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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.

Background In epithelial ovarian cancer (EOC), 15-20% of the tumors do not respond to first-line chemotherapy (paclitaxel with platinum-based therapy), and in recurrences this number increases. Our aim is to determine the feasibility of cell proliferation assays of tumor cells isolated from malignant ascites to predict in vitro chemotherapy sensitivity, and to correlate these results with clinical outcome. Materials and methods Ascites was collected from twenty women with advanced EOC. Cell samples were enriched for tumor cells and EOC origin was confirmed by intracellular staining of CK7, surface staining of CA125 and EpCAM, and HE4 gene expression. In vitro sensitivity to chemotherapy was determined in cell proliferation assays using intracellular ATP content as an indirect measure of cell number. In vitro drug response was quantified by calculation of the drug concentration at which cell growth was inhibited with 50%. Clinical outcome was determined using post-treatment CA125 level. Results Cell samples of twenty patients were collected, of which three samples that failed to proliferate were excluded in the analysis (15%). Three other samples were excluded, because clinical outcome could not be determined correctly. In twelve of the fourteen remaining cases (86%) in vitro drug sensitivity and clinical outcome corresponded, while in two samples (14%) there was no correspondence. Conclusions Our study demonstrates the feasibility of drug sensitivity tests using tumor cells isolated from ascites of advanced EOC patients. Larger observational studies are required to confirm the correlation between the in vitro sensitivity and clinical outcome.

Indoleamine 2,3-dioxygenase (IDO1) is a key regulator of immune suppression by catalyzing the oxidation of L-tryptophan. IDO1 expression has been related to poor prognosis in several cancers and to resistance to checkpoint immunotherapies. We describe the characterization of a novel small molecule IDO1 inhibitor, NTRC 3883-0, in a panel of biochemical and cell-based assays, and various cancer models. NTRC 3883-0 released the inhibitory effect of IDO1 on CD8-positive T cell proliferation in co-cultures of IDO1-overexpressing cells with healthy donor lymphocytes, demonstrating its immune modulatory activity. In a syngeneic mouse model using IDO1-overexpressing B16F10 melanoma cells, NTRC 3883-0 effectively counteracted the IDO1-induced modulation of L-tryptophan and L-kynurenine levels, demonstrating its in vivo target modulation. Finally, we studied the expression and activity of IDO1 in primary cell cultures established from the malignant ascites of ovarian cancer patients. In these cultures, IDO1 expression was induced upon stimulation with IFNγ, and its activity could be inhibited by NTRC 3883-0. Based on these results, we propose the use of ascites cell-based functional assays for future patient stratification. Our results are discussed in light of the recent discontinuation of clinical trials of more advanced IDO1 inhibitors and the reconsideration of IDO1 as a valid drug target.

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.

In epithelial ovarian cancer (EOC), 15-20% of tumors do not respond to first-line chemotherapy, consisting of platinum-based therapy plus paclitaxel, and in recurrences the response rate is even lower. PARP inhibitors synergize with platinum therapy and have been approved for platinum sensitive EOC. Clinical trials with immunotherapies, such as PD-1/PD-L1 blockade, have so far not been successful. There is a great need of novel therapies with improved long term treatment outcome, as well as diagnostic tools and biomarkers to predict chemotherapy response in the clinic. We have used malignant ascites collected from patients with EOC, to identify new biomarkers of chemotherapy response and to support small molecule drug discovery programs on the tryptophan metabolizing enzymes TDO and IDO1 with clinically relevant assays. Modulation of TDO or IDO1 activity with small molecule inhibitors decreases anti-tumor immune response by inducing T cell anergy. Ascites was gathered from eighteen patients with advanced stage EOC. Cells were isolated by centrifugation and tumor cells were separated from immune cells by overnight adherence to tissue culture plates. Both cell fractions were characterized for the expression of immune and cancer cell markers by flow cytometry and/or quantitative PCR. TDO and IDO1 activity was determined using a functional assay and small molecule inhibitors. All samples expressed CA125 or HE4, or both ovarian cancer markers. Flow cytometry analysis of eleven adherent cell samples with anti-EpCAM or CA125 confirmed their EOC origin. Five of six samples showed high cell surface expression of PD-L1. Adherent cell samples were cultured over 2 - 3 passages and further characterized for TDO and IDO1 expression and activity. All cultured cell samples stained negative for immune cell markers (CD45) and only a small fraction (less than 10%) stained positive for a fibroblast marker. Two samples out of eighteen showed high IDO1 gene expression. However, only after stimulation with IFNγ, the tryptophan metabolizing activity in these samples was detectable, and could be inhibited with selective IDO1 inhibitors. In contrast, four samples expressed relatively high TDO and detectable endogenous tryptophan metabolizing activity, which could be inhibited by selective TDO inhibitors. The immune cell composition of the ascites, the expression and activity of TDO, IDO1 and PD-L1 were related to the clinical outcome of the patients from which the ascites was derived. Citation Format: Wilhelmina van Riel, Antoon van Doornmalen, Winfried Mulder, Diep Vu, Jelle Dylus, Judith den Ouden, Anne van Altena, Rogier Buijsman, Guido Zaman. High TDO and IDO1 expression in ovarian cancer-associated cells isolated from malignant ascites [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5604.

