for isolating and purifying nucleic acids from biological tissue or bodily fluid, reagents for performing assays on the isolated and purified nucleic acid, instructions for use, and reference values or the means for obtaining reference values in a control sample for the included genes.
  • a preferred kit for patient classification with regard to disease activity and clinical manifestations would include probes for at least one gene from each of the MR signatures described herein.
  • the kit would include reagents for testing for Synaptophysin, for example.
  • reagents for testing for Synaptophysin for example.
  • Such a kit could include antibodies that recognize the peptide of interest, reagents for isolating and/or purifying protein from a biological tissue or bodily fluid, reagents for performing assays on the isolated and purified protein, instructions for use, and reference values or the means for obtaining reference values for the quantity or level of peptides in a control sample.
  • a preferred kit for monitoring treatment to disease activity would include probes from at least one gene from each of the MR signatures described herein.
  • Such a kit could include antibodies that recognize the peptide of interest, reagents for isolating and/or purifying protein from a biological tissue or bodily fluid, reagents for performing assays on the isolated and purified protein, instructions for use, and reference values or the means for obtaining reference values for the quantity or level of peptides in a control sample.
  • a preferred kit for diagnosing or prognosing CRPC and/or the development of super- resistant prostate tumors, and/or castration resistant prostate cancer with neuroendocrine differentiation (CRPC-NE) or CRPC-NE likelihood would include probes for at least one gene from each of the determinative signatures, such as any combination of those from Table S4 or Table S8, or corresponding protein.
  • kits may further or optionally, include one or more antibodies for detecting one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron- specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), and any combination thereof, and optionally, any one or more of TP53, PTEN, SOX11 and/or RBI, and further optionally, wherein any one or more of the antibodies or probes are detectably labeled.
  • Synaptophysin Synaptophysin
  • CgA chromogranin A
  • NSE neuron- specific enolase
  • DDC 1-DOPA Decarboxylase
  • kits suitable for use by a medical specialist may be prepared to determine the presence or amount of a desired gene or protein activity, expression or signature gene amplification in suspected cancer cells or biopsy or tumor samples.
  • One class of such kits will contain at least the labeled target or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive,” “sandwich,” “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • the kits comprise one or more PCR primers described herein.
  • test kit may be prepared for the determination and quantitation of a desired target or protein in cells or a cellular or biopsy sample, comprising:
  • the diagnostic test kit may comprise:
  • test kit may be prepared and used for the purposes stated above, and comprises:
  • an assay system for screening potential drugs effective to modulate the activity or expression of the target or gene signature may be prepared and is provided.
  • the target may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the target activity of the cells, or in the proliferation or division of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known target.
  • target can include any of the following: any of the genes (including any single or combinations) identified in Table S4 and/or Table S8, any corresponding protein of these genes; alone or in combination with one or more
  • neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron- specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), and any combination thereof, and optionally, any one or more of TP53, PTEN, SOX11 and/or RBI.
  • biomarkers disclosed herein can be used as the basis for drug screening assays and research tools.
  • polypeptides and proteins encoded by the transcripts in the gene signatures, described herein can be used in drug screening assays, free in solution, or affixed to a solid support. All of these forms can be used in binding assays to determine if agents being tested form complexes with the peptides, proteins or fragments, or if the agent being tested interferes with the formation of a complex between the peptide or protein and a known ligand.
  • the present invention provides for methods and assays for screening agents for treatment of prostate cancer, comprising contacting or incubating the test agent with a polypeptide or protein encoded by a gene in one of the gene signatures described herein and detecting the presence of a complex between the polypeptide and the agent or the presence of a complex between the polypeptide and a ligand, by methods known in the art.
  • the polypeptide or fragment is typically labeled. Free polypeptide is separated form that in the complex, and the amount of free or uncomplexed polypeptide is measured. This measurement indicates the amount of binding of the test agent to the polypeptide or its interference with the binding of the polypeptide to a ligand.
  • High throughput screening can also be used to screen for therapeutic agents.
