Effects of tozasertib on histone H3 phosphorylation (indicating aurora kinase A and B activity), cell cycle distribution, and apoptosis (indicated by BAX activation) in neuroblastoma cells.

<p>A) Numbers of cells expressing phosphorylated histone H3 were determined flow cytometry; * P<0.05 relative to untreated control; B) Representative histograms indicating cell cycle distribution in neuroblastoma cells after tozasertib treatment, arrows indicate additional peaks with high DNA content, possibly indicating endoreduplication; C) Numbers of cells with activated BAX expressed as fold change relative to control as determined by flow cytometry using an antibody specific for activated BAX after 24 h of tozasertib treatment. * P<0.05 relative to untreated control.</p

Effects of tozasertib and alisertib on neuroblastoma cell viability.

<p>A) IC₅₀ values determined after 120 h of incubation by MTT assay; B) tozasertib IC₅₀ values in the presence of the ABCB1 inhibitor zosuquidar (5 µM); C) Average IC₅₀ values in high and low ABCB1-expressing cells, * P<0.05 compared to low ABCB1-expressing cells; D) IC₅₀ values in UKF-NB-3 cells, UKF-NB-3 cells transduced with a lentiviral vector encoding for ABCB1 (UKF-NB-3^ABCB1), and UKF-NB-3 cells transduced with a control vector (UKF-NB-3^control), * P<0.05 compared to UKF-NB-3.</p

Effects of tozasertib on the viability of neuroblastoma cells in combination with the MDM2 inhibitor nutlin-3.

<p>Neuroblastoma cells were treated for five days with tozasertib, nutlin-3, or their combination. Cell viability was determined by MTT assay. The drug concentrations were: UKF-NB-3, tozasertib 6 nM, Nutlin-3 0.625 µM; IMR-32, tozasertib 6 nM, Nutlin-3 1.25 µM; UKF-NB-3^rCDDP¹⁰⁰⁰, tozasertib 6 nM, Nutlin-3 1.25 µM; UKF-NB-3^rDOX²⁰, tozasertib 156 nM, Nutlin-3 2.5 µM; UKF-NB-3^rNutlin^10µM, tozasertib 156 nM, Nutlin-3 5 µM; UKF-NB-3^rVCR¹⁰, tozasertib 156 nM, Nutlin-3 5 µM. * P<0.05 relative to either single treatment.</p

Tozasertib-induced apoptosis in UKF-NB-3 cells transduced with a lentiviral vector encoding shRNA directed against p53 (UKF-NB-3^p53shRNA) and UKF-NB-3 transduced with a control vector expressing non-targeting scrambled shRNA (UKF-NB-3^scr) as indicated by BAX activation, caspase 3/7 activation, and determination of sub-G1 cells.

<p>* P<0.05 relative to UKF-NB-3^scr.</p

Role of p53 signalling in response to aurora kinase inhibition.

<p>A) tozasertib-induced p53 and p21 expression as indicated by Western blot after 24 h of incubation; B) tozasertib- and alisertib-induced expression of p53 target genes in UKF-NB-3 cells in which p53 was depleted using a lentiviral vector encoding shRNA directed against p53 (UKF-NB-3^p53shRNA) or in UKF-NB-3 cells transduced with a control vector encoding non-targeting scramble shRNA (UKF-NB-3^scr) as indicated by qPCR after 24 h. Expression levels are presented as fold change relative to non-treated controls. * P<0.05 relative to non-treated control; C) tozasertib and alisertib concentrations that reduce UKF-NB-3, UKF-NB-3^scr, and UKF-NB-3^p53shRNA viability by 50% (IC₅₀). * P<0.05 relative to UKF-NB-3 cells.</p

Genomic array data.

<p>A: The respective breakpoints and FISH clones at ACTR2 and inside RAF1 of der(2)t(2;3)(p14;p25) in KARPAS-1106P as revealed by genomic arrays expands the FISH data. B: Color coded plots of FARAGE (purple), KARPAS-1106P (pink), MEDB-1 (blue), U-2940 (green)—show genomic copy number (solid) and LOH (barred) at 12 loci (1p12, 2p15, 2p16, 6q23, 7p22, 7q31, 8q24, 9p21, 9p24, 16p13, 15q23, 19q13, together with OMIM genes below. Copy number polymorphic regions (<a href="http://dgv.tcag.ca/dgv/app/home" target="_blank">http://dgv.tcag.ca/dgv/app/home</a>) are shown between, listing gains (blue), losses (red), and copy number neutral alterations, such as inversions (gray).</p

Spectral Karyotyping (SKY): FARAGE (A), KARPAS-1106P (B), MEDB-1 (C) and U-2940 (D).

<p>Inverse DAPI G-banding shown left of corresponding pseudocolored SKY. Arrows show deletions (white), duplications (red), and translocations (ochre). After trypsin G-banding (not shown) analyses were extended to prepare consensus karyotypes. Individual metaphases sometimes departed from consensus karyotypes, e.g. note loss of Y-chromosome in MEDB-1. Note absence of key cytogenetic rearrangements and relative lack of rearrangement when compared to cHL cell lines. Thus the salient cytogenetic features of PMBL are essentially negative with respect to neighboring entities, cHL and PMBL.</p

Fluorescence in situ hybridization (FISH) Analysis.

<p>A-D: Shows the cytogenetic configuration of focal deletions affecting 16p13 including the SOCS1 locus (arrows) in three PMBL cell lines: monoallelic in FARAGE, biallelic in KARPAS1106P and U-2940 effected by t(16;16) rearrangement, but absent in MEDB-1. E/F: Shows deletions affecting the PTPN1 locus in KARPAS-1106P (E) and MEDB-1 (F). Twin partial chromosome 20 long-arm deletions in KARPAS-1106P effect PTPN1 monosomy, while in MEDB-1 where the gene is mutated this locus escapes deletion despite proximity to a translocation breakpoint therein. G-I: Shows analysis of der(3)t(2;3)(p14;p25) in KARPAS-1106P. The respective breakpoints at 2p14 and 3p25 were placed close to ACTR2 (within clone RP11-441L10) and RAF1 (within RP11-148M13). Coordinates and labelling scheme are shown below. Coordinates (MBp) are from HG19. FISH was performed using tilepath BAC clones.</p

Gene expression at genomically rearranged loci.

<p>A: Microarray expression data for genes implicated at CNV (mainly deletions). Note, for example gene silencing accompanying focal biallelic deletions at multiple loci: including, CD58 at 1p12 in KARPAS-1106P; MSH6 and FBXO11 at 2p26 in FARAGE; TNFAIP3, AIMP2 at 6q23 in U2940; EIF2AK1 at 7p22 in U-2940; CDKN2A at 9p21 in KARPAS-1106P and U-2940; CD274 at 9p24 in U-2940; SOCS1 at 16p13 in KARPAS-1106P and U-2940; and KIAA0355 at 19q13 in U-2940. B: Shows qPCR expression of select target genes at recurrent PMBL amplicons (2p15, 9p24) and a deletion (16p13) set against TBP reference in cell lines FARAGE, KARPAS-1106P, MEDB-1, and U-2940 (PMBL) shown blue, alongside reference SU-DHL-8 (DLBCL) shown red. Diamonds indicate undetectable expression. Quantitative data were verified by twofold or more biological replication.</p