Thomas Harding

Discovery of a Mutant-Selective Covalent Inhibitor of EGFR that Overcomes T790M-Mediated Resistance in NSCLC

UNLABELLEDnPatients with non-small cell lung cancer (NSCLC) with activating EGF receptor (EGFR) mutations initially respond to first-generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, frequently driven by the EGFR(T790M) mutation. CO-1686 is a novel, irreversible, and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR, including T790M, while exhibiting minimal activity toward the wild-type (WT) receptor. Oral administration of CO-1686 as single agent induces tumor regression in EGFR-mutated NSCLC tumor xenograft and transgenic models. Minimal activity of CO-1686 against the WT EGFR receptor was observed. In NSCLC cells with acquired resistance to CO-1686 in vitro, there was no evidence of additional mutations or amplification of the EGFR gene, but resistant cells exhibited signs of epithelial-mesenchymal transition and demonstrated increased sensitivity to AKT inhibitors. These results suggest that CO-1686 may offer a novel therapeutic option for patients with mutant EGFR NSCLC.nnnSIGNIFICANCEnWe report the preclinical development of a novel covalent inhibitor, CO-1686, that irreversibly and selectively inhibits mutant EGFR, in particular the T790M drug-resistance mutation, in NSCLC models. CO-1686 is the fi rst drug of its class in clinical development for the treatment of T790M-positive NSCLC, potentially offering potent inhibition of mutant EGFR while avoiding the on-target toxicity observed with inhibition of the WT EGFR.

Vascular Endothelial Cell Growth Factor Receptor 3-Mediated Activation of Lymphatic Endothelium Is Crucial for Tumor Cell Entry and Spread via Lymphatic Vessels

Lymphangiogenic growth factors vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to promote lymphatic metastasis by inducing tumor-associated lymphangiogenesis. In this study, we have investigated how tumor cells gain access into lymphatic vessels and at what stage tumor cells initiate metastasis. We show that VEGF-C produced by tumor cells induced extensive lymphatic sprouting towards the tumor cells as well as dilation of the draining lymphatic vessels, suggesting an active role of lymphatic endothelial cells in lymphatic metastasis. A significant increase in lymphatic vessel growth occurred between 2 and 3 weeks after tumor xenotransplantation, and lymph node metastasis occurred at the same stage. These processes were blocked dose-dependently by inhibition of VEGF receptor 3 (VEGFR-3) signaling by systemic delivery of a soluble VEGFR-3-immunoglobulin (Ig) fusion protein via adenoviral or adeno-associated viral vectors. However, VEGFR-3-Ig did not suppress lymph node metastasis when the treatment was started at a later stage after the tumor cells had already spread out, suggesting that tumor cell entry into lymphatic vessels is a key step during tumor dissemination via the lymphatics. Whereas lymphangiogenesis and lymph node metastasis were significantly inhibited by VEGFR-3-Ig, some tumor cells were still detected in the lymph nodes in some of the treated mice. This indicates that complete blockade of lymphatic metastasis may require the targeting of both tumor lymphangiogenesis and tumor cell invasion.

Stable antibody expression at therapeutic levels using the 2A peptide.

Therapeutic monoclonal antibodies (mAbs) are currently being developed for the treatment of cancer and other diseases. Despite clinical success, widespread application of mAb therapies may be limited by manufacturing capabilities. In this paper, we describe a mAb delivery system that allows continuous production of a full-length antibody at high-concentrations in vivo after gene transfer. The mAb is expressed from a single open reading frame by linking the heavy and light chains with a 2A self-processing peptide derived from the foot-and-mouth disease virus. Using this expression system, we generated a recombinant adeno-associated virus vector encoding the VEGFR2-neutralizing mAb DC101 (rAAV8-DC101). A single dose of rAAV8-DC101 resulted in long-term expression of >1,000 μg/ml of DC101 in mice, demonstrating significant anti-tumor efficacy. This report describes the first feasible gene therapy approach for stable delivery of mAbs at therapeutic levels, which may serve as an attractive alternative to direct injection of mAbs.