  • Small peptides or molecules can be synthesized and bound to a surface and contacted with the polypeptides encoded by the gene signature transcripts, and washed. The bound peptide is visualized and detected by methods known in the art.
  • Antibodies to the polypeptides can also be used in competitive drug screening assays.
  • the antibodies compete with the agent being tested for binding to the polypeptides.
  • the antibodies can be used to find agents that have antigenic determinants on the polypeptides, which in turn can be used to develop monoclonal antibodies that target the active sites of the polypeptides.
  • the invention also provides for polypeptides to be used for rational drug design where structural analogs of biologically active polypeptides can be designed. Such analogs would interfere with the polypeptide in vivo, such as by non-productive binding to target.
  • the three-dimensional structure of the protein is determined by any method known in the art including but not limited to x-ray crystallography, and computer modeling. Information can also be obtained using the structure of homologous proteins or target-specific antibodies.
  • agents can be designed which act as inhibitors or antagonists of the polypeptides, or act as decoys, binding to target molecules non-productively and blocking binding of the active polypeptide.
  • Polypeptides encoded by any of the differentially expressed transcripts of the gene signatures described herein can be used. Additionally, testing can be done as described above using the proteins or fragments of the proteins described herein.
  • a further embodiment of the present invention is gene constructs comprising any one of the differentially expressed transcripts and a vector. These gene construct can be used for testing of therapeutic agents as well as basic research regarding prostate cancer, the development of CRPC, the development of super-resistant tumors, and tumor metastasis in general. These gene constructs can also be used to transform host cells can be transformed by methods known in the art.
  • the resulting transformed cells can be used for testing for therapeutic agents as well as basic research regarding prostate cancer.
  • cells can be transformed with any one of the differentially expressed transcripts, and contacted with a test agent.
  • the resulting expression of the transcript can be detected and compared to the expression of the transcript in the cell before contact with the agent.
  • the expression of the transcripts in host cells can be detected and measured by any method known in the art, including but not limited to, reporter gene assays.
  • genes constructs as well as the host cells transformed with these gene constructs can also be the basis for transgenic animals for testing both as research tools and for therapeutic agents.
  • Such animals would include but are not limited to, nude mice.
  • Phenotypes can be correlated to the genes and looked at in order to determine the genes effect on the animals as well as the change in phenotype after administration or contact with a potential therapeutic agent.
  • the gene constructs and host cells transformed with the gene constructs can also be administered to murine models of prostate cancer, for analyzing test agents as well as basic research. Any of the differentially expressed transcripts of the gene signatures found in Tables and described herein can be used.
  • the Nkx3.1 CreERT2 driver is a knock-in allele in which a tamoxifen-inducible CreERT2 cassette is placed under the transcriptional control of the endogenous Nkx3.1 promoter, resulting in heterozygous inactivation of Nkx3.1 and thereby resulting in pre-invasive phenotypes (19).
  • Induction of Cre activity by tamoxifen administration enables temporal control of gene deletion in mature adult prostate, as well as spatial restriction to prostatic luminal epithelial cells (19,22), which are a cell of origin for prostate cancer (23), and enables lineage-tracing to define the cellular origin of tumor phenotypes.
  • the CRPC phenotype in the NP and NPp53 mice shows appropriate expression of epithelial cytokeratins, consistent with the luminal phenotype of prostate adenocarcinoma, and has a high proliferative index (Fig. 1C; Table S2).
  • AR displays primarily nuclear localization in prostate tumor cells of hormonally-intact mice, but is more diffusely localized in NP and NPp53 CPRC (Fig. 1C).
  • mice MR signatures representing the transition to CRPC in the NP or NPp53 mice (NP CRPC versus N and NPp53 CRPC versus N, respectively, with N representing the control Nkx3.1 CreERT2 mice), or comparing the CRPC in NPp53 and NP mice (NPp53 CRPC versus NP CRPC).
