John E. Monahan

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of ‘personalized’ therapeutic regimens.

Interfering with Resistance to Smoothened Antagonists by Inhibition of the PI3K Pathway in Medulloblastoma

Resistance of medulloblastoma to Smo antagonists can be delayed or prevented by specific drug combinations. An End Run Against Tumor Resistance Cancer cells are as clever as microbes. Mustering their considerable abilities to rapidly replicate and evolve, both cancer cells and bacteria quickly develop resistance to the drugs we use to fight them. Modern medicine confronts a growing population of pathogens that cannot be treated by our usual antibiotics, and oncologists must be prepared with second- and third-line therapies, because tumors that retreat from initial drug treatments often return with renewed vigor. Buonamici et al. confront this problem in their study of a new class of cancer therapeutic agents now in clinical trials—antagonists of a membrane protein called Smoothened (Smo). The Smo receptor normally regulates a developmental pathway but is abnormally activated in medulloblastoma (a malignant brain tumor) and basal cell carcinoma of the skin. Medulloblastomas in mice respond well to these Smo antagonists but soon become resistant, these authors find. If, however, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is added to the initial drug cocktail, resistance is delayed or even prevented. In some cancers, the Smo receptor is active even when its ligand is absent, conferring dependence of the tumor on the downstream Hedgehog signaling pathway, which ultimately regulates gene expression through the Gli transcription factors. Treatment of Smo-addicted tumors in mice with Smo antagonists ultimately leads to development of resistance, although tumor growth is inhibited for a while. The authors found that the tumors eluded the drug in several ways: The genes for the Gli transcription factors were sometimes amplified, compensating for loss of pathway stimulation. In other resistant tumors, there were point mutations in the Smo receptor itself that allowed reactivation of the pathway. In yet another group of tumors, by examining which genes were up-regulated, the authors found activation of a completely different signaling pathway—the PI3K pathway. Further experiments in medulloblastoma-bearing mice revealed that resistance could be delayed or even prevented by including a PI3K inhibitor along with the Smo antagonist in the initial treatment that tumor-bearing animals received. The PI3K inhibitor alone had no effect. By looking at resistance mechanisms to Smo antagonists before the drug is used in the clinic, the results of this study will better arm oncologists against the molecular defenses that cancers may commandeer to evade this drug. And by identifying a drug combination that delays or even combats development of resistance when used as a first-line treatment in clinical trials, these results could ultimately improve the lives of patients with medulloblastoma or other cancers that depend on Smo for their survival. The malignant brain cancer medulloblastoma is characterized by mutations in Hedgehog (Hh) signaling pathway genes, which lead to constitutive activation of the G protein (heterotrimeric guanosine triphosphate–binding protein)–coupled receptor Smoothened (Smo). The Smo antagonist NVP-LDE225 inhibits Hh signaling and induces tumor regression in animal models of medulloblastoma. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed several resistance mechanisms. We noted chromosomal amplification of Gli2, a downstream effector of Hh signaling, and, more rarely, point mutations in Smo that led to reactivated Hh signaling and restored tumor growth. Analysis of pathway gene expression signatures also, unexpectedly, identified up-regulation of phosphatidylinositol 3-kinase (PI3K) signaling in resistant tumors as another potential mechanism of resistance. Probing the relevance of increased PI3K signaling, we demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance. Our findings may be useful in informing treatment strategies for medulloblastoma.

An F876L Mutation in Androgen Receptor Confers Genetic and Phenotypic Resistance to MDV3100 (Enzalutamide)

UNLABELLEDnCastration-resistant prostate cancer (CRPC) is the most aggressive, incurable form of prostate cancer. MDV3100 (enzalutamide), an antagonist of the androgen receptor (AR), was approved for clinical use in men with metastatic CRPC. Although this compound showed clinical efficacy, many initial responders later developed resistance. To uncover relevant resistant mechanisms, we developed a model of spontaneous resistance to MDV3100 in LNCaP prostate cancer cells. Detailed characterization revealed that emergence of an F876L mutation in AR correlated with blunted AR response to MDV3100 and sustained proliferation during treatment. Functional studies confirmed that AR(F876L) confers an antagonist-to-agonist switch that drives phenotypic resistance. Finally, treatment with distinct antiandrogens or cyclin-dependent kinase (CDK)4/6 inhibitors effectively antagonized AR(F876L) function. Together, these findings suggest that emergence of F876L may (i) serve as a novel biomarker for prediction of drug sensitivity, (ii) predict a withdrawal response to MDV3100, and (iii) be suitably targeted with other antiandrogens or CDK4/6 inhibitors.nnnSIGNIFICANCEnWe uncovered an F876L agonist-switch mutation in AR that confers genetic and phenotypic resistance to the antiandrogen drug MDV3100. On the basis of this fi nding, we propose new therapeutic strategies to treat patients with prostate cancer presenting with this AR mutation.

