WO2020214718A1 - Gènes de signature rrm2 utilisés comme marqueurs pronostiques chez des patients atteints d'un cancer de la prostate - Google Patents

Gènes de signature rrm2 utilisés comme marqueurs pronostiques chez des patients atteints d'un cancer de la prostate Download PDF

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WO2020214718A1
WO2020214718A1 PCT/US2020/028338 US2020028338W WO2020214718A1 WO 2020214718 A1 WO2020214718 A1 WO 2020214718A1 US 2020028338 W US2020028338 W US 2020028338W WO 2020214718 A1 WO2020214718 A1 WO 2020214718A1
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rrm2
genes
cancer
prostate cancer
cells
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Ying MAZZU
Philip W. Kantoff
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Memorial Sloan Kettering Cancer Center
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    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/44—Multiple drug resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present technology is directed to methods for identifying cancers associated with poor prognosis (e.g ., prostate cancer, breast cancer, ovarian cancer, lung cancer, or liver cancer) in a patient in need thereof.
  • the methods disclosed herein are useful in determining whether a patient will benefit from or is predicted to be responsive to treatment with ribonucleotide reductase inhibitors based on detecting elevated expression levels of specific RRM2 signature genes.
  • Prostate cancer is a heterogenous disease and the third leading cause of cancer death among American men.
  • Clinical decision-making has been largely driven by clinical and pathologic variables, such as tumor stage, Gleason score, and serum prostate-specific antigen (PSA) levels (Falzarano and Magi-Galluzzi, Adv Anat Pathol 18, 159-164 (2011); Gleason and Mellinger, J Urol 111, 58-64 (1974)).
  • PSA serum prostate-specific antigen
  • Inhibition of androgen receptor (AR) signaling is the mainstay of therapy for recurrent or advanced prostate cancer but is limited in its utility because of acquired resistance (Attard et al, Lancet 387, 70-82 (2016)).
  • the present disclosure provides RRM2 signature genes associated with aggressive subtypes of prostate cancer as well as other types of cancer (e.g ., breast cancer, ovarian cancer, lung cancer, or liver cancer), which are useful for predicting patient prognosis and guiding treatment decisions.
  • Such methods would aid in predicting the responsiveness of individual patients to a particular drug regimen (e.g., ribonucleotide reductase inhibitors) and the identification of optimal therapeutic strategies at the outset.
  • the present disclosure provides a method for detecting an aggressive subtype of prostate cancer in a subject in need thereof comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the subject compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists of ARL6IP1, ASF IB, ATAD2, ATAD5, AUNIP, AURKA, AURKB, BLM, BORA, BUB1, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU, CEP152,
  • PIMREG PIMREG, PKMYT1, PLK1, PLK4, POLQ, POT1, PPIF, PRC1, PSMC3IP, PSRC1, RAD51, RAD 51 API, RAD54B, RAD54L, RFC3, RMI2, SHCBP1, SKA1, SKA 3, SPAG5, SPC25, STIL, STAIN/, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRF1, ZNF273, ZNF695, and ZWINT.
  • the method further comprises detecting an increase in expression levels of at least one gene selected from among ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MK167, PTTG1, UBE2T, and NUF2 in the test sample compared to the healthy control subject or the reference sample.
  • the present disclosure provides a method for detecting an aggressive subtype of prostate cancer in a subject in need thereof comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the subject compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists oiANLN, BIRC5, CCNB1, CDC20,
  • the aggressive subtype of prostate cancer is PCS1 or luminal B, but not PCS2, PCS3, luminal A, or basal.
  • the prostate cancer may be localized prostate cancer or metastatic castration-resistant prostate cancer and/or comprises tumors with immunosuppressive M2 macrophages, and/or regulatory T cells (Tregs).
  • the aggressive subtype of prostate cancer is associated with a high Gleason score, a high incidence of recurrence, and/or lethality.
  • the present disclosure provides a method for identifying a cancer associated with poor prognosis in a subject in need thereof comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the subject compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists of ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MKI67, PTTG1, and UBE2T , and wherein the cancer is breast cancer, ovarian cancer, lung cancer, or liver cancer.
  • the present disclosure provides a method for identifying enzalutamide resistance in a subject with castration-resistant prostate cancer comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the subject compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists oiAUPKB, MIS18A, MK167, CENPF, PPIF, FANCI, TPX2, CDKN3, KIF2C, K1F11 and CCNB2.
  • the method further comprises detecting an increase in expression levels of at least one gene selected from among TACC3, NUSAP1, INSIG1, GPSM2, KPNA2, CKS1B, ZNF273, STMNl, PADS I API, and FANCD2.
  • the expression levels are detected via RNA-seq, northern blotting, microarrays, dot or slot blots, fluorescent in situ hybridization, reverse transcription polymerase chain reaction (RT-PCR), ribonuclease protection assay (RPA), real-time quantitative RT-PCR, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), enzyme-linked immunosorbent assay (ELISA), immunoprecipitation,
  • RT-PCR reverse transcription polymerase chain reaction
  • RPA ribonuclease protection assay
  • HPLC high-performance liquid chromatography
  • LC/MS liquid chromatography-mass spectrometry
  • ELISA enzyme-linked immunosorbent assay
  • the present disclosure provides a method for selecting a prostate cancer patient for treatment with a ribonucleotide reductase inhibitor comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the patient compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists oiARL6IPl, ASF1B, ATAD2, ATAD5, AUNIP, AURKA, AURKB, BLM, BORA, BUB1, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CEN
  • CEP 152 CIT, CKAP2, CKAP5, CKS1B, CKS2, CTSV, DBF4, DDIAS, DEPDC1, DEPDC1B, DNA2, DNMT3B, DONSON, DSCC1, EZH2, FAM72D, FAM83D, FANCD2, FANCI, GINS1, GINS2, GPC2, GPSM2, GTSE1, HJURP, HMGB2, HMMR, INSIG1, KIF11, KIF15, KIF18B, K1F20A, KIF20B, KIF23, K1F4A, KPNA2, MAD2L1, MIS18A, MIS18BP1, MNS1, MYBL2, NCAPG, NCAPG2, NCAPH, NUSAP1, ORC1, PARP2, PBK, PCLAF, PIF1, PIGA,
  • PIMREG PIMREG, PKMYT1, PLK1, PLK4, POLQ, POT1, PPIF, PRC1, PSMC3IP, PSRC1, RAD51, RAD 51 API, RAD54B, RAD54L, RFC 3, RMI2, SHCBP1, SKA1, SKA 3, SPAG5, SPC25, STIL, STAIN/, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRF1, ZNF273, ZNF695, and ZWINT ; and administering to the patient an effective amount of a ribonucleotide reductase inhibitor.
  • the method further comprises detecting an increase in expression levels of at least one gene selected from among ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MK167, PTTG1, UBE2T ⁇ and NUF2 in the test sample compared to the healthy control subject or the reference sample.
  • the present disclosure provides a method for selecting a prostate cancer patient for treatment with a ribonucleotide reductase inhibitor comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the patient compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists oiANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MK167, PTTG1, and UBE2T , and administering to the patient an effective amount of a ribonucleotide reductase inhibitor.
  • the prostate cancer may be localized prostate cancer or metastatic castration-resistant prostate cancer, and/or has a PCS1 or a luminal B subtype.
  • the present disclosure provides a method for selecting a cancer patient for treatment with a ribonucleotide reductase inhibitor comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the patient compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists of ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MK167, PTTG1, and UBE2T , and wherein the cancer is breast cancer, ovarian cancer, lung cancer, or liver cancer; and administering to the patient an effective amount of a ribonucleotide reductase inhibitor.
  • ribonucleotide reductase inhibitors include, but are not limited to, hydroxyurea (HU), 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), GTI2040, COH3, COH4, COH20, COH29, gemcitabine, ribonucleotide reductase inhibitor compounds described in US 7,956,076 and RRM2-specific inhibitory nucleic acids.
  • the RRM2-specific inhibitory nucleic acid is a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.
  • the methods of the present technology further comprise sequentially, simultaneously, or separately administering an effective amount of a cytokine and/or a monoclonal antibody.
  • cytokines include, but are not limited to, interferon a, interferon b, interferon g, complement C5a, IL-2, TNF alpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7
  • the monoclonal antibody targets CTLA-4 (e.g., ipilimumab, tremelimumab), PD-1 (e.g., cemiplimab, nivolumab, pembrolizumab), PDL1 (e.g., avelumab, durvalumab, atezolizumab, Envafolimab, BMS- 936559, CK-301, CS-1001, SHR-1316, CBT-502, BGB-A333), TIM-3 (e.g., LY3321367, TSR-022, MBG453), BTLA, or VEGF (e.g., bevacizumab, ranibizumab).
  • CTLA-4 e.g., ipilimumab, tremelimumab
  • PD-1 e.g., cemiplimab, nivolumab, pembrolizumab
  • PDL1 e.
  • FIGS. 1A-1G show PPM2 function is disease-state specific.
  • Figure 1A shows a schematic of the experimental design. Transcriptomic changes induced by PPM2
  • FIG. 1B shows volcano plots depicting transcriptomic changes induced by RRM2 overexpression in LNCaP (left panel) and PC-3 (right panel) cells.
  • Figure 1C shows Venn diagrams depicting the overlap of genes upregulated (left) and downregulated (right) with RRM2 overexpression in LNCaP and PC-3 cells (FDR ⁇ 0.05, -1.5 > FC > 1.5).
  • Figure ID shows Venn diagrams (left) depicting the overlap of GO analysis of genes upregulated by RRM2 overexpression.
  • Figure IE shows enrichment of A ⁇ /2-upregul ated genes from LNCaP in epithelial mesenchymal transition (EMT).
  • Figure IE shows enrichment of RRM2 -upregulated genes from LNCaP in angiogenesis.
  • GSEA results are from LNCaP cells, and the Venn diagrams show the overlap between pathway genes and genes upregulated in LNCaP and PC-3 cells that overexpress RRM2.
  • Figure 1G shows GO enrichment of common genes deregulated in LNCaP -RRM2/C4-2-siRRM2 (left) and PC-3-RRM2/C4-2- si RRM2 (right).
  • Figures 2A-2D show integration of prostate cancer cell line transcriptomic data with clinical outcomes.
  • Figure 2A depicts Venn diagrams (Figure 2A, Top Panel) depicting the overlap of genes with expression that positively correlated with RRM2 levels in TCGA (Figure 2A, Left Panel), Kumar ( Figure 2A, Middle Panel), and SU2C/PCF ( Figure 2A, Right Panel) cohorts with upregulated genes in LNCaP -RRM2 or PC-3 -RRM2 cells.
  • Figure 2B plots show the genes with expression that correlates with RRM2 expression level in each prostate cancer cohort.
  • RRM2 signature the 626 genes with expression that correlated with RRM2 levels in the 3 clinical cohorts ( Figure 2B, Left Panel) were compared with genes upregulated in PC-3 -RRM2 or LNCaP-A’/riTC’ ( Figure 2B, Right Panel) to identify RRM2 signature (126 genes). Clinical significance of expression of the 126-gene RRM2 signature in the TCGA cohort. Samples were ranked based on expression of the 126-gene RRM2 signature, and Kaplan-Meier curves were used to estimate survival differences between patients in the top and bottom 25th percentiles of expression (Figure 2C). The log-rank test was calculated to determine significance.
  • Figure 2D shows association between RRM2 signature (126 genes) level with Gleason score (Figure 2D, Left Panel) and lethality (Figure 2D, Right Panel) in the Setlur cohort.
  • Figure 3 shows RRM2 levels are highly correlated with PCS1 gene expression. Correlation of RRM2 level with PCS gene expression in TCGA ( Figure 3, Left Panel), Taylor ( Figure 3, Middle Panel), and SU2C/PCF ( Figure 3, Right Panel) cohorts. Each individual patient sample is indicated by a single column ( Figure 3, Top Plot) and a single dot ( Figure 3, Bottom Plot).
  • PCS scores were calculated with GSVA using the ssGSEA method, and the values were compared to RRM2 mRNA expression levels divided by quantiles. The differences between pairs are statistically significant except for those labeled as N.S. (not significant).
  • Figures 4A-4F demonstrate Clinical significance of the 12-gene RRM2 sub signature.
  • Figure 4A Left Panel shows a GSEA plot (left) demonstrating high enrichment of PCS 1 genes in the RRM2 signature.
  • Figure 4A Right Panel, Venn diagrams depict the overlap between genes in the RRM2 signature with PCS1 (right) and PAM50 genes (left) and PCS2 and PCS3 genes (right).
  • the 12 genes shared by PCS1, PAM50, and RRM2 signature comprise the 12-gene sub-signature.
  • Figure 4B shows correlation between expression of the 12-gene signature with disease free survival in Taylor (Figure 4B, Left Panel) and TCGA cohorts ( Figure 4B, Right Panel).
  • Figure 4C demonstrates correlation of the 12-gene signature ssGSEA score with Gleason score (Figure 4C, Left Panel) and lethality (Figure 4C, Right Panel) in the Setlur cohort.
  • Figure 4D shows correlation between 12-gene signature expression and probability of overall survival (OS) were analyzed in breast and lung cancer and
  • Figure 4E shows correlation for ovarian and liver cancer. Samples were ranked based on expression of the 12-gene sub-signature, and Kaplan-Meier curves were used to estimate survival differences between patients in the top and bottom 25th percentiles of expression. The log-rank test was calculated to determine significance.
