Targeting CCNE1 amplified ovarian and endometrial cancers by combined inhibition of PKMYT1 and ATR

Abstract

Ovarian cancers (OVCAs) and endometrial cancers (EMCAs) with CCNE1-amplification are often resistant to standard of care treatment and represent an unmet clinical need. Previously, synthetic-lethal screening identified loss of the CDK1 regulator, PKMYT1, as synthetically lethal with CCNE1-amplification. We hypothesized that CCNE1-amplification associated replication stress will be more effectively targeted by combining the PKMYT1 inhibitor, lunresertib (RP-6306), with the ATR inhibitor, camonsertib (RP-3500/RG6526). Low dose combination RP-6306 with RP-3500 synergistically increased cytotoxicity more in CCNE1 amplified compared to non-amplified cells. Combination treatment produced durable antitumor activity and increased survival in CCNE1 amplified patient-derived and cell line-derived xenografts. Mechanistically, low doses of RP-6306 with RP-3500 increase CDK1 activation more so than monotherapy, triggering rapid and robust induction of premature mitosis, DNA damage and apoptosis in a CCNE1-dependent manner. These findings suggest that targeting CDK1 activity by combining RP-6306 with RP-3500 is a novel therapeutic approach to treat CCNE1-amplifed OVCAs and EMCAs.

Keywords: ATR; CCNE1 amplification; PKMYT1.

Figure 1.. Combination PKMYT1i-ATRi is synergistic in CCNE1 amplified OVCA and EMCA cells.

(A-B) Detection of cells response to monotherapy of PKMYT1i, RP6306 (A) and ATRi, RP-3500 (B) with MTT assay. CCNE1^AMP in blue, CCNE1^GAIN in orange, and CCNE1^LOW in black. (C) Cell viability analysis of the indicated CCNE1 amplified, gain, and low/neutral cell lines after treatment at indicated doses. Monotherapy for PKMYT1i, RP6306, is highlighted in blue and ATRi, RP3500, is highlighted in red. Combinations are highlighted in purple. Assays were normalized by doubling time such that cells doubled at least twice. n=3; Mean was presented. Most synergistic dose found was compared (right) and highlighted in red in the table (bottom right). Growth inhibition relative to DMSO control of indicated cells after treatment with pink highlighted doses. n=3; Mean ± S.D. (D) Coefficient of drug interaction (CDI) relative to fraction affected (Fa) plot of the indicated cell lines. CCNE1^AMP in Green, CCNE1^GAIN in orange, and CCNE1^LOW in black. CDI<1 synergy with CDI<0.7 significant synergy, CDI=1 additive, CDI>1 antagonistic. Plot of CDI versus CCNE1 copy number at indicated dose highlighted in pink (right). (E) Colony formation Analysis of PKMYT1i-ATRi combination in CCNE1 amplified OVCA and EMCA cells with RP-6306 (31.3nM), RP-3500 (6.25nM) or combination for 10 days. (F) Cell viability detection of PKMYT1i-ATRi combination on CCNE1 amplified HGSOC organoids. Organoids were developed from CCNE1 amplified, BRCA1 mutant HGSOC WO-58 and characterized with ovarian cancer marker PAX8 and epithelial marker CK7 by immunofluorescence (IF) and immunohistochemistry (IHC). P53 expression was detected by IHC. WO-58 organoids were treated with RP-6306 (250nM), RP-3500 (50nM), or both for 10 days and measured with CCK8 assay. n=3; Mean + SD. ****P<0.0001, ***P<0.001, *P<0.05, NS: P>0.05.

Figure 2.. Combination PKMYT1i-ATRi is synergistic in OVCA and EMCA cells depending on CCNE1 level.

