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Enitociclib delivers robust inhibition of RNA polymerase II Ser2 phosphorylation for up to 48 hours in cell lines. A, The gene expression of MYC, and MCL1 as determined by RNA-seq expressed as transcripts per million (TPM) from parental untreated SU-DHL-4 and SU-DHL-10 DLBCL cells. B, SU-DHL-4 and SU-DHL-10 DLBCL cells were seeded 18 hours prior to treatment with one of three CDK9 inhibitors: enitociclib (0.25 or 1 µmol/L), atuveciclib (1 µmol/L), or KB-0742 (1 µmol/L). Following the 4-hour treatment, the cells were washed, and the incubation continued for up to 48 hours. Samples were collected 4 hours before treatment (−4), immediately after treatment (0), and at 1, 2, 4, 8, 12, 16, 24, and 48 hours after washout for RNA-seq, qPCR, and Western blotting analysis. C, Western blots of phosphorylated Ser2 RNA polymerase II (p-Ser2-RNApolII), total RNApolII and HSP90 ( D ) quantification of p-Ser2-RNApolII protein levels (p-Ser2) in SU-DHL-4 and SU-DHL-10 DLBCL cells normalized to HSP90 as a ratio to DMSO matched timepoint.

Journal: Cancer Research Communications

Article Title: Enitociclib, a Selective CDK9 Inhibitor, Induces Complete Regression of MYC+ Lymphoma by Downregulation of RNA Polymerase II Mediated Transcription

doi: 10.1158/2767-9764.CRC-23-0219

Figure Lengend Snippet: Enitociclib delivers robust inhibition of RNA polymerase II Ser2 phosphorylation for up to 48 hours in cell lines. A, The gene expression of MYC, and MCL1 as determined by RNA-seq expressed as transcripts per million (TPM) from parental untreated SU-DHL-4 and SU-DHL-10 DLBCL cells. B, SU-DHL-4 and SU-DHL-10 DLBCL cells were seeded 18 hours prior to treatment with one of three CDK9 inhibitors: enitociclib (0.25 or 1 µmol/L), atuveciclib (1 µmol/L), or KB-0742 (1 µmol/L). Following the 4-hour treatment, the cells were washed, and the incubation continued for up to 48 hours. Samples were collected 4 hours before treatment (−4), immediately after treatment (0), and at 1, 2, 4, 8, 12, 16, 24, and 48 hours after washout for RNA-seq, qPCR, and Western blotting analysis. C, Western blots of phosphorylated Ser2 RNA polymerase II (p-Ser2-RNApolII), total RNApolII and HSP90 ( D ) quantification of p-Ser2-RNApolII protein levels (p-Ser2) in SU-DHL-4 and SU-DHL-10 DLBCL cells normalized to HSP90 as a ratio to DMSO matched timepoint.

Article Snippet: Genetic status and gene expression level for MYC and MCL1 was obtained from CrownBioscience.

Techniques: Inhibition, Phospho-proteomics, Gene Expression, RNA Sequencing, Incubation, Western Blot

Enitociclib depletes mRNA transcripts, MYC and MCL1; downregulates Myc and Mcl-1 protein levels and activates cell death in vitro MYC ( A ) and MCL1 ( B ) mRNA levels were determined using qPCR normalized to 18s rRNA from SU-DHL-10 and SU-DHL-4 DLBCL cells treated with indicated concentration of CDK9 inhibitor for 4 hours. The protein levels of Myc ( C ) and Mcl-1 ( D ) pretreatment and at indicated times after treatment washout were quantified relative to GAPDH in SU-DHL-10 and SU-DHL-4 cells. The inset shows Myc protein levels in SU-DHL-10 cells until 8 hours after treatment. E, The protein levels of cPARP is quantified from SU-DHL-10 DLBCL cells pretreatment and at indicated times after treatment.