  • Figure 4F shows median survival time compared between cases with low or high expression of the 12-gene panel.
  • Figure 5A-5B shows inhibition of RRM2 specifically targets genes that define poor prognostic subtypes of prostate cancer.
  • Figure 5A shows supervised hierarchical clustering of prostate cancer cases in the Taylor ( Figure 5A, Top Panel), Grasso ( Figure 5A, Middle Panel), and Kumar ( Figure 5A, Bottom Panel) cohorts, based on expression of PCS genes. Genes deregulated with RRM2 overexpression (PC-3 -RRM2) and inhibition of RRM2 (by COH29) are shown.
  • Figure 5B shows supervised hierarchical clustering of prostate cancer cases from the Kumar cohort, based on expression of PAM50 classifier genes. Genes deregulated with RRM2 overexpression (PC-3 -RRM2) and inhibition of RRM2 (by COH29) are shown.
  • Figure 6A-6E demonstrates that RRM2 overexpression contributes to enzalutamide (ENZ) resistance and an immunosuppressive TIME.
  • Figure 6A shows unsupervised hierarchical clustering of single-cell RNA-seq data from 77 CTCs from 13 patients with CRPC treated with enzalutamide (from GSE67980) based on expression of 21 genes from the RRM2 signature. Genes with expression that was significantly upregulated in the ENZ- resistant CTCs of Group II are shown in red (11-gene panel). * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001.
  • Figure 6B shows profiling of immune cells by CIBERSORT in the TCGA; Figure 6C in Taylor, and Figure 6D in SU2C/PCF cohorts.
  • Figure 6E shows assessment of cell viability in LNCaP-Empty Vector (EV) and LNC&P-RRM2 cells treated with ENZ. Values represent the mean ⁇ standard error of 3 independent experiments. **, p ⁇ 0.01 vs the control group.
  • Figures 7A-7B show that a panel derived from RRM2 signature could be useful in predicting enzalutamide resistance in the CTCs of patients with CRPC.
  • High expression of the 11-gene signature is significantly associated with poor clinical outcomes.
  • Gleason score and lethality in the Setlur cohort is shown in Figure 7A and disease-free survival in the TCGA cohort is shown in Figure 7B.
  • FIG. 8 shows LM22 gene signatures for the TCGA prostate cancer cohort.
  • LM 22 is a gene signature matrix array that contains signature profiles for 22 distinct immune cell types along the columns of the matrix.
  • Figure 9 shows LM22 gene signatures for the Taylor prostate cancer cohort. High infiltration of immunosuppressive immune cells is suggestive of dysfunctional or exhausted cytotoxic T cells in RRM2 -high tumors.
  • Figure 10 shows LM22 gene signatures for the SU2C/PCF prostate cancer cohort. High infiltration of immunosuppressive immune cells is suggestive of dysfunctional or exhausted cytotoxic T cells in RRM2- high tumors.
  • Figures llA-llO illustrates the oncogenic roles of RRM2 in prostate cancer and that knockdown of RRM2 inhibits tumorogenesis in PC cells.
  • Figure 11A shows dNTP production in small interfering RNA (siRNA) transfected cells. dNTP was detected at both 48 hours and 72 hours post-transfection of siRNAs in C4-2 cells.
  • Figure 11B shows siRRM2-induced DNA damage. DNA damage marker activation was monitored in LNCaP and C4-2 cells using Musemulti-color DNA damage kit.
  • Figure 11C shows activation of H2A.X confirmed by immunoblotting.
  • Figure 11D shows analysis of cell proliferation in transfected cells.
  • Figure HE shows analysis of cell cycle in transfected cells.
  • Figure 11F shows apoptosis detected by Annexin V assays and immunoblots.
  • Figure 11G illustrates dNTP production in empty vector (EV)/RRM2-overexpressing PC-3 cells (PC3-EV; PC3- RRM2).
  • Figure 11H shows cell proliferation in stable cells.
  • Figure 1 II shows soft agar assays of stable PC-3 cells. The colony numbers were normalized to those in the control cells.
  • Figure 11 J shows wound healing assays after the scratch done for 24 hours.
  • Figure 11 K demonstrates invasion assays after cells were plated for 48 hours.
  • Figure 11L shows EMT marker expression detected by qPCR in both EV- and RRM2-expressing PC-3 cells.
  • Figure 11M shows EMT marker expression detected by immunoblots in both EV- and RRM2-expressing PC-3 cells.
  • Figure 11N shows invasion assays after multiple siRNAs were transfected in PC3-RRM2 cells.
  • Figure llO shows that inhibition of RRM2 expression inhibited dNTP production.
  • Figure 11 values represent the mean ⁇ S.E. of three independent experiments. *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.0001 vs control groups treated with empty vector (EV) or with nonspecific (siNS) siRNA.
  • Figure 12A-12I demonstrate that overexpression of RRM2 positively associates with poorer clinical outcomes in multiple prostate cancer clinical cohorts.
  • Figures 12A-12C show the correlation of gene alteration with the fraction of genome altered (FGA) and Gleason grades in TCGA cohort was visualized in Figure 12A. The statistical quantitation was shown in Figure 12B (fraction of genome alteration, FGA) and Figure 12C (clinical Gleason score).
  • Figure 12D shows the correlation of RRM2 level with Gleason grade in tumor and matched normal tissues in the PHS/HPFS cohorts.
  • Figure 12E shows the correlation of RRM2 expression with tumor progression in Taylor cohorts.
  • Figure 12F shows the correlation of RRM2 expression with tumor progression in Grasso cohorts.
  • FIG. 12G graphically illustrates the association of RRM2 expression and the disease-free survival in the TCGA, Taylor, and Glinsky cohorts.
  • Figure 12H shows the correlation of RRM2 with the risk of lethal prostate cancer over long-term follow-up, independent from clinical characteristics and Gleason grade, in the combined HPFS and PHS prostate cancer cohorts. The odd ratios for lethal disease were adjusted for Gleason score. ****, pO.OOOl vs comparator groups.
  • Figure 121 shows that higher levels of RRM2 expression is significantly correlated with lethality in PHS and HPFS cohorts.
  • Figures 13A-13F show transcriptomic analysis unraveled the molecular mechanism of RRM2 function.
  • Figure 13A shows gene set enrichment analysis (GSEA) of GSEA
  • MYC targets Figure 13A, Left Panel shows MYC_up_Vl_up gene set
  • E2F targets Figure 13A, Second Panel from left shows hallmark_E2F_targets gene set
  • cell cycle Figure 13A, Third Panel from left shows Module_54 gene set
  • p53 pathway Figure 13A, Fourth Panel from left shows hallmark_p53 _pathway
  • apoptosis Figure 13A, Right Panel is hallmark apoptosis.
  • “Up-gene” signifies up-regulated genes and“Down-gene” signifies down-regulated genes.
  • Figure 13B shows multiple targets of pathways were validated by quantitative reverse transcription PCR (qRT-PCR).
  • Figures 13C-13D show siRRM2 -regulated gene profiling and the correlation of these genes with disease-free survival (DFS) in Taylor cohort.
  • Figure 13E shows significant enrichment in EMT and Angiogenesis gene sets in RRM2-overexpressing PC-3 cells.
  • Figure 13F shows RRM2-regulated 126-gene profiling and correlation with the clinical outcome in clinical cohorts. 126 genes were revealed by overlapping 1230 up-regulated genes in PC3-RRM2 cells with 627 common genes positively correlated with RRM2 overexpression in three prostate cancer cohorts (TCGA, Kumar, and SU2C/PCF). The correlation of these genes with disease-free survival was analyzed in the Taylor cohort.
  • Figures 14A-14J demonstrate the tumor suppressive role of COH29 (RRM2 inhibitor).
  • Figure 14A shows dNTP production in COH29-treated C4-2 cells. Two doses of COH29 were used in C4-2 cells, and dNTP production was detected after 24 hours of treatment.
  • Figure 14B shows that DNA damage was induced by COH29.
  • Figure 14C further illustrates activation of DNA damage markers.
  • Figure 14C Left Panel shows COH29 (RRM2 inhibitor) percent of total DNA damage in C-42 and LnCap cells.
  • Figure 14C Right Panel depicts immunoblot assay results where DNA damage markers were activated by COH29 in PC cells.
  • Figure 14D shows a cell proliferation assay where COH29 significantly inhibited PC cell grown in a dose-dependent manner.
  • Figure 14E shows cell cycle analysis after 48 hours of COH29 treatment.
  • Figure 14F shows COH29 induced apoptosis, where COH29 led to remarkable S phase arrest and apoptosis in PC cells.
  • Figure 14G shows the global mRNA changes induced by COH29 in C4-2 cells, after 48 hours of treatment.
  • Figure 14H shows GSEA analysis of mRNA profiling in 20 mM of COH29- treated cells. The histograms showed the distribution of select top GSEA molecular signatures. Up-gene (COH29 induced up-regulated genes); Down-gene (COH29-induced down-regulated genes). NES, normalized enrichment score; FDR, false discovery rate.
  • Figures 141- J show identification of targeted genes by inhibition of RRM2. Genes affected by inhibition of RRM2 in cells were overlapped with genes correlated with RRM2 overexpression in Taylor cohort to reveal 33 down-regulated genes and 12 up-regulated genes (Figure 141). Figure 14J shows these gene panels were validated in three additional prostate cancer cohorts. The Figure 14 values represent the mean ⁇ S.E. of three independent experiments. *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001 vs control groups.
  • Figures 15A-15J- show phospho-proteomic changes induced by inhibition of RRM2 and COH29 efficacy in vivo.
  • Figure 15A and Figure 15B show phosphokinase array analysis after 24-hour COH29 treatment in C4-2 cells.
  • Figure 15A whole-cell lysates were collected for human phosphor-kinase array analysis. Each membrane contains kinase- specific antibodies (number indicated).
  • Figure 15B relative phosphorylation of spots was quantified by Image J software and the value of vehicle (0 mM) was set up as“1”. Densities of individual dots corresponding to a phosphorylated kinase were measured by Image J software, and comparison between siNS and siRRM2 group was performed.
  • Figure 15C shows validation of phospho-kinase array by immunoblots.
  • siRNA-treated groups cell lysates were collected after transfection for 48 hours. The representative blots for each condition are shown and the values represent the mean ⁇ S.E. of two independent
  • Figure 15D shows druggable RRM2 signatures.
  • Figure 15D Left Panel shows the top three drugs from ToppGene analysis and COH29.
  • Figure 15D Right Panel shows numbers of genes down-regulated by docetaxel (in PC-3 cells) and docetaxel + ADT (in patients).
  • Figures 15E shows evidence of antitumor effects of COH29 in vivo.
  • Figure 15G shows corresponding data by weight. Values are means ⁇ S.E. ***P ⁇ 0.001 versus vehicle mice.
  • Figures 15H-15I show regulation of key genes by COH29 in vivo. Multiple genes regulated by COH29 were assessed in xenograft tumors by immunohistochemistry staining (Figure 15H) or
  • Figure 15J shows that the body weight of mice carrying tumor was not affected by COH29 treatment.
  • Figures 16A-16N show transcriptional activation of RRM2.
  • Figure 16A shows H3K27Ac ChIP-Seq in tissues.
  • the binding signal on RRM2 enhancer (1Mb region) is quantified in Figure 16B.
  • Figure 16C shows the strategy to identify RRM2- targeting transcription factors (TFs).
  • Human TFs were selected in the genes positively correlated with RRM2 expression in prostate cancer cohorts. Some TFs appeared in four/three/two cohorts.
  • Figure 16D show the correlation of FOXM1 and RRM2 in PHS/HPFS cohorts.
  • Figure 16E demonstrates FOXM1 binding on RRM2 promoter in cancer cells.
  • FIG. 16F shows FOXM1 or H3K4me3-ChIP-PCR on RRM2 promoter.
  • RRM2 promoter activity is shown as regulated by FOXM1.
  • the reporters without (R2-0K) or with (R2-3K) 3kb RRM2 promoter sequence were transfected in siRNA-treated 22Rvl cells.
  • Figure 16H and Figure 161 show inhibition of RRM2 expression by siFOXMl in 22Rvl and C4-2 cells.
  • Figure 16J shows inhibition of FOXM1 targets by FDI-6 (20 mM) in 22Rvl cells.
  • Figure 16K demonstrates that H2K27Ac ChIP in tissues showed RRM2 was more transcriptionally activated in PC tumors than in normal tissues.
  • Figure 16L shows high level of FoxMl expression is highly correlated with metastasis of PC.
  • Figure 16M Left Panel graphically shows knockdown of FoxMl inhibited RRM2 mRNA and protein expression;
  • Figure 16M Right Panel demonstrates the same in immunoblot results.
  • Figure 16N shows that inhibition of FoxMl inhibited RRM2 promoter activity.
  • the Figure 16 values represent the mean ⁇ S.E. of three independent experiments. *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001 vs control groups..
  • Figures 17A-17C show transcriptome changes induced by knockdown of RRM2 in C4-2 cells.
  • Figure 17A shows the global transcriptomic changed induced by knockdown of RRM2 in C4-2 cells.
  • Figure 17B is a GO analysis showing that biological process and pathways were affected by inhibition of RRM2 expression in PC cells.
  • GSEA analysis shows the key networks were affected by inhibition of RRM2.
  • Figures 18A-18B show further transcriptomic changes induced by COH29 in C4-2 cells.
  • Figure 18A Top Panel is a GO analysis showing biological processes regulated by COH29 treatment.
