Inhibition of Janus Kinase 1 synergizes docetaxel sensitivity in prostate cancer cells

Abstract Prostate cancer (PCa) is the second most common malignancy and is the fifth leading cause of cancer mortality among men globally. Docetaxel‐based therapy remains the first‐line treatment for metastatic castration‐resistant prostate cancer. However, dose‐limiting toxicity including neutropenia, myelosuppression and neurotoxicity is the major reason for docetaxel dose reductions and fewer cycles administered, despite a recent study showing a clear survival benefit with increased total number of docetaxel cycles in PCa patients. Although previous studies have attempted to improve the efficacy and reduce docetaxel toxicity through drug combination, no drug has yet demonstrated improved overall survival in clinical trial, highlighting the challenges of improving the activity of docetaxel monotherapy in PCa. Herein, we identified 15 lethality hits for which inhibition could enhance docetaxel sensitivity in PCa cells via a high‐throughput kinome‐wide loss‐of‐function screen. Further drug‐gene interactions analyses identified Janus kinase 1 (JAK1) as a viable druggable target with existing experimental inhibitors and FDA‐approved drugs. We demonstrated that depletion of endogenous JAK1 enhanced docetaxel‐induced apoptosis in PCa cells. Furthermore, inhibition of JAK1/2 by baricitinib and ruxolitinib synergizes docetaxel sensitivity in both androgen receptor (AR)–negative DU145 and PC3 cells, but not in the AR‐positive LNCaP cells. In contrast, no synergistic effects were observed in cells treated with JAK2‐specific inhibitor, fedratinib, suggesting that the synergistic effects are mainly mediated through JAK1 inhibition. In conclusion, the combination therapy with JAK1 inhibitors and docetaxel could be a useful therapeutic strategy in the treatment of prostate cancers.


| INTRODUC TI ON
Prostate cancer (PCa) is the second most common cancer in men with an estimated worldwide incidence and mortality of 1. Docetaxel-based therapy is the current first-line chemotherapy for metastatic castration-resistant prostate cancer (CRPC) with a response rate ranging from 17% to 33%. [4][5][6] Dose-limiting toxicity, including neutropenia, myelosuppression and neurotoxicity, is the major challenge that cause docetaxel dose reductions and eventually fewer cycles administered, despite recent study showing a clear association of survival benefit with docetaxel in PCa and total number of cycles administered. 6 Several studies have attempted to improve the efficacy and reduce the toxicity of docetaxel through combination therapies, but have failed to improve the overall survival of PCa in clinical trials. [7][8][9][10][11][12][13] Thus, the discovery of new druggable targets that could enhance docetaxel sensitivity in PCa is imperative.
Recent studies have demonstrated that 'druggable' pathways that regulate the survival of PCa cells can be identified through comprehensive loss-of-function RNA interference (RNAi) screens. [14][15][16] Herein, we describe a systematic and comprehensive approach in employing a high-throughput kinome-wide shRNA screen coupled with in silico drug-gene interaction analyses to uncover druggable targets that could enhance docetaxel sensitivity in PCa cells. We  The RWPE-1 normal prostate cell line was acquired from ATCC and grown in keratinocyte serum-free medium consisting of 5 ng/mL of recombinant epidermal growth factor and 0.05 mg/mL of bovine pituitary extract (Invitrogen, Carlsbad, CA, USA). All cells were maintained in their logarithmic growth and kept in a humidified 37°C, 5% CO 2 incubator. normalized against NS controls. The sensitivity index (SI) for each shRNA was calculated as described previously [17][18][19][20] : SI = (Rc/Cc*Cd/ Cc)-(Rd/Cc), where Rc is the viability of cells following shRNA transduction (shRNA only), Rd is the viability of cells in following shRNA transduction and docetaxel treatment (shRNA + docetaxel), Cc is the in cells treated with JAK2-specific inhibitor, fedratinib, suggesting that the synergistic effects are mainly mediated through JAK1 inhibition. In conclusion, the combination therapy with JAK1 inhibitors and docetaxel could be a useful therapeutic strategy in the treatment of prostate cancers.

| Protein isolation and Western blot analysis
Protein lysates from the cells were extracted in an ice-cold lysis buffer (1% NP-40, 1 mM DTT, protease inhibitors and phosphatase inhibitor I and II cocktails in PBS as described previously). 14,15 A total protein of 50μg was loaded for immunoblotting. Primary antibodies against JAK1 and JAK2 were obtained from Cell Signaling Technology, MA, USA, and β-actin was purchased from Santa Cruz Biotechnology, CA, USA.  (Table S1). 21

| Lentiviral production and transduction
Lentiviral shRNA constructs targeting JAK1 were purchased from GE Healthcare Dharmacon Inc with target sequences shown in Table S2.