Inhibition of Lymphogenous Metastasis Using Adeno-Associated Virus-Mediated Gene Transfer of a Soluble VEGFR-3 Decoy Receptor

The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival in many types of cancer. It has recently been shown that the lymphangiogenic growth factor, vascular endothelial growth factor-C (VEGF-C), and its receptor, VEGF receptor-3 (VEGFR3), may play a pivotal role in the promotion of metastasis to regional lymph nodes. In this study, human prostate and melanoma tumor models that preferentially metastasize to the lymph nodes following s.c. tumor cell implantation were established from lymph node metastases via in vivo selection. Melanoma tumor cell sublines established from lymph node metastasis express higher amounts of VEGF-C than the parental tumor cells. The inhibition of tumor-derived VEGF-C with a soluble VEGFR3 decoy receptor, sVEGFR3-Fc, expressed via a recombinant adeno-associated viral vector, potently blocks tumor-associated lymphangiogenesis and tumor metastasis to the lymph nodes, when the treatment was initiated before the tumor implantation. In addition, sVEGFR3-Fc serum levels required for efficient blockade of lymph node metastases are strictly dependent on the VEGF-C levels generated by the primary tumor. Recombinant adeno-associated virus-mediated gene transfer of sVEGFR3-Fc may represent a feasible therapeutic strategy for blockade of lymphogenous metastasis.

Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 Part 1): an international, multicentre, open-label, phase 2 trial

BACKGROUNDnPoly(ADP-ribose) polymerase (PARP) inhibitors have activity in ovarian carcinomas with homologous recombination deficiency. Along with BRCA1 and BRCA2 (BRCA) mutations genomic loss of heterozygosity (LOH) might also represent homologous recombination deficiency. In ARIEL2, we assessed the ability of tumour genomic LOH, quantified with a next-generation sequencing assay, to predict response to rucaparib, an oral PARP inhibitor.nnnMETHODSnARIEL2 is an international, multicentre, two-part, phase 2, open-label study done at 49 hospitals and cancer centres in Australia, Canada, France, Spain, the UK, and the USA. In ARIEL2 Part 1, patients with recurrent, platinum-sensitive, high-grade ovarian carcinoma were classified into one of three predefined homologous recombination deficiency subgroups on the basis of tumour mutational analysis: BRCA mutant (deleterious germline or somatic), BRCA wild-type and LOH high (LOH high group), or BRCA wild-type and LOH low (LOH low group). We prespecified a cutoff of 14% or more genomic LOH for LOH high. Patients began treatment with oral rucaparib at 600 mg twice per day for continuous 28 day cycles until disease progression or any other reason for discontinuation. The primary endpoint was progression-free survival. All patients treated with at least one dose of rucaparib were included in the safety analyses and all treated patients who were classified were included in the primary endpoint analysis. This trial is registered with ClinicalTrials.gov, number NCT01891344. Enrolment into ARIEL2 Part 1 is complete, although an extension (Part 2) is ongoing.nnnFINDINGSn256 patients were screened and 206 were enrolled between Oct 30, 2013, and Dec 19, 2014. At the data cutoff date (Jan 18, 2016), 204 patients had received rucaparib, with 28 patients remaining in the study. 192 patients could be classified into one of the three predefined homologous recombination deficiency subgroups: BRCA mutant (n=40), LOH high (n=82), or LOH low (n=70). Tumours from 12 patients were established as BRCA wild-type, but could not be classified for LOH, because of insufficient neoplastic nuclei in the sample. The median duration of treatment for the 204 patients was 5·7 months (IQR 2·8-10·1). 24 patients in the BRCA mutant subgroup, 56 patients in the LOH high subgroup, and 59 patients in the LOH low subgroup had disease progression or died. Median progression-free survival after rucaparib treatment was 12·8 months (95% CI 9·0-14·7) in the BRCA mutant subgroup, 5·7 months (5·3-7·6) in the LOH high subgroup, and 5·2 months (3·6-5·5) in the LOH low subgroup. Progression-free survival was significantly longer in the BRCA mutant (hazard ratio 0·27, 95% CI 0·16-0·44, p<0·0001) and LOH high (0·62, 0·42-0·90, p=0·011) subgroups compared with the LOH low subgroup. The most common grade 3 or worse treatment-emergent adverse events were anaemia or decreased haemoglobin (45 [22%] patients), and elevations in alanine aminotransferase or aspartate aminotransferase (25 [12%]). Common serious adverse events included small intestinal obstruction (10 [5%] of 204 patients), malignant neoplasm progression (10 [5%]), and anaemia (nine [4%]). Three patients died during the study (two because of disease progression and one because of sepsis and disease progression). No treatment-related deaths occurred.nnnINTERPRETATIONnIn patients with BRCA mutant or BRCA wild-type and LOH high platinum-sensitive ovarian carcinomas treated with rucaparib, progression-free survival was longer than in patients with BRCA wild-type LOH low carcinomas. Our results suggest that assessment of tumour LOH can be used to identify patients with BRCA wild-type platinum-sensitive ovarian cancers who might benefit from rucaparib. These results extend the potential usefulness of PARP inhibitors in the treatment setting beyond BRCA mutant tumours.nnnFUNDINGnClovis Oncology, US Department of Defense Ovarian Cancer Research Program, Stand Up To Cancer-Ovarian Cancer Research Fund Alliance-National Ovarian Cancer Coalition Dream Team Translational Research Grant, and V Foundation Translational Award.

Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients

Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment.

Secondary Somatic Mutations Restoring RAD51C and RAD51D Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma

High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51CIn vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations.Significance: Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. Cancer Discov; 7(9); 984-98. ©2017 AACR.See related commentary by Domchek, p. 937See related article by Quigley et al., p. 999See related article by Goodall et al., p. 1006This article is highlighted in the In This Issue feature, p. 920.

A Quantitative Chemotherapy Genetic Interaction Map Reveals Factors Associated with PARP Inhibitor Resistance

SUMMARY Chemotherapy is used to treat most cancer patients, yet our understanding of factors that dictate response and resistance to such drugs remains limited. We report the generation of a quantitative chemical-genetic interaction map in human mammary epithelial cells charting the impact of the knockdown of 625 genes related to cancer and DNA repair on sensitivity to 29 drugs, covering all classes of chemotherapy. This quantitative map is predictive of interactions maintained in other cell lines, identifies DNA-repair factors, predicts cancer cell line responses to therapy, and prioritizes synergistic drug combinations. We identify that ARID1A loss confers resistance to PARP inhibitors in cells and ovarian cancer patients and that loss of GPBP1 causes resistance to cisplatin and PARP inhibitors through the regulation of genes involved in homologous recombination. This map helps navigate patient genomic data and optimize chemotherapeutic regimens by delineating factors involved in the response to specific types of DNA damage.

Abstract 1876: Preclinical efficacy of fibroblast growth factor ligand trap HGS1036 in lung carcinoma models with genomic amplification of FGFR1

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnLung cancer is the most common cause of cancer-related death in industrialized countries. In 2010, it is estimated that cancer of the lung and bronchus will be responsible for 157,300 deaths in the US (Jermal et al. 2010). A number of recent publications have reported the genomic amplification of the fibroblast growth factor receptor 1 gene (FGFR1) in lung cancer (Weiss et al. 2010; Voortman et al. 2010). Subtype analysis indicates that approximately 22% of squamous non-small cell lung cell carcinomas (NSCLC) and 27-33% of small cell lung cancer (SCLC) have amplification of the FGFR1 gene. Amplification of the FGFR1 gene in lung cancer cell lines is associated with over-expression of the FGFR1 receptor protein and increased signaling through the FGFR1 pathway resulting in increased cell proliferation and tumorigenesis. HGS1036 (formerly FP-1039) is a soluble fusion protein consisting of the extracellular domain of human FGFR1 isoform α-IIIc linked to the Fc region of human immunoglobulin G1 (IgG1). HGS1036 acts as a ligand “trap” that sequesters multiple fibroblast growth factor (FGF) family ligands, blocking their ability to bind to and activate multiple FGF receptors. We investigated the preclinical efficacy of HGS1036 in preclinical lung cancer models with genomic FGFR1 amplification as a novel therapeutic strategy. A panel of lung cancer cell lines (n=5) were identified that displayed genomic FGFR1 amplification on chromosome 8. Two of the cell lines, DMS53 and DMS114, were derived from the tumors of patients with SCLC. The other three cell lines, NCI-H1581, NCI-H520 and NCI-H1703, were derived from the tumors of patients with NSCLC. Examination of in vitro drug sensitivity indicated that all 5 cell lines were sensitive to HGS1036. Treatment of NCI-H1581, NCI-H520, NCI-H1703, DMS53 and DMS114 cells with HGS1036 in vitro resulted in a 52%, 71%, 46%, 47% and 82% reduction in cell proliferation, respectively, as determined by CellTiterGlo. Sensitivity to HGS1036 in vitro generally correlated with FGFR1 copy number and total FGFR1 protein expression. HGS1036 treatment of mice bearing FGFR1 amplified lung cancer xenografts resulted in tumor growth inhibition in all five FGFR1-amplified models examined (p=<0.01). The degree of tumor growth inhibition, as assessed by area-under-the-curve analysis, varied from 31-84% depending on the FGFR1 amplified lung model examined. These experiments demonstrate that lung tumor cell lines with genomic FGFR1 amplification can be sensitive to FGF-ligand blockade by HGS1036 and provide preclinical rationale for the clinical study of HGS1036 in the treatment of lung cancer.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1876. doi:1538-7445.AM2012-1876