  • Figure 18A Bottom Panel is a GO analysis showing pathways regulated by COH29 treatment.
  • Figure 18B shows COH29 regulated genes were correlated with DSF for Taylor cohort.
  • Figures 19A-19B show phosphoproteomic changes induced by inhibition of RRM2 by siRNA in C4-2 cells.
  • Figure 19A shows knockdown of RRM2 inhibited AKT/mTOR signaling.
  • Figure 19B graphically demonstrates the result of knockdown of RRM2 inhibited AKT/mTOR signaling, STAT signaling, SFK signaling, and control after 24 hour COH29 treatment in C4-2 cells.
  • Figures 20A-20B show inhibition of RRM2 targets poor-prognosis subtypes of PC.
  • RRM2 expression is highly associated with PCS1 subtyping, rather than PCS2 and PCS3.
  • L is low level
  • M is medium level
  • H is high level.
  • Figure 20B shows that inhibition of RRM2 by siRNA or COH29 target majority of PAM50 genes.
  • the heatmap also showed the overlapping of PCS 1 signatures and basal like genes in PAM50.
  • Figures 21A-21D show further changes induced by knockdown of RRM2 or overexpression of RRM2.
  • Figure 21 A shows GO analysis demonstrating biological process were affected by overexpression of RRM2 expression in PC cells.
  • Figure 21B shows GO analysis demonstrating biological pathways were affected by overexpression of RRM2 expression in PC cells.
  • Figure 21C shows enrichment of RRM2 regulated genes in EMT gene sets and
  • Figure 21D shows enrichment of RRM2 regulated genes in angiogenesis gene sets.
  • Figure 22A-22D show methylation of miR-193b may contribute to
  • FIG. 22A shows RRM2 may be direct targets of miR- 193b.
  • Figure 22C shows DNMT inhibitor (5-Aza) and HD AC inhibitor (SAHA) significantly restored miR-193b expression in 22RV1 in which methylation of miR-193b is observed.
  • Figure 22D graphically shows Anti-miR-193b (A- 193b) partially blocked the 5-Aza-induced repression of RRM2, suggesting up-regulation of miR-193b by 5-Aza could induce down-regulation of RRM2 in PC cells. **p ⁇ 0.01;
  • FIG. 23 shows the RRM2 signature and the derived sub-signatures
  • RRM2 signature includes 126 genes and was created by intersecting upregulated genes in PC3- RRM2 cells with positively correlated with RRM2 overexpression in three cohorts (TCGA, Kumar, and SU2C/PF).
  • 50-gene panel includes common genes between RRM2 126-gene panel and PCS1 subtpying.
  • 14 genes are shared by PCS1 subtype and PAM50 classifiers
  • 12-gene panel includes common genes among RRM2 126-gene panel, PCS1 subtyping, and PAM50 classifier
  • 11 -gene panel includes upregulated genes in ENZ-resistant CTCs.
  • 126-gene panel was applied in single cell RNA-seq profiling of CTCs from patients
  • the present disclosure identifies RRM2- regulated signature genes that are significantly correlated with clinical outcomes.
  • the present disclosure further demonstrates that targeting RRM2 specifically inhibits the expression of genes in the PC SI and luminal B signatures, and thus provide guidance on treatment decisions in relevant prostate cancer patient populations.
  • the term“about” in reference to a number is generally taken to include numbers that fall within a range of l%-5% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context.
  • the term“adapter” refers to a short, chemically synthesized, nucleic acid sequence which can be used to ligate to the end of a nucleic acid sequence in order to facilitate attachment to another molecule.
  • the adapter can be single-stranded or double- stranded.
  • An adapter can incorporate a short (typically less than 50 base pairs) sequence useful for PCR amplification or sequencing.
  • the“administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or
  • Administration includes self-administration and the administration by another.
  • nucleic acid amplification methods refer to methods that increase the representation of a population of nucleic acid sequences in a sample.
  • Nucleic acid amplification methods include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two- step multiplexed amplifications, rolling circle amplification (RCA), recombinase- polymerase amplification (RPA)(TwistDx, Cambridge, UK), transcription mediated amplification, signal mediated amplification of RNA technology, loop-mediated isothermal amplification of DNA, helicase-dependent amplification, single primer isothermal amplification, and self
  • a bait is a type of hybrid capture reagent that retrieves target nucleic acid sequences for sequencing.
  • a bait can be a nucleic acid molecule, e.g., a DNA or RNA molecule, which can hybridize to (e.g., be complementary to), and thereby allow capture of a target nucleic acid.
  • a bait is an RNA molecule (e.g., a naturally-occurring or modified RNA molecule); a DNA molecule (e.g., a naturally-occurring or modified DNA molecule), or a combination thereof.
  • a bait in other embodiments, includes a binding entity, e.g., an affinity tag, that allows capture and separation, e.g., by binding to a binding entity, of a hybrid formed by a bait and a nucleic acid hybridized to the bait.
  • a bait is suitable for solution phase hybridization.
  • “bait set” refers to one or a plurality of bait molecules.
  • cancer or“tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term“cancer” includes premalignant, as well as malignant cancers.
  • nucleic acid sequence refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3’ end of the other, is in“antiparallel association.”
  • sequence“5'-A-G-T-3”’ is complementary to the sequence “3’-T-C-A-5.”
  • Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA).
  • duplex stability need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases.
  • Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the
  • a complement sequence can also be an RNA sequence complementary to the DNA sequence or its complement sequence, and can also be a cDNA.
  • control is an alternative sample used in an experiment for comparison purpose.
  • a control can be "positive” or “negative.”
  • A“control nucleic acid sample” or“reference nucleic acid sample” as used herein, refers to nucleic acid molecules from a control or reference sample.
  • the reference or control nucleic acid sample is a wild type or a non-mutated DNA or RNA sequence.
  • the reference nucleic acid sample is purified or isolated ( e.g ., it is removed from its natural state).
  • the reference nucleic acid sample is from a non-tumor sample, e.g., a blood control, a normal adjacent tumor (NAT), or any other non- cancerous sample from the same or a different subject.
  • a non-tumor sample e.g., a blood control, a normal adjacent tumor (NAT), or any other non- cancerous sample from the same or a different subject.
  • Detecting refers to determining the presence of a mutation in a nucleic acid or polypeptide of interest in a sample and/or measuring changes in expression levels of a nucleic acid or polypeptide of interest in a sample. Detection does not require the method to provide 100% sensitivity.
  • Analysis of nucleic acid markers can be performed using techniques known in the art including, but not limited to, sequence analysis, and electrophoretic analysis. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al, Methods Mol.
  • Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Additionally, next generation sequencing methods can be performed using commercially available kits and instruments from companies such as the Life Technologic s/Ion Torrent PGM or Proton, the Illumina HiSEQ or MiSEQ, and the Roche/454 next generation sequencing system.
  • Detectable label refers to a molecule or a compound or a group of molecules or a group of compounds used to identify a nucleic acid or protein of interest.
  • the detectable label may be detected directly. In other embodiments, the detectable label may be a part of a binding pair, which can then be subsequently detected. Signals from the detectable label may be detected by various means and will depend on the nature of the detectable label. Detectable labels may be isotopes, fluorescent moieties, colored substances, and the like. Examples of means to detect detectable labels include but are not limited to spectroscopic, photochemical, biochemical, immunochemical,
  • electromagnetic, radiochemical, or chemical means such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means.
  • the term“effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g ., an amount which results in the prevention of, or a decrease in a disease or disorder or one or more signs or symptoms associated with a disease or disorder (e.g., prostate cancer).
  • the amount of a composition administered to the subject will depend on the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the therapeutic compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder.
  • a“therapeutically effective amount” of a compound refers to compound levels in which the physiological effects of a disease or disorder are, at a minimum, ameliorated.
  • Gene refers to a DNA sequence that comprises regulatory and coding sequences necessary for the production of an RNA, which may have a non-coding function (e.g., a ribosomal or transfer RNA) or which may include a polypeptide or a polypeptide precursor.
  • RNA Ribonucleic acid
  • polypeptide Ribonucleic acid
  • the RNA or polypeptide may be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.
  • “Gleason score” refers to an approach for describing prostate cancer based on the microscopic appearance of the cancer cells in a biopsy sample and how quickly they are likely to grow and metastasize. Most prostate cancers contain cells that are different grades. The Gleason score is calculated by adding together the two grades of cancer cells that make up the largest areas of the biopsied tissue sample and usually ranges from 6 to 10. The lower the Gleason score, the more the cancer cells look like normal cells and are likely to grow and spread slowly. The Gleason score is used to help plan treatment and determine prognosis.
  • hybridize refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs.
  • Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 15- 100 nucleotides in length, more preferably 18-50 nucleotides in length. Nucleic acid hybridization techniques are well known in the art.
  • Hybridization and the strength of hybridization is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (T m ) of the formed hybrid.
  • T m thermal melting point
  • specific hybridization occurs under stringent hybridization conditions.
  • An oligonucleotide or polynucleotide e.g., a probe or a primer
  • a probe or a primer that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.
  • the term“library” refers to a collection of nucleic acid sequences, e.g., a collection of nucleic acids derived from whole genomic, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments, or a combination thereof.
  • a portion or all of the library nucleic acid sequences comprises an adapter sequence.
  • the adapter sequence can be located at one or both ends.
  • the adapter sequence can be useful, e.g., for a sequencing method (e.g., an NGS method), for amplification, for reverse transcription, or for cloning into a vector.
  • the library can comprise a collection of nucleic acid sequences, e.g., a target nucleic acid sequence (e.g., a prostate tumor nucleic acid sequence), a reference nucleic acid sequence, or a combination thereof.
  • the nucleic acid sequences of the library can be derived from a single subject.
  • a library can comprise nucleic acid sequences from more than one subject (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more subjects).
  • two or more libraries from different subjects can be combined to form a library having nucleic acid sequences from more than one subject.
  • the subject is human having, or at risk of having, a cancer or tumor.
  • A“library nucleic acid sequence” refers to a nucleic acid molecule, e.g., a DNA, RNA, or a combination thereof, that is a member of a library.
  • a library nucleic acid sequence is a DNA molecule, e.g., genomic DNA or cDNA.
  • a library nucleic acid sequence is fragmented, e.g., sheared or enzymatically prepared, genomic DNA.
  • the library nucleic acid sequences comprise sequence from a subject and sequence not derived from the subject, e.g., adapter sequence, a primer sequence, or other sequences that allow for identification, e.g.,“barcode” sequences.
  • Massively parallel sequencing refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a high throughput parallel fashion (e.g., greater than 10 3 , 10 4 , 10 5 or more molecules are sequenced simultaneously).
  • the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment.
  • Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M.
  • oligonucleotide refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the
  • oligonucleotide The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position. Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group.
  • Oligonucleotides of the method which function as primers or probes are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 25 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide.
  • oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, restriction endonuclease digestion of plasmids or phage DNA, reverse transcription, PCR, or a combination thereof.
  • the oligonucleotide may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
  • the term“primer” refers to an oligonucleotide, which is capable of acting as a point of initiation of nucleic acid sequence synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a target nucleic acid strand is induced, i.e., in the presence of different nucleotide triphosphates and a polymerase in an appropriate buffer (“buffer” includes pH, ionic strength, cofactors etc.) and at a suitable temperature.
  • buffer includes pH, ionic strength, cofactors etc.
  • One or more of the nucleotides of the primer can be modified for instance by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
  • a primer sequence need not reflect the exact sequence of the template.
  • a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand.
  • primer includes all forms of primers that may be synthesized including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, and the like.
  • forward primer as used herein means a primer that anneals to the anti-sense strand of dsDNA.
  • A“reverse primer” anneals to the sense-strand of dsDNA.
  • primer pair refers to a forward and reverse primer pair (i.e ., a left and right primer pair) that can be used together to amplify a given region of a nucleic acid of interest.
  • Probe refers to nucleic acid that interacts with a target nucleic acid via hybridization.
  • a probe may be fully complementary to a target nucleic acid sequence or partially complementary. The level of complementarity will depend on many factors based, in general, on the function of the probe.
  • a probe or probes can be used, for example to detect the presence or absence of a mutation in a nucleic acid sequence by virtue of the sequence characteristics of the target. Probes can be labeled or unlabeled, or modified in any of a number of ways well known in the art.
  • a probe may specifically hybridize to a target nucleic acid. Probes may be DNA, RNA or a RNA/DNA hybrid.
  • Probes may be oligonucleotides, artificial chromosomes, fragmented artificial chromosome, genomic nucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleic acid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA), locked nucleic acid, oligomer of cyclic heterocycles, or conjugates of nucleic acid. Probes may comprise modified nucleobases, modified sugar moieties, and modified internucleotide linkages. A probe may be used to detect the presence or absence of a target nucleic acid. Probes are typically at least about 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100 nucleotides or more in length.
  • a“sample” refers to a substance that is being assayed for the presence of a mutation in a nucleic acid of interest. Processing methods to release or otherwise make available a nucleic acid for detection are well known in the art and may include steps of nucleic acid manipulation.
  • a biological sample may be a body fluid or a tissue sample.
  • a biological sample may consist of or comprise blood, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, and the like. Fresh, fixed or frozen tissues may also be used. In one embodiment, the sample is preserved as a frozen sample or as formaldehyde- or
  • FFPE paraffin-embedded tissue preparation
  • the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample.
  • a matrix e.g., an FFPE block or a frozen sample.
  • Whole blood samples of about 0.5 to 5 ml collected with EDTA, ACD or heparin as anti-coagulant are suitable.
