Extended Data Fig. 11: Pharmacokinetic and Pharmacodynamic Studies of Cyclin A/B RxL Inhibitors in SCLC Cell-Line Xenograft and Patient-Derived Xenograft Models.
From: Targeting G1–S-checkpoint-compromised cancers with cyclin A/B RxL inhibitors
a, b, Body weights of mice enrolled in the NCI-H69 (a) or NCI-H1048 (b) xenografts efficacy treatment studies treated for 14 days with vehicle, cyclin A/B RxL inhibitor (CIRc-028, 100 mg/kg IV QD). For a, n = 10 independent mice per arm. For b, n = 10 independent mice for vehicle and n = 8 independent mice for CIRc-028 arm. QD=Every day. c, Representative IHC micrographs of cell pellets from NCI-H1048 cells treated with CIRc-004 at 200 nM or DMSO (vehicle) overnight and then stained for phospho-KNL1 to validate the phospho-KNL1 antibody for IHC. Magnification=20x, scale bar = 50 µm. d, Biochemical activity of the orally bioavailable cyclin A/B RxL macrocyclic inhibitor (CIRc-014) for cyclin A1/Cdk2, cyclin E1/Cdk2 and cyclin B/CDK1 complexes measured by Fluorescence Polarization. CIRc-014 was used for in vivo efficacy studies in SCLC PDX models in Fig. 5g–n. e, Heat map of z-scores from RNA-seq data of NCI-H1048 cells treated with cyclin A/B RxL inhibitor (CIRc-004 or CIRc-014 at 200 nM), inactive enantiomer (CIRc-005 at 200 nM) or DMSO for 24 h. Data is sorted for Log2FoldChange of CIRc-004 (replicate 1 and 2) vs. DMSO (replicate 1 and 2) showing genes with Padj<0.05 which was 150 top up-regulated and down-regulated genes. Relevant up-regulated genes shown on the right. f, Bar graphs showing top significantly enriched Hallmark pathways (padj<0.05) calculated using differentially expressed genes from bulk RNA-seq experiment in e comparing CIRc-014 vs. DMSO in NCI-H1048 cell line. For e, f, n = 2 biological replicates. g, Principal component analysis (PCA) from RNA-seq data integrating RNA-seq data from DFCI-393 and DFCI-402 PDX models with 81 primary SCLC human samples from George et al. Nature 20151. Bar scale shows neuroendocrine score (see Methods). h,i, Body weights of mice enrolled in the DFCI-393 SCLC PDX (h) or DFCI-402 SCLC PDX (i) efficacy studies treated for 28 days with CIRc-014 (100 mg/kg PO TID for DFCI-393 and 100 mg/kg PO BID for DFCI-402) or vehicle. For DFCI-393 PDX in h, n = 10 independent mice for vehicle and n = 9 independent mice for CIRc-014. For DFCI-402 PDX in i, n = 10 independent mice per arm for both vehicle and CIRc-014. TID=three times a day; BID=two times a day. j, Plasma concentration of CIRc-014 from NSG mice bearing DFCI-393 PDX tumours treated with CIRc-014 100 mg/kg PO TID in the pharmacodynamic study in Fig. 5k–n. Mice were dosed for 4 days and plasma was collected at the times indicated after the last dose. Unbound concentrations (nM) at indicated time points shown on the right. Data are mean +/− SEM. n = 2 for 1-hour time point; n = 3 independent mice for 30-min, 2 and 4-hour timepoints; and n = 6 mice for 8-hour timepoint. k, l, Plasma concentration of CIRc-028 at the end of the efficacy studies where CIRc-028 was dosed 100 mg/kg IV QD for 14 days in CDX models of NCI-H69 model (k) shown in Fig. 5a or NCI-H1048 model (l) shown in Fig. 5b. Unbound concentrations (nM) at indicated time points shown on the right. Data are mean +/− SEM. For k, n = 5 mice for each time point. For l, n = 8 mice for each time point. For j, study run in NSG mice and NOD SCID % plasma protein binding (PPB) used to estimate unbound fraction. For k-l, studies run in Athymic Nude and BALB/c Nude mice, respectively, and % PPB from each strain was used to estimate unbound fraction. Plasma was collected from mice after the last dose and CIRc-014 or CIRc-028 concentrations were determined by LC-MS/MS. m, Heat map of z-scores from RNA-seq data of DFCI-393 human SCLC PDX from Fig. 5k–n treated with CIRc-014 (100 mg PO TID) or vehicle for 4 days. Values are sorted for Log2FoldChange of CIRc-014 treated tumours (n = 6 tumours from independent mice) vs. vehicle treated tumours values (n = 5 tumours from independent mice) showing genes with Padj<0.05 which was 39 differentially expressed genes. Relevant up-regulated genes shown on the right. The NCI-H1048 cell line treated with cyclin A/B RxL inhibitors (CIRc-004, CIRc-014 at 200 nM), inactive enantiomer (CIRc-005 at 200 nM), or DMSO (vehicle) as shown in Extended Data Fig. 11e is included as a benchmark control showing overlap of genes upregulated in DFCI-393 PDX tumours after CIRc-014 and NCI-H1048 cell lines treated with cyclin A/B RxL inhibitors (CIRc-004 and CIRc-014). n, Bar graphs showing top significantly enriched Hallmark pathways (padj<0.05) calculated using differentially expressed genes from bulk RNA-seq experiment in m comparing CIRc-014 treated DFCI-393 PDX tumours (n = 6 tumours from independent mice) to vehicle treated DFCI-393 PDX tumours (n = 5 tumours from independent mice).