High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response

Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses.

FGFR Genetic Alterations Predict for Sensitivity to NVP-BGJ398, a Selective Pan-FGFR Inhibitor

UNLABELLEDnPatient stratification biomarkers that enable the translation of cancer genetic knowledge into clinical use are essential for the successful and rapid development of emerging targeted anticancer therapeutics. Here, we describe the identification of patient stratification biomarkers for NVP-BGJ398, a novel and selective fibroblast growth factor receptor (FGFR) inhibitor. By intersecting genome-wide gene expression and genomic alteration data with cell line-sensitivity data across an annotated collection of cancer cell lines called the Cancer Cell Line Encyclopedia, we show that genetic alterations for FGFR family members predict for sensitivity to NVP-BGJ398. For the first time, we report oncogenic FGFR1 amplification in osteosarcoma as a potential patient selection biomarker. Furthermore, we show that cancer cell lines harboring FGF19 copy number gain at the 11q13 amplicon are sensitive to NVP-BGJ398 only when concomitant expression of β-klotho occurs. Thus, our findings provide the rationale for the clinical development of FGFR inhibitors in selected patients with cancer harboring tumors with the identified predictors of sensitivity.nnnSIGNIFICANCEnThe success of a personalized medicine approach using targeted therapies ultimately depends on being able to identify the patients who will benefit the most from any given drug. To this end, we have integrated the molecular profiles for more than 500 cancer cell lines with sensitivity data for the novel anticancer drug NVP-BGJ398 and showed that FGFR genetic alterations are the most significant predictors for sensitivity. This work has ultimately endorsed the incorporation of specific patient selection biomakers in the clinical trials for NVP-BGJ398.

Oncogenic PIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and PI3K pathway-independent mechanisms

PIK3CA gain-of-function mutations are a common oncogenic event in human malignancy, making phosphatidylinositol 3-kinase (PI3K) a target for cancer therapy. Despite the promise of targeted therapy, resistance often develops, leading to treatment failure. To elucidate mechanisms of resistance to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing human PIK3CAH1047R. Notably, most PIK3CAH1047R-driven mammary tumors recurred after PIK3CAH1047R inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including focal amplification of Met or Myc (also known as c-Met and c-Myc, respectively). Whereas Met amplification led to tumor survival dependent on activation of endogenous PI3K, tumors with Myc amplification became independent of the PI3K pathway. Functional analyses showed that Myc contributed to oncogene independence and resistance to PI3K inhibition. Notably, PIK3CA mutations and c-MYC elevation co-occur in a substantial fraction of human breast tumors. Together, these data suggest that c-MYC elevation represents a potential mechanism by which tumors develop resistance to current PI3K-targeted therapies.

Pharmacological and genomic profiling identifies NF-κB–targeted treatment strategies for mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive malignancy that is characterized by poor prognosis. Large-scale pharmacological profiling across more than 100 hematological cell line models identified a subset of MCL cell lines that are highly sensitive to the B cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin. Sensitive MCL models exhibited chronic activation of the BCR-driven classical nuclear factor-κB (NF-κB) pathway, whereas insensitive cell lines displayed activation of the alternative NF-κB pathway. Transcriptome sequencing revealed genetic lesions in alternative NF-κB pathway signaling components in ibrutinib-insensitive cell lines, and sequencing of 165 samples from patients with MCL identified recurrent mutations in TRAF2 or BIRC3 in 15% of these individuals. Although they are associated with insensitivity to ibrutinib, lesions in the alternative NF-κB pathway conferred dependence on the protein kinase NIK (also called mitogen-activated protein 3 kinase 14 or MAP3K14) both in vitro and in vivo. Thus, NIK is a new therapeutic target for MCL treatment, particularly for lymphomas that are refractory to BCR pathway inhibitors. Our findings reveal a pattern of mutually exclusive activation of the BCR–NF-κB or NIK–NF-κB pathways in MCL and provide critical insights into patient stratification strategies for NF-κB pathway–targeted agents.

