Adavosertib Enhances Antitumor Activity of Trastuzumab Deruxtecan in HER2-Expressing Cancers

Abstract

Purpose: Cyclin E (CCNE1) has been proposed as a biomarker of sensitivity to adavosertib, a Wee1 kinase inhibitor, and a mechanism of resistance to HER2-targeted therapy.

Experimental design: Copy number and genomic sequencing data from The Cancer Genome Atlas and MD Anderson Cancer Center databases were analyzed to assess ERBB2 and CCNE1 expression. Molecular characteristics of tumors and patient-derived xenografts (PDX) were assessed by next-generation sequencing, whole-exome sequencing, fluorescent in situ hybridization, and IHC. In vitro, CCNE1 was overexpressed or knocked down in HER2+ cell lines to evaluate drug combination efficacy. In vivo, NSG mice bearing PDXs were subjected to combinatorial therapy with various treatment regimens, followed by tumor growth assessment. Pharmacodynamic markers in PDXs were characterized by IHC and reverse-phase protein array.

Results: Among several ERBB2-amplified cancers, CCNE1 co-amplification was identified (gastric 37%, endometroid 43%, and ovarian serous adenocarcinoma 41%). We hypothesized that adavosertib may enhance activity of HER2 antibody-drug conjugate trastuzumab deruxtecan (T-DXd). In vitro, sensitivity to T-DXd was decreased by cyclin E overexpression and increased by knockdown, and adavosertib was synergistic with topoisomerase I inhibitor DXd. In vivo, the T-DXd + adavosertib combination significantly increased γH2AX and antitumor activity in HER2 low, cyclin E amplified gastroesophageal cancer PDX models and prolonged event-free survival (EFS) in a HER2-overexpressing gastroesophageal cancer model. T-DXd + adavosertib treatment also increased EFS in other HER2-expressing tumor types, including a T-DXd-treated colon cancer model.

Conclusions: We provide rationale for combining T-DXd with adavosertib in HER2-expressing cancers, especially with co-occuring CCNE1 amplifications. See related commentary by Rolfo et al., p. 4317.

Figure 1.

DXd is synergistic in combination with adavosertib and induces expression of cyclin E in vitro. A, Effects of cyclin E overexpression on cell sensitivity to adavosertib and T-DXd. HER2+ HCC-1954 cells were transfected with a viral expression cassette for cyclin E. In the absence or presence of doxycycline, cells were treated with adavosertib or T-DXd at individual dose ranges, or their combination, for 72 hours. Immunoblotting was performed to detect cyclin E, pCHK1, and γH2AX. Cell viability was measured using SRB assay. Drug IC₅₀s and combination index (CI) were calculated using CalcuSyn. B, Effects of cyclin E knockdown on cell sensitivity to adavosertib and T-DXd. HER2+ MKN7 cells were transfected with CCNE1 shRNA virus. Immunoblotting was performed to detect cyclin E and pCHK1. Cell viability assays with adavosertib and T-DXd were performed as described above. C, Immunoblot of cell-cycle regulatory proteins in MKN7 cells following treatment with DXd and T-DXd for 6, 12, 24, and 48 hours. D, Combination index of T-DXd and adavosertib measured by cell viability assay in a panel of 11 cell lines. E, Apoptosis assay of DXd and T-DXd in combination with adavosertib in MKN7 cells using Annexin V labeling. F, Immunoblot of cyclin E, γH2AX, and pCHK1 in MKN7 cells following treatment with DXd and T-DXd in combination with adavosertib for 24 or 96 hours. CCNE1, cyclin E1; DXd, deruxtecan; ERBB2, Erb-B2 receptor tyrosine kinase 2; T-DXd, trastuzumab deruxtecan.

Figure 2.

In vivo activity of T-DXd in combination with adavosertib in HER2 low (1/2+) and high (3+) expressing gastroesophageal cancers. A, Adavosertib (60 mg/kg p.o. 5on/2off) enhanced tumor growth inhibition when combined with T-DXd (10 mg/kg i.v. every 3 weeks) in a HER2 low, cyclin E amplified gastroesophageal cancer PDX model PDX.003.204. Right panel demonstrates HER2 IHC and immunoblotting for HER2, Cyclin E1, and actin. B, Adavosertib (60 mg/kg p.o. 5on/2off) in combination with T-DXd (10 mg/kg i.v. every 3 weeks) led to enhanced tumor regression in a HER2 low, cyclin E amplified gastroesophageal cancer PDX model PDX.003.213, with an ERBB2 V777 L and G778A mutation. C, T-DXd (10 mg/kg IV every 3 weeks) induced durable tumor regression alone and in combination with adavosertib in an ERBB2 amplified and HER2 overexpressing gastroesophageal PDX cancer model PDX.003.230. Adavosertib alone also demonstrated significant antitumor activity. D and E, T-DXd (10 mg/kg i.v. every 3 weeks) induced durable tumor regression in an ERBB2 amplified and HER2 overexpressing gastroesophageal PDX cancer model PDX.003.227 with concomitant cyclin E amplification/expression alone and when T-DXd was combined with adavosertib (60 mg/kg p.o. 5on/2off) (D). Antitumor activity of lower dose T-DXd (1 mg/kg i.v. every 3 weeks) was enhanced with the combination with adavosertib (60 mg/kg p.o. 5on/2off) (E). PDX, patient-derived xenograft; T-DXd, trastuzumab deruxtecan.

Figure 3.

In vivo activity of T-DXd in combination with adavosertib in other HER2 high (3+) expressing tumors. A, T-DXd (3 mg/kg i.v. every 3 weeks) induced durable tumor regression in an ERBB2 amplified/HER2 overexpressing and CCNE1 amplified endometrial neuroendocrine carcinoma PDX, PDX.003.368. B and C, Adavosertib (60 mg/kg p.o. 5on/2off) in combination with T-DXd (3 and 10 mg/kg i.v. every 3 weeks) led to enhanced tumor regression in a ERBB2 amplified/HER2 overexpressing colorectal cancer model PDX.003.396, which was developed from a patient who had progressed after treatment with T-DXd. PDX, patient-derived xenograft; T-DXd, trastuzumab deruxtecan.

Figure 4.

Pharmacodynamic effects of adavosertib in combination with T-DXd following 10-day treatment. Mice were treated for 10 days with adavosertib, T-DXd, or T-DXd + adavosertib, or were untreated (N = 5). A, PD marker assessment by IHC for adavosertib, T-DXd, and adavosertib + T-DXd tumors assessing phosphorylated Chk1, phosphorylated Kap1, γH2AX, Ki67, and cleaved caspase 3 (CC3). B, Differential expression of proteins assessed by RPPA revealed alterations in expression of several proteins involved in G₂–M transition, including Aurora-A, cyclin B, PLK1, CDC25, and phosphorylated CDK1. C, Morphologic features on H&E staining observed following 10-day treatments with adavosertib, T-DXd, or T-DXd + adavosertib. CCNE1, Cyclin E; ERBB2, Erb-B2 receptor tyrosine kinase 2; H&E, hematoxylin and eosin; PD, pharmacodynamic.