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Triple-negative breast cancers with amplification of JAK2 at the 9p24 locus demonstrate JAK2-specific dependence

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Science Translational Medicine  13 Apr 2016:
Vol. 8, Issue 334, pp. 334ra53
DOI: 10.1126/scitranslmed.aad3001
  • Fig. 1. JAK2AMP is associated with a decreased response to NAC and poor patient survival.

    (A) JAK2AMP breast tumor samples, detected by NGS, were validated by FISH and are represented as the average JAK2/centromere 9 (CEN9) ratio per cell (at least 30 cells counted for each case). Red bars show JAK2AMP cases and blue bars represent nonamplified controls. (B) Representative FISH images of JAK2/9p24 (red) and CEN9 (green). Four cases are depicted, with the upper left demonstrating a patient tumor with normal JAK2 (two copies). The remaining three cases showed JAK2 gains/amplification. Scale bars, 20 μm. (C and D) Kaplan-Meier curves assessing recurrence-free survival (RFS) and overall survival (OS) in patients with JAK2AMP and JAK2NML cancers [progression-free survival: hazard ratio (HR), 0.19; 95% confidence interval (CI), 0.05 to 0.73; overall survival: HR, 0.1; 95% CI, 0.02 to 0.44]. (E) IL6 mRNA in JAK2AMP (n = 4) and JAK2NML (n = 32) tumors quantified by NanoString analysis. Bars represent mean IL-6 mRNA expression. (F) RNA in situ analysis of JAK2AMP tumors for IL6 (red) and JAK2 (green). Scale bars, 20 μm. (G) Quantification of JAK2/IL6 RNAscope in three high-power fields across three JAK2AMP tumors, showing that single-positive cells are more common than dual-positive cells. (H) Individual data points for each of three JAK2AMP tumors analyzed by RNA in situ hybridization. Each point shows the number of single- or double-positive cells observed in each high-power field (n = 3 fields per sample).

  • Fig. 2. Chemotherapy enriches the JAK2AMP tumor cell population.

    (A) Comparison of JAK2 amplification rates in 68 TNBCs treated with NAC versus TCGA (primary basal-like). (B) Amplifications in JAK2 occur primarily in BLBC. Data were obtained through the cBio Web site for TCGA data access. Luminal, n = 324; basal, n = 81. (C) Enrichment of JAK2 gene copy number in longitudinal samples (pretherapy biopsy, post-NAC surgical specimen, and metastatic biopsy in two patients with TNBC). (D) JAK2/CEN9 copy number ratio across a series of 22 PDXs. PDX tumors (all annotations are truncated for readability and actual identifiers are BCM-XXXX, except for MC-1) were stained as a tissue microarray (TMA), with one to three independent cores (biological replicates) per PDX model. Bars represent means + SEM of the JAK2/CEN9 ratios for all PDXs. At least 30 cells were counted and averaged for each core. Samples in red are JAK2-amplified. Samples in patterned pink are matched PDXs from the same TNBC, established before (2147) and after (2277) chemotherapy. (E) JAK2 FISH copy number counts (relative to centromere 9 signal) at the single-cell level in matched untreated (2147) and postchemotherapy (2277) PDXs. Clinically amplified PDX (4013) and FISH from an untreated patient (primary JAK2AMP) demonstrating high level of amplification are plotted for comparison. The population of JAK-amplified cells was enriched in the PDX model established after chemotherapy treatment. P value represents result of a two-tailed t test.

  • Fig. 3. JAK2 drives a STAT3-independent program in JAK2AMP TNBC cell lines.