  • GSEA cross-species gene set enrichment analysis
  • abiraterone is widely used for treatment of advanced prostate cancer (1), it has not been extensively investigated in preclinical studies in vivo. Furthermore, although originally described as a CYP17A1 inhibitor that blocks androgen biosynthesis, abiraterone has also been shown to be metabolized to a potent AR antagonist (29,30), and thus can potentially inhibit androgen signaling at multiple levels. To perform preclinical studies, we first optimized dosing and scheduling parameters using strain-matched mice (Figs. 8A-C), which led us to select a dose of 200 mg/kg/day, which is similar to the dosage used for other mouse strain backgrounds (e.g., (31)).
  • abiraterone differed considerably between NP CRPC and NPp53 CRPC (Fig. 2A-E).
  • NPp53 CRPC has a latent potential for non-adenocarcinoma phenotypes that is augmented by abiraterone treatment.
  • ssGSEA single-sample GSEA
  • the data described above define a subgroup of abiraterone-treated NPp53 CRPC with accelerated tumor phenotypes and distinct molecular profiles that are highly enriched for genes expressed in human CRPC. Based on their distinct histopathological and molecular phenotypes, we term these Group 2 abiraterone-treated NPp53 CRPC as "exceptional non- responders” to distinguish them from the Group 1 "non-responder" NPp53 CRPC tumors and the "responder” NP CRPC tumors. Thus, in subsequent analyses, we consider these NPp53 CRPC "exceptional non-responder” and “non-responder” groups separately, and compare these to the NP CRPC "responder” group.
  • non-responders are phenotypically and molecularly similar to the vehicle- treated controls, and have signatures that are essentially unchanged post-treatment (Fig. 3A, Scenario 2).
  • Fig. 3A, Scenario 3 Experiments were designed to illustrate that "exceptional non-responders” would display an enhancement of the MR signature (Fig. 3A, Scenario 3), which might be associated with adverse treatment response and/or adverse disease outcome in human CRPC.
  • these phenotypic and computational analyses define a sub-group of "exceptional non-responders" in the mouse that are conserved with more aggressive variants of human CRPC, including CRPC-NE.
  • the molecular drivers of this phenotype namely the adverse treatment-response MRs, are conserved with human CRPC, enriched in patients that develop CRPC-NE, and associated with adverse outcome for human prostate cancer. Therefore, these adverse treatment-response MRs may help identify patients with aggressive prostate cancer and/or who are predisposed to fail treatment with abiraterone.
  • Neuroendocrine differentiation mTP53 -deficient CRPC is mediated in part by SOX11
  • SOX11 a member of the SoxC subclass of HMG-box transcriptional regulators that functions in a wide range of neural and mesenchymal progenitors during organogenesis and is also a pan- neuronal differentiation factor (36-38).
  • Soxll was also the most up-regulated gene in the TRAMP mouse model of prostate cancer in the transition from adenocarcinoma to neuroendocrine disease (40).
  • Soxll expression was also up-regulated in mouse NPp53 CRPC relative to NP CRPC (p ⁇ 0.01, t-test), and particularly in the exceptional non-responders (Fig. 13A).
  • Soxll expression was up-regulated in mouse epithelial cell lines established from these NPp53 tumors relative to lines from NP tumors (p ⁇ 0.01, t-test), and was correlated with expression of the neuroendocrine marker neuron-specific enolase (NSE) (p ⁇ 0.001, t-test; Fig. 4D).
  • NSE neuroendocrine marker neuron-specific enolase
  • NPp53 CRPC shares features in common with human CRPC-NE
  • Synaptophysin-positive (Syn + ) cells in tumors from NP and NPp53 mice that were hormonally-intact, castrated, and castrated with abiraterone treatment Figs. 5A-B; Table S5A.
  • NPp53 CRPC neuroendocrine differentiation
  • Sox11 a known target of TP53
  • SoxC factors such as Soxll function later to promote neuronal differentiation (36,53).
  • Sox2 may act early in the emergence of CRPC-NE to promote epithelial plasticity (51), whereas Soxl l may act at subsequent stages to promote neuroendocrine differentiation.