(A) ZIP synergy scores at various dose combinations of RP-6306 and RP-3500 in FT282-hTERT p53^R175H parental (WT) and CCNE1-overexpressing (CCNE1-O/E) cells. Score ≥10 (red color) represents synergy, ≤−10 (green) represents antagonism. Values were obtained by analyzing mean data from 3 independent biological replicates with SynergyFinder. (B) Growth inhibition relative to DMSO control of parental and CCNE1-overexpressing cells after treatment with the indicated dose of RP-6306, RP-3500 or the combination of both. n=3; Mean + SD. (C-D) Cell viability detection of SNU685 cells in response to RP-6306 monotherapy (C) and RP-3500 monotherapy (D). n=3; Mean ± SD. Highlighted dose showing statistical difference in CCNE1 induced SNU685 cells with or without CCNE1 induction. (E-F) Cell viability analysis of the indicated SNU685 CCNE1 inducible cells ± doxycycline lines after treatment at the indicated doses. Doxycycline: 1µg/ml. Monotherapy for PKMYT1i, RP6306, is highlighted in blue, for ATRi, RP3500, is highlighted in red, Combinations are highlighted in purple for PKMYT1i-ATRi. Assays were normalized by doubling time such that cells doubled at least twice. n=3; Mean± SD. Growth inhibition relative to DMSO control of parental and CCNE1-overexpressing cells after treatment with indicated doses in pink (middle). Coefficient of drug interaction (CDI) relative to fraction affected (Fa) plot of the indicated cell lines is indicated to the right of each bar graph. CDI<1 synergy with CDI<0.7 significant synergy, CDI=1 additive, CDI>1 antagonistic. Red dot corresponds to doses highlighted in pink. (G) Measurement of drugs combinations in WO-20 CCNE1^inducible cells with or without Cyclin E1 induction. n=4; Mean + SD. (H-I) Colony formation analysis of WO-20 CCNE1^inducible cells (H) and SNU685 CCNE1^inducible (I) in response to RP-6306 (31.3nM), RP-3500 (6.25nM) or combination for 10 days. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, NS: P>0.05.

Figure 3.. Combination of PKMYTi-ATRi synergistically suppress CCNE1 amplified OVCA and EMCA PDXs growth.

(A-C) Tumor volume growth was measured weekly in (A) EMCA WU-115 (B) OVCA WO-19 and (C) OVCA WO-77 CCNE1 amplified PDX models treated with the indicated drugs. RP-6306 was given oral BID on days 1–5 and RP-3500 was given oral QD on a 3 days on / 4 days off schedule until tumor progression (tumor volume >1000 mm³). Survival rate (middle panel) was analyzed at the end of each experiment. The toxicity of drugs was revealed by the mice body weights (right panel) changes. (D) Tumor growth (left) and body weight change (right) of OVCAR3 xenografts in mice treated with either RP-6306, RP-3500 or both. RP-3500 was given oral QD and RP-6306 was given oral BID, both given on a 3 day on / 4 day off schedule for 24 days (n=8). Tumor growth and percent body weight change shown is mean ± SEM. Longitudinal tumor growth was analyzed by linear mixed effects modeling with type II ANOVA and pairwise comparisons across groups. Data were analyzed for overall survival using Mantel-Cox log-rank test. Body weight shown is mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.

Figure 4.. Dual inhibition of PKMYT1 and ATR induced DNA damage and cell apoptosis in CCNE1 amplified cancers.

(A) Representative QIBC plots of γH2AX nuclear intensity, EdU incorporation and DNA content (DAPI) in FT282-hTERT p53^R175H parental (WT) and CCNE1-overexpressing (CCNE1-O/E) cells treated with RP-6306 (63 nM), RP-3500 (50 nM) or combination of both treated for 48 h. (B) QIBC quantitation of FT282-hTERT p53^R175H parental (WT, left) and CCNE1-overexpressing (CCNE1-O/E, right) EdU⁻/pan-γH2AX⁺ cell in response to the indicated RP-6306/RP-3500 combinations treated for 48 h. n=3; Mean + SD. (C) Detection of γH2AX⁺ cells by flow cytometry in indicated cells after treated with RP-6306 (250nM), RP-3500 (50nM), or combination of both treated for 24 h. n=3; Mean + SD. (D-E) Whole cell lysates of OVCAR3 (D) and KLE (E) cells were treated with RP-6306 (250nM), RP-3500 (50nM), or both for the indicated times and immunoblotted with γH2AX and Actin antibodies. Actin is loading control. (F) OVCAR3 tumor-bearing mice were administered RP-6306 (5 mg/kg) orally BID, RP-3500 (5 mg/kg) orally QD or combination of both for 3 days, sacrificed 2h post last treatment and tumor tissue was prepared for FFPE. Tumor tissues were stained with γH2AX antibodies (left) and the percentage of γH2AX strong positive tissue (right) present in the tumor area was quantified by HALO software. n=4; Mean + SD. Scale bar: 100 µm. (G) Flow cytometry quantification of apoptotic cells with Annexin V and propidium iodide (PI) staining of the indicated cells after treated with drugs RP-6306 (250nM), RP-3500 (50nM), or both for 72 hrs. n=3; Mean + SD (H) SNU685 CCNE1 inducible cells were treated and detected with apoptotic cells same as (G). n=3; Mean + SD. (I-J) Whole cell lysates of OVCAR3 (I) and KLE (J) cells were treated with RP-6306 (250nM), RP-3500 (50nM), or both for the indicated times and immunoblotted with cCas3 and Actin antibodies. Actin is loading control. ****P<0.0001, **P<0.01, *P<0.05, NS: P>0.05.

