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Letters to the Editor
activated by an N-RAS mutation5 and the phosphoryla- tion of the S6 ribosomal protein downstream of PI-3 kinase/AKT were not inhibited (Online Supplementary Figure S1). For the in vivo studies, a subline of INA-6 was used.5,8 In general, 25x106 INA-6.Tu1 cells were injected intraperitoneally into approximately 8-week-old female SCID/beige (C.B.-17.Cg-Prkdcscid Lystbg/Crl) mice (Charles River, Sulzfeld, Germany). All animal experiments were performed in strict adherence to German laws for animal welfare and were approved by the governmental authorities of Schleswig-Holstein. Animals were kept under specified pathogen-free condi- tions with free access to food and water in a light-dark cycle of 12 hours.
Blocking one single survival pathway may not be suffi- cient to eradicate myeloma cells in their tumor environ- ment.1 The choice of the anti-apoptotic Mcl-1 protein as a second target is based on the knowledge that Mcl-1 is a critical survival factor for myeloma cells and is upregu- lated by IL-6 produced in the bone marrow microenvi- ronment in a STAT3-dependent manner.9-11 An additional pathway leading to Mcl-1 upregulation, involving phos- phatase of regenerating liver (PRL)-3, has recently been identified.12 S63845, provided by Novartis, is a potent and selective BH3-mimetic with higher affinity for human than for murine Mcl-1.13
Ruxolitinib and S63845 were used in combination and the effects in vitro and in animal studies compared with those of the single agents. INA-6.Tu1 cell growth in vitro was dose-dependently inhibited by both drugs with a sig- nificantly greater effect in combination at higher concen- trations (Figure 2A). For the in vivo study, INC424 was freshly formulated in 0.5% w/v methylcellulose (Sigma- Aldrich, M0430) in sterile water every 3 to 4 days. S63845 was freshly dissolved in 2% D-α-tocopherol polyethylene glycol 1000 succinate (vitamin E-TPGS) (Sigma-Aldrich) in 0.9% sodium chloride solution shortly before every application. SCID/beige mice were inoculat- ed with INA-6.Tu1 cells as described above and treated for 10 consecutive days starting 1 day after injection of the cells (Figure 2B). Ruxolitinib was administered by oral gavage (60 mg/kg body weight) twice daily with a 6 h interval between the two doses. S63845 was injected intravenously at the dose of 25 mg/kg on days 1, 4, 7 and 10 according to the scheduling described previously.13 Mice were monitored regularly for signs of tumor growth. The survival time was defined as the time between cell inoculation and the day of sacrifice, which occurred before tumor burden caused paraplegia, cachex- ia, or any other signs of suffering. Animals without any signs of tumors were sacrificed at the end of the experi- ment on day 98 (Figure 2B). Treatment was well tolerated in all groups, as indicated by no body weight losses dur- ing the first 20 days (Online Supplementary Figure S2).
The Kaplan-Meier survival analysis (Figure 2C) shows that all mice of the control group (n=8) developed overt plasmacytomas and had to be sacrificed before day 40. The median survival time in this group was 23 days. A significant delay in tumor growth was observed in four out of seven mice treated with ruxolitinib, while three mice did not show any signs of tumors until the end of the experiment on day 98, resulting in a significantly pro- longed median survival time of 56 days (P<0.0001 by the log-rank test). Treatment of mice with the Mcl-1 inhibitor (n=8) prevented tumor growth in 50% of the animals and significantly prolonged the median survival time com- pared to that of the control group (P<0.0001). In mice treated with single agents, tumor growth seen in some of the animals was not caused by the development of drug
resistance, as the sensitivity to both ruxolitinib and S63845 was retained in explanted tumors (Online Supplementary Figure S3). Remarkably, none of the mice treated with the combination (n=6) showed any signs of disease; at day 98 the experiment was terminated and animals were found to be tumor-free. The combination therapy was significantly superior to treatment with rux- olitinib alone, as determined by the log-rank test (P=0.0325).
In INA-6-bearing mice, human soluble IL-6 receptors (sIL-6R) accumulate in the blood representing a tool for the detection of minimal residual disease.5 sIL-6R levels were measured in the serum taken from all mice at the time of sacrifice (Figure 2D). Mice with overt plasmacy- tomas, i.e., all mice of the control group and four mice each in the ruxolitinib and in the S63845 treatment groups, had measurable sIL-6R levels of up to 180 ng/mL (by enzyme-linked immunosorbent assay; Diaclone, Besançon, France). sIL-6R was not detected in any of the mice with long-term survival. These results strongly indi- cate that these mice were indeed free of INA-6 tumors.
Ruxolitinib is currently in early clinical evaluation for patients with relapsed/refractory multiple myeloma in combination with steroids, immunomodulatory drugs and proteasome inhibitors. Likewise, clinical trials with the highly selective Mcl-1 inhibitor S64315 (MIK665), a molecule resembling S63845, as well as other Mcl-1 inhibitors are underway.14 Inhibitors of JAK1/2 and Bcl-2 family proteins are synergistic in myeloid malignancies and are currently being evaluated.15 In myeloma, simulta- neously targeting JAK/STAT3 and Mcl-1 may either dis- turb one single signaling pathway or, more likely, block more than one pathway to efficiently control myeloma cell growth in vivo. The use of ruxolitinib with an Mcl-1 inhibitor in clinical studies is warranted.
Renate Burger, Anna Otte, Jan Brdon, Matthias Peipp and Martin Gramatzki
Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, University Medical Center Schleswig- Holstein and University of Kiel, Kiel, Germany
Correspondence:
RENATE BURGER - r.burger@med2.uni-kiel.de doi:10.3324/haematol.2020.276865
Received: November 30, 2020.
Accepted: April 13, 2021.
Pre-published: April 22, 2021.
Disclosures: no conflicts of interest to disclose.
Contributions: RB designed and conducted experiments, analyzed data, and wrote the manuscript; AO was responsible for the design and institutional approval of the animal experiments; JB conducted the animal experiments; MP helped with the animal experiments, reviewed the manuscript and discussed the results; MG supervised the research project, revised the manuscript and discussed the results.
Acknowledgments: the authors thank Kathrin Richter, Tanja Ahrens, Anna Böttiger, and the staff of the animal facility in Kiel for excellent technical assistance. Our special thanks go to Thomas Radimerski from Novartis, Basel, Switzerland, who supported us with the provision of the compounds and very helpful discussions.
References
1. Mughal TI, Girnius S, Rosen ST, et al. Emerging therapeutic para- digms to target the dysregulated JAK/STAT pathways in hematolog- ical malignancies. Leuk Lymphoma. 2014;55(9):1968-1979.
2. Quintás-Cardama A, Vaddi K, Liu P, et al. Preclinical characteriza-
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