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Purpose: Anti-EGF receptor (EGFR) antibodies and small-molecule tyrosine kinase inhibitors have shown activity in epithelial tumors; however, agents that work by blocking the EGFR growth signal are ineffective when the oncogenic stimulus arises downstream, such as in tumors with KRAS mutations. Antibodies of the IgG1 subclass can also kill tumor cells directly through antibody-dependent cell-mediated cytotoxicity (ADCC), and the efficacy of this is determined by the interaction of the Fc portion of the target cell–bound antibody and Fc receptors present on immune effector cells.Experimental Design: We report the development of GA201, a novel anti-EGFR monoclonal antibody with enhanced ADCC properties. GA201 was derived by humanization of the rat ICR62 antibody. The Fc region of GA201 was glycoengineered to contain bisected, afucosylated carbohydrates for enhanced binding to FcγRIIIA.Results:In vitro binding of GA201 to EGFR inhibited EGF ligand binding, EGFR/HER2 heterodimerization, downstream signaling, and cell proliferation to a similar extent as cetuximab. However, GA201 exhibited superior binding to both the low- and high-affinity variants of FcγRIIIA. This resulted in significantly enhanced induction of ADCC compared with cetuximab against both KRAS-wild-type and -mutant tumor cells lines. This enhanced ADCC translated into superior in vivo efficacy in a series of mouse xenograft models. Efficacy of GA201 was further increased when administered in combination with chemotherapy (irinotecan).Conclusions: These data suggest that GA201 may be more effective than cetuximab in patients with EGFR-positive solid tumors and may also represent a first-in-class treatment of patients with KRAS-mutated tumors. Clin Cancer Res; 19(5); 1126–38. ©2012 AACR.

Supplemental Materials and Methods

PDF file - 307K, ADCC activity of GA201 using PMBCs as effector cells at E:T ratios of 1:1, 5:1 (24 hour LDH release measured) and 10:1 (4 hour LDH release measured). Multiple ADCC experiments were performed using a variety of different effector:target cell ratios (E:T). Figure 3 in the manuscript displays the results of ADDC assays using NK-92 cells as effectors at an E:T ratio of 3:1 and using human peripheral blood mononuclear cells (PBMCs) as effectors at an E:T ratio of 25:1. These E:T ratios represent the optimal experimental conditions that generated robust ADCC signal read outs. Supplementary Fig. S1 shows additional ADCC results for the MKN45 cell line using human PBMCs as effector cells at an E:T ratios of 1:1, 5:1 and 10:1. The data shows that under all the tested conditions GA201 exhibited superior cell killing compared with cetuximab. The overall ADCC activity with both antibodies using PMBCs at an E:T ratio of 1:1 was substantially lower than at 5:1 or 10:1.

The clinically aggressive alveolar rhabdomyosarcoma (RMS) subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here, we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacologic combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as top compound in a screen from 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS.Significance:These findings show that Aurora kinase A and Bcl-2 family proteins are potential targets for FP-RMS.

The clinically aggressive alveolar rhabdomyosarcoma (RMS) subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here, we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacologic combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as top compound in a screen from 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS. Significance: These findings show that Aurora kinase A and Bcl-2 family proteins are potential targets for FP-RMS.

Long noncoding RNAs (lncRNAs) are widely dysregulated in cancer, yet their functional roles in cellular disease hallmarks remain unclear. Here we employ pooled CRISPR deletion to perturb all 831 lncRNAs in KRAS-mutant non-small cell lung cancer (NSCLC), and measure their contribution to proliferation, chemoresistance and migration across two cell backgrounds. Integrative analysis of this data outperforms conventional “dropout” screens in identifying cancer genes, while prioritising disease-relevant lncRNAs with pleiotropic and background-independent roles. Altogether 60 high-confidence oncogenic lncRNAs are active in NSCLC, the majority identified here for the first time, and which tend to be amplified and overexpressed in tumours. A follow-up antisense oligonucleotide (ASO) screen shortlisted two candidates, Cancer Hallmarks in Lung LncRNA (CHiLL 1&2), whose knockdown consistently suppressed cancer hallmarks in a variety of 2D and 3D tumour models. Molecular phenotyping reveals that CHiLL 1&2 control cellular-level phenotypes via distinct transcriptional networks converging on common oncogenic pathways. In summary, this work reveals a multi-dimensional functional lncRNA landscape underlying NSCLC that contains potential therapeutic vulnerabilities.

