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A.A. Cortés et al.
receptor and, therefore, constitutively activate the JAK/STAT pathway.13,14
Ruxolitinib, a JAK1/JAK2 inhibitor, is the first and only drug approved by the European Medicines Agency for the treatment of PMF, post-PV MF, and post-ET MF15 and is the first-line treatment for MF. Results of the COMFORT- I and II clinical trials showed that ruxolitinib produced a reduction in spleen volume, improved MF-related symp- toms, and was associated with prolonged overall survival of patients compared with controls.15
Despite the beneficial effect of ruxolitinib, a high per- centage of patients lose their response at some point dur- ing treatment, and others are refractory or present a sub- optimal response. Because of this, the use of drug combi- nations might increase the effectiveness of the treatment and response time, and overcome treatment resistance. Indeed, numerous studies have used this premise, and a number of combinations have been tested in clinical trials with varying success. For instance, whereas the combina- tion of ruxolitinib with simtuzumab (clinicaltrials.gov iden- tifier: 01369498) produced no clinical benefit,16 and the combination with lenalidomide (clinicaltrials.gov identifier: 01375140) had to be terminated early because the efficacy objectives were not achieved,17 the combination with danazol (clinicaltrials.gov identifier: 01732445) achieved a hematologic stabilization but did not increase the response to ruxolitinib.18 Other combinations including ruxolitinib with buparlisib (clinicaltrials.gov identifier: 01730248) or with panobinostat (clinicaltrials.gov identifiers: 01693601 and 01433445) are currently under evaluation in clinical trials. In this scenario, the objective of the present study was to develop a drug combination that enhances the effect of ruxolitinib in the treatment of MF.
The proposed combination in this work, ruxolitinib, nilotinib and prednisone, is the result of testing 17 drugs with ruxolitinib to evaluate the best therapy for MF. We hypothesized that this combination would be synergic through a decrease in the proinflammatory status by rux- olitinib and prednisone19 and the known antifibrogenic effect of nilotinib,20 and would result in a better histologi- cal response.
Methods
Primary samples and cell lines
Peripheral blood (PB) samples were collected from MF patients and from healthy donors after obtaining informed consent in accordance with the guidelines of the 12 Octubre Hospital ethics committee and the Declaration of Helsinki. The diagnosis of MF was based on 2016 World Health Organization criteria.21 PB mononuclear cells (PBMCs) were isolated from 6-10 mL of PB by density gradient centrifugation (Ficoll-PaqueTM PLUS, GE Healthcare, Little Chalfont, UK) and were cultured (0.4x106 cells/mL) in Methocult TM GF_H4535 supplemented with 20 ng/mL IL-3 and 50 ng/mL Stem Cell Factor (both from StemCell Technologies, Vancouver, Canada) at 37°C in a humidified atmos- phere containing 5% CO2. For the drug screening study, samples from 9 patients were used; age range was 49-83 years, there were 5 males and 4 females, and 6 of them harbored the JAK2-V617F mutation (Online Supplementary Table S1). For synergy studies, all patients (aged 66-83 years) had the JAK2-V617F mutation (3 males and 2 females). No patient had been treated previously (Online Supplementary Table S2).
The BA/F3 wild-type (BA/F3 WT), BA/F3 with JAK2-V617F
Table 1. Drugs used in the screening to search for the best combination with ruxolitinib.
Drug
Ruxolitinib
Nilotinib Bosutinib Ponatinib Midostaurin Sorafenib Buparlisib Dactolisib Everolimus Sonidegib SB 431542 LCL161 Bortezomib Panobinostat HSP990 Prednisone Anagrelide Danazol
Target
Jak 1/2 inhibitor
PDGF-R. c-kit and BCR/ABL inhibitor Src/Abl kinase inhibitor
BCR/ABL inhibitor
FLT3 inhibitor
Multikinase inhibitor
PI3K inhibitor PI3K/Akt/mTOR inhibitor
mTOR inhibitor
SMO inhibitor
Inhibitor of TGF-β receptor
SMAC mimetic
Proteosome inhibitor Deacetylase histone inhibitor
HSP90 inhibitor Immunosuppressant
PDE3 immunosuppressant Antigonadotropic and anti-estrogenic activity
mutation (BA/F3 V617F JAK2), and WEHI cell lines were kindly provided by Dr Quintás-Cardama (MD Anderson Cancer Center, Houston, TX, USA). The WEHI cell line, which produces IL-3, was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Biowest, Nuaillé, France) with 10% heat-inactivated fetal bovine serum (FBS). BA/F3 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Biowest, Nuaillé, Francia) with 10% FBS plus 10% conditioned medium from WEHI cells. The SET2 cell line (DSMZ, Braunschweig, Germany), which harbors the JAK2-V617F mutation, was cultured in RPMI 1640 with 20% FBS. The HS27a human BM mesenchymal cell line (DSMZ) was cultured in DMEM with 10% FBS.
Dose response and synergy analysis
A total of 10,000-20,000 cells of the different cell lines were seeded per well in 96-well plates in the presence of the drugs alone (Table 1 and Online Supplementary Table S3) or in combination with ruxolitinib. Dimethyl sulfoxide (DMSO) was used as vehicle. After 48-72 hours (h), cell viability was measured by flow cytom- etry with Annexin V-phycoerythrin (Biolegend, San Diego, CA, USA) or the metabolic WST-8 assay (Cell Counting Kit - 8 BioChemika; Sigma-Aldrich). Drugs were purchased from Sigma- Aldrich (St. Louis, MO, USA), Tocris (Bristol, UK) or kindly donat- ed by Novartis (Basel, Switzerland).
Peripheral blood mononuclear cells were treated as follows: a) directly delivering the drugs to methylcellulose solid culture at the outset of the experiment (ex vivo, model A); or b) after 14 days of methylcellulose culture (ex vivo, model B). In model B, colony- forming cells were collected, washed with phosphate buffer saline (PBS) and cultured in RPMI with 10% FBS at 15,000 cells per well in 96-well plates in the presence of the drugs alone (Table 1) or in combination with ruxolitinib, for 72 h. DMSO was used as vehicle at a maximum concentration of 0.5%. Flow cytometry to measure myeloid cell viability was performed with monoclonal antibodies against CD45-allophycocyanin-Cy7, CD13-allophycocyanin and Annexin V-phycoerythrin (all from Biolegend, San Diego, CA, USA) using the ExviTech automated flow cytometry plataform.
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haematologica | 2019; 104(5)