Fig. 6: PKMYT1i-ATRi leads to premature mitosis in CCNE1 amplified OVCA and EMCA.
From: Targeting CCNE1 amplified ovarian and endometrial cancers by combined inhibition of PKMYT1 and ATR
A QIBC quantitation of FT282-hTERT p53R175H parental (WT, left) CCNE1-overexpressing (CCNE1-O/E, middle) and OVCAR3 (right) cells with percent of EdU+/cyclin B-pS126+ as a function of time after addition of RP-6306 (250 nM), RP-3500 (100 nM) or combination of both. B Whole cell lysates of FT282-hTERT p53R175H CCNE1-overexpressing (CCNE1-O/E) cells treated with RP-3500 (100 nM), RP-6306 (250 nM) or both for the indicated times were immunoblotted with CDK1, CDK1-pT14, CHK1, CHK1-pS345, CDC25B, CDC25B-pS151 and Actinin specific antibodies Actinin is used as loading control. C, D Whole cell lysates of KLE (C) and OVCAR3 (D) cells treated with RP-6306 (250 nM), RP-3500 (50 nM), or both for the indicated times were immunoblotted with CDK1, CDK1-pT14, CHK1, CHK1-pS345 and Actin specific antibodies. E, F Tumor tissue from WO-77 (E) tumor-bearing mice from Fig. 3C at end of treatment or WU-115 (F) tumor-bearing mice administered RP-6306 (10 mg/kg) orally BID, RP-3500 (5 mg/kg) orally QD or combination of both for 10 days and sacrificed 2 h post last treatment was prepared for FFPE Tumor tissues were stained with CDK1-pT14 antibodies (left) and the percentage of CDK1-pT14 strong-positive tissue (right) present in the tumor area was quantified by HALO software. n = 3,3,3,3,4,4 (E), n = 6,5,5,5 (F) Mean ± SD. Scale bar: 200 µm. G Growth inhibition relative to DMSO control of RPE1-hTERT TP53-/- parental, BRCA1-/-, ATM-/- and CCNE1-overexpressing (CCNE1-2A-GFP) cells after treatment with the indicated dose of RP-6306, RP-3500 or the combination of both. n = 3; Mean + SD. Significance determined by one-way ANOVA followed by Tukey’s multiple comparisons test in for (A, E, G), and Students’ t test in (F).
