Figure 4

KIF11 is a universal target across all ATRT-subtypes and KIF11-targeting with ispinesib inhibited proliferation in a panel of 7 ATRT cell lines. KIF11 gene expression in (a) Discovery Cohort of 10 patient tumors, 8 patient-derived tumor cell lines, 10 normal brain control tissues (RNA-Seq), Validation Cohort 1 of patient tumors of three ATRT subtypes—SHH-ATRTs, MYC-ATRTs and TYR-ATRTs (n = 49, Microarray Affymetrix U133plus2.0, GSE70678¹⁸), (b) Validation Cohort 2 of 18 patient tumors with long or short survival outcome (GSE28026¹⁹, Affymetrix U133plus2.0), and (c) KIF11 relative gene expression in our panel of 7 patient-derived ATRT cell lines. For Discovery Cohort, KIF11 was highly expressed in patient tumors (Fold change 24.4, p < 0.0001, Student T-Test) and tumor cell lines compared to normal brain controls. For Validation Cohort 1, patient tumors of all 3 molecular ATRT-subtypes exhibited high KIF11 expression, with SHH-ATRTs demonstrating the highest KIF11 expression compared to MYC-ATRTs and TYR-ATRTs (fold change 1.4, p = 0.04, Student T-Test). For Validation Cohort 2, KIF11 expression was referenced to GAPDH (housekeeping gene), KIF11 was highly expressed in patient groups of both long and short survival outcome (p = 0.4, unpaired T-test with Welch's Correction). (d) Immunohistochemistry of patient tumors from Validation Cohort 3 similarly demonstrated KIF11 expression, showing Patient Tumor 1 with approximately 10–20% staining (red arrows indicate mitoses, green arrows indicate apoptotic bodies). KIF11 protein expression co-localized with mitoses. KIF11 (target of ispinesib) was expressed on different cell populations within patient tumor, and not restricted to mitoses, indicating ispinesib targeted not only mitoses (actively dividing cells) but also other tumor cell populations within the tumor. (e) Targeted KIF11 inhibition: Dose–response effect of ispinesib on 7 ATRT cell lines. Values are shown as gradations of green (lowest dose) to purple (highest dose).