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Introduction: Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified or mutated across various cancer types. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. All three TKIs bind and inhibit the kinase domain of HER2 but differ both in the mechanism of binding and in specificity for other HER family members. Direct comparisons to differentiate the pre-clinical efficacy of the three TKIs have been limited to small-scale studies and novel biomarkers of response to further define appropriate patient populations are required. Methods: In this study, the anti-proliferative effects of the three TKIs were compared using a 115-cancer cell line panel, including 12 breast cancer cell lines and 22 cell lines harbouring point mutations or amplifications of EGFR, HER2, or HER3. Hierarchical clustering analysis was carried out to compare the IC50 “fingerprint” of the three TKIs to 168 other anti-cancer agents. Novel markers of TKI sensitivity and resistance were identified through cross-analysis of each drug response profile with mutation, copy number variation, and gene expression data. Results: All three TKIs were effective against HER2-positive breast cancer models; neratinib showed the most potent activity, followed by tucatinib and lapatinib respectively (Table 1). Neratinib displayed the greatest anti-proliferative activity in HER2-mutant and EGFR-mutant cell lines. Clustering analysis revealed that the anti-proliferative profile of tucatinib was most similar to trastuzumab, while neratinib and lapatinib were most like other HER family inhibitors. Mutation and gene expression analysis identified potential markers of response for each TKI. High expression of four genes (HER2, VTCN1, CDK12, and RAC1) correlated with response to all three TKIs. DNA damage repair genes were significantly associated with resistance to the HER2-targeted TKIs. BRCA2 mutation was correlated with neratinib and tucatinib response, and high gene expression of ATM, BRCA2, and BRCA1 were all associated with neratinib resistance. Conclusions: Neratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed possible mechanisms that may be exploited using combinatorial strategies involving CDK inhibitors, immunotherapies, and targeting DNA repair pathways. Table: IC50 values for neratinib, lapatinib, and tucatinib in the HER2+ breast cancer cell linesIC50 values (nM)Cell linesNeratinibLapatinibTucatinibAU-56520294125BT-4745926229HCC195413814262122MDA-MB-453306228445928SKBR3715222 Citation Format: Neil T Conlon, Jeffrey J Kooijman, Suzanne JC van Gerwen, Winfried R Mulder, Guido JR Zaman, Irmina Diala, Lisa D Eli, Alshad S Lalani, John Crown, Denis Collins. Comparative analysis of anti-proliferative effects and gene profiling of lapatinib, neratinib, and tucatinib [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-06.

BackgroundHuman epidermal growth factor 2 (HER2/ERBB2) is frequently amplified/mutated in cancer. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. Direct comparisons of the preclinical efficacy of the TKIs have been limited to small-scale studies. Novel biomarkers are required to define beneficial patient populations.MethodsIn this study, the anti-proliferative effects of the three TKIs were directly compared using a 115 cancer cell line panel. Novel TKI response/resistance markers were identified through cross-analysis of drug response profiles with mutation, gene copy number and expression data.ResultsAll three TKIs were effective against HER2-amplified breast cancer models; neratinib showing the most potent activity, followed by tucatinib then lapatinib. Neratinib displayed the greatest activity inHER2-mutant andEGFR-mutant cells. High expression ofHER2,VTCN1,CDK12, andRAC1correlated with response to all three TKIs. DNA damage repair genes were associated with TKI resistance.BRCA2mutations were correlated with neratinib and tucatinib response, and high expression ofATM,BRCA2, andBRCA1were associated with neratinib resistance.ConclusionsNeratinib was the most effective HER2-targeted TKI againstHER2-amplified, -mutant, andEGFR-mutant cell lines. This analysis revealed novel resistance mechanisms that may be exploited using combinatorial strategies.

The fibroblast growth factor receptors (FGFRs) 1-4 are receptor tyrosine kinases (RTKs) involved in activation of essential cellular processes such as differentiation, proliferation and migration. Since alterations in FGFRs are common in multiple cancers, including breast cancer, non-small cell lung cancer and endometrial cancer, several kinase inhibitors targeting FGFRs are in clinical development. Erdafitinib, a pan-FGFR inhibitor, has been approved as second-line treatment of locally advanced or metastatic urothelial carcinoma harboring genetic FGFR2 or FGFR3 alterations. Although erdafitinib is very effective against these tumors, progression-free survival lasts only a few months, indicating that resistance also develops fast. Insight into the evolved resistance mechanism is crucial for the development of improved therapies. In this study we generated cell lines resistant to erdafitinib by prolonged culturing of the endometrial carcinoma cell line AN3 CA, harboring FGFR2 gain-off-function mutation N549K, to increasing doses of erdafitinib. To gain insight into the developed resistance, the expression of genes that have previously been reported to be involved in resistance against FGFR inhibitors was analyzed by qPCR and immunoblot. In addition, anti-proliferative effects of target inhibition by small molecules was evaluated. To get an unbiased view on altered gene expression, RNA sequencing (RNA-seq) was performed, followed by Gene-Set-Enrichment Analysis (GSEA). Alterations in FGFR1-4 and cancer hotspot gene sequences were detected by DNA sequencing. Occurrence of resistance to erdafitinib was confirmed in proliferation assays by a decreased response of the erdafitinib-resistant cell lines compared to the parental line. In addition, cross-resistance to other FGFR inhibitors infigratinib, pemigatinib, derazantinib and AZD4547 was observed. Although mRNA expression was altered for several RTKs previously reported to be involved in resistance to FGFR inhibitors, such as EGFR, ERBB2/3 and c-MET, involvement in resistance to erdafitinib could be excluded, as no change in response was observed in proliferation assays with their associated targeted inhibitors. RNA-seq and GSEA indicated upregulation of c-Myc target genes in erdafitinib-resistant cell lines. The involvement of c-Myc in the developed resistance was further confirmed by increased response to BET-bromodomain inhibitors JQ1 and I-BET-762, which indirectly target c-Myc. Furthermore, DNA sequencing identified novel mutations in coding regions of FGFR1 and KRAS genes. In conclusion, we show that multiple factors contribute to the development of resistance against erdafitinib in an FGFR2-mutant endometrial carcinoma cell line. BET-bromodomain inhibitors are of potential interest as therapeutic agents to overcome resistance against FGFR inhibitors. Citation Format: Wilhelmina E VAN RIEL, Janneke J. Melis, Demi H. Vogels, Winfried R. Mulder, Jeffrey J. Kooijman, Rogier C. Buijsman, Guido J. Zaman. Altered response to BET-bromodomain inhibitors JQ1 and I-BET-762 targeting c-Myc in erdafitinib-resistant endometrial carcinoma cell line AN3 CA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1080.