  • sensitivity is a measure of the ability of a method to detect a preselected sequence variant in a heterogeneous population of sequences.
  • a method has a sensitivity of S % for variants of F % if, given a sample in which the preselected sequence variant is present as at least F % of the sequences in the sample, the method can detect the preselected sequence at a preselected confidence of C %, S % of the time.
  • the term“separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
  • sequential therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
  • oligonucleotide primer means that the nucleotide sequence of the primer has at least 12 bases of sequence identity with a portion of the nucleic acid to be amplified when the oligonucleotide and the nucleic acid are aligned.
  • An oligonucleotide primer that is specific for a nucleic acid is one that, under the stringent hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and more preferably at least 98% sequence identity.
  • a method has a specificity of X % if, when applied to a sample set of NTotai sequences, in which X-rme sequences are truly variant and CN ⁇ L true are not truly variant, the method selects at least X % of the not truly variant as not variant.
  • a method has a specificity of 90% if, when applied to a sample set of 1,000 sequences, in which 500 sequences are truly variant and 500 are not truly variant, the method selects 90% of the 500 not truly variant sequences as not variant.
  • Exemplary specificities include 90, 95, 98, and 99%.
  • hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5xSSC, 50 mM NaEhPCri, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5x Denharfs solution at 42° C. overnight; washing with 2x SSC, 0.1% SDS at 45° C; and washing with 0.2x SSC, 0.1% SDS at 45° C.
  • stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.
  • target sequence and“target nucleic acid sequence” refer to a specific nucleic acid sequence to be detected and/or quantified in the sample to be analyzed.
  • Treating” or“treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.
  • treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
  • PIMREG PIMREG, PKMYT1, PLK1, PLK4, POLQ, POT1, PPIF, PRC1, PSMC3IP, PSRC1, RAD51, RAD 51 API, RAD54B, RAD54L, RFC3, RMI2, SHCBP1, SKA1, SKA 3, SPAG5, SPC25, STIL, STAIN/, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRF1, ZNF273, ZNF695, ZWINT, ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55,
  • KIF2C, MELK, MKI67, PTTG1, UBE2T , and Nl //’2- may be detected by a variety of methods known in the art. Non-limiting examples of detection methods are described below.
  • the detection assays in the methods of the present technology may include purified or isolated DNA (genomic or cDNA), RNA or protein or the detection step may be performed directly from a biological sample without the need for further DNA, RNA or protein
  • PIMREG PIMREG, PKMYT1, PLK1, PLK4, POLQ, POT1, PPIF, PRC1, PSMC3IP, PSRC1, RAD51, RAD 51 API, RAD54B, RAD54L, RFC3, RMI2, SHCBP1, SKA1, SKA 3, SPAG5, SPC25, STIL, STAIN I, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRF1, ZNF273, ZNF695, ZWINT, ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55,
  • KIF2C, MELK, MKI67, PTTG1, UBE2T, and NUF2 can be detected by the use of nucleic acid amplification techniques that are well known in the art.
  • the starting material may be genomic DNA, cDNA, RNA or mRNA.
  • Nucleic acid amplification can be linear or exponential.
  • Specific variants or mutations may be detected by the use of amplification methods with the aid of oligonucleotide primers or probes designed to interact with or hybridize to a particular target sequence in a specific manner, thus amplifying only the target variant.
  • Non-limiting examples of nucleic acid amplification techniques include polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR, ligase chain reaction (see Abravaya, K. et al., Nucleic Acids Res. (1995), 23:675-682), branched DNA signal amplification (see Urdea, M. S. et al., AIDS (1993), 7(suppl 2):S11- S14), amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA) (see Kievits, T.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • nested PCR see Abravaya, K. et al., Nucleic Acids Res. (1995), 23:675-682
  • Oligonucleotide primers for use in amplification methods can be designed according to general guidance well known in the art as described herein, as well as with specific requirements as described herein for each step of the particular methods described.
  • oligonucleotide primers for cDNA synthesis and PCR are 10 to 100 nucleotides in length, preferably between about 15 and about 60 nucleotides in length, more preferably 25 and about 50 nucleotides in length, and most preferably between about 25 and about 40 nucleotides in length.
  • Tm of a polynucleotide affects its hybridization to another polynucleotide (e.g ., the annealing of an oligonucleotide primer to a template polynucleotide).
  • another polynucleotide e.g ., the annealing of an oligonucleotide primer to a template polynucleotide.
  • the oligonucleotide primer used in various steps selectively hybridizes to a target template or polynucleotides derived from the target template (i.e., first and second strand cDNAs and amplified products).
  • target template i.e., first and second strand cDNAs and amplified products.
  • hybridization occurs when two polynucleotide sequences are substantially complementary (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary). See Kanehisa, M., Polynucleotides Res. (1984), 12:203, incorporated herein by reference. As a result, it is expected that a certain degree of mismatch at the priming site is tolerated. Such mismatch may be small, such as a mono-, di- or tri -nucleotide. In certain embodiments, 100% complementarity exists.
  • Probes are capable of hybridizing to at least a portion of the nucleic acid of interest or a reference nucleic acid (i.e., wild-type sequence). Probes may be an oligonucleotide, artificial chromosome, fragmented artificial chromosome, genomic nucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleic acid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA), locked nucleic acid, oligomer of cyclic heterocycles, or conjugates of nucleic acid. Probes may be used for detecting and/or capturing/purifying a nucleic acid of interest.
  • probes can be about 10 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 75 nucleotides, or about 100 nucleotides long.
  • longer probes are possible. Longer probes can be about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 750 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 7,500 nucleotides, or about 10,000 nucleotides long.
  • Probes may also include a detectable label or a plurality of detectable labels.
  • the detectable label associated with the probe can generate a detectable signal directly.
  • the detectable label associated with the probe can be detected indirectly using a reagent, wherein the reagent includes a detectable label, and binds to the label associated with the probe.
  • detectably labeled probes can be used in hybridization assays including, but not limited to Northern blots, Southern blots, microarray, dot or slot blots, and in situ hybridization assays such as fluorescent in situ hybridization (FISH) to detect a target nucleic acid sequence within a biological sample.
  • FISH fluorescent in situ hybridization
  • Certain embodiments may employ hybridization methods for measuring expression of a polynucleotide gene product, such as mRNA. Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al.
  • Detectably labeled probes can also be used to monitor the amplification of a target nucleic acid sequence.
  • detectably labeled probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a function of time.
  • probes include, but are not limited to, the 5'- exonuclease assay (TAQMAN® probes described herein (see also U.S. Pat. No. 5,538,848) various stem- loop molecular beacons (see for example, U.S. Pat. Nos.
  • the detectable label is a fluorophore.
  • Suitable fluorescent moieties include but are not limited to the following fluorophores working individually or in combination: 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid
  • EDANS 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate
  • Lucide Yellow VS N-(4-anilino-l- naphthyl)maleimide; anthranilamide; Black Hole QuencherTM (BHQTM) dyes (biosearch Technologies);
  • BODIPY dyes BODIPY® R-6G, BOPIPY® 530/550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4- methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol- sulfonephthal
  • Detector probes can also comprise sulfonate derivatives of fluorescenin dyes with S03 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (commercially available for example from Amersham).
  • Detectably labeled probes can also include quenchers, including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).
  • quenchers including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).
  • Detectably labeled probes can also include two probes, wherein for example a fluorophore is on one probe, and a quencher is on the other probe, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on the target alters the signal signature via a change in fluorescence.
  • interchelating labels such as ethidium bromide, SYBR® Green I (Molecular Probes), and PicoGreen® (Molecular Probes) are used, thereby allowing visualization in real-time, or at the end point, of an amplification product in the absence of a detector probe.
  • real-time visualization may involve the use of both an intercalating detector probe and a sequence-based detector probe.
  • the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction, and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction.
  • the amount of probe that gives a fluorescent signal in response to an excited light typically relates to the amount of nucleic acid produced in the amplification reaction.
  • the amount of fluorescent signal is related to the amount of product created in the amplification reaction. In such embodiments, one can therefore measure the amount of amplification product by measuring the intensity of the fluorescent signal from the fluorescent indicator.
  • Primers or probes can also be prepared that are complementary and specific for the nucleotide sequence of ARL6IP1, ASF IB, ATAD2, ATAD5, AUNIP, AURKA, AURKB, BLM, BORA, BUB1, BUB1B, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU, CEP152, CIT, CKAP2, CKAP5, CKS1B, CKS2, CTSV, DBF4, DDIAS, DEPDC1, DEPDC1B, DNA2, DNMT3B, DONSON, DSCC1, EZH2, FAM72D, FAM83D, FANCD2, FANCI, GINS1, GINS2, GPC2, GPSM2, GTSE1, HJURP, HMGB2, HMMR, INSIG1, KIF11, KIF15, KIF18B, K1F20A, KIF20
  • detection can occur through any of a variety of mobility dependent analytical techniques based on the differential rates of migration between different nucleic acid sequences.
  • mobility-dependent analysis techniques include electrophoresis, chromatography, mass spectroscopy, sedimentation, for example, gradient centrifugation, field-flow fractionation, multi-stage extraction techniques, and the like.
  • mobility probes can be hybridized to amplification products, and the identity of the target nucleic acid sequence determined via a mobility dependent analysis technique of the eluted mobility probes, as described in Published PCT Applications
  • detection can be achieved by various microarrays and related software such as the Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other
  • detection can comprise reporter groups that are incorporated into the reaction products, either as part of labeled primers or due to the incorporation of labeled dNTPs during an amplification, or attached to reaction products, for example but not limited to, via hybridization tag complements comprising reporter groups or via linker arms that are integral or attached to reaction products.
  • unlabeled reaction products may be detected using mass spectrometry.
  • high throughput, massively parallel sequencing employs sequencing-by-synthesis with reversible dye terminators.
  • sequencing is performed via sequencing-by-ligation.
  • sequencing is single molecule sequencing. Examples of Next Generation Sequencing techniques include, but are not limited to pyrosequencing, Reversible dye-terminator sequencing, oligonucleotide ligation chemistry, proton detection, or phospholinked fluorescent nucleotide chemistry etc.
  • the Ion TorrentTM (Life Technologies, Carlsbad, CA) amplicon sequencing system employs a flow-based approach that detects pH changes caused by the release of hydrogen ions during incorporation of unmodified nucleotides in DNA replication.
  • a sequencing library is initially produced by generating DNA fragments flanked by sequencing adapters. In some embodiments, these fragments can be clonally amplified on particles by emulsion PCR. The particles with the amplified template are then placed in a silicon semiconductor sequencing chip. During replication, the chip is flooded with one nucleotide after another, and if a nucleotide complements the DNA molecule in a particular microwell of the chip, then it will be incorporated.
  • a proton is naturally released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH.
  • the pH of the solution then changes in that well and is detected by the ion sensor. If homopolymer repeats are present in the template sequence, multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.
  • the 454TM GS FLXTM sequencing system (Roche, Germany), employs a light- based detection methodology in a large-scale parallel pyrosequencing system.
  • Pyrosequencing uses DNA polymerization, adding one nucleotide species at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates.
  • adapter-ligated DNA fragments are fixed to small DNA-capture beads in a water-in-oil emulsion and amplified by PCR (emulsion PCR).
  • PCR emulsion PCR
  • Each DNA-bound bead is placed into a well on a picotiter plate and sequencing reagents are delivered across the wells of the plate.
  • the four DNA nucleotides are added sequentially in a fixed order across the picotiter plate device during a sequencing run.
  • nucleotide flow millions of copies of DNA bound to each of the beads are sequenced in parallel.
  • nucleotide complementary to the template strand is added to a well, the nucleotide is incorporated onto the existing DNA strand, generating a light signal that is recorded by a CCD camera in the instrument.
  • DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed.
  • RT -bases reversible terminator bases
  • non-incorporated nucleotides are washed away.
  • the DNA can only be extended one nucleotide at a time.
  • a camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.
  • Helicos's single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. At each cycle, DNA polymerase and a single species of fluorescently labeled nucleotide are added, resulting in template-dependent extension of the surface-immobilized primer-template duplexes. The reads are performed by the Helioscope sequencer. After acquisition of images tiling the full array, chemical cleavage and release of the fluorescent label permits the subsequent cycle of extension and imaging.
  • electrophoretic sequencing relies on incorporation of nucleotides by a DNA polymerase to determine the base sequence.
  • a DNA library with affixed adapters is denatured into single strands and grafted to a flow cell, followed by bridge amplification to form a high-density array of spots onto a glass chip.
  • Reversible terminator methods use reversible versions of dye-terminators, adding one nucleotide at a time, detecting fluorescence at each position by repeated removal of the blocking group to allow polymerization of another nucleotide.
  • the signal of nucleotide incorporation can vary with fluorescently labeled nucleotides, phosphate- driven light reactions and hydrogen ion sensing having all been used. Examples of SBS platforms include Illumina GA and HiSeq 2000.
  • the MiSeq® personal sequencing system (Illumina, Inc.) also employs sequencing by synthesis with reversible terminator chemistry.
  • the sequencing by ligation method uses a DNA ligase to determine the target sequence.
  • This sequencing method relies on enzymatic ligation of oligonucleotides that are adjacent through local complementarity on a template DNA strand.
  • This technology employs a partition of all possible oligonucleotides of a fixed length, labeled according to the sequenced position.
