Article Figures & Data
Figures
- Figure 1.
Cancer cell sensitivity profile of prexasertib. Prexasertib was evaluated for efficacy in a panel of more than 300 adult and pediatric cancer cell lines. Average EC50 values for proliferation for a subset of these lines after prexasertib treatment is displayed here and are grouped by histology. Several pediatric tumor types, including neuroblastoma, exhibited sensitivity to prexasertib treatment over two cell doublings, with EC50 values within the clinically achievable range based on the average plasma concentration 24 hours postinfusion (Cavg, 24) reported in the phase I trial in adult patients with solid tumors (Hong 2016). NS, not specified.
- Figure 2.
Prexasertib reduces neuroblastoma cell proliferation and inhibits CHK1 autophosphorylation. A, Neuroblastoma cell lines, PANC-1, and primary melanocytes were assayed for proliferation after 72 hours of prexasertib treatment and EC50 values were calculated. Experiments were repeated in triplicate, and error bars represent SEM. B, After 24 or 48 hours of treatment with 50 nmol/L prexasertib, cells were lysed, and the indicated total and phosphorylated proteins were assessed by Western blot analysis.
- Figure 3.
Prexasertib-induced double-strand DNA breaks leads to neuroblastoma cell death in vitro. KELLY, NBL-S, PANC-1, and primary melanocytes were incubated with DMSO or 50 nmol/L prexasertib for 24 hours and subsequently fixed. Cells were immunostained for γH2AX (green) and cleaved PARP (red). DNA was stained with Hoescht 33342 (blue). Representative single channel and composite images taken with a 20× objective using the appropriate filters are shown. Experiments were repeated at least twice.
- Figure 4.
CHK1 is the functional target of prexasertib in neuroblastoma. CHK1 and CHK2 were knocked down individually and in combination with siRNA for 72 hours, then treated with 50 nmol/L prexasertib for an additional 24 hours. A, Whole-cell lysates were analyzed by Western blot analysis for expression and/or phosphorylation of the indicated proteins. B, Effects of siRNA against CHK1 or CHK2 with or without additional prexasertib treatment on cell proliferation were evaluated by CellTiter Glo in KELLY (top) and PANC-1 (bottom). Error bars represent SEM from technical triplicates.
- Figure 5.
Neuroblastoma xenografts rapidly regress during prexasertib treatment. KELLY (A) or IMR-32 (C) xenograft models were treated subcutaneously with vehicle or 10 mg/kg prexasertib twice daily following a 3 day on, 4 day off dosing schedule for 4 weeks (n = 5 for all arms). KELLY (B) and IMR-32 (D) xenografts were harvested after 6 days of vehicle or prexasertib treatment and subjected to an immunofluorescence-based tumor health panel. Cell proliferation, cell death, and vessel-positive area were measured by Ki67, TUNEL, and MECA-32 immunostaining, respectively. Average percent positive area (= 100 × marker + phantoms/total Hoescht + phantoms) for each group (KELLY: n = 5/group; IMR-32: n = 6/group) ± SEM is displayed below representative images taken at a 10X magnification. Vehicle:
; prexasertib starting at 200 mm3 average tumor volume: ; prexasertib starting at 500 mm3 average tumor volume: . Thin and thick dashed lines represent dosing period for 200 mm3 and 500 mm3 starting tumor volume, respectively. *, P < 0.05; ***, P < 0.0001.
Tables
- Table 1.
EC50 (μmol/L) comparison of prexasertib and SOC in pediatric cancer cell lines
Cell line Cancer type Prexasertib Doxorubicin Cisplatin Gemcitabine MOLT-4 <0.001 0.026 1.121 0.014 MOLT-3 Acute lymphocytic leukemia <0.001 0.006 0.227 0.003 CCRF-CEM <0.001 0.036 1.443 0.017 RD-ES Ewing sarcoma 0.003 0.267 2.944 0.070 SK-NM-C <0.001 0.074 0.214 0.010 DAOY Medulloblastoma <0.001 0.072 1.170 0.035 D238 <0.001 0.138 2.407 0.007 KELLY <0.001 0.030 1.660 0.002 TGW Neuroblastoma 0.001 0.190 3.499 0.029 IMR-32 <0.001 0.010 0.005 0.004 SH-SY5Y <0.001 0.038 0.420 0.034 SJSA1 0.001 >0.200 13.260 >0.200 HOS Osteosarcoma <0.001 0.040 6.774 >0.200 SAOS-2 0.001 0.043 1.445 0.002 Y79 Retinoblastoma 0.001 0.028 1.383 0.001 A204 Rhabdoid <0.001 0.027 2.400 0.003 TE 381.T 0.001 0.026 2.040 0.003 SJCRH30 Rhabdomyosarcoma <0.001 0.007 1.384 0.001 RD <0.001 0.013 0.781 0.002
Supplementary Data
- Supplemental Materials and Methods - Supplemental methodology, specifically the in vitro cord formation assay and the enzyme-linked immunosorbent assay.
- Supplemental Data - Supplemental Table 1. Cell culture conditions. Supplemental Table 2. Antibody details. Supplemental Table 3. Catalog information for Dharmacon RNAi constructs. Supplemental Table 4. EC50 values of prexasertib in pediatric cell lines included in the cancer cell line sensitivity panel. Supplemental Table 5. Quantification of percent positive cells for each immunostain following prexasertib treatment. Supplemental Figure 1. Prexasertib induces caspase activation in NB cell lines. Supplemental Figure 2. Prexasertib promotes neuroblastoma cell death by inducing DNA damage. Supplemental Figure 3. Inhibition of CHK1 promotes phosphorylation of RPA32/2. Supplemental Figure 4. Assessment of siRNA-mediated knockdown of CHK1 on total protein and phosphorylation status. Supplemental Figure 5. No significant weight loss was observed during prexasertib treatment. Supplemental Figure 6. Combination with doxorubicin does not enhance prexasertib efficacy in vivo. Supplemental Figure 7. Vasculature markers are more prevalent in prexasertib-treated neuroblastoma xenografts. Supplemental Figure 8. Prexasertib reduces VEGF endothelial cell cord formation in vitro, but does not affect established cords. Supplemental Figure 9. Neuroblastoma cell lines drive endothelial cord formation in vitro.
Volume 23, Issue 15