  • Sox2 expression is reduced in the NPp53 CRPC relative to NP CRPC (Fig. 13A).
  • POU3F2 POU3F2
  • MYCN MYCN
  • these activities of MYCN contrast with those of MFC (c-Myc), which collaborates with Pten loss- of-function in mouse models to generate highly aggressive adenocarcinoma and metastasis but not neuroendocrine differentiation (57).
  • transdifferentiation has been implicated as a potential cause of drug resistance in a clinical setting for non-small cell lung cancer with alterations of TP53 and RBI (59,60).
  • Our current findings provide direct experimental evidence for transdifferentiation in mediating drug resistance, and extend the generality of this mechanism to other tumor types and cancer drivers.
  • perturbations of key conserved pathways that regulate cellular plasticity and differentiation in normal developmental contexts may represent significant mechanisms for driving drug resistance in cancer.
  • Illumina HiSeq 2500 platform a complete list of differentially expressed genes is provided in Dataset 1 (61). Published human datasets used in these studies for cross-species analyses are described in Table S 1. Master regulator analysis was performed using the MAster Regulator INference algorithm (MARINa) to interrogate a human prostate cancer interactome, as described (27). Cross-species gene GSEA and single sample GSEA (ssGSEA) were done using "humanized" mouse signatures and human MR signatures, as described (27).
  • MARINa MAster Regulator INference algorithm
  • ssGSEA single sample GSEA
  • Luminal cells are favored as the cell of origin for prostate cancer.
  • Sock E Rettig SD, Enderich J, Bosl MR, Tamm ER, Wegner M. Gene targeting reveals a widespread role for the high-mobility-group transcription factor Soxl l in tissue remodeling. Mol Cell Biol 2004;24(15):6635-44 doi 10.1128/MCB.24.15.6635- 6644.2004.
  • Sequist LV Waltman BA, Dias-Santagata D, Digumarthy S, Turke AB, Fidias P, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med 2011;3(75):75ra26 doi 10.1126/scitranslmed.3002003.
  • Niederst MJ Sequist LV, Poirier JT, Mermel CH, Lockerman EL, Garcia AR, et al. RB loss in resistant EGFR mutant lung adenocarcinomas that transform to small-cell lung cancer. Nat Commun 2015;6:6377 doi 10.1038/ncomms7377.
  • Supplemental Table S8 is a subset of differentially expressed genes associated with super resistant prostate tumors, or "exceptional non-responders”. Detecting differential expression of any one or combination of these genes (from Table S4 and/or Table S8) or their corresponding protein/s will be useful for diagnosing or prognosing a prostate cancer patient with CRPC, or with super resistant prostate tumors, or castration resistant prostate cancer with neuroendocrine differentiation (CRPC-NE) or CRPC-NE likelihood, such that the appropriate treatment options can then be made.
  • CRPC-NE neuroendocrine differentiation
  • Such treatment options include alternatives to anti-androgen treatment when CRPC and/or the development of super-resistant prostate tumors is prognosed,
  • it may be determined to administer to the patient an effective amount of one or any combination of the following: androgen synthesis inhibitors (TAK700), androgen receptor (AR) inhibitors (ARN-509, ODM-201, and EZN-4176), AR DNA binding domain inhibitors (EPI-001), selective AR downregulators or SARDs (AZD-3514), including agents that inhibit both androgen synthesis and receptor binding ( ⁇ -001/galeterone).
  • detecting one or more neuroendocrine markers including Synaptophysin (Syn), chromogranin A (CgA), neuron-specific enolase (NSE), and 1-DOPA Decarboxylase (DDC), or any combination thereof, can be combined with detecting differential expression from any of the markers from Table S4 or Table S8 (or the corresponding protein/s) for diagnosing or prognosing a prostate cancer patient with CRPC, with super resistant prostate tumors, or castration resistant prostate cancer with neuroendocrine differentiation (CRPC-NE) or CRPC-NE likelihood.