  • Oligonucleotides are annealed and ligated and the preferential ligation by DNA ligase for matching sequences results in a dinucleotide encoded color space signal at that position (through the release of a fluorescently labeled probe that corresponds to a known nucleotide at a known position along the oligo).
  • This method is primarily used by Life Technologies’ SOLiDTM sequencers.
  • the DNA is amplified by emulsion PCR.
  • the resulting beads, each containing only copies of the same DNA molecule, are deposited on a solid planar substrate.
  • SMRTTM sequencing is based on the sequencing by synthesis approach.
  • the DNA is synthesized in zero-mode wave-guides (ZMWs)-small well-like containers with the capturing tools located at the bottom of the well.
  • the sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution.
  • the wells are constructed in a way that only the fluorescence occurring at the bottom of the well is detected.
  • the fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.
  • the present disclosure provides RRM2 signature genes associated with aggressive subtypes of prostate cancer as well as other types of cancer (e.g ., breast cancer, ovarian cancer, lung cancer, or liver cancer), which are useful for predicting patient prognosis and guiding treatment decisions.
  • Such methods would aid in predicting the responsiveness of individual patients to a particular drug regimen (e.g., ribonucleotide reductase inhibitors) and the identification of optimal therapeutic strategies at the outset.
  • the present disclosure provides a method for detecting an aggressive subtype of prostate cancer in a subject in need thereof comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the subject compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists of ARL6IP1, ASF IB, ATAD2, ATAD5, AUNIP, AURKA, AURKB, BLM, BORA, BUB1, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU, CEP152,
  • PIMREG PIMREG, PKMYT1, PLK1, PLK4, POLQ, POT1, PPIF, PRC1, PSMC3IP, PSRC1, RAD51, RAD 51 API, RAD54B, RAD54L, RFC3, RMI2, SHCBP1, SKA1, SKA 3, SPAG5, SPC25, STIL, STAIN/, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRF1, ZNF273, ZNF695, and ZWINT.
  • the method further comprises detecting an increase in expression levels of at least one gene selected from among ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MK167, PTTG1, UBE2T ⁇ and NUF2 in the test sample compared to the healthy control subject or the reference sample.
  • the present disclosure provides a method for detecting an aggressive subtype of prostate cancer in a subject in need thereof comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the subject compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists oiANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MK167, PTTG1, and UBE2T
  • the aggressive subtype of prostate cancer is PCS1 or luminal B, but not PCS2, PCS3, luminal A, or basal.
  • the prostate cancer may be localized prostate cancer or metastatic castration-resistant prostate cancer and/or comprises tumors with immunosuppressive M2 macrophages, and/or regulatory T cells (Tregs).
  • the aggressive subtype of prostate cancer is associated with a high Gleason score, a high incidence of recurrence, and/or lethality.
  • the present disclosure provides a method for identifying a cancer associated with poor prognosis in a subject in need thereof comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the subject compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists of ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MKI67, PTTG1, and UBE2T , and wherein the cancer is breast cancer, ovarian cancer, lung cancer, or liver cancer.
  • the present disclosure provides a method for identifying enzalutamide resistance in a subject with castration-resistant prostate cancer comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the subject compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists oiAURKB, MIS18A, MK167, CENPF, PPIF, FANCI, TPX2, CDKN3, KIF2C, K1F11 and CCNB2.
  • the method further comprises detecting an increase in expression levels of at least one gene selected from among TACC3, NUSAP1, INSIG1, GPSM2, KPNA2, CKS1B, ZNF273, STMNl, RAD51AP1, and FANCD2.
  • the expression levels are detected via RNA-seq, northern blotting, microarrays, dot or slot blots, fluorescent in situ hybridization, reverse transcription polymerase chain reaction (RT-PCR), ribonuclease protection assay (RPA), real-time quantitative RT-PCR, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), enzyme-linked immunosorbent assay (ELISA), immunoprecipitation,
  • RT-PCR reverse transcription polymerase chain reaction
  • RPA ribonuclease protection assay
  • HPLC high-performance liquid chromatography
  • LC/MS liquid chromatography-mass spectrometry
  • ELISA enzyme-linked immunosorbent assay
  • test sample comprises biopsied tumor tissue or circulating tumor cells.
  • subject is human.
  • the methods disclosed herein rely on detection via high throughput massively parallel sequencing of a large number of diverse genes, e.g., ARL6IP1, ASF1B, ATAD2, ATAD5, AUNIP, AURKA, AURKB, BLM, BORA, BUB1, BUB1B, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU, CEP 152, CIT, CKAP2, CKAP5, CKS1B, CKS2, CTSV, DBF4, DDIAS, DEPDC1, DEPDC1B, DNA2, DNMT3B, DONSON, DSCC1, EZH2, FAM72D, FAM83D, FANCD2, FANCI, GINS1, GINS2, GPC2, GPSM2, GTSE1, HJURP, HMGB2, HMMR, INSIG1, KIF11, K1F
  • the methods featured in the present technology are used in a multiplex, multi-gene assay format, e.g., assays that incorporate multiple signals from a large number of diverse genetic alterations in a large number of genes.
  • a single primer or one or both primers of a primer pair comprise a specific adapter sequence (also referred to as a sequencing adapter) ligated to the 5’ end of the target specific sequence portion of the primer.
  • This sequencing adapter is a short oligonucleotide of known sequence that can provide a priming site for both
  • adapters allow binding of a fragment to a flow cell for next generation sequencing.
  • Any adapter sequence may be included in a primer used in the present technology.
  • the employed primers do not contain adapter sequences and the amplicons produced are subsequently ( i.e . after amplification) ligated to an oligonucleotide sequencing adapter on one or both ends of the amplicons.
  • all forward amplicons i.e., amplicons extended from forward primers that hybridized with antisense strands of a target nucleic acid
  • all forward amplicons contain the same adapter sequence and all reverse amplicons (i.e., amplicons extended from reverse primers that hybridized with sense strands of a target segment) contain an adapter sequence that is different from the adapter sequence of the forward amplicons.
  • the adapter sequences further comprise an index sequence (also referred to as an index tag, a“barcode” or a multiplex identifier (MID)).
  • the adapter sequences are P5 and/or P7 adapter sequences that are recommended for Illumina sequencers (MiSeq and HiSeq). See, e.g., Williams- Carrier et al., Plant J., 63(1): 167-77 (2010).
  • the adapter sequences are PI, A, or Ion XpressTM barcode adapter sequences that are recommended for Life
  • PIMREG PIMREG, PKMYT1, PLK1, PLK4, POLQ, POT1, PPIF, PRC1, PSMC3IP, PSRC1, RAD51, RAD 51 API, RAD54B, RAD54L, RFC3, RMI2, SHCBP1, SKA1, SKA 3, SPAG5, SPC25, STIL, STAIN l, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRF1, ZNF273, ZNF695, ZWINT, ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55,
  • KIF2C, MELK, MKI67, PTTG1, UBE2T , and NUF2 from more than one sample are sequenced. In some embodiments, all samples are sequenced simultaneously in parallel.
  • amplicons derived from a single sample may further comprise an identical index sequence that indicates the source from which the amplicon is generated, the index sequence for each sample being different from the index sequences from all other samples.
  • index sequences permits multiple samples to be pooled per sequencing run and the sample source subsequently ascertained based on the index sequence.
  • the Access ArrayTM System Fludigm Corp., San Francisco, CA
  • the Apollo 324 System Wang, CA
  • indexed amplicons are generated using primers (for example, forward primers and/or reverse primers) containing the index sequence.
  • primers for example, forward primers and/or reverse primers
  • Such indexed primers may be included during library preparation as a“barcoding” tool to identify specific amplicons as originating from a particular sample source.
  • the adapter sequence and/or index sequence gets incorporated into the amplicon (along with the target-specific primer sequence) during amplification. Therefore, the resulting amplicons are sequencing-competent and do not require the traditional library preparation protocol.
  • the presence of the index tag permits the differentiation of sequences from multiple sample sources.
  • the amplicons may be amplified with non-adapter-ligated and/or non-indexed primers and a sequencing adapter and/or an index sequence may be subsequently ligated to one or both ends of each of the resulting amplicons.
  • the amplicon library is generated using a multiplexed PCR approach.
  • indexed amplicons from more than one sample source are quantified individually and then pooled prior to high throughput sequencing.
  • index sequences permits multiple samples (i.e., samples from more than one sample source) to be pooled per sequencing run and the sample source subsequently ascertained based on the index sequence.
  • Multipleing is the pooling of multiple adapter-tagged and indexed libraries into a single sequencing run. When indexed primer sets are used, this capability can be exploited for comparative studies. In some embodiments, amplicon libraries from up to 48 separate sources are pooled prior to sequencing.
  • the amplicons are sequenced using high throughput, massively parallel sequencing (i.e., next generation sequencing).
  • high throughput, massively parallel sequencing i.e., next generation sequencing.
  • Methods for performing high throughput, massively parallel sequencing are known in the art.
  • the high throughput massive parallel sequencing is performed using 454TM GS FLXTM
  • high throughput massively parallel sequencing may be performed using a read depth approach.
  • the present disclosure provides a method for selecting a prostate cancer patient for treatment with a ribonucleotide reductase inhibitor comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the patient compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists oiARL6IPl, ASF1B, ATAD2, ATAD5, AUNIP, AURKA, AURKB, BLM, BORA, BUB1, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU,
  • CEP 152 CIT, CKAP2, CKAP5, CKS1B, CKS2, CTSV, DBF4, DDIAS, DEPDC1, DEPDC1B, DNA2, DNMT3B, DONSON, DSCC1, EZH2, FAM72D, FAM83D, FANCD2, FANCI, GINS1, GINS2, GPC2, GPSM2, GTSE1, HJURP, HMGB2, HMMR, INSIG1, KIF11, KIF15, KIF18B, K1F20A, KIF20B, KIF23, K1F4A, KPNA2, MAD2L1, MIS18A, MIS18BP1, MNS1, MYBL2, NCAPG, NCAPG2, NCAPH, NUSAP1, ORC1, PARP2, PBK, PCLAF, PIF1, PIGA,
  • PIMREG PIMREG, PKMYT1, PLK1, PLK4, POLQ, POT1, PPIF, PRC1, PSMC3IP, PSRC1, RAD51, RAD 51 API, RAD54B, RAD54L, RFC 3, RMI2, SHCBP1, SKA1, SKA 3, SPAG5, SPC25, STIL, STAIN/, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRF1, ZNF273, ZNF695, and ZWINT ; and administering to the patient an effective amount of a ribonucleotide reductase inhibitor.
  • the method further comprises detecting an increase in expression levels of at least one gene selected from among ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MK167, PTTG1, UBE2T ⁇ and NUF2 in the test sample compared to the healthy control subject or the reference sample.
  • the present disclosure provides a method for selecting a prostate cancer patient for treatment with a ribonucleotide reductase inhibitor comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the patient compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists oiANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MK167, PTTG1, and UBE2T , and administering to the patient an effective amount of a ribonucleotide reductase inhibitor.
  • the prostate cancer may be localized prostate cancer or metastatic castration-resistant prostate cancer, and/or has a PCS1 or a luminal B subtype.
  • the present disclosure provides a method for selecting a cancer patient for treatment with a ribonucleotide reductase inhibitor comprising detecting an increase in expression levels of each of a plurality of genes in a test sample obtained from the patient compared to a healthy control subject or a reference sample, wherein the plurality of genes comprises, consists essentially of, or consists oiANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP55, KIF2C, MELK, MK167, PTTG1, and UBE2T , and wherein the cancer is breast cancer, ovarian cancer, lung cancer, or liver cancer; and administering to the patient an effective amount of a ribonucleotide reductase inhibitor.
  • ribonucleotide reductase inhibitors include, but are not limited to, hydroxyurea (HU), 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), GTI2040, COH3, COH4, COH20, COH29, gemcitabine, ribonucleotide reductase inhibitor compounds described in US 7,956,076 and RRM2-specific inhibitory nucleic acids.
  • the RRM2-specific inhibitory nucleic acid is a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.
  • the methods of the present technology further comprise sequentially, simultaneously, or separately administering an effective amount of a cytokine and/or a monoclonal antibody.
  • cytokines include, but are not limited to, interferon a, interferon b, interferon g, complement C5a, IL-2, TNF alpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7
  • the monoclonal antibody targets CTLA-4 (e.g., ipilimumab, tremelimumab), PD-1 (e.g., cemiplimab, nivolumab, pembrolizumab), PDL1 (e.g., avelumab, durvalumab, atezolizumab, Envafolimab, BMS- 936559, CK-301, CS-1001, SHR-1316, CBT-502, BGB-A333), TIM-3 (e.g., LY3321367, TSR-022, MBG453), BTLA, or VEGF (e.g., bevacizumab, ranibizumab).
  • CTLA-4 e.g., ipilimumab, tremelimumab
  • PD-1 e.g., cemiplimab, nivolumab, pembrolizumab
  • PDL1 e.
  • kits of the present disclosure are useful in methods for identifying a cancer associated with poor prognosis (e.g., breast cancer, ovarian cancer, lung cancer, or liver cancer) and/or detecting aggressive subtypes of prostate cancer in a subject in need thereof.
  • the kits of the present technology are also useful for identifying enzalutamide resistance in a subject with castration-resistant prostate cancer.