Introduction: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. First-line therapy in advanced EOC is surgery in combination with platinum-based chemotherapy and paclitaxel. 15-20% of patients do not respond and in 80% of advanced cases, the disease recurs within three years. PARP inhibitors synergize with platinum therapy and have been approved for platinum sensitive EOC. Clinical trials with immunotherapies, such as PD-1/PD-L1 blockade, have so far not been successful. Currently, the only approved companion diagnostic is BRCA gene mutations for PARP inhibitors. More diagnostic assays to predict the clinical response to chemo- or immunotherapies are needed. We have developed a biomarker discovery platform using ascites of ovarian cancer patients. Experimental procedures Ascites was gathered from patients by punction or during debulking surgery. Cells were collected by centrifugation and characterized by flow cytometry using specific antibodies. Genomic DNA was sequenced using Illumina cancer gene panels. Gene expression was analyzed by quantitative PCR (qPCR). Cellular activity of the tryptophan metabolizing enzymes IDO1 and TDO was measured with NFK Green [1]. Levels of L-tryptophan and its metabolite L-kynurenine in ascites fluid and blood were determined with LC-MS/MS. In vitro data were related to tumor histopathology and clinical response data. Results: Low passage cell samples from twenty patients were profiled for sensitivity to various cytotoxic agents and targeted anti-cancer therapies in cell proliferation assays. In parallel the mutation status of fifty cancer genes including BRCA1 and 2 was assessed by DNA sequencing. The expression of genes implicated in resistance to chemotherapy (CCNE1, ABCB1) or immunotherapy (PD-L1, IDO1, TDO) was determined with qPCR. The immune status of ascites was analyzed by measuring the relative proportion of different immune cell populations, i.e., cytotoxic and regulatory T cells, monocytes, dendritic and natural killer cells. The expression of the immune suppressive markers PD-L1, IDO1 and TDO was related to the immune cell composition of the ascites, kynurenine-tryptophan ratio, and clinical response data. A tumor cell sample derived from a patient with low grade serous ovarian cancer (LGSOC) was heterozygous for an oncogenic NRAS mutation and was much more sensitive to MEK inhibitors than other samples not harboring the mutation. The cells also expressed IDO1 and high levels of PD-L1 at the cell surface. Two other samples derived from high grade serous ovarian cancer (HGSOC) expressed high PD-L1 and one also IDO1. Several HGSOC samples expressed TDO. One HGSOC sample showed high expression of the ABCB1 gene, encoding the multidrug transporter P-glycoprotein. The sample was relatively resistant to paclitaxel, a known substrate of P-glycoprotein Conclusion: Our study shows that in vitro drug sensitivity assays with primary patient samples can be used to confirm or identify predictive drug response biomarkers. In an ongoing study, in which hundred patients with HGSOC will be included, the in vitro drug response of tumor cells from ascites to first-line cytotoxic anti-cancer agents will be determined and compared to the clinical response of patients bearing specific genomic biomarkers. [1] Seegers et al. (2014) J Biomol Screen 19, 1266-1272 Citation Format: Guido J.R. Zaman, Judith E. den Ouden, Jelle Dylus, Antoon M. van Doornmalen, Winfried R. Mulder, Diep Vu-Pham, Suzanne J.C. van Gerwen, Joost C.M. Uitdehaag, Rogier C. Buijsman, Leon F. Massuger, Anne M. van Altena. A precision medicine platform to predict the clinical response to chemo- and immunotherapy for epithelial ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A044. doi:10.1158/1535-7163.TARG-19-A044