  • Synaptophysin Syn
  • CgA chromogranin A
  • NSE neuron-specific enolase
  • DDC 1-DOPA Decarboxylase
  • PTEN or any combination of these markers may also be useful in combination with any of the above described markers for diagnosing or prognosing a prostate cancer patient with CRPC, with super resistant prostate tumors, or castration resistant prostate cancer with neuroendocrine differentiation (CRPC-NE) or CRPC-NE likelihood.
  • CRPC-NE neuroendocrine differentiation
  • detecting expression of SOX11 may also be useful in combination with any of the above described markers for diagnosing or prognosing a prostate cancer patient with CRPC, with super resistant prostate tumors, or castration resistant prostate cancer with neuroendocrine differentiation (CRPC-NE) or CRPC-NE likelihood.
  • CRPC-NE neuroendocrine differentiation
  • mice contains a tamoxifen-inducible Cre under the control of the Nkx3.1 promoter, resulting in heterozygous deletion of Nkx3.1 in the germline and, following delivery of tamoxifen, conditional gene recombination specifically in prostate luminal epithelial cells ( 14, 15). Th U allele was crossed with a conditional Pten allele to generate the ⁇ mice (NP mice) as described in (16), and a conditional Trp53 allele (17) to generate the
  • NPp53 mice For lineage tracing, NP and NPp53 mice were crossed with a Rosa-CAG-LSL-EYFP-WPRE reporter allele, which contains a conditionally-activatable enhanced YFP (EYFP) under the control of the Rosa locus (18).
  • EYFP conditionally-activatable enhanced YFP
  • mice were androgen- ablated by surgical castration as in (16). Tumor-bearing mice were monitored daily to assess body condition as described in (19), and were euthanized if their Body Condition Score was ⁇ 1.5, as per IACUC guidelines.
  • Abiraterone-acetate was provided by Johnson & Johnson. Optimal dosage, pharmacokinetic profile, and optimal scheduling were determined using non-tumor bearing littermates to control for strain background, based on published studies in other mouse strain backgrounds (2,3). To determine dosage tolerance, abiraterone-acetate was diluted to the desired concentration in corn oil, and administrated by oral gavage at a dose of 50 or 200 mg/kg once daily for 3 weeks. Body weight and animal condition were recorded daily throughout the dosing period and compared to the vehicle control group.
  • mice were administered a single dose of abiraterone-acetate (200 mg/kg) at time 0, and then sacrificed at individual time points up to 24 hours following drug delivery.
  • abiraterone-acetate 200 mg/kg
  • prostate tissues and serum were collected and levels of abiraterone, the active form of abiraterone-acetate, were determined by mass spectrometry.
  • mice were induced with tamoxifen at 3 months of age; after tumor induction, the mice underwent castration (or mock surgery) at 10 or 7 months, respectively.
  • Mice were randomly enrolled in the vehicle or abiraterone treatment groups, which was initiated two months following castration. Treatment was continued for four weeks, except in cases where the mouse had to be euthanized early due to poor body condition score (as above). During the last week of treatment, mice were treated for 7 consecutive days and dissected within 24 hours of the last treatment.
  • MRI Magnetic Resonance Imaging
  • Bruker Biospec 9.4T Tesla Small Animal MR Imager located within the mouse barrier in the Herbert Irving Cancer Center Small Animal Imaging facility.
  • MRI imaging was done using a Bruker ICON 1 Tesla desktop MRI scanner, which was also located within the mouse barrier.
  • MRI was done immediately prior to initiation of treatment and then immediately following the last drug treatment. Images were generated using Para Vision 6 software for preclinical MRI (Bruker) and reviewed by an MRI physicist (YS).
  • Immunohistochemical and immunofluorescence staining were done on 3 ⁇ paraffin sections as previously described (15). Briefly, sections were deparaffinized in xylene, followed by antigen retrieval in antigen unmasking solution (Vector Labs). Slides were blocked in 10% normal goat serum, then incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hour.
