CASC5 (E4A5L) Rabbit mAb 26687T from Cell Signaling Technology

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Nature
August 20 2025

Fig 1: Pharmacokinetic and Pharmacodynamic Studies of Cyclin A/B RxL Inhibitors in SCLC Cell-Line Xenograft and Patient-Derived Xenograft Models.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). Source Data
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Nature
August 20 2025

Fig 2: Cyclin A/B RxL inhibitors exhibit on-target anti-tumour activity in SCLC mouse models in vivo.a,b, Tumour volume curves of NCI-H69 (a) and NCI-H1048 (b) xenografts in athymic nude mice treated with vehicle or cyclin A/B RxL inhibitor (CIRc-028) dosed at 100 mg per kg (mpk) intravenously (i.v.) once per day for 14 days. n = 10 mice per arm (a), and n = 10 (vehicle) and n = 8 (CIRc-028) mice (b). Data are mean ± s.e.m. Statistical analysis was performed using two-way analysis of variance (ANOVA); P < 0.0001. c,d, Representative IHC analysis of p-KNL1 (c) and cleaved caspase-3 (d) in NCI-H69 tumours 18 h after treatment. e,f, Quantification of c (e) and d (f). n = 5 tumours from independent mice per group. Data are mean ± s.d. Statistical significance calculated using unpaired two-tailed Student’s t-tests. g, Schematic for DFCI-393 and DFCI-402 PDX studies with oral cyclin A/B RxL inhibitor CIRc-014. h, mRNA expression of SCLC transcription factors and neuroendocrine markers by RNA-seq. i,j, Tumour volume curves of DFCI-393 (i) and DFCI-402 (j) PDXs treated with CIRc-014 (100 mg per kg orally three times per day (i) or twice per day (j)) or vehicle for 28 days. The dashed lines indicate the start and end of treatment. For i, n = 10 (vehicle) and n = 9 (CIRc-014) mice. For j, n = 10 mice per arm. Data are mean ± s.e.m. Statistical analysis was performed using two-way ANOVA; P < 0.0001. k,l, Representative IHC analysis of p-KNL1 (k) and cleaved caspase-3 (l) in DFCI-393 tumours treated with CIRc-014 (100 mg per kg orally three times per day) for 4 days. m,n, Quantification of k (m) and l (n). For m and n, n = 5 (vehicle) and 6 (CIRc-014) tumours from independent mice. Data are mean ± s.d. Statistical significance was calculated using unpaired two-tailed students t-tests. o, The proposed mechanism: in cancers with compromised G1–S checkpoint (for example, RB1 and TP53 loss) and consequently high E2F activity, cyclin A binds to activating E2Fs through RxL motifs to dampen E2F activity, and cyclin B binds to MYT1 to restrain CDK1 activity during G2. Cyclin A/B RxL inhibitors disrupt both interactions: (1) blocking cyclin A–E2F1 repression increases replication stress; (2) blocking cyclin B–MYT1 binding allows cyclin B to form a neomorphic complex with CDK2, driving RxL-independent phosphorylation (of, for example, stathmin), SAC activation and mitotic death. The diagrams in g and o were created with BioRender. Scale bars, 50 µm (c,d), and 100 µm (×20) and 50 µm (×40) (k,l).Source Data
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Nature
August 20 2025

Fig 3: Cyclin A/B RxL Inhibitors Promote SAC Activation in Sensitive SCLC Cell Lines which Requires Loss of RB1 and TP53.a, Representative images from time-lapse fluorescent microscopy of NCI-H1048 cells expressing H2B-GFP treated with 20 nM cyclin A/B RxL inhibitor (CIRc-004) over 10 h (yellow star: arrested mitotic nuclei, purple star: normal mitotic nuclei, red star: apoptotic nuclei). Magnification = 20x, scale bar 10 µm. b, Dot plot showing time (mins) from chromosome condensation to cytokinesis completion in CIRc-004 vs vehicle treated cells. Cells that failed to complete cytokinesis by experimental end point are shown in red. n = 50 total mitotic nuclei per condition were counted from 2 independent experiments. Statistical significance was calculated using unpaired, two-tailed students t-test. c, Representative phospho-KNL1, DAPI and anti-centromere antibody (ACA) confocal microscopy images of NCI-H1048 cells treated with cyclin A/B RxL inhibitor (CIRc-004) at 20 nM or DMSO for 24 h. Magnification = 63x, scale bar = 10 µm. d-e, Dot plot measuring fluorescence intensity of phospho-KNL1 per mitotic cell (d) or number of phospho-KNL1 foci per mitotic cell (e) of NCI-H1048 cells from c n = 45 mitotic cells from 3 biological replicates. f, Immunoblot analysis of the indicated human SCLC cell lines (NCI-H1048, NCI-H446, and NCI-H69) and insensitive human NSCLC cell lines (A549, HCC4006, and NCI-H1299) treated for 24 h with CIRc-004 at 200 nM or DMSO. Note, this immunoblot contains the same lysates from Fig. 2i and is included to ensure that the differences in phospho-KNL1 levels after CIRc-004 treatment between cell lines are not a consequence of being loaded on independent immunoblots. g-h, Immunoblot analysis comparing p-KNL1 levels of insensitive NCI-H82 (g) and RPE1 (h) cell lines after treatment with CIRc-004 for 24 h. NCI-H1048 cells (a sensitive model) are included as a benchmark control. i-j, Bar graphs of significantly enriched Hallmark pathways (Padj <0.05) from RNA-seq comparing CIRc-004 vs. DMSO (i) and CIRc-005 vs. DMSO (j). For, i, j, n = 2 biological replicates. k, Representative histograms of flow cytometric analysis of PI stained RPE1 cells transduced with indicated sgRNAs targeting RB1 and TP53 (sgRB1, sgTP53) or a non-targeting control (sgCtrl) and then treated with CIRc-004 or DMSO for 72 h. n = 3 biological independent experiments. l, Immunoblot analysis of indicated RPE1 cells and NCI-H1048 treated with CIRc-004 or DMSO for 72 h. Representative immunoblot from 3 independent experiments is shown. Where indicated, *=p < 0,05, **=p < 0.01, ***=p < 0.001, ****=p < 0.0001. Source Data

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CASC5 (E4A5L) Rabbit mAb

Cell Signaling Technology's CASC5 (E4A5L) Rabbit mAb is a Rabbit monoclonal antibody. The CASC5 (E4A5L) Rabbit mAb Antibody was generated using KNL1 as the antigen. It reacts with Homo Sapiens, and Human. This antibody has been shown to work in applications such as: Precipitation, Immunocytochemistry, Immunofluorescence, Immunoprecipitation, and Western Blot.

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CASC5 (E4A5L) Rabbit mAb from Cell Signaling Technology

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CASC5 (E4A5L) Rabbit mAb from Cell Signaling Technology

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