  • kits of the present technology comprise reagents (e.g., target-specific primers, probes, antibodies, and any combinations thereof) for detecting alterations in mRNA or polypeptide expression levels of any one or more of ARL6IP1, ASF IB, A TAD 2, A TADS, AUNIP, AURKA, AURKB, BLM, BORA, BUB l, BUB IB, CCNA2, CCNB2, CCNE!, CDCA3, CDCA5, CDCA8, CDK1,
  • K1F2C K1F2C, MELK, MK167, PTTG1, UBE2T, and NUF2, and instructions for use.
  • Kits of the present technology comprise one or more primer pairs or probes that selectively hybridize and are useful in amplifying one or more of the genes selected from the group consisting of ARL6IP1, ASF IB, ATAD2, AT AD 5, AUNIP, AURKA , AURKB, BLM, BORA, BUBl, BUBIB, CCNA2 , CCNB2, CCNE1, CDCA3, CDCA5 , CDCA8, CDK1 ,
  • RAF 5 I API, RAD54B, RAD 541,, RFC 3, RMI2 , SHCBP1, SKA1, SKA3, SPAG5, SPC25, SELL, STMNl, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRFl, ZNF273, ZNF695, ZWINT, ANLN, B1RC5, CCNB1, CDC20, CDC6, CENPF, CEP 55 ,
  • kits of the present technology comprise a single primer pair and/or probe that hybridizes to an exon of a single gene selected from the
  • RAT 5 IAP I, RAD54B, RAD54L, RFC 3, RML2, SHCBP1, SKA I, SKA 3, SPAG5, SPC25, STIL, STMNL, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRFl, ZNF273, ZNF695, ZWINT, ANLN, BIRC5, CCNBI, CDC20, CDC6, CENPF, CEP55,
  • KLF2C KLF2C, MELK, MK167, PITCH, UBE2T and NUF2.
  • kits of the present technology compri se multiple primer pairs and/or probes that hybridize to one or more exons of a single gene selected from the group consisting of ARL61PL, ASF LB, A TAD 2, ATAD5, AUNIP, AURKA, AURKB, BLM, BOM, BlIBl, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, ( DC AH, CDK1,
  • kits of the present technology comprise multiple primer pairs and/or probes comprising a single primer pair or probe that specifically hybridizes to an exon of a single gene for each of ARL6IP1, ASF IB, ATAD2, ATAD5,
  • AUNIP AURKA, AURKB, BUM, BORA, BUB1, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU,
  • CEP 152 CIT, CKAP2, CKAP5, CKS1B, CKS2, CTSV, DBF4, DDIAS, DEPDC1, DEPDC1B, DNA2, DNMT3B, DONSON, DSCC1, EZH2, FAM72D, FAM83D, FANCD2, FANCI, GINS1, GINS2, GPC2, GPSM2, GTSE1, HJURP, HMGB2, HMMR, INSIG1, KIFll, KIF15, KIF18B, K1F20A, KIF20B, KIF23, K1F4A, KPNA2, MAD2L1, MIS18A, MIS18BP1, MNS1, MYBL2, NCAPG, NCAPG2, NCAPH, NUSAPl, ORC1, PARP2, PBK, PCLAF, PIF1, PIGA,
  • PIMREG PIMREG, PKMYT1, PLKl, PLK4, POLQ, POT1, PPIF, PRC1, PSMC3IP, PSRC1, RAD51, RAD 51 API, RAD54B, RAD54L, RFC3, RMI2, SHCBP1, SKA1, SKA 3, SPAG5, SPC25, STIL, STMN1, TACC3, TK1, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRF1, ZNF273, ZNF695, ZWINT, ANLN, BIRC5, CCNB1, CDC20, CDC6, CENPF, CEP 55,
  • kits of the present technology comprise multiple primer pairs and/or probes comprising more than one primer pair or probe that hybridizes to one or more exons for each of ARL6IP1, ASF1B, ATAD2, ATAD5, AUNIP, AURKA, A URKB, BLM BORA, BUBl, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU, CEP 152, CIT, CKAP2, CKAP5, CKS1B, CKS2, CTSV, DBF4, DDIAS, DEPDC1, DEPDC1B, DNA2, DNMT3B, DONSON, DSCC1, EZH2, FAM72D, F AMS 3D, FANCD2, FANCI, GINS/, GINS2, GPC2, GPSM2, GTSE1, HJURP, HMGB2, HMMR, INS
  • kits of the present technology can comprise primer pairs and/or probes that recognize and specifically hybridize to one or more exons of one or more genes selected from the group con si sti ng A RINK' / ASF IB, ATAD2,
  • kits of the present technology comprise one or more antibodies that selectively bind to at least one polypeptide encoded by one or more of the genes selected from the group consisting of ARL6TPI, ASF IB, ATAD2, ATAD5, A UNIP, AURKA, AIJRKB, BLM, BOM, BUB1, BUB IB, CCNA2, CCNB2, CCNEl, GDC A3, GDC AS.
  • GDC A 8 CDK1, CDKN3, GDT!, GENRE, CENPH, CENPM, CENPP, CENPU, CEP 152, CIT, CKAP2, CKAP5, CKSIB, CKS2, CTSV, DBF 4, DDIAS, DEPDCI, DEPDC1B, DNA2, DNMT3B, DONSON, DSCC1, EZTI2, FAM72D, FAM83D, FANCD2, FANC!, G1NS1, G1NS2, GPC2, GPSM2, GTSEI, HJURP, HMGB2, HMMR, INSICil, KIEL 1, KILLS, K1F18B, KIF20A, KLF20B, K1F23, KIF4A, KPNA2, MAD2L1, MTSI8A, MIS18BP1, MNS1, MYBL2, NCAPG, NCAPG2, NCAPH, NUSAPl, ORC1, PARP2, PBK, PCLAF, PIF1, PIGA, PIMR
  • kits of the present technology comprise a plurality of antibodies that selectively bind to each of ARL6IP 1, ASF IB, A TAD2, A TAD5, LI ⁇ N ⁇ R, AURKA , AIJRKB, BIM BORA , BUB l, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, GDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU, CEP 152,
  • KJF2C KJF2C
  • MELK MKI67
  • PTTG1 PTTG1
  • UBE2T UBE2T
  • NUF2 NUF2
  • kits further comprise buffers, enzymes having polymerase activity, enzymes having polymerase activity and lacking 5'— >3’ exonuclease activity or both 5'— >3’ and 3’— >5' exonuclease activity, enzyme cofactors such as magnesium or manganese, salts, chain extension nucleotides such as deoxynucleoside triphosphates (dNTPs), modified dNTPs, nuclease-resistant dNTPs or labeled dNTPs, necessary to carry out an assay or reaction, such as amplification and/or detection of altered expression levels of target nucleic acid sequences corresponding to ARL6IP1, ASF IB, A TAD2, A TADS, A UNIP, AURKA, AURKB, BUM, BORA, BUBl, BUB IB, CCNA2, CCNB2, CCNE1, ( DC A 3. CDCA5, CDCA8, CDK1, CDKN3, CDT!, C
  • PIMREG PIMREG, PKMYT1, PLK1, PLK4, POLQ, POT1, PPIF, PRCl, PSMC31P, PSRC1, RAD51, RADS 1 API, RAD54B, RAD54L, RFC 3, RM12, SIR PPL SKA 1, SKA3, SPAG5, SPC25, SUL, STMNl, TACC3, TK1, TOP 2 A, TPX2 , TRIM59 , TUBA IB, UBE2S, WDR62, WEE l, ZGRF1, ZNF273, ZNF695, ZWINT, ANLN, B1RC5, CCNB1, CDC20, CDC6, CENPF, CEP 55,
  • kits of the present technology further comprise a positive control nucleic acid sequence and a negative control nucleic acid sequence to ensure the integrity of the assay during experimental runs.
  • a kit may further contain a means for comparing the levels and/or activity of one or more of ARL61PI, ASF IB, ATAD2, ATAD5,
  • a UNIP A UREA. AURKB, BUM, BORA, BUB I, BUB IB, CCNA2, CCNB2, CCNEI, CDCA3, CDCA5, CU-( AS CDKl, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPIJ,
  • PIMREG PIMREG, PKMYT1, PLK1, PIE 4.
  • POLO POT 1, PP1F, PRC!, PSMC3IP, PSRC1, RAD51, RADS 1 API, RAD54B, RAD54L, RFC 3, RM12, SHCBP1, SKA1, SKA 3.
  • SPAG5 SPC25, ST1L, STMNl, TACC3, TKl, TOP 2 A, TPX2, TRIM59, TUBA IB, UBE2S, WDR62, WEE1, ZGRF1, ZNF273, ZNF695, ZWINT, ANLN, BIRC5, CCNBl, CDC20, CDC6, CENPF, CEP 55,
  • the kit may also comprise instructions for use, software for automated analysis, containers, packages such as packaging intended for commercial sale and the like.
  • kits of the present technology can also include other necessary reagents to perform any of the NGS techniques disclosed herein.
  • the kit may further comprise one or more of: adapter sequences, barcode sequences, reaction tubes, ligases, ligase buffers, wash buffers and/or reagents, hybridization buffers and/or reagents, labeling buffers and/or reagents, and detection means.
  • the buffers and/or reagents are usually optimized for the particular amplification/detection technique for which the kit is intended. Protocols for using these buffers and reagents for performing different steps of the procedure may also be included in the kit.
  • kits of the present technology may include components that are used to prepare nucleic acids from a tumor test sample for the subsequent amplification and/or detection of altered expression levels of target nucleic acid sequences corresponding to ARL6IP1, ASF IB, ATAD2, ATAD5, AUNIP, AURKA, AURKB, BLM, BORA, BUB1, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU, CEP152, CIT, CKAP2, CKAP5, CKS1B, CKS2, CTSV, DBF4, DDIAS, DEPDC1, DEPDC1B, DNA2, DNMT3B, DONSON, DSCC1, EZH2,
  • sample preparation components can be used to produce nucleic acid extracts from tissue samples.
  • the test samples used in the above-described methods will vary based on factors such as the assay format, nature of the detection method, and the specific tissues, cells or extracts used as the test sample to be assayed.
  • Methods of extracting nucleic acids from samples are well known in the art and can be readily adapted to obtain a sample that is compatible with the system utilized.
  • Automated sample preparation systems for extracting nucleic acids from a test sample are commercially available, e.g., Roche Molecular Systems’ COBAS AmpliPrep System, Qiagen's BioRobot 9600, and Applied Biosystems' PRISMTM 6700 sample preparation system.
  • the kit can comprise, e.g. , 1) a first antibody attached to a solid support, which binds to a polypeptide encoded by a target gene selected from the group consisting of ARL6IP1, ASF IB, ATAD2, ATAD5, AUNIP, AURKA, AURKB, BLM, BORA, BUB1, BUB1B, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3,
  • a target gene selected from the group consisting of ARL6IP1, ASF IB, ATAD2, ATAD5, AUNIP, AURKA, AURKB, BLM, BORA, BUB1, BUB1B, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, CDCA8, CDK1, CDKN3,
  • kits of the present technology comprise one or more antibodies that selectively bind to at least one polypeptide encoded by one or more of the genes selected from the group consisting of ARL61P1, ASF IB, ATAD2, AT AD 5, AIJNIP, AURKA, AURKB, BLM, BOM, BlJBl, BUB IB, CCNA2, CCNB2, CCNE1, CDCA3, CDCA5, COCAS, CDKl, CDKN3, CDT1, CENPE, CENPH, CENPM, CENPP, CENPU, CEP 152, C1T, CKAP2, CKAP5, CKS1B, CKS2, CTSV, DBF4, DD1AS, DEPDC1, DEPDCIB, DNA2, DNA4T3B, DOS SOS, DSCC1 , EZH2, FAM72D, FAM83D, FANCD2, FANCl GINS1, GINS2, GPC2, GPSM2, GTSEl, HJUR
  • MIS ISA M1S18BP1, MNS1, MY B 1.2.
  • kits of the present technology comprise a plurality of antibodies that selectively bind to each of ARL6IPJ, ASF IB, ATAD2, ATAD5, A UNTP, AURKA, AURKB, BLM, BOM, BUBl, BUB IB, CCNA2, CCNB2, CCNE1, ( DC A 3. CDCA5, CDCA8, CDKl, CDKN3, CDTl, CENPE, CENPH, CENPM, CENPP, CENPU, CEP 152,
  • PIMREG PIMREG, PKMYT1, PLKl , PLKl, POLQ, POTl, PPIF, PRCl, PSMC31P, PSRC1, MD51, RADS 1 API, MU 54P. RAD54L, RFC 3, RS 112.
  • SHCBPI SKA 1, SKA3, SPAGS, SPC25, SI!..
  • STMNl TACC3, TK1, TOP2A, TPX2, TRIM59, TUBA IB, 1 FIDS.
  • the kit can also comprise, e.g., a buffering agent, a preservative or a protein- stabilizing agent.
  • the kit can further comprise components necessary for detecting the detectable-label, e.g., an enzyme or a substrate.
  • the kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
  • the above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for
  • the kit may further comprise a second container which holds a diluent suitable for diluting the antibody compositions towards a higher volume. Suitable diluents include, but are not. limited to, the pharmaceutically acceptable excipient of the antibody composition and a saline solution.
  • the containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle).
  • the kit may further comprise more containers comprising a
  • buffer such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts.
  • aCGH array comparative genomic hybridization
  • BCR biochemical recurrence
  • dbGaP database of Genotypes and Phenotypes
  • GEO Gene Expression Omnibus
  • NCI GDC National Cancer Institute Genomic Data Commons
  • OS overall survival
  • PRAD prostate adenocarcinoma
  • RNA seq RNA sequencing
  • RPPA reverse phase protein array
  • SU2C/PCF Stand Up To Cancer/Prostate Cancer Foundation
  • TCGA The Cancer Genome Atlas
  • WES whole exome sequencing
  • LNCaP (RRID: CVCL 0395) and PC-3 (RRID: CVCL 0035) cells were purchased from ATCC (Manassas, VA).