  • frozen tissues were weighed and added to 60°C water containing deuterated internal standards, heated to 60°C for 10 minutes, and homogenized using a tissue homogenizer (Precellys; Bertin, Rockville, MD); supernatant was extracted twice with hexane (ethyl acetate [80:20 v/v]), and the organic layer was dried (SpeedVac; Thermo Scientific,Waltham, MA), derivatized with 0.025 M hydroxylamine hydrochloride for 24 hours at room temperature to form oximes, and quantified using liquid chromatography electrospray-ionization tandem mass spectrometry.
  • tissue homogenizer Precellys; Bertin, Rockville, MD
  • supernatant was extracted twice with hexane (ethyl acetate [80:20 v/v]), and the organic layer was dried (SpeedVac; Thermo Scientific,Waltham, MA), derivatized with 0.025 M hydroxylamine hydrochloride for 24 hours at room
  • the lower limit of detection for steroids was 0.49 pg/sample for progesterone, androstenedione, testosterone and DHT; 0.98 pg/sample for DHEA and androsterone, and 1.96 pg/sample for pregnenolone.
  • the lower limit of detection for abiraterone was 0.06 ng/sample in serum and 1.2 ng/sample for tissue. Limits of quantification in serum were 0.02 ng/ml for progesterone, androstenedione, testosterone and DHT; 0.04 ng/ml for DHEA and androsterone, 0.08ng/ml for pregnenolone, and 0.005 ng/dL for abiraterone.
  • Limits of quantification in tissue were 0.02 pg/mg for progesterone, androstenedione, DHT, and testosterone, 0.05 ng/ml for DHEA and androsterone, 0.11 pg/mg for pregnenolone, and 0.06 pg/gm for abiraterone.
  • Cell lines were generated from NP or NPp53 prostate tumors in our laboratory in the Fall of 2013 as described (22). Two independent cell lines were obtained from two independent tumors each from N and NPp53 mice; to avoid clonal variation, we used a pooled cell population for these studies. Each cell line was confirmed to be negative for a multi-species mycoplasma test (Mycoplasma Detection Kit, Cat# MP70114, Fisher), which was performed in early 2014. The cell lines were confirmed to have the correct genotypes using a commercial source (TransnetYX, Inc). Further characterization of the cell lines included analyses of growth rate using MTT assays, and expression of relevant markers by Western blot analyses.
  • Lentiviral particles were made using the 2nd generation packaging vectors, psPAX2 and pMD2.G (Addgene) in HEK-293T cells (ATCC), and concentrated using the Lenti-X Concentrator reagent (Clonetech), as described previously (6).
  • Mouse cell lines were infected with the lentiviruses and selected for 5 days using 6 g/ml of puromycin.
  • qPCR quantitative real-time PCR
  • total RNA was isolated using a MagMAX-96 Total RNA Isolation Kit (Life Technologies, Grand Island, NY).
  • cDNA was prepared using Superscript III First-Strand Synthesis SuperMix for qRT-PCR kit from Thermo Fisher Scientific according to manufacturer's instructions.
  • RNA sequencing was performed at the JP Sulzberger Columbia Genome Center at Columbia University Medical Center.
  • a TruSeq RNA Sample Prep Kit v2 (Illumina) was used for library preparation followed by sequencing (30 million single-end reads) on an Illumina HiSeq 2500.
  • RNAseq data raw counts were normalized and the variance was stabilized using DESeq2 package (Bioconductor) in R-studio 0.99.902, R v3.3.0 (The R Foundation for Statistical Computing, ISBN 3-900051-07-0).
  • the raw and normalized data files are deposited in Gene Expression Omnibus (GEO) GSE92721. Differentially expressed genes are listed in Dataset 1.
  • GEO Gene Expression Omnibus
  • Differential gene expression signatures were defined as a list of genes ranked by their differential expression between any two phenotypes of interest (e.g., abiraterone treated vs. vehicle treated; castration-resistant vs. intact etc.), using 2-tailed Welch t-test.
  • mouse gene signatures were mapped to their corresponding human orthologs based on the homoloGene database (NCBI); all analyses comparing mouse and human genes were done using mouse "humanized" signatures.