    (A) Analysis of CCLE data via the cBio portal to identify cell lines with JAK2 copy alterations. Solid red boxes, amplified; solid pink, gained; light blue, shallow deletion; dark blue, deep deletion; outlined pink, mRNA overexpression (ESR1 only). (B) FISH for JAK2 (red) and CEN9 (green) for breast cancer cell lines SUM159PT (JAK2NORMAL), HCC-70 (JAK2GAIN), HCC-38 (JAK2GAIN), and MDA-436 (JAK2AMP). Scale bars, 20 μm. (C) Cells were treated with 10% fetal bovine serum (FBS) ± 1 μM ruxolitinib for 24 hours and analyzed by immunoblot with the indicated antibodies. Cell line names in pink are JAK2GAIN and cell line names in red are JAK2AMP. (D) The indicated TNBC cell lines were treated with increasing doses of the JAK2-specific inhibitor BSK805 or ruxolitinib for 24 hours and analyzed by immunoblot. (E) TNBC cell lines were transfected with siControl (siCON), siJAK1, or siJAK2 or treated with 1 μM ruxolitinib. Two different sequences for each of JAK1 and JAK2 were used to confirm specificity. Cells were harvested 72 hours after siRNA transfection or 24 hours after ruxolitinib treatment and analyzed by immunoblot with the indicated antibodies. (F) The indicated cell lines were treated with serum-free or 10% serum–containing medium for 16 hours and then treated with 1 μM of BSK805 or ruxolitinib for 1 hour; cells were next stimulated with oncostatin M (OSM; 50 ng/ml) for 30 min, followed by cell harvest, preparation of lysates, and immunoblot analysis.

  • Fig. 4. JAK2 knockdown abrogates tumorsphere expansion after chemotherapy.

    (A) HCC-38 cells stably transduced with doxycycline-inducible shRNA targeting JAK2 (two independent sequences) or shNTC were grown in 10% FBS and treated with vehicle (control), IC50 of paclitaxel (50 nM) ± doxycycline (100 ng/ml), or doxycycline for 4 days and allowed to recover in fresh medium for 3 days; cells were then trypsinized and assessed for their ability to form mammospheres. Colony numbers are expressed relative to untreated controls. (B) On day 7, colonies were stained with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and photographed. Scale bars, 200 μm. (C and D) Experiments identical to (A) and (B) were carried out in MDA-436 cells (paclitaxel IC50, 150 nM). (E) Immunoblot analysis demonstrating doxycycline-inducible knockdown of JAK2 at 72 hours in HCC-38 and MDA-436 cells. (F) MDA-436 and HCC-38 cells were grown in 10% FBS and treated with vehicle (control), paclitaxel (at the cell line IC50) ± 5 μM BSK805, or 5 μM BSK805 alone for 4 days and then allowed to recover in fresh medium for 3 days. Colonies were quantitated as described above. All experiments were replicated at least twice (n = 3). (G) Limiting dilution assay results for MDA-436 or HCC-38 cells treated with shNTC + doxycycline or shJAK2 + doxycycline, or parental cells treated with BSK805 (1 μM) or vehicle control. Cells were pretreated for 1 week before inoculation, and treatment was maintained in the mice for 1 week after orthotopic (no. 4 mammary fat pad) injection. Data are presented as the number of tumors (palpable at 30 days for MDA-436 and either palpable or confirmed microscopically after biopsy at 60 days for HCC-38) formed out of the number of mice inoculated. P value represents the χ2 assay result. Statistics were performed using the protocol at http://bioinf.wehi.edu.au/software/elda/.

  • Fig. 5. Pharmacological JAK2 inhibition in vivo abrogates tumor-initiating potential after chemotherapy.

    (A and B) Female athymic mice were injected with MDA-436 or HCC-38 cells in the no. 4 mammary fat pad. Mice bearing tumors ≥150 mm3 were randomized to treatment with vehicle, paclitaxel [20 mg/kg per day × 4 doses intraperitoneally (i.p.)], or paclitaxel (20 mg/kg per day × 4 doses i.p.) + BSK805 [100 mg/kg per day orally (p.o.)]. Paclitaxel doses are represented by arrows. Tumor volumes were measured twice weekly. Two complete responses to dual therapy were achieved in mice bearing HCC-38 tumors. Bars represent means ± SEM. Differences were analyzed by one-way analysis of variance (ANOVA) with Tukey’s contrasts. (C) Representative images of mammospheres from treated tumors in (A) and (B). Scale bars, 200 μm. (D) Quantification of mammospheres from tumors harvested at the end of treatment. Bars represent means + SEM for n = 9 measurements. (E) Severe combined immunodeficient (SCID)/beige mice implanted with PDX model PDX4013 were randomized to treatment with vehicle (intraperitoneal saline and oral gavage with oral suspension agent), paclitaxel (20 mg/kg per day × 4 doses i.p.), BSK805 (80 mg/kg per day p.o.), or paclitaxel (20 mg/kg per day × 4 doses i.p.) + BSK805 (80 mg/kg per day p.o.). Tumor volumes were measured twice weekly. (F) Image of JAK2-FISH in the PDX4013 model demonstrating gene amplification. Scale bar, 20 μm.