Abstract 2597: Preclinical efficacy of FP-1039 (FGFR1: Fc) in endometrial carcinoma models with activating mutations in FGFR2

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnEndometrial cancer is the most common gynecological cancer in industrialized countries, with an estimated incidence of approximately 42,000 cases in the United States in 2009. The majority of patients with endometrial cancer are cured with hysterectomy; however, for patients with non-resectable or recurrent disease, there is no curative treatment. A number of publications have recently reported the identification of somatic mutations in the fibroblast growth factor receptor 2 (FGFR2) in 15-16% of endometrial carcinomas. The most common FGFR2 mutation observed was S252W (∼7 % of all cases), which is identical to the germline activating mutation in FGFR2 seen in Apert Syndrome, a congenital human skeletal disorder. Structure-function studies of S252W FGFR2 demonstrate that the mutant receptor has altered ligand specificity and enhanced affinity for multiple FGFs, compared to wild-type FGFR2. FP-1039 is a soluble fusion protein consisting of the extracellular domain of human fibroblast growth factor receptor 1 (FGFR1) isoform α-IIIc linked to the Fc region of human immunoglobulin G1 (IgG1). FP-1039 acts as a ligand “trap” that sequesters multiple fibroblast growth factor (FGF) family ligands, blocking their ability to bind to and activate multiple FGF receptors. FP-1039 is currently being investigated in a single agent Phase I dose-escalation study in patients with advanced solid malignancies (Tolcher et al., AACR-NCI-EORTC 2009). We have examined the in vitro and in vivo sensitivity of FGFR2 S252W bearing mutant (MFE-280 and MFE-319) and wild-type (w.t.; HEC-1-B) human endometrial carcinoma cell lines to FP-1039. Addition of FP-1039 to MFE-280 and MFE-319 cells in tissue culture resulted in a 49% and 14% reduction, respectively, in cell proliferation as determined by 3H thymidine incorporation. FP-1039 had no effect on HEC-1-B cell proliferation in vitro. FP-1039 treatment of mice bearing HEC-1-B and MFE-319 xenografts resulted in a 22% and 23% decrease (p=<0.01) in tumor volume, respectively, as assessed by area-under-the-curve analysis. FP-1039 administration in the MFE-280 xenograft model produced a dramatic 95% reduction in tumor growth (p<0.001) and a corresponding increase in median survival time (MST) when compared to vehicle-treated controls. These experiments demonstrate that tumor cell lines bearing the S252W FGFR2 mutation can be exquisitely sensitive to FGF ligand inhibition by FP-1039. The cellular mechanisms responsible for the differential sensitivity of S252W FGFR2-bearing endometrial carcinoma cell lines to FGF ligand-binding blockade are not understood and are currently being investigated. These data provide a preclinical rationale for the clinical study of FP-1039 as a single agent for the treatment of non-resectable or recurrent endometrial carcinoma in patients with tumors bearing the S252W mutation.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2597.