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V.G. Ramakrishnan et al.
suggesting a role in disease progression.7,10 Furthermore, >70% of patients have RAS/RAF mutations present at relapse.11 It follows that directly targeting RAS/RAF/MEK/ERK in MM could be a promising strate- gy. However, MEK inhibition is merely cytostatic in MM in vitro.12 Furthermore, a clinical trial evaluating MEK inhibitor monotherapy in MM showed limited efficacy.13 Thus, it appears that for MEK inhibitors to be relevant in MM, they must be combined with other agents.
Recently, histone deacetylase (HDAC) inhibitors have shown significant activity in numerous tumor types, both in vitro and in the clinic.14 In fact, the pan-HDAC inhibitor LBH589 (panobinostat) was recently approved for treating relapsed/refractory MM patients in combination with bortezomib.15 As chemotherapeutic agents, HDAC inhibitors have been shown to inhibit cell survival and pro- liferation and enhance immune-mediated cytotoxicity.14,15
We hypothesized that LBH589 could induce enhanced apoptosis when combined with MEK inhibition in MM. Our hypothesis stemmed from two considerations: (i) MEK inhibitors induce apoptosis in several other RAS/RAF mutated cancers,16,17 suggesting MM-specific resistance factors, and (ii) HDAC inhibitors kill MM cells through several known mechanisms, including modulation of the pro- and anti-apoptotic BCL-2 family members, which often mediate chemoresistance.15,18-20
In the present study, we show that MEK inhibition with AZD6244 (selumetinib), when combined with LBH589, synergistically drives intrinsic apoptotic cell death in MCL- 1 “primed” RAS/RAF mutated MM cell lines. Mechanistically, MEK inhibition increases BIM levels; LBH589 acts as a de facto MCL-1 and BCL-XL inhibitor, dis- sociating BIM:MCL-1 and BIM:BCL-XL complexes. In con- trast, we demonstrate that LBH589 synergizes with the BH3 mimetic ABT-199 (venetoclax) in BCL-2 “primed” cell lines, which are resistant to the AZD6244/LBH589 combi- nation. Finally, we show that concomitant inhibition of HDAC1 and HDAC2 is sufficient to synergize with either MEK or BCL-2 inhibition in the same distinct MM cell lines.
Given that refractoriness to whole classes of drugs (e.g. proteasome inhibitors) is the final common endpoint for nearly all patients with MM,21 the agents in this study are felicitous because they work via alternative mechanisms of action, are already approved or in clinical development, and offer the tantalizing prospect of targeted therapy guid- ed by RAS/RAF mutational status and MCL-1/BCL-2 func- tional dependence.
Methods
Ethics
This study was approved by the Mayo Clinic Institutional Review Board. Patients’ cells were collected after informed con- sent, in adherence to the Declaration of Helsinki.
Multiple myeloma cell lines and patients’ cells
DOX40, H929, KMS11, KMS18, KMS28BM, MM1S, MM1R, OPM1, OPM2, RPMI8226 and U266 were obtained (see Online Supplementary Methods).19,22 Briefly, all cell lines were cultured in RPMI 1640 medium (Mediatech Inc., Manassas, VA, USA) con- taining 10% fetal bovine serum (Mediatech, Inc.). Freshly obtained bone marrow aspirates from MM patients were collected after informed consent, then CD138+ or bone marrow stromal cells were sorted and cultured as previously described.19,22
Reagents
ABT-199 was generously provided by Abbvie (Chicago, IL, USA). AZD6244, MEK162, SCH772984, SAHA, LBH589, MS275 and FK228 were purchased from Selleckchem (Houston, TX, USA). Tubacin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions were made in dimethylsulfoxide, aliquoted and stored at −20°C.
MTT, proliferation, and apoptosis assays
Cellular viability was measured using 3-(4, 5-dimethylthiazol-2- yl)-2, 5-diphenyl tetrasodium bromide (MTT) (Chemicon International Inc., Temecula, CA, USA) colorimetric assays at the indicated time points. Proliferation arrest assays were completed using 3H-thymidine uptake as previously described.23 Apoptosis of patients’ cells was assayed using annexin/propidium iodide (PI) as previously described.22,23 Briefly, cells were washed twice with annexin binding buffer (ABB: 10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and then 100 μL cells (107/mL) were stained for 15 min at room temperature with 3 μL of annexin V-FITC (Caltag, Burlingame, CA, USA), then washed with ABB and re- suspended in 500 μL of ABB with 5 μL of 1 mg/mL PI (Sigma- Aldrich, St. Louis, MO, USA). The samples were then run on a Canto flow cytometer (BD Biosciences, San Jose, CA). All experi- ments with MM cell lines were performed in triplicate.
Western blotting
MM cell lines were lysed with NP40 buffer, 1 mM phenyl- methyl-sulfonyl-fluoride (PMSF), protease inhibitor cocktail (PIC), and 1 mM HALT Phosphatase Inhibitor (Thermo Fisher Scientific, Rockford, IL, USA). Protein concentrations were measured using the BCA assay (Thermo Fisher). First, 20-25 μg were loaded on 4- 20% Tris-Glycine gels and transferred onto polyvinylidene difluo- ride membranes (Bio-Rad, Hercules, CA , USA). Antibodies for acetylated histone 3, BAK, BAX, BCL-2, BCL-XL, BIM, caspase 8, caspase 9, ERK, GAPDH, HDAC1, HDAC2, HDAC6, MCL-1, PARP, p-BCL-2 (S70), pERK, p-MCL-1 (S64), and p-MCL-1 (T163) were purchased from Cell Signaling Technology (Danvers, MA, USA) and used for probing as previously described.19,22 All western blot experiments were performed in triplicate with a representa- tive blot shown.
Immunoprecipitation
Proteins (100-150 μg) were incubated in a total volume of 500 μL of NP40 buffer, 1 mM PMSF, 1 mM PIC, 1 mM HALT Phosphatase Inhibitor (Thermo Fisher Scientific), and a 1:100 dilution of the following primary antibodies for 4 h: BAX 6A7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (Millipore Sigma, Burlington, MA, USA), and BCL-2, BCL-XL, BIM and MCL-1 (Cell Signaling Technology). Samples were then incubated with ChIP-Grade Protein G Magnetic Beads (Cell Signaling Technology) for 12 h, washed five times, boiled in 2x Laemmli Sample Buffer dye (Bio-Rad) at 100 oC for 5 min, then loaded on 4-20% Tris-Glycine gels (Bio-Rad) and probed as described above. All experiments were performed in triplicate with a representative blot shown.
Short interfering RNA transfection
Short interfering (si)RNA for BIM, BAX, BAK, HDAC1, HDAC2 and HDAC6 were purchased from Thermo Fisher Scientific. ERK1 and ERK2 siRNA were purchased from Santa Cruz Biotechnology. siRNA were electroporated into MM cell lines using the Lonza nucleofector kit V (Lonza, Basel, Switzerland). The manufacturer’s G-15 program was used for KMS18 and OPM2; O-23 was used for MM1S and KMS28. All experiments were performed in triplicate.
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haematologica | 2019; 104(10)