Journal: Cancer Research Communications

Article Title: Enitociclib, a Selective CDK9 Inhibitor, Induces Complete Regression of MYC+ Lymphoma by Downregulation of RNA Polymerase II Mediated Transcription

doi: 10.1158/2767-9764.CRC-23-0219

Figure Lengend Snippet: Enitociclib depletes mRNA transcripts, MYC and MCL1; downregulates Myc and Mcl-1 protein levels and activates cell death in vitro MYC ( A ) and MCL1 ( B ) mRNA levels were determined using qPCR normalized to 18s rRNA from SU-DHL-10 and SU-DHL-4 DLBCL cells treated with indicated concentration of CDK9 inhibitor for 4 hours. The protein levels of Myc ( C ) and Mcl-1 ( D ) pretreatment and at indicated times after treatment washout were quantified relative to GAPDH in SU-DHL-10 and SU-DHL-4 cells. The inset shows Myc protein levels in SU-DHL-10 cells until 8 hours after treatment. E, The protein levels of cPARP is quantified from SU-DHL-10 DLBCL cells pretreatment and at indicated times after treatment.

Article Snippet: Genetic status and gene expression level for MYC and MCL1 was obtained from CrownBioscience.

Techniques: In Vitro, Concentration Assay

Enitociclib treatment results in complete regression of MYC -overexpressing SU-DHL-10 lymphoma growth and mechanism of action confirmed in vivo . A, Growth curves where red arrows indicate dosing days and tumor volumes of SU-DHL-10 tumors treated with either vehicle [30 or 60% PEG400, 10% ethanol (EtOH), water for infusion] or enitociclib at 10 or 15 mg/kg (once weekly, i.v., n = 12/group) for 3 weeks. On days 16 and 20, the T/C ratios for 10 and 15 mg/kg enitociclib were 0.19 and 0.005, respectively. Unpaired t test were performed using comparison with the corresponding vehicle treatment: **, P = 0.011; ***, P < 0.001. B, Body weight changes were observed after application of enitociclib followed by recovery before the next application. C, Phosphorylated RNA polymerase II protein levels at residue Ser2 (p-Ser2) and Ser5 (p-Ser5) were quantified MYC and MCL1 mRNA levels ( D ) assessed by qPCR and Myc, Mcl-1, cPARP protein levels (E) were quantified from in vivo xenograft of SU-DHL-10 DLBCL cells at predose and indicated times postdose in vivo . Error bars denote SD.

Journal: Cancer Research Communications

Article Title: Enitociclib, a Selective CDK9 Inhibitor, Induces Complete Regression of MYC+ Lymphoma by Downregulation of RNA Polymerase II Mediated Transcription

doi: 10.1158/2767-9764.CRC-23-0219

Figure Lengend Snippet: Enitociclib treatment results in complete regression of MYC -overexpressing SU-DHL-10 lymphoma growth and mechanism of action confirmed in vivo . A, Growth curves where red arrows indicate dosing days and tumor volumes of SU-DHL-10 tumors treated with either vehicle [30 or 60% PEG400, 10% ethanol (EtOH), water for infusion] or enitociclib at 10 or 15 mg/kg (once weekly, i.v., n = 12/group) for 3 weeks. On days 16 and 20, the T/C ratios for 10 and 15 mg/kg enitociclib were 0.19 and 0.005, respectively. Unpaired t test were performed using comparison with the corresponding vehicle treatment: **, P = 0.011; ***, P < 0.001. B, Body weight changes were observed after application of enitociclib followed by recovery before the next application. C, Phosphorylated RNA polymerase II protein levels at residue Ser2 (p-Ser2) and Ser5 (p-Ser5) were quantified MYC and MCL1 mRNA levels ( D ) assessed by qPCR and Myc, Mcl-1, cPARP protein levels (E) were quantified from in vivo xenograft of SU-DHL-10 DLBCL cells at predose and indicated times postdose in vivo . Error bars denote SD.

Article Snippet: Genetic status and gene expression level for MYC and MCL1 was obtained from CrownBioscience.

Techniques: In Vivo, Comparison, Residue