  • C4-2 (LNCaP C4-2, RRID: CVCL_4782) cells were obtained from VitroMed (Burlington, NC).
  • lentiviral vectors encoding RRM2 were infected in LNCaP and PC-3 cells, and stable cell lines were generated and maintained using puromycin selection. Efficiency of overexpression was verified by qPCR and Western blot.
  • SMARTpool siRNAs (Dharmacon, Lafayette, CO) were used for transfection with RNAiMAX (Thermo Fisher Scientific) to knock down target gene expression.
  • RNAiMAX Thermo Fisher Scientific
  • cells were transduced with lentiviral vectors encoding RRM2 and selected by treatment with puromycin as described previously (Mazzu et ak, 2019). Efficiency of knockdown and overexpression was verified after two or three days by qPCR and Western blot.
  • RNA sequencing was performed by 50 million 2x50bp reads at the Memorial Sloan Kettering Cancer Center Integrated Genomics Operation, and data were analyzed in Partek Flow software (St. Louis, MI). The data are available from GEO
  • PCS scores were calculated with gene set variation analysis (GSVA) using single sample GSEA (ssGSEA) (Barbie et al., 2009). Briefly, PCS signature scores were defined by the quantification of the composite expression of each gene in the signature in each sample. A z-score was computed for the expression of each gene in each sample by subtracting the pooled mean from the RNA-seq expression values and dividing by the pooled standard deviation. The overall survival analysis with the 12-gene signature was performed using KM plotter (www.kmplot.com/mirpower) (Lanczky et al., 2016).
  • samples were categorized as RRM2 high (upper quantile) or low (lower quantile) based on mRNA expression.
  • the fraction of TILs in TCGA cases was determined with a machine-learning algorithm that uses digital hematoxylin and eosin (H&E) slides (Saltz et al., 2018).
  • the abundance of immune cell fractions in each sample was determined using CIBERSORT and LM22, a validated leukocyte gene signature matrix (Newman et al., 2015).
  • Results are reported as mean ⁇ standard deviation. Comparisons between groups were performed using an unpaired two-sided Student’s t test or Wilcoxon rank-sum test (p ⁇ 0.05 was considered significant). Disease-free survival was examined using the Kaplan-Meier method.Patients were divided into 2 groups (upper and lower quartile based on RRM2 expression or RRM2 signature score), and Kaplan-Meier curves were generated for each group. The log-rank test was used to determine significance. Cox proportional hazard regression was performed, adjusting for clinical and demographic factors. The significance of the correlation between gene expression and enzalutamide resistance was analyzed by Fisher’s exact test. The significance of the differences in the abundance of immune cell types between groups was determined using Wilcoxon’ s rank-sum test with Benjamini-Hochberg correction. Statistical analysis was completed using R version 3.4.3. Data Accessibility
  • RNA-seq data are available from the Gene Expression Omnibus (GEO:
  • GSE117921 GEO: GSE117922, GEO: GSE117923, GEO: GSE117924).
  • RRM2 function is disease-state specific in prostate cancer
  • PCS1 is the most aggressive and lethal, and PCS1 tumors progress more rapidly to metastatic disease than PCS2 or PCS3 tumors (You et al., 2016).
  • the FOXM1 pathway was recently reported as the master regulator of the PCS1 subtype (Ketola et al., 2017). It was also previously reported that RRM2 is not only a target of FOXM1 but also regulates the FOXM1 pathway (Mazzu et al., 2019). Furthermore, RRM2 is one of the most highly expressed genes in the PCS1 signature.
  • ssGSEA was performed to determine the correlation between RRM2 expression level and PCS score in multiple prostate cancer cohorts in order to determine if overexpression of RRM2 could contribute to the development of PCS 1 tumors.
  • scores of PCS1, PCS2, and PCS3 gene expression were calculated using ssGSEA.
  • RRM2 was removed from the PCS1 signature to avoid a false positive correlation.
  • RRM2 may be a driver of PCS1 tumors
  • PCS genes were compared to genes downregulated with RRM2 inhibition and genes upregulated with RRM2 overexpression. Strikingly, COH29 treatment specifically inhibited the expression of most PCS1 genes and also targeted PCS2 genes (Figure 5A). In addition to the PCS signatures, the PAM50 classifier was also applied in the analysis. In the Kumar cohort, some separation was observed between basal and luminal subtypes ( Figure 5B). The majority of PCS 1 genes overlapped with genes upregulated in luminal B tumors; these cases have the poorest clinical prognoses (Zhao et al., 2017). Genes targeted by RRM2 inhibition or genes upregulated by RRM2 overexpression in PC-3 cells were highly enriched in luminal B genes ( Figure 5B). Together, these data suggest that RRM2 is a driver of the aggressive prostate cancer subtypes and that inhibition of RRM2 could specifically target the subtypes of prostate cancer with the worst prognosis.
  • the RRM2 signature may predict enzalutamide resistance in prostate cancer circulating tumor cells
  • Circulating tumor cells detach from the primary or secondary tumor sites and invade the bloodstream, and they have been reported to be useful prognostic biomarkers to aid prostate cancer diagnosis, treatment decision-making, and patient follow-up (Chung et al., 2019; De Laere et al., 2019; Nimir et al., 2019).
  • the prognostic value of CTCs collected by the epithelial marker-dependent method CellSearch has been established in the context of metastatic PC (Hegemann et al., 2016). Given the prognostic significance of the RRM2 signature in prostate cancer, here it was further investigated whether the RRM2 signature had clinical significance in prostate cancer CTCs.
  • RRM2 overexpression creates an immunosuppressive immune microenvironment in prostate cancer
  • TIME tumor-immune microenvironment
  • CIBERSORT analysis was applied, a method of estimating the composition and abundance of immune cells from tumor biopsies (Newman et al., 2015).
  • There was also greater infiltration of antitumor CD8+ T cells (p 0.031) in RRM2- high tumors.
  • the signature scores of the 22 types of immune cells in the LM22 signature in the three prostate cancer cohorts are shown ( Figures 8-10).
  • prostate cancer classification systems PCS and PAM50 are significantly better at identifying aggressive and resistant cases of prostate cancer.
  • PCS classification system was developed and validated in 4,600 samples from patients with prostate cancer.
  • TCGA genomic subtypes e.g., ERG, ETV1/4, SPOP, FOXA1, and others
  • PCS1 is highly enriched with the SPOP subtype, whereas PCS2 tumors were overrepresented in ERG cancers (You et al., 2016).
  • GRID Genomic Resource Information Database
  • RRM2 is a master driver of poor prognosis prostate cancer identified by both the PCS1 andPAM50 classification systems.
  • RRM2 is essential for DNA synthesis and repair by producing deoxyribonucleotide triphosphates (dNTPs). Its level is rigorously regulated during the cell cycle and delayed degradation may lead to genomic instability (D'Angiolella et al., 2012).
  • RRM2 is expressed at low levels in normal prostate tissue, but increased expression of RRM2 is highly correlated with poor clinical outcomes in prostate cancer (Huang et al., 2014; Mazzu et al., 2019).
  • RRM2 is an oncogene in prostate cancer cells, regulates multiple oncogenic signaling pathways, and promotes EMT and angiogenesis (Mazzu et al., 2019). Although common pathways were activated by RRM2 overexpression in LNCaP and PC-3 cells, the majority of the upregulated genes were different ( Figure 2D-2F), suggesting that RRM2- regulated genes may be disease-state specific.
  • the PAM50 classifier was recently reported as a pan-carcinoma luminal/basal subtyping across epithelial tumors, and luminal B tumors were more sensitive to the ribonucleotide reductase inhibitor gemcitabine than the other subtypes (Zhao et al., 2019).
  • gemcitabine-induced amplification of RRM2 is a mechanism of gemcitabine resistance (Duxbury et al., 2004; Zhou et al., 2001)
  • RRM2-specific inhibitors e.g., COH29
  • RRM2 overexpression may contribute to AR antagonist resistance, suggesting that inhibition of RRM2 may delay the development of resistance.
  • immunosuppressive M2 macrophages which may contribute to immune escape.
  • the association between RRM2 overexpression and changes in the TIME by histologic staining in prostate cancer tissue will be validated.
  • Patients with RRM2- high tumors may be good candidates to receive immunotherapy because of increased TIL infiltration.
  • Combination treatment of RRM2 inhibitors with immunomodulators to stimulate cytotoxic T cells and inhibit immunosuppressive cells may sensitize these tumors to immunotherapies.
  • RRM2 is a master driver of aggressive prostate cancer subtypes.
  • Targeting RRM2 may be an effective therapeutic option to reprogram the TIME and treat the subtypes of prostate cancer with poor prognosis.
  • EXAMPLE 2 RRM2 SIGN A TORE AS A PROGNOSTIC MARKER FOR PROSTA TE
  • RRM2 ribonucleotide reductase small subunit M2
  • FOXM1 is identified as the driver of RRM2 overexpression in prostate cancer.
  • COH29 an RRM2 inhibitor
  • RRM2 oncogenic activity of RRM2 in PC cells was assessed by inhibiting or overexpressing RRM2.
  • COH29 an RRM2 inhibitor
  • Normal human prostate cell lines PWR-E1, PZ-HPV-7, RWPE-1) and human PC cell lines(LNCaP, 22Rvl, DU145, and PC-3) were purchased from ATCC (Manassas, VA).
  • C4-2 cells were obtained from VitroMed (Burlington, NC).
  • E006AA cells were provided by John T. Isaacs (The Johns Hopkins University School of Medicine, Baltimore, MD) and the LAPC-4 cell line was provided by Charles Sawyers (Memorial Sloan Kettering Cancer Center [MSK], New York, NY).
  • Normal prostate cell lines were cultured in Keratinocyte Serum-Free Medium (K-SFM) (Kit Cat Number: 17005-042; Thermo Fisher Scientific).
  • SMARTpool siRNA were obtained from Dharmacon and transfected with
  • RNAiMAX from Invitrogen (Carlsbad, CA). Cells were harvested 48 hours or 72 hours after transfection for protein and mRNA analysis. Lentiviral vectors encoding RRM2 were purchased from GeneCopoeia (Rockville, MD) and transfected with psPAX2 packaging and pMD2.G envelope plasmid to HEK293FT cells for 2 days using Lipofectamine 3000
  • PC cells were infected with viral supernatants in the presence of 8 pg/ml polybrene. Stable cells were generated using puromycin selection. Efficiency of knockdown and overexpression was verified by qPCR and western blot.
  • RNA sequencing was performed by 50 million 2x50bp reads in the MSK Integrated Genomics Operation Core. RNA sequencing data were analyzed by Partek Inc. (St. Louis, MO). The data are available from GEO
  • Phospho-kinase arrays were applied according to the manufacturer’s instructions. Briefly, protein was extracted from cells treated with siRNAs or inhibitors. Changes in kinase activity were visualized by chemiluminescence on
  • Cells were treated with siRNAs or inhibitors. Cell viability was assessed using CellTiter-Gloluminescent cell viability assay from Promega (Madison, WI). At 72 hours post-transfection, cell cycle and apoptosis were detected using the Muse cell cycle assay kit and the Muse Annexin V and dead cell kit (EMD Millipore, Burlington, MA). DNA damage was detected at 48 hours post-transfection of siRNAs or at 24 hours treatment of inhibitors in cells by using Muse multi-color DNA damage kit (EMD Millipore).
  • EMD Millipore Muse multi-color DNA damage kit
  • Soft agar assays were performed in 6-well tissue culture plates by placing cells (2-5 xl04/well) in 2 ml of 0.3% soft-agar above a 2-ml layer of 0.5% agar. After two weeks' incubation, cells were stained with 1 mg/ml MTT in medium for 1 hour. Colonies were detected and counted using GelCount technology (Oxford Optronix Inc., Oxford, UK).
  • Photographs of each scratch were taken at 24 hours after scratching.
  • Matrigel invasion assays were performed in Matrigel invasion chamber (Fisher Scientific) with cells on the top of chambers in serum-free media. 10% FBS in the lower chamber was used as chemo-attractant. After indicated times, cells in the bottom chamber were fixed in methanol and stained with crystal violet, photographed, and counted under phase-contrast microscopy.
  • ChIP Chromatin immunoprecipitations
  • Radical prostatectomy tissue was prepared and ChIP-Seq performed with 6-ug antibodies to AR and H3K27Ac antibody.
  • DNA sequencing libraries were prepared using the ThruPLEX-FD Prep Kit (Rubicon Genomics, Ann Arbor, MI). Libraries were sequenced using 50-base pair reads on the Illumina platform (Illumina, San Diego, CA) at Dana-Farber Cancer Institute. Related data analysis for ChIP-Seq and peak calling followed the same protocol as reported. The quantitation of signals on the 1-MB region of enhancer of RRM2 was performed from 4 normal prostate tissue samples and 4 tumor samples. The data are available from GEO (GSE118845).
  • ChIP-qPCR was performed using the ChIP-IT High Sensitivity Kit (Active Motif, #53040) with ChIP-grade H3K4me3, FOXM1 antibodies and native IgG, according to the manufacturer's instructions. DNA was analyzed via qPCR. All ChIP experiments were completed with at least two biological replicates.