  • ssGSEA single sample GSEA
  • individual abiraterone-treated samples were compared to the pooled group of vehicle-treated samples, such that the fold- change value for each gene was estimated as the gene's expression in a single treated sample over the average of the gene's expression levels in the vehicle-treated samples. If fold change (FC) fell below 1, the resulting fold change was defined as -1/FC.
  • Single-sample signatures were then defined by a list of genes ranked by their fold-change between a single treated sample and a pooled group of vehicle-treated samples. Each single-sample signature was compared to the human Best et al. signature (9) using Gene set enrichment analyses (GSEA).
  • GSEA Gene set enrichment analyses
  • Master regulator analysis was performed using the MAster Regulator INference algorithm (MARINa) (25) by interrogating a human prostate cancer interactome (26) with "humanized” mouse signatures or with human signatures.
  • Gene set enrichment analyses was done using "humanized” mouse signatures or “humanized” mouse master regulator signatures as described (27) or using human gene or human MR signatures, where appropriate.
  • Sboner et al. dataset 13 in which cancer specific survival data (i.e., death due to prostate cancer) is available.
  • Corresponding survival analysis was estimated using the transcriptional activity levels of MRs, which were estimated as enrichment of MR transcriptional targets (from the human prostate cancer interactome) in each data sample from the Sboner et al. dataset.
  • k-means clustering was done on the activity levels of the MRs to cluster patients into two groups: one group having similarity in overall activity to the adverse response MRs and one group having no similarity.
  • Kaplan-Meier analysis was done using the "surv” and "coxph” functions from the survcomp package in R-studio 0.99.902, R v3.3.0; the p- value was estimated using log-rank test.
  • Lam HM McMullin R, Nguyen HM, Coleman I, Gormley M, Gulati R, et al.

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Abstract

L'invention concerne des méthodes et des marqueurs permettant de déterminer le développement d'un cancer de la prostate résistant à la castration et aux traitements associés. Dans certains modes de réalisation, la présente invention concerne une méthode de dépistage d'un cancer de la prostate résistant à la castration (CRPC) et/ou de tumeurs de la prostate super-résistantes chez un sujet diagnostiqué comme atteint d'un cancer de la prostate, la méthode comprenant le dosage des niveaux d'expression génique d'un ou de plusieurs gènes, ou des niveaux protéiques correspondants, dans un échantillon provenant du sujet pour obtenir un profil d'expression d'essai, la comparaison dudit profil d'expression d'essai des gènes ou protéines avec un profil d'expression de référence desdits gènes ou protéines, une expression différentielle ou des niveaux protéiques différentiels indiquant la présence d'un CRPC et/ou le développement de tumeurs de la prostate super-résistantes. Un kit comprenant une ou plusieurs sondes pour détecter lles niveaux d'expression d'un ou de plusieurs gènes est en outre décrit.
PCT/US2017/038803 2016-06-22 2017-06-22 Transdifférenciation servant de mécanisme de résistance au traitement pour le cancer de la prostate résistant à la castration Ceased WO2017223344A1 (fr)

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EP4103752A4 (fr) * 2020-02-11 2024-05-15 Dana-Farber Cancer Institute, Inc. Méthodes et compositions pour l'identification du cancer de la prostate neuroendocrinien résistant à la castration
WO2022266413A1 (fr) * 2021-06-17 2022-12-22 Mayo Foundation For Medical Education And Research Évaluation et traitement du cancer de la prostate
CN116699148A (zh) * 2022-02-28 2023-09-05 中国科学院分子细胞科学卓越创新中心 预测前列腺癌患者响应cyp17a抑制剂治疗的方法和试剂盒
WO2024055010A1 (fr) * 2022-09-08 2024-03-14 Board Of Regents, The University Of Texas System Compositions pour le traitement du cancer et leurs méthodes d'utilisation
CN119716058A (zh) * 2024-12-12 2025-03-28 复旦大学附属眼耳鼻喉科医院 特异性结合nse蛋白或其编码基因的物质在制备arn诊断产品或预后产品中的用途

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