Fig 2: Selective CDK7 inhibition with YKL-5-124 leads to aberrant cell cycle progression in NB cells. (A) Analysis of target engagement in NB cells following THZ1 or YKL-5-124 treatment. Cells were treated with THZ1, YKL-5-124 or DMSO for 6 h at the indicated doses and cell lysates were incubated with 1 μM of biotinylated THZ1 (bio-THZ1) overnight. Western blot (WB) analysis of CDK7 and CDK12 in the immunoprecipitate (PD). (B) Dose-response curves for NB cells treated with increasing concentrations of YKL-5-124 or THZ1 for 72 h. Cytotoxicity is reported as percent cell viability relative to DMSO-treated cells. Data represent mean ± SD; n = 3. (C) Flow cytometry analysis of EdU staining in NB cells treated with 100 nM THZ1, 100 nM YKL-5-124 or DMSO for 24 h (left). The percentages of living cells in each phase of the cell cycle are shown. Quantification of staining (right), data represents mean ± SD; n = 3. (D) WB analysis of pCDK1 (Thr161), pCDK2 (Thr160) and total CDK1/2 in NB cells treated with YKL-5-124 or THZ1 at indicated doses for 6 h.

Fig 3: CDK7 is a direct transcriptional target of MITF and c-MYC. a Time course of CDK7 mRNA expression in 501 mel cells transfected with siMITF1 vs. control siRNA under treatment with phorbol ester (TPA). Data represent mean ± SD from technical triplicates (two-tailed paired t-test). b Immunoblot analysis of MITF, c-MYC and CDK7 protein expression in 501 mel cells under TPA treatment. Lower and upper arrowheads indicate non-phosphorylated (54 kd) and phosphorylated (60 kd) MITF forms, respectively. Actin used as loading control. c ChIP-seq tracks of MITF (marked red) and c-MYC (marked blue) binding signals at CDK7 gene locus in primary melanocytes and primary melanomas, melanoma cell lines and non-melanocytic cancer cells, respectively. Green arrowhead indicates E box consensus sequence −86 base pairs upstream of the transcriptional start site of CDK7. GEO accession numbers of corresponding data sets are listed under Materials and methods. d ChIP, in vivo occupancy of MITF at CDK7 promoter in 501 mel cells in the presence or absence of forced c-MYC expression (OE overexpression) compared to empty vector (EV) including intron sequence and IgG controls. Mean ± SD from technical triplicates. e ChIP, in vivo occupancy of exogenous c-MYC (OE) at CDK7 promoter in 501 mel cells under siSCR or siMITF1 transfection. Intron sequence and IgG were used as controls. Mean ± SD is presented from technical triplicates. d, e Two-tailed paired t-test. a, b, d, e Experiments were performed twice and biological replicates revealed very similar results

Fig 4: The combination of YKL-5-124 and JQ1 leads to improved therapeutic efficacy in vivo. (A) Tumor volumes of IMR-32 NB xenografts in NSG mice treated with 2.5 mg/kg YKL-5-124, 25 mg/kg JQ1, JQ1 and YKL-5-124 or vehicle control (5DW +10% DMSO + 10% HP-beta-cyclodextrin) QOD including weekends for 24 days. Mean ± SEM values are presented. (n=5, all arms) ***P < 0.001, as compared to vehicle. (B) Kaplan-Meier survival curves for the experiment described in (A) but with mice with BCS<2 excluded. ***P < 0.001, ****P < 0.0001 (log-rank test). (C) Immunohistochemical (IHC) analysis of morphology (hematoxylin & eosin, H&E), proliferation (Ki67), apoptosis (cleaved caspase 3) and MYCN in tumors harvested from animals treated as described in (A) for 7 days. Magnification, 40X. (D) Tumor volumes of PDX tumors in NSG mice treated with the indicated agents at similar doses as in (A) or vehicle control three times per week for 28 days (14 total treatments). Mean ± SEM values are presented. (vehicle n=8, other arms n=6). (E) Kaplan-Meier survival curves for the experiment described in (C) **P < 0.01, ns, not significant (log-rank test). (F) Analysis of target engagement in tumors from mice treated as described in (C) Lysates prepared from homogenized tumor tissue were incubated with bio-THZ1 for 24 h at 4°C and immunoprecipitated proteins analyzed for CDK7 by WB.

Fig 5: Model of TFIIH-CAK regulation by MITF and c-MYC. In the melanocytic lineage, the microphthalmia-associated transcription factor (MITF) determines transcriptional homeostasis and genomic integrity through regulation of the transcription factor II H (TFIIH) and the CDK-activating kinase (CAK) complex. (I) MITF controls general transcription and UVR-induced nucleotide excision repair by targeting the transcription initiation complex through direct transactivation of GTF2H1 as a core element of TFIIH. (II) The melanocyte master regulator MITF and its structural homolog c-MYC control the expression of CDK7, the catalytic subunit of CAK complex, which has a dual role in transcription and cell cycle activities. (III) MITF controls MYC activities by transactivation of fuse binding protein (FUBP2) involved in pulse regulation of MYC. In addition, MYC is likely affected by MITF-dependent effects on proliferation