Page 162 - Haematologica Vol. 110 - January 2025

ARTICLE - Targeted CDK7 and BRD4 inhibition in myeloma
In our previous investigation, we delineated distinct regu- latory axes controlled by E2F1 and BRD4 in MM, with E2F predominately regulating growth/proliferation genes at active promoters and BRD4 mainly affecting enhancer-regulated tissue-specific genes.16 Intriguingly, E2F1 genetic depletion potentiated the anti-MM effect of JQ1 in vitro and in vivo.16,17 We recently uncovered CDK7, a kinase with a dual role in cell cycle progression and transcription,16 as a major driver of E2F1 activity in MM.17,18 Indeed, its suppression by the small molecule YKL-5-12417,19 exerted therapeutic effects in MM by mitigating cell cycle CDK plasticity and perturbing the E2F1 transcriptional program in an Rb-dependent manner.17 Given that E2F1 and BRD4 operate in two distinct regulatory axes in MM, we hypothesized that simultaneously targeting E2F1 (via CDK7 or CDK4/6 small molecule inhibitors) and BRD4 (via BET inhibitors) would be more effective at reducing tumor growth compared to targeting only one or the other with a single agent. Indeed, the data reported in this study suggest that the combination therapy halts tumor cell growth and viability, offering a promising therapeutic strategy to improve outcomes for patients with MM while minimizing the risk of resistance and toxicity associated with the use of high doses of single agents.
Methods
Cells
Human MM cell lines (MM1s, JJN3, OPM2, LR5, ANBL6WT, H929, AMO1, SKMM1, XG1, KMS12BM and IM9) and Walden- ström macroglobulinemia (WM) cell lines (BCWM1, MWCL1 and RPCIWM1) were cultured in RPMI-1640 (Gibco or KeyGEN BioTECH) supplemented with 10% fetal bovine serum (Gibco or Bio-channel), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin, with 2.5 ng/mL of interleukin-6 in ANBL6 and XG1 cells.
In accordance with the Declaration of Helsinki and under the oversight of the Ethics Consultation Service at Dana Farber Cancer Institute, primary MM cells and bone marrow mononuclear cells were isolated from bone marrow aspirates of myeloma patients using Ficoll-Hypaque density gradient sedimentation and anti-CD138 microbead separation. Pri- mary WM cells were separated from bone marrow samples of WM patients with anti-CD19 microbeads. Peripheral blood mononuclear cells were isolated from healthy donors using Ficoll-hypaque density gradient sedimentation and activated with 20 μg/mL phytohemagglutinin (InvivoGen, #inh-phap). Bone marrow stromal cells were established from mono- nuclear cells isolated via Ficoll-Hypaque density gradient centrifugation from bone marrow specimens of MM patients, as previously described.20,21
Reagents
YKL-5-124 was a kind gift from Nathanael S. Gray. JQ1, palbociclib, ARV-825 and I-BET151 were purchased from
Y. Yao et al. MedChemexpress. Compounds were dissolved in dimethyl
sulfoxide unless otherwise stated.
Cell viability, cell cycle and apoptosis assays
Cell viability was detected by CellTiter-Glo assay (Promega, G7572) or Cell Counting Kit-8 (VICMED). The cell cycle was measured by flow cytometric assay following staining with propidium iodide (BD Biosciences, 564907) and then ana- lyzed by ModFit software. Apoptosis was evaluated by flow cytometric analysis following staining with APC annexin-V (BD Biosciences, 561012) and DAPI (BD Biosciences, 564907).
RNA sequencing
AMO1 cells were treated with YKL-5-124 (50 nM) or JQ1 (200 nM) alone or in combination for 24 hours. RNA was extracted using a RNeasy Plus Mini Kit (QIAGEN, #74136) according to the manufacturer’s instructions, and then subjected to bulk RNA sequencing. RNA-sequencing analysis was performed using the HG19 ERCC human reference genome with HG19 ERCC gene annotations. Gene set enrichment analysis was performed via the computational platform of the Broad Institute with the Hallmark gene signature.
Murine xenograft model of human multiple myeloma or Waldenström macroglobulinemia
SCID mice were purchased from Charles River Laboratories. All animal studies were approved by and conducted accord- ing to the protocols of the Animal Ethics Committee of the Dana-Farber Cancer Institute. The mice were irradiated by 200 cGy and then inoculated subcutaneously in the right or left flank with 5×106 MM or WM cells. Tumor growth was measured in two dimensions by caliper, and volume was calculated using the formula: V=0.5×a×b2, where “a” and “b” represent the length and width of the tumor. The body weight of the animal was measured every week throughout the study to monitor the toxicity of drugs.
Statistical analysis
All values are displayed as mean ± the standard devia- tion. The statistical significance of differences between experimental variables was analyzed using the Student t test, or analysis of variance for multiple comparisons, with Prism GraphPad software. The significance of the P value is *P<0.05, **P<0.01, and ***P<0.001.
Results
Dual E2F1 and BRD4 inhibition leads to synergistic anti- myeloma effects
We evaluated the effect of dual inhibition of CDK7 and BET bromodomain proteins with low doses of YKL-5-124 and JQ1, respectively, in an extensive array of cell lines (N=11), to account for the molecular heterogeneity of MM cells and their varying response to therapy. We observed
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