Figure 5.. PKMYT1i-ATRi combination resulted double strand DNA in CCNE1 amplified OVCA and EMCA.

(A) Representative γH2AX and RPA nuclear intensity QIBC plots of FT282-hTERT p53^R175H CCNE1-overexpressing (CCNE1-O/E) and OVCAR3 cells treated with the indicated conditions. (B-C) QIBC quantitation of FT282-hTERT p53^R175H CCNE1-overexpressing (CCNE1-O/E, B) and OVCAR3 (C) cells with percent of only pan-γH2AX⁺, only pan-RPA⁺ or both pan-γH2AX⁺/pan-RPA⁺ as a function of time after addition of RP-3500 (100 nM), RP-3500 (100 nM) and hydroxyurea (2 mM), RP-6306 (500 nM) or RP-6306 (500 nM) and RP-3500 (100 nM). (D) QIBC quantitation of FT282-hTERT p53^R175H parental (WT, left) CCNE1-overexpressing (CCNE1-O/E, middle) and OVCAR3 (right) cells with percent of EdU⁺/ pHH3⁺ as a function of time after addition of RP-6306 (250 nM), RP-3500 (100 nM) or combination of both. (E) Measurement pHH3⁺ cells in the indicated cells after treated with 50 nM RP-3500 (50 nM), 250nM RP-6306 (250 nM) or combination for 24 h. n=3; Mean + SD. ****P<0.0001, NS: P>0.05.

Figure 6.. PKMYT1i-ATRi leads to premature mitosis in CCNE1 amplified OVCA and EMCA.

(A) QIBC quantitation of FT282-hTERT p53^R175H 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 p53^R175H CCNE1-overexpressing (CCNE1-O/E) cells treated with RP-3500 (100nM), RP-6306 (250nM) 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 (250nM), RP-3500 (50nM), or both for the indicated times were immunoblotted with CDK1, CDK1-pT14, CHK1, CHK1-pS345 and Actin specific antibodies (E) Tumor tissue from WO-77 tumor-bearing mice from figure 3C was prepared for FFPE at end of treatment. Tumor tissues were stained with CDK1-pT14 antibodies (left) and the percentage of CDK1-pT14 positive tissue (right) present in the tumor area was quantified by HALO software. n=4; Mean ± SD (). Detection of pCDK1-T14 level in WO-77 with RP-6306, RP-3500 or combination of both treatments by IHC. The intensity of pCDK1-T14 positive signals were quantified by HALO software. Scale bar: 200 µm. (F) 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.. ***P<0.001, **P<0.01, *P<0.05, NS: P>0.05.

Figure 7.. Schematic model of targeting CCNE1 amplified cancers with dual inhibition of PKMYT1 and ATR.

(A-B) Model of synergy between PKMYT1 and ATR inhibition in CCNE1-amplified or overexpressing cells. (A) CCNE1 amplification or overexpression in cells causes replication stress and S-phase elongation. To delay induction of mitosis until DNA replication is complete CDK1 activity is inhibited by increased PKMYT1 inhibitory phosphorylation on CDK1-Thr-14 and decreased CDC25 phosphatase activity via ATR-CHK1 signaling. (B) Inhibition of PKMYT1 (lunresertib) reduces CDK1 The14 phosphorylation and inhibition of ATR (camonsertib) increases CDC25 activity resulting in rapid and robust S-phase CDK1 activation and premature mitosis with synergistic induction of DNA damage and cell death.