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

Abstract

Amplifications at 9p24 have been identified in breast cancer and other malignancies, but the genes within this locus causally associated with oncogenicity or tumor progression remain unclear. Targeted next-generation sequencing of postchemotherapy triple-negative breast cancers (TNBCs) identified a group of 9p24-amplified tumors, which contained focal amplification of the Janus kinase 2 (JAK2) gene. These patients had markedly inferior recurrence-free and overall survival compared to patients with TNBC without JAK2 amplification. Detection of JAK2/9p24 amplifications was more common in chemotherapy-treated TNBCs than in untreated TNBCs or basal-like cancers, or in other breast cancer subtypes. Similar rates of JAK2 amplification were confirmed in patient-derived TNBC xenografts. In patients for whom longitudinal specimens were available, JAK2 amplification was selected for during neoadjuvant chemotherapy and eventual metastatic spread, suggesting a role in tumorigenicity and chemoresistance, phenotypes often attributed to a cancer stem cell-like cell population. In TNBC cell lines with JAK2 copy gains or amplification, specific inhibition of JAK2 signaling reduced mammosphere formation and cooperated with chemotherapy in reducing tumor growth in vivo. In these cells, inhibition of JAK1-signal transducer and activator of transcription 3 (STAT3) signaling had little effect or, in some cases, counteracted JAK2-specific inhibition. Collectively, these results suggest that JAK2-specific inhibitors are more efficacious than dual JAK1/2 inhibitors against JAK2-amplified TNBCs. Furthermore, JAK2 amplification is a potential biomarker for JAK2 dependence, which, in turn, can be used to select patients for clinical trials with JAK2 inhibitors.

Figure 1. JAK2^AMP is associated with a lower response to NAC and poor patient survival

A) Validation by FISH of JAK2 amplifications detected by NGS in tumor samples. B) Representative images of JAK2 FISH9p24 region (JAK2) in red and for centromere9 centromere in green. Four cases are depicted, with the upper left demonstrating a patient tumor with normal (2 copies) of JAK2. The remaining 3 cases demonstrated JAK2 gains/amplification. C,D) Kaplan-Meier curves assessing recurrence free survival (RFS) and overall survival (OS) in patients with JAK2^AMP and JAK2^NML cancers (PFS, HR=0.19, 95% CI, 0.05 to 0.73; OS, HR=0.1, 95% CI, 0.02 to 0.44) E) IL6 mRNA in JAK2^AMP and JAK2^NML tumors, quantified by nanoString analysis. F) RNA in situ analysis of a JAK2^AMP tumor for IL6 (red) and JAK2 (green).

Figure 2. Chemotherapy enriches the JAK2^AMP tumor cell population

A) Comparison of JAK2 amplification rates in 68 post-NAC TNBCs versus TCGA (primary basal-like) B) Amplifications in JAK2 occur primarily in basal-like breast cancer. Data were obtained through the cBIO website for TCGA data access. Luminal: n=324; Basal: n=81. C) Enrichment of JAK2 gene copy number in longitudinal samples (pre-therapy biopsy, post-NAC surgical specimen and metastatic biopsy in two patients with TNBC). D) JAK2 copy number across a series of 22 patient-derived TNBC xenografts (PDX) using a JAK2:CEP9 ratio as described in Methods. 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 post-chemotherapy (2277) PDXs. Clinically amplified PDX (4013) and FISH from an untreated patient (1° JAK2 amp) demonstrating high level of amplification are plotted for comparison. The population of JAK2-amp cells was enriched in the PDX model established after chemotherapy treatment. P-value represents result of a two-tailed t-test.

Figure 3. JAK2 drives a STAT3-independent program in JAK2^AMP TNBC cell lines

A) Analysis of CCLE data via the cBio portal to identify cell lines JAK2 copy alterations. B) JAK2 FISH in red and for chromosome 9 centromere in green for breast cancer cell lines SUM-159PT (JAK2^NORMAL), HCC-70 (JAK2^GAIN), HCC-38 (JAK2^GAIN) and MDA-436 (JAK2^AMP)). C) Cells were treated with 10% FBS ± 1 µM ruxolitinib for 24 h and analyzed by immunoblot with the indicated antibodies. D) SUM159PT cells were treated increasing doses of the JAK2-specific inhibitor BSK805 or ruxolitinib for 24 h and analyzed by immunoblot. E) TNBC cell lines were transfected with siControl (siCON), siJAK1, siJAK2, or treated with 1 µM ruxolitinib. Two different sequences for each JAK1 and JAK2 were used to confirm specificity. Cells were harvested 72 h after siRNA transfection or 24 h after ruxolitinib treatment and analyzed by immunoblot with the indicated antibodies.

Figure 4. JAK2 knockdown abrogates tumorsphere expansion after chemotherapy

A) HCC38 cells stably-transduced with doxycycline-inducible expression of shRNA targeting JAK2 (two independent sequences) or a non-targeting shRNA control (shNTC) were grown in 10% FBS and treated with vehicle (control), IC₅₀ of paclitaxel (50 nM) ± 100 ng/ml doxycycline or doxycycline for 4 days and allowed to recover in fresh media for 3 days; cells were then trypsinized and assessed for their ability to form mammospheres. B) On day 7, colonies were stained with MTT and photographed. Colony numbers are expressed relative to untreated controls (scale bars: 200 µm.) C–D) Experiments identical to (A–B) were carried out in MDA-436 cells (paclitaxel IC₅₀ 150nM). E) Immunoblot analysis demonstrating doxycycline-inducible knockdown of JAK2 at 72 h in HCC38 and MDA-436 cells. F) MDA-436 and HCC38 cells were grown in 10% FBS and treated with vehicle (control), paclitaxel (at the respective IC₅₀) ± 5 µM BSK805 or 5 µM BSK805 alone for 4 days and allowed to recover in fresh media for 3 days; colonies were quantitated as described above. All experiments were replicated at least twice (n=3).

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

Female athymic mice were injected with MDA-436 (A) or HCC-38 (B) cells in the number 4 mammary fat pad. Mice bearing tumors ≥150 mm³ were randomized to treatment with vehicle, paclitaxel (20 mg/kg/d i.p. 4 times) or paclitaxel (20 mg/kg/d i.p. 4 times) + BSK805 (100 mg/kg/day, p.o.). Tumor volumes were measured twice weekly. Two complete responses to dual therapy were achieved in mice bearing HCC38 tumors. Bars represent mean ± SEM (*p<0.001 vs. vehicle, #p<0.05 vs. paclitaxel). Inset: At the end of treatment, tumors were dissociated to a single cell suspension and plated in a mammosphere assay. After 12 days, mammosphere growth was imaged and quantified. Differences were analyzed by one-way ANOVA with Tukey’s contrasts (*p<0.05; **p<0.01). C) Western blot analysis of tumors harvested at the end of treatment for the indicated antibodies. D) Representative images of mammospheres from treated tumors in (A) and (B). E) Quantification of mammospheres from tumors harvested at the end of treatment. F) SCID-beige mice implanted with PDX model PDX4013 were randomized to treatment with vehicle, paclitaxel (20 mg/kg/d i.p. 4 times), BSK805 (80 mg/kg/day, p.o.), or paclitaxel (20 mg/kg/d i.p. 4 times) + BSK805 (80 mg/kg/day, p.o.). Tumor volumes were measured twice weekly. G) Image of JAK2-FISH in the PDX4013 model demonstrating amplification.