  • siRNAs were transfected in cells for 24 hours and 500 ng of RRM2 promoter reporters (GeneCopoeia, Rockville, MD) containing Okb or 3kb promoter sequence of human RRM2 were cotransfected with Lipofectamine 2000
  • the template:primer complex was extended by HIV-1 RT to generate one additional nucleotide extension product (“P+1”) for one of four dNTPs contained in the dNTP samples extracted from the cells.
  • P+1 nucleotide extension product
  • the molar amount of the P+1 product is equal to that of each dNTP contained in the extracted samples, which allowed us to calculate and compare the amounts of the cellular dNTPs for the different treatments.
  • the dNTP amounts were normalized to lxlO 6 cells for the comparisons.
  • NOG-SCID mice were implanted subcutaneously with C4-2 cells. After palpable tumors developed (typically 100 mm3), six mice per group received either vehicle or COH29 (200 mg/kg) by oral gavage twice a day for 3 weeks. Tumors were measured twice a week using calipers. Tissues were collected for immunohistochemistry (IHC) staining and protein assays. Multiple proteins were assessed by IHC using antibodies of H2A.x (1 : 1000), MYC (1 : 100), RRM2 (1 :2500), cleaved-caspase3 (1 :300), prostate-specific antigen (PSA) (1 :2000) (Table 4). All animal care was in accordance with the guidelines of the Institutional Animal Care and Use Committee at MSK.
  • IHC immunohistochemistry
  • Results are reported as mean ⁇ S.E. Comparisons between groups were performed using an unpaired two-sided Student’s t test or Wilcoxon test (p ⁇ 0.05 was considered significant). Disease-free survival was examined for time since diagnosis. Cox proportional hazard regression was performed adjusting for clinical and demographic factors.
  • RRM2 functions as an oncogene in PC cells
  • RRM2 is highly expressed in most PC cell lines tested, regardless of androgen receptor (AR) status.
  • PC cell lines LNCaP, C4-2, 22Rvl, and E006AA
  • LNCaP normal prostate tissue
  • C4-2, 22Rvl, and E006AA PC-3 cells
  • RRM2 protein expression was further detected by IHC staining in a PC tissue microarray which included 9 cases of PC adenocarcinoma and 1 leiomyosarcoma, plus 2 tissue samples from normal prostates.
  • dNTP production (dATP, dCTP and dTTP) was significantly inhibited by knockdown of RRM2 (siRRM2) ( Figure 11 A).
  • siRRM2 induced DNA damage with significant activation of DNA damage markers, including upstream ATM and downstream H2A.X phosphorylation ( Figure 11B, 11C).
  • siRRM2 led to cell growth inhibition, S phase arrest, and apoptosis ( Figure 11D, HE, and 11F).
  • Similar phenotypes induced by siRRM2 were also detected in 22Rvl cells, which express AR-V7, and E006AA cells, which do not express AR. Therefore, the tumor suppressive effects of RRM2 inhibition are not related to AR status and are not cell line specific.
  • N-cadherin (CDH2) and vimentin (VIM) were not affected by increased RRM2 expression, while the EMT markers (SNAI1 and SLUG) were up-regulated in PC3-RRM2 cells ( Figure 11L).
  • Pcadherin (CDH3) a cell-to- cell adhesion molecule
  • E-cadherin (CDH1) expression was increased about 5-fold ( Figure 11L-11M).
  • CDH3 has been observed in the development and progression of multiple cancers including PC (27). CDH3 and CDH1 co-regulate collective migration in breast cancer (Ribeiro, 2013).
  • RRM2 may promote collective migration by upregulating key regulators such as CDH1, CDH3, and SNAIl.
  • key regulators such as CDH1, CDH3, and SNAIl.
  • COH29 inhibited the androgen response gene set (Figure 14H). Compared to the siRNA approach, COH29 induced wider transcriptomic changes. Here, GO analysis was applied by using genes whose expression were similarly altered (219 down-regulated genes; 106 up-regulated genes) with COH29 and siRRM2 treatment. The top five inhibited pathways were PLK, MYC activation, FOXM1, Aurora B, and telomerase pathways, while p53 downstream pathway and MYC repressive pathway were activated with both approaches to inhibit RRM2 activity. The major inhibited biological processes included cell cycle, RNA processing, response to DNA damage, and chromosome organization-related processes. Intriguingly, besides apoptosis, DNA damage, and cell proliferation, the processes for signal transduction, regulation of kinase activity, and inhibition of phosphorylation were activated by inhibition of RRM2.
  • Phospho-proteomic changes are induced by modulating RRM2 expression in PC cells
  • RRM2 inhibitor COH29 has antitumor effect in vivo
  • COH29- inhibited AR may not be directly associated with inhibition of RRM2.
  • COH29 has antitumor effects in vivo in a human PC xenograft model.
  • RRM2 is transcriptionally activated by FOXM1 in PC
  • TFs transcription factors
  • a correlation matrix from the TCGA (primary tumor samples) and SU2C/PCF (metastatic samples) cohorts not only showed the relationships between RRM2 and all potential RRM2- targeting TFs but also showed the correlations among the TFs. It revealed different correlation patterns for the TCGA and SU2C/PCF cohorts, indicating that disease state may contribute to the specificity of RRM2 -targeting TFs in PC. The subset of RRM2- targeting TFs which correlate with significant clinical outcome should be more meaningful and could facilitate the development of future therapeutic strategies. Among 13 TFs, both FOXM1 and E2F8 were shared in all the cohorts (Fig. 6B). FOXM1 has been reported to be one of the major drivers in PC (46).
  • FOXM1 binding region was first identified to the RRM2 promoter from published FOXM1 ChIP-Seq. In breast cancer cells and HeLa cells, there were two major FOXM1 binding peaks in the promoter of RRM2 ( Figure 16E). Although no FOXM1 ChIP-Seq data in PC cells is available, ChIP-Seq of transcriptional activation marks (including H3K4me3, H3K27Ac, and POLR2A) in multiple PC cell lines provided supporting evidence that the FOXM1 -binding regions of RRM2 promoter are activated.
  • siFOXMl led to approximately 50% reduction of RRM2 mRNA and protein expression (Figure 16H-16I).
  • a small molecule inhibitor of FOXMl FDI- 6
  • FOXMl targets including RRM2, MYC, PSA, and CENPF ( Figure 16J).
  • FOXMl can transcriptionally activate RRM2 expression by directly binding to the promoter.
  • RRM2 could be required to compensate for the DNA repair deficiency induced by deletion of DNA repair genes (e.g., CHEK2 or ATM).
  • DNA repair genes e.g., CHEK2 or ATM.
  • CGS clinical Gleason sum
  • RRM2 activated atypical EMT progression by up-regulating E-cadherin (CDH1) and P-cadherin (CDH3).
  • RNA-Seq analysis in siRRM2 or COH29 treated PC cells provided a global assessment of RRM2-regulated transcriptome changes.
  • GSEA analysis revealed that inhibition of RRM2 could activate biological processes including cell cycle checkpoint, DNA damage response, and apoptotic signaling.
  • COH29 treatment could target genes highly enriched in PC.
  • RRM2-regulated gene signature (from RNA-Seq datasets) to TCGA and Taylor cohorts was applied. Intriguingly, the RRM2 signature was highly correlated with metastasis and disease free survival (p ⁇ 0.001). Furthermore, inhibition of RRM2 specifically targets poor prognostic luminal subtypes (PCS1 subtype; LumBin PAM50 classifier). Besides transcriptome changes, protein kinase arrays showed that AKT/mTOR and SFK STAT signaling were repressed by inhibition of RRM2. These oncogenic signaling pathways are crucial for EMT program.
  • Amplification of RRM2 is rare in PC and transcriptional activation of RRM2 may play a major role in overexpression of RRM2.
  • H3K27acChIP-Seq from tissues revealed more activated RRM2 promoter in PC than in normal prostate.
  • 13 potential RRM2 -targeting transcription factors (TFs) were identified by integrating clinical cohorts and a TF database. They showed a positive correlation with RRM2 expression in PC cohorts.
  • FOXM1 was reported to be the master driver of the aggressive luminal subtype of PC. It is further revealed that FOXM1 expression is associated with clinical outcomes.
  • the ChlP- PCR and luciferase reporter assays provided evidence of physical binding of FOXM1 to the RRM2 promoter in PC cells. Knockdown of FOXM1 significantly repressed RRM2 mRNA and protein levels. FOXM1 -regulated transcriptional activation contributes to overexpression ofRRM2. Intriguingly, COH29 can also repress FOXM1 expression, which leads to transcription repression of RRM2.
  • FIG. 17A shows the global transcriptomic changed induced by knockdown of RRM2 in C4-2 cells.
  • Figure 17B shows GO analysis indicating that biological process and pathways were affected by inhibition of RRM2 expression in PC cells.
  • GSEA analysis shows the key networks were affected by inhibition of RRM2.
  • Figure 18 shows further transcriptomic changes induced by COH29 in C4-2 cells.
  • FIG. 18A Top Panel is a GO analysis showing biological processes regulated by COH29 treatment.
  • Figure 18A Bottom Panel is a GO analysis showing pathways regulated by COH29 treatment.
  • Figure 18B shows COH29 regulated genes were correlated with DSF for Taylor cohort.
  • FIG. 19A shows knockdown of RRM2 inhibited AKT/mTOR signaling.
  • Figure 19B graphically demonstrates the result of knockdown of RRM2 inhibited AKT/mTOR signaling, STAT signaling, SFK signaling, and control after 24 hour COH29 treatment in C4-2 cells.
  • RRM2 also targets poor-prognosis subtypes of PC.
  • RRM2 expression is highly associated with PCS1 subtyping, rather than PCS2 and PCS3.
  • L is low level
  • M is medium level
  • H is high level.
  • Figure 20B shows that inhibition of RRM2 by siRNA or COH29 target majority of PAM50 genes.
  • the heatmap also showed the overlapping of PCS1 signatures and basal like genes in PAM50.
  • FIG. 21A shows GO analysis demonstrating certain biological process were affected by overexpression of RRM2 expression in PC cells, in LNCaP and PC- 3 cells.
  • Figure 21B shows GO analysis demonstrating biological pathways affected by overexpression of RRM2 expression in PC cells.
  • Figure 21C shows enrichment of RRM2 regulated genes in EMT gene sets and
  • Figure 21D shows enrichment of RRM2 regulated genes in angiogenesis gene sets.
  • FIG. 22A indicates RRM2 may be direct targets of miR- 193b.
  • Figure 22C shows DNMT inhibitor (5-Aza) and HD AC inhibitor (SAHA) significantly restored miR-193b expression in 22RV1 in which methylation of miR-193b is observed.
  • Figure 22D graphically shows Anti-miR-193b (A- 193b) partially blocked the 5-Aza-induced repression of RRM2, suggesting up-regulation of miR-193b by 5-Aza could induce down-regulation of RRM2 in PC cells.
  • RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine. Oncogene 23, 1539-1548.
  • Ketola K., Munuganti, R.S.N., Davies, A., Nip, K.M., Bishop, J.L., Zoubeidi, A., 2017. Targeting Prostate Cancer Subtype 1 by Forkhead Box Ml Pathway Inhibition. Clin Cancer Res 23, 6923- 6933.
  • miRpower a web-tool to validate survival-associated miRNAs utilizing expression data from 2178 breast cancer patients.
  • RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance. Science 349, 1351-1356.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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Abstract

La présente invention concerne des procédés d'identification de cancers associés à un mauvais pronostic (par exemple, un cancer de la prostate, un cancer du sein, un cancer de l'ovaire, un cancer du poumon ou un cancer du foie) chez un patient qui en a besoin. Les procédés décrits dans la description de l'invention sont utiles pour déterminer si un patient bénéficiera d'un traitement avec des inhibiteurs de la ribonucléotide réductase sur la base de la détection de niveaux d'expression élevés de gènes de signature RRM2 spécifiques.
PCT/US2020/028338 2019-04-16 2020-04-15 Gènes de signature rrm2 utilisés comme marqueurs pronostiques chez des patients atteints d'un cancer de la prostate WO2020214718A1 (fr)

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WO2022253604A1 (fr) * 2021-06-03 2022-12-08 Metacurum Biotech Ab Biomarqueurs et leurs utilisations
EP4253567A1 (fr) * 2022-03-31 2023-10-04 OncoAssure Limited Procédé de prédiction du risque d'un cancer agressif ou récurrent
WO2023186985A1 (fr) 2022-03-31 2023-10-05 Oncoassure Limited Procédé de prédiction du risque d'un cancer agressif ou récurrent
WO2023196978A3 (fr) * 2022-04-08 2023-11-16 Rutgers, The State University Of New Jersey Programme myc en tant que marqueur de réponse à l'enzalutamide dans la prostate
CN115998729A (zh) * 2022-11-17 2023-04-25 贵州大学 邻甲酚酞在制备blm解旋酶抑制剂或抗癌药物中的应用
CN115998729B (zh) * 2022-11-17 2024-04-26 贵州大学 邻甲酚酞在制备blm解旋酶抑制剂或抗癌药物中的应用
CN116590415A (zh) * 2023-05-18 2023-08-15 南方医科大学南方医院 一种基于组蛋白修饰基因特征开发的前列腺癌预后风险评估模型及应用
CN116590415B (zh) * 2023-05-18 2023-11-14 南方医科大学南方医院 一种基于组蛋白修饰基因特征开发的前列腺癌预后风险评估模型及应用

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