| Detection of apoptosis by Annexin V flow cytometry
All floating and attached cells were stained for cell apoptosis assay using a PE Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) as described previously. 14,15 The samples were quantitated using a FACSCalibur flow cytometer and analysed by CellQuest Pro software (version 5.1.1; BD Biosciences, San Jose, CA, USA).

| Statistical analysis
All results were presented as mean ± standard deviation (s.d.) from at least three independent experiments. Statistical significance was determined by Student's independent t test through SPSS (version 19.0 INC, Chicago, IL). A P-value <0.05 was considered statistically significant.

| DU145 and PC3 AR-negative PCa cells are inherently resistant to docetaxel
We first determined the sensitivity of a panel of PCa cells and normal prostate epithelial cancer cells against docetaxel. As shown in Figure 1A and Table S3, the AR-positive LNCaP PCa cells and RWPE-1 normal prostate epithelial cells were significantly more sensitive to docetaxel with an IC 50 of <0.16 nM and 0.88 ± 0.12 nM, respectively, while the AR-negative DU145 and PC3 cells were inherently resistant to docetaxel with IC 50 of >10 nM. shRNA screen scatter plot. Sensitivity Index (SI) was plotted on the y-axis against 3109 corresponding shRNAs on the x-axis. The red dot represents lethality hit (ie genes when knock-down enhances docetaxel sensitivity) and green dots represent rescue hit (ie genes when knock-down confers docetaxel resistance). (C) Protein-protein interaction network and cluster analysis of the 15 lethality hits using STRING. The identified clusters (by k-means) are coloured in red (cluster 1), green (cluster 2) and blue (cluster 3). Nodes represent proteins. The solid and the dotted lines indicate connections within the same and different clusters, respectively. (D) Drug-gene interaction network. Data mining of potential inhibitors interacting with the lethality hits were extracted from the DGIdb database. Node size represents the number of interactions

| JAK1 as a potential druggable and repurposing candidate to enhance docetaxel sensitivity in PCa cells
To identify novel druggable targets that could enhance docetaxel sensitivity in PCa, we queried the lethality hits against the Drug Gene Interaction Database (DGIdb) and identified JAK1 and IGF1R as druggable targets with existing FDA-approved or experimental inhibitors ( Figure 1D). We note that monoclonal antibodies against IGF1R have already shown poor efficacy and increased docetaxel toxicity in PCa clinical trials. 53,54 In contrast, JAK inhibitors were shown to possess good safety profiles in randomized controlled trials and their long-term extension studies have been demonstrated in various immune-mediated diseases such as rheumatoid arthritis and psoriatic arthritis. 55,56 In addition, recent studies in PCa model also demonstrated that JAK1/2 inhibitors suppress the immune escape of castration-resistant prostate cancer CRPC) to natural killer (NK) cells, 57 inhibit PCa metastasis 37 and inhibit progression of CRPC. 38 Hence, JAK1 represents a promising druggable target for repurposing existing drugs to enhance docetaxel sensitivity in PCa.

| Depletion of endogenous JAK1 enhances docetaxel sensitivity in PCa cells
To directly validate the effects of JAK1 inhibition in enhancing docetaxel sensitivity in PCa cells, we generated stable pools of JAK1-depleted isogenic cell lines in DU145 and PC3 cells.   Figure 3B; Tables 3-5). In contrast, the JAK2 inhibitor, fedratinib, exhibited mainly additive effects with docetaxel in DU145 and antagonistic effects in PC3 cells (Tables 3 and 4).

| JAK1/2 inhibitors synergize docetaxel sensitivity in AR-negative DU145 and PC3 PCa cells
Taken together, our findings demonstrate that inhibition of JAK1 synergized docetaxel sensitivity in AR-negative PCa cells.

| CON CLUS IONS
In conclusion, we showed that inhibition of JAK1 synergizes with docetaxel sensitivity in AR-negative PCa cells via inhibition of STAT3 signalling. Overall, our findings suggest that combination therapy with JAK1/2 inhibitors and docetaxel may be a useful approach for treating patients with advanced PCa and warrant further investigation in the future in vivo models.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the supplementary material of this article.