Comprehensive mapping of p53 pathway alterations reveals an apparent role for both SNP309 and MDM2 amplification in sarcomagenesis

Purpose: Reactivation of p53 tumor suppressor activity in diseases such as soft-tissue sarcoma is considered an attractive means of targeted therapy. By systematically assessing alterations affecting the p53 pathway, we aimed to (a) classify sarcoma subtypes, (b) define a potential role in malignancy, and (c) identify potential patient biomarkers in this heterogeneous disease. Experimental Design: We have mapped mutational events in a panel of 192 benign or malignant bone and soft-tissue sarcomas. Analyses included TP53 and CDKN2A mutational and SNP status, MDM2 and MDM4 amplification and MDM2 SNP309 status. Results: We found an inverse relationship between MDM2 amplification and TP53 mutations, with a predominantly wild-type CDKN2A background. A high rate of point mutations in TP53 was observed uniquely in leiomyosarcoma, osteosarcoma, and MFH. Both MDM2 and MDM4 were also amplified in a subtype-specific manner, which was frequently seen as a coamplification event. We have also analyzed the risk allele frequencies for MDM2 SNP309, and show that the G allele was strongly associated with both liposarcomas and MDM2 amplification. Conclusions: Our data emphasize the critical role of p53 inactivation in sarcomagenesis, whereby different pathway alterations may be related to the heterogeneity of the disease. Moreover, we observed a strong association of malignancy with TP53 mutation, or MDM2 amplification and the presence of a G allele in SNP309, especially in lipoma versus liposarcoma. We propose, therefore, that MDM2 markers along with TP53 sequencing should be considered as patient biomarkers in clinical trials of sarcomas using MDM2 antagonists. Clin Cancer Res; 17(3); 416–26. ©2010 AACR.

A drug resistance screen using a selective MET inhibitor reveals a spectrum of mutations that partially overlap with activating mutations found in cancer patients

The emergence of drug resistance is a primary concern in any cancer treatment, including with targeted kinase inhibitors as exemplified by the appearance of Bcr-Abl point mutations in chronic myeloid leukemia (CML) patients treated with imatinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. To predict resistance mutations in the receptor tyrosine kinase MET that could emerge during inhibitor treatment in patients, we conducted a resistance screen in BaF3 TPR-MET cells using the novel selective MET inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of tyrosine 1230 but also included other missense mutations and partially overlapped with activating MET mutations that were previously described in cancer patients. Cocrystallization of the MET kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972, as previously reported for multiple other selective MET inhibitors. A second resistance screen in the same format with the MET inhibitor AMG 458 yielded a distinct spectrum of mutations rich in F1200 alterations, which is consistent with a different predicted binding mode. Our findings suggest that amino acid substitutions in the MET kinase domain of cancer patients need to be carefully monitored before and during treatment with MET inhibitors, as resistance may preexist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a condition that may be addressed by MET inhibitors with alternative binding modes.

The Specificity of the FOXL2 c.402C>G Somatic Mutation: A Survey of Solid Tumors

Background A somatic mutation in the FOXL2 gene is reported to be present in almost all (97%; 86/89) morphologically defined, adult-type, granulosa-cell tumors (A-GCTs). This FOXL2 c.402C>G mutation changes a highly conserved cysteine residue to a tryptophan (p.C134W). It was also found in a minority of other ovarian malignant stromal tumors, but not in benign ovarian stromal tumors or unrelated ovarian tumors or breast cancers. Methodology/Principal Findings Herein we studied other cancers and cell lines for the presence of this mutation. We screened DNA from 752 tumors of epithelial and mesenchymal origin and 28 ovarian cancer cell lines and 52 other cancer cell lines of varied origin. We found the FOXL2 c.402C>G mutation in an unreported A-GCT case and the A-GCT-derived cell line KGN. All other tumors and cell lines analyzed were mutation negative. Conclusions/Significance In addition to proving that the KGN cell line is a useful model to study A-GCTs, these data show that the c.402C>G mutation in FOXL2 is not commonly found in a wide variety of other cancers and therefore it is likely pathognomonic for A-GCTs and closely related tumors.