  • Table 1. Clinical characteristics of cohort.

    n = 111; median age, 48 years.

    No.%
    Menopausal status
      Premenopausal5751.4
      Postmenopausal5448.6
    Node status
      Positive7164.0
      Negative4036.0
    Stage
      II98.1
      III10291.9
    Anthracyclines
      Yes10695.5
      No54.5
    Taxane
      Yes5852.3
      No5347.7
    JAK2 amplification7/6810.2
  • Table 2. Clinical comparison of patients by JAK2 status.

    Characteristics of patients with complete tNGS data. OS, overall survival; RFS, recurrence-free survival.

    JAK2-amplifiedJAK2-normal
    No.7 (10.9%)57 (89.9%)
    Median age39.947.6
    Premenopausal5 (71.4%)33 (57.9%)
    Miller-Payne
    I4 (57.1%)9 (15.8%)*
    II1 (14.2%)15 (26.3%)
    III0 (0%)5 (8.8%)
    IV0 (0%)6 (10.5%)
    V0 (0%)5 (8.8%)
    Unknown2 (28.7%)17 (29.8%)
    OS (median)10.824.4
    RFS (median)7.921.8

    *P = 0.027, two-tailed Fisher’s exact test.

    P = 0.002, two-sided log-rank test.

    P = 0.015, two-sided log-rank test.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/8/334/334ra53/DC1

      Fig. S1. Focal and nonfocal amplifications at 9p24/JAK2 exist in ER-negative breast cancer.

      Fig. S2. JAK2 copy number is associated with JAK2 gene expression.

      Fig. S3. Ruxolitinib has low impact on gene expression in vitro.

      Fig. S4. Ruxolitinib does not reduce cell viability or enhance chemosensitivity in vitro.

      Fig. S5. JAK2 knockdown reduces mammosphere potential in HCC-38 and MDA-436 cells.

      Fig. S6. Pharmacological inhibition of JAK2 reduces mammosphere formation after chemotherapy selection in vitro.

      Fig. S7. JAK2 inhibition overcomes taxane resistance in PDXs with JAK2GAIN.

    • Supplementary Material for:

      Triple-negative breast cancers with amplification of JAK2 at the 9p24 locus demonstrate JAK2-specific dependence

      Justin M. Balko,* Luis J. Schwarz, Na Luo, Mónica V. Estrada, Jennifer M. Giltnane, Daniel Dávila-González, Kai Wang, Violeta Sánchez, Phillip T. Dean, Susan E. Combs, Donna Hicks, Joseph A. Pinto, Melissa D. Landis, Franco D. Doimi, Roman Yelensky, Vincent A. Miller, Phillip J. Stephens, David L. Rimm, Henry Gómez, Jenny C. Chang, Melinda E. Sanders, Rebecca S. Cook, Carlos L. Arteaga*

      *Corresponding author. E-mail: carlos.arteaga{at}vanderbilt.edu (C.L.A.); justin.balko{at}vanderbilt.edu (J.M.B.)

      Published 13 April 2016, Sci. Transl. Med. 8, 334ra53 (2016)
      DOI: 10.1126/scitranslmed.aad3001

      This PDF file includes:

      • Fig. S1. Focal and nonfocal amplifications at 9p24/JAK2 exist in ER-negative breast cancer.
      • Fig. S2. JAK2 copy number is associated with JAK2 gene expression.
      • Fig. S3. Ruxolitinib has low impact on gene expression in vitro.
      • Fig. S4. Ruxolitinib does not reduce cell viability or enhance chemosensitivity in vitro.
      • Fig. S5. JAK2 knockdown reduces mammosphere potential in HCC-38 and MDA-436 cells.
      • Fig. S6. Pharmacological inhibition of JAK2 reduces mammosphere formation after chemotherapy selection in vitro.
      • Fig. S7. JAK2 inhibition overcomes taxane resistance in PDXs with JAK2GAIN.

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