Response to ERBB3-Directed Targeted Therapy in NRG1-Rearranged Cancers

Abstract

NRG1 rearrangements are oncogenic drivers that are enriched in invasive mucinous adenocarcinomas (IMA) of the lung. The oncoprotein binds ERBB3-ERBB2 heterodimers and activates downstream signaling, supporting a therapeutic paradigm of ERBB3/ERBB2 inhibition. As proof of concept, a durable response was achieved with anti-ERBB3 mAb therapy (GSK2849330) in an exceptional responder with an NRG1-rearranged IMA on a phase I trial (NCT01966445). In contrast, response was not achieved with anti-ERBB2 therapy (afatinib) in four patients with NRG1-rearranged IMA (including the index patient post-GSK2849330). Although in vitro data supported the use of either ERBB3 or ERBB2 inhibition, these clinical results were consistent with more profound antitumor activity and downstream signaling inhibition with anti-ERBB3 versus anti-ERBB2 therapy in an NRG1-rearranged patient-derived xenograft model. Analysis of 8,984 and 17,485 tumors in The Cancer Genome Atlas and MSK-IMPACT datasets, respectively, identified NRG1 rearrangements with novel fusion partners in multiple histologies, including breast, head and neck, renal, lung, ovarian, pancreatic, prostate, and uterine cancers.Significance: This series highlights the utility of ERBB3 inhibition as a novel treatment paradigm for NRG1-rearranged cancers. In addition, it provides preliminary evidence that ERBB3 inhibition may be more optimal than ERBB2 inhibition. The identification of NRG1 rearrangements across various solid tumors supports a basket trial approach to drug development. Cancer Discov; 8(6); 686-95. ©2018 AACR.See related commentary by Wilson and Politi, p. 676This article is highlighted in the In This Issue feature, p. 663.

©2018 American Association for Cancer Research.

Figures

FIGURE 1. Clinical response to anti-ERBB3 monoclonal antibody therapy in a patient with an advanced NRG1-rearranged non-small cell lung cancer

A. A never smoker with advanced invasive mucinous adenocarcinoma was treated with four lines of systemic therapy (chemotherapy and immune therapy) with no response to any of these treatments. His tumor was subsequently found to harbor an in-frame CD74-NRG1 fusion that includes the EGF-like extracellular domain that serves a binding site for ERBB3. Targeted therapy with GSK2849330, an anti-ERBB3 monoclonal therapeutic antibody, was initiated on a phase 1 clinical trial. B. A durable (19 months) and confirmed partial response (RECIST version 1.1) was achieved that exceeded the duration of disease control achieved on all four prior systemic therapy regimens in aggregate. Substantial disease shrinkage of a right lower lobe mass was noted early (by week 6 as shown) in the course of therapy.

FIGURE 2. Cell lines with aberrant expression of NRG1 are exquisitely sensitive to downregulation of ERBB3 signaling

A. Expression of the DOC4-NRG1 fusion was confirmed by RT-PCR (left panel) and by DNA sequencing of the PCR amplicon (right panel) in MDA-MB-175-VII cells. B. Copy number breakpoints affecting NRG1 in HCC-95, a human lung cancer cell line, are depicted using normal human DNA as reference. The outer circle depicts human autosomes (–22), chromosomes X and Y, and the mitochondrial genome (M). Chromosome 8, zooming into the amplified NRG1 locus (inset), is shown. Genome-wide distribution of log2 ratios is represented in concentric circles as gains (red) and losses (blue), each data point representing a probe from the array according to the locations in the human genome (NCBI 36). C. Quantitative RT-PCR was used to determine the levels of NRG1 mRNA in MDA-MB-175-VII and HCC-95 using MCF-7 and HCC827 as control cell lines for comparison. D. Cells were treated with GSK2849330 for 1 hour then stimulated with heregulin (HRGB1) for 30 minutes before preparation of whole-cell extracts for Western blotting with the antibodies described. A representative blot is shown. E. Cells were left untreated, or treated with 10 μg/mL GSK2849330 or non-specific IgG and then measured for growth by CTG assays at the indicated time points. The y-axis represents the mean ± SD of relative luciferase units (RLU) measured in triplicate. F. Cells were infected with lentivirus containing non-targeting (NT) or shRNAs targeting the genes shown and then examined for relative number of cells remaining after 7 days of infection (top panel) or relative caspase 3/7 enzymatic activity (bottom panel). Results represent the mean ± SD of two experiments in which each condition was assayed in duplicate. *significantly different from NT-shRNA, p<0.05, two tailed t-test

FIGURE 3. In vivo growth inhibition elicited by afatinib and GSK2849330 in a CLU-NRG1-rearranged patient-derived xenograft (PDX) mouse model

A. A panel of patient-derived xenograft (PDX) models was profiled by RNA-seq. An ovarian cancer PDX model, OV-10-0050, showed a high level of NRG1 mRNA expression, resulting from a novel CLU-NRG1 intragenic rearrangement (inset) where Exon 2 of CLU (chromosome 8) was fused with exon 6 or NRG1 (chromosome 8) with retention of the extracellular epidermal growth factor (EGF)-like domain in the fusion product, as shown. B. Female BALB/c nude mice were subcutaneously implanted with CLU-NRG1-rearranged tumors and treated with afatinib at 15 mg/kg daily for 28 days, resulting in a significant inhibition of tumor growth compared to the vehicle arm (n=6, top panel). The same PDX mouse model was treated with anti-ERBB3 monoclonal antibody, GSK2849330 at 25 mg/kg biweekly for 35 days, resulting in a dramatic response of complete tumor regression compared to vehicle- or IgG-treated controls (n=10, bottom panel). Changes from baseline tumor volume (TV), as measured by [TV(final) – TV(initial)]*100/TV(initial), are shown in the waterfall plots. C. Pharmacodynamic changes in tumor tissues as measured 4 hours after a single dose of treatment with afatinib, vehicle, GSK2849330, or IgG control (n=3) are shown.

FIGURE 4. NRG1 rearrangements are found in multiple solid tumors

A. A schematic of CD74-NRG1, the most commonly identified genomic rearrangement identified in lung cancers in this series, is shown. The CD74 gene (chromosome 5q) is disrupted and inverted downstream of exon 5 and subsequently ligated to a position upstream of exon 6 of the NRG1 gene. B. Structural features of fusions, involving the indicated 5′ and 3′ chromosomal partners, identified by next-generation DNA sequencing (MSK-IMPACT, n=17,485 tumors profiled) or targeted RNA sequencing (MSK-Solid Fusion Assay) are shown. The EGF-like domain is maintained in all fusions identified. C. Fusion junction exons, the associated chromosomal partners, and corresponding transcripts (Ensembl database version 75) are shown for all fusions detected by analyzing The Cancer Genome Atlas (TCGA) dataset (n=8,984 tumors analyzed), with the EGF-like domain indicated, wherever applicable. NRG1 fusion partners are not drawn to scale. The asterisk indicates that two distinct fusions were found in the same tumor sample. D.NRG1 RNA-seq expression is plotted across fusion-positive TCGA cancer types (x-axis) with fusion-positive samples indicated in red. Note that only one NRG1 expression value is plotted for the lung squamous cell carcinoma (LUSC) sample with two distinct fusions. BRCA (breast invasive carcinoma); HNSC (head and neck squamous cell carcinoma); KIRC (kidney renal clear cell carcinoma); LUAD (lung adenocarcinoma); OV (ovarian serous cystadenocarcinoma); PAAD (pancreatic adenocarcinoma); PRAD (prostate adenocarcinoma); UCS (uterine carcinosarcoma)