Ewing sarcoma tumorigenesis is tightly linked to epigenetic deregulation. Indeed, the aberrant fusion transcription factor and tumor driver EWS-FLI1 produces extensive rewiring of the cancer cell epigenome. At enhancers containing canonical ETS motifs, the fusion protein represses gene expression but, when binding to GGAA repeats, EWS-FLI1 induces transcription by recruiting activating epigenetic regulators such as the acetyltransferase p300 and the BAF chromatin remodeling complex. The nucleosome remodeling and deacetylase (NuRD) complex is an ATP-dependent multi-subunit complex that modulates chromatin architecture and regulates DNA damage repair, genome stability and gene expression. In Ewing sarcoma, NuRD subunits, such as LSD1, have been suggested to interfere with EWS-FLI1 activity and prevent tumor progression. Hence, we here aimed to comprehensively study the role of NuRD in Ewing sarcoma pathogenesis. First, we performed a NuRD-centered, negative selection, CRISPR/Cas9 screen and identified the chromatin remodeling helicase CHD4 as essential for Ewing sarcoma tumor cell survival. Validation assays using two doxycycline-inducible shRNAs targeting CHD4 demonstrated that silencing of this remodeler in fact drastically reduces tumor cell proliferation and completely prevents colony formation. This cell proliferation impairment was caused by an induction of cell death by apoptosis and not by cell cycle arrest. Surprisingly, this cell death phenotype was not linked to impaired fusion protein activity. CHD4 and NuRD, despite primarily locating to enhancers and super-enhancers in Ewing sarcoma cells, did not preferentially localize to GGAA repeats, unlike EWS-FLI1. Moreover, RNA sequencing assays performed upon CHD4 silencing showed that this ATPase does not regulate the EWS-FLI1 signature. As CHD4 is a chromatin remodeler able to move nucleosomes along the DNA, we performed ATAC sequencing experiments to investigate changes in chromatin status that could explain the dependency of Ewing sarcoma cells on CHD4 for survival. We observed that CHD4 depletion from Ewing sarcoma cells causes a drastic and global chromatin relaxation which renders tumor cells prone to DNA damage and increasingly sensitive to DNA damaging agents. Interestingly, augmented sensitivity to DNA damage was not observed by silencing CHD4 in non-tumorigenic human fibroblasts. Finally, CHD4 depletion also reduced Ewing sarcoma tumor growth in vivo and prolonged mice survival. In conclusion, we demonstrate for the first time that CHD4 is crucial for Ewing sarcoma cell survival and highlight this helicase as a promising therapeutic target for Ewing sarcoma with potential for combination therapy with drugs inducing DNA damage. Importantly, this work has initiated ongoing efforts to develop first-in-class small molecules specifically targeting CHD4 which will be crucial for the future validation of this ATPase as a viable target with clinical application. Citation Format: Joana Graca Marques, Blaz Pavlovic, Quy Ai Ngo, Gloria Pedot, Michaela Roemmele, Larissa Volken, Marco Wachtel, Beat W. Schäfer. The NuRD subunit CHD4 is essential for ewing sarcoma cell survival as it regulates global chromatin architecture [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B010.

Purpose: Anti-EGF receptor (EGFR) antibodies and small-molecule tyrosine kinase inhibitors have shown activity in epithelial tumors; however, agents that work by blocking the EGFR growth signal are ineffective when the oncogenic stimulus arises downstream, such as in tumors with KRAS mutations. Antibodies of the IgG1 subclass can also kill tumor cells directly through antibody-dependent cell-mediated cytotoxicity (ADCC), and the efficacy of this is determined by the interaction of the Fc portion of the target cell–bound antibody and Fc receptors present on immune effector cells. Experimental Design: We report the development of GA201, a novel anti-EGFR monoclonal antibody with enhanced ADCC properties. GA201 was derived by humanization of the rat ICR62 antibody. The Fc region of GA201 was glycoengineered to contain bisected, afucosylated carbohydrates for enhanced binding to FcγRIIIA. Results: In vitro binding of GA201 to EGFR inhibited EGF ligand binding, EGFR/HER2 heterodimerization, downstream signaling, and cell proliferation to a similar extent as cetuximab. However, GA201 exhibited superior binding to both the low- and high-affinity variants of FcγRIIIA. This resulted in significantly enhanced induction of ADCC compared with cetuximab against both KRAS-wild-type and -mutant tumor cells lines. This enhanced ADCC translated into superior in vivo efficacy in a series of mouse xenograft models. Efficacy of GA201 was further increased when administered in combination with chemotherapy (irinotecan). Conclusions: These data suggest that GA201 may be more effective than cetuximab in patients with EGFR-positive solid tumors and may also represent a first-in-class treatment of patients with KRAS-mutated tumors. Clin Cancer Res; 19(5); 1126–38. ©2012 AACR.

Over the last decades, it has become increasingly clear that tumors are characterized by inter-individual heterogeneity, which can account as one of the prominent reasons for treatment failure. To address this problem, personalized precision medicine approaches need to be applied such as comprehensive genomic profiling to identify actionable driver mutations in individual tumors. Unfortunately, genomics alone is not sufficient in tumors that are driven by mutated but otherwise undruggable targets and a typically low mutational burden. This is a characteristic of many pediatric malignancies including rhabdomyosarcoma (RMS), the most common childhood soft-tissue sarcoma. Hence, we aim to develop an in vitro drug-profiling platform to identify and prioritize treatment strategies for RMS patients. To this end, we generated a panel of patient-derived xenografts (PDXs) including some diagnostic and relapse samples. We then screened 18 different culture conditions to identify suitable parameters to establish in vitro primary cultures of PDX tumors. Interestingly, addition of fetal calf serum to cell culture media has a detrimental effect on viability of most primary RMS cell cultures (PRCCs). In contrast, defined serum-free conditions allow to grow primary cultures for several passages that closely preserve the clonal composition and phenotypic characteristics of the parental tumor, as assessed by genomic and copy number analysis. Pharmacologic profiles of PRCCs using a targeted drug library of more than 200 compounds revealed patient-specific vulnerabilities, among them an unexpected sensitivity to AKT inhibitors in some fusion-positive RMS. Interestingly, hierarchical clustering of drug sensitivities clustered PAX3-FOXO1 fusion-positive tumors together and separated them from fusion-negative RMS. Moreover, a screen to establish effective drug combinations in a highly resistant high-risk relapse sample using standard chemotherapeutics (doxorubicin, etoposide, vincristine) together with our targeted compound library revealed the BH3-only mimetic ABT-263 as the top-scoring drug capable of resensitizing recurrent PRCCs to first-line treatment. Resensitizing with ABT-263 was not a patient-specific vulnerability as it was observed in several additional PRCCs. Mechanistically, genetic loss-of-function validation experiments revealed that this occurs via blockade of the BCL-XL-MCL-1 axis and is dependent on upregulation of the BH3-only protein NOXA. Taken together, our study provides an in vitro tool kit to prioritize actionable drug targets or combinatorial options for RMS patients for whom conventional therapies are failing. Citation Format: Beat W. Schaefer, Gabriele Manzella, Michaela Roemmele, Luduo Zhang, Joëlle Tchinda, Felix Niggli, Marco Wachtel. Development of an in vitro drug-profiling platform for functional guidance of treatment decisions and identification of vulnerabilities in chemoresistant relapsed rhabdomyosarcoma tumors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B01.

Purpose: Imgatuzumab (GA201) is a novel anti-EGFR mAb that is glycoengineered for enhanced antibody-dependent cellular cytotoxicity (ADCC). Future treatment schedules for imgatuzumab will likely involve the use of potentially immunosuppressive drugs, such as premedication therapies, to mitigate infusion reactions characteristic of mAb therapy and chemotherapy combination partners. Because of the strong immunologic component of mode of action of imgatuzumab, it is important to understand whether these drugs influence imgatuzumab-mediated ADCC and impact efficacy. Experimental Design: We performed a series of ADCC assays using human peripheral blood mononuclear cells that were first preincubated in physiologically relevant concentrations of commonly used premedication drugs and cancer chemotherapies. The ability of common chemotherapy agents to enhance the efficacy of imgatuzumab in vivo was then examined using orthotopic xenograft models of human cancer. Results: A majority of premedication and chemotherapy drugs investigated had no significant effect on the ADCC activity of imgatuzumab in vitro. Furthermore, enhanced in vivo efficacy was seen with imgatuzumab combination regimens compared with single-agent imgatuzumab, single-agent chemotherapy, or cetuximab combinations. Conclusions: These data indicate that medications currently coadministered with anti-EGFR therapies are unlikely to diminish the ADCC capabilities of imgatuzumab. Further studies using syngeneic models with functional adaptive T-cell responses are now required to fully understand how chemotherapy agents will influence a long-term response to imgatuzumab therapy. Thus, this study and future ones can provide a framework for designing imgatuzumab combination regimens with enhanced efficacy for investigation in phase II trials. Clin Cancer Res; 22(10); 2453–61. ©2015 AACR.