Alex Pearson

FGFR1 Amplification Drives Endocrine Therapy Resistance and Is a Therapeutic Target in Breast Cancer

Amplification of fibroblast growth factor receptor 1 (FGFR1) occurs in approximately 10% of breast cancers and is associated with poor prognosis. However, it is uncertain whether overexpression of FGFR1 is causally linked to the poor prognosis of amplified cancers. Here, we show that FGFR1 overexpression is robustly associated with FGFR1 amplification in two independent series of breast cancers. Breast cancer cell lines with FGFR1 overexpression and amplification show enhanced ligand-dependent signaling, with increased activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase-AKT signaling pathways in response to FGF2, but also show basal ligand-independent signaling, and are dependent on FGFR signaling for anchorage-independent growth. FGFR1-amplified cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1, suggesting that FGFR1 overexpression also promotes endocrine therapy resistance. FGFR1 signaling suppresses progesterone receptor (PR) expression in vitro, and likewise, amplified cancers are frequently PR negative, identifying a potential biomarker for FGFR1 activity. Furthermore, we show that amplified cancers have a high proliferative rate assessed by Ki67 staining and that FGFR1 amplification is found in 16% to 27% of luminal B-type breast cancers. Our data suggest that amplification and overexpression of FGFR1 may be a major contributor to poor prognosis in luminal-type breast cancers, driving anchorage-independent proliferation and endocrine therapy resistance.

Integrative molecular profiling of triple negative breast cancers identifies amplicon drivers and potential therapeutic targets

Triple negative breast cancers (TNBCs) have a relatively poor prognosis and cannot be effectively treated with current targeted therapies. We searched for genes that have the potential to be therapeutic targets by identifying genes consistently overexpressed when amplified. Fifty-six TNBCs were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH), of which 24 were subjected to genome-wide gene expression analysis. TNBCs were genetically heterogeneous; no individual focal amplification was present at high frequency, although 78.6% of TNBCs harboured at least one focal amplification. Integration of aCGH and expression data revealed 40 genes significantly overexpressed when amplified, including the known oncogenes and potential therapeutic targets, FGFR2 (10q26.3), BUB3 (10q26.3), RAB20 (13q34), PKN1 (19p13.12) and NOTCH3 (19p13.12). We identified two TNBC cell lines with FGFR2 amplification, which both had constitutive activation of FGFR2. Amplified cell lines were highly sensitive to FGFR inhibitor PD173074, and to RNAi silencing of FGFR2. Treatment with PD173074 induced apoptosis resulting partly from inhibition of PI3K-AKT signalling. Independent validation using publicly available aCGH data sets revealed FGFR2 gene was amplified in 4% (6/165) of TNBC, but not in other subtypes (0/214, P=0.0065). Our analysis demonstrates that TNBCs are heterogeneous tumours with amplifications of FGFR2 in a subgroup of tumours.

Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer.

ESR1 mutations evolve during the treatment of metastatic breast cancer. An evolving problem A large number of breast cancers express the estrogen receptor, making them susceptible to hormonal treatments. Unfortunately, these tumors can develop mutations in the estrogen receptor gene (ESR1) and become resistant to hormonal therapies that were previously effective. Schiavon et al. used three independent cohorts of breast cancer patients to demonstrate that these mutations only evolved in cases where hormonal therapy was started late in the course of the disease, after development of metastasis, and not during the initial course of treatment. If these findings are confirmed in prospective clinical trials, then they will explain why starting hormonal treatment early decreases the risk of subsequent resistance to hormonal therapy. Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high–sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor–positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer.

FGFR signaling promotes the growth of triple-negative and basal-like breast cancer cell lines both in vitro and in vivo.

Purpose: The oncogenic drivers of triple-negative (TN) and basal-like breast cancers are largely unknown. Substantial evidence now links aberrant signaling by the fibroblast growth factor receptors (FGFR) to the development of multiple cancer types. Here, we examined the role of FGFR signaling in TN breast cancer. Experimental Design: We examined the sensitivity of a panel of 31 breast cancer cell lines to the selective FGFR inhibitor PD173074 and investigated the potential mechanisms underlying sensitivity. Results: TN breast cancer cell lines were more sensitive to PD173074 than comparator cell lines (P = 0.011), with 47% (7/15) of TN cell lines showing significantly reduced growth. The majority of TN cell lines showed only modest sensitivity to FGFR inhibition in two-dimensional growth but were highly sensitive in anchorage-independent conditions. PD173074 inhibited downstream mitogen-activated protein kinase and PI3K–AKT signaling and induced cell-cycle arrest and apoptosis. Basal-like breast cancer cell lines were found to express FGF2 ligand (11/21 positive) and, similarly, 62% of basal-like breast cancers expressed FGF2, as assessed by immunohistochemistry compared with 5% of nonbasal breast cancers (P < 0.0001). RNA interference targeting of FGF2 in basal-like cell lines significantly reduced growth in vitro and reduced down stream signaling, suggesting an autocrine FGF2 signaling loop. Treatment with PD173074 significantly reduced the growth of CAL51 basal-like breast cancer cell line xenografts in vivo. Conclusions: Basal-like breast cancer cell lines, and breast cancers, express autocrine FGF2 and show sensitivity to FGFR inhibitors, identifying a potential novel therapeutic approach for these cancers. Clin Cancer Res; 17(16); 5275–86. ©2011 AACR.

Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1-CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301-13. ©2016 AACR.

High-Level Clonal FGFR Amplification and Response to FGFR Inhibition in a Translational Clinical Trial

UNLABELLED FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control. Signaling in low-level FGFR1-amplified cancers is more restricted to MAPK signaling, limiting sensitivity to FGFR inhibition. Finally, we show that circulating tumor DNA screening can identify high-level clonally amplified cancers. Our data provide a mechanistic understanding of the distinct pattern of oncogene addiction seen in highly amplified cancers and demonstrate the importance of clonality in predicting response to targeted therapy. SIGNIFICANCE Robust single-agent response to FGFR inhibition is seen only in high-level FGFR-amplified cancers, with copy-number level dictating response to FGFR inhibition in vitro, in vivo, and in the clinic. High-level amplification of FGFR2 is relatively rare in gastric and breast cancers, and we show that screening for amplification in circulating tumor DNA may present a viable strategy to screen patients. Cancer Discov; 6(8); 838-51. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

Parallel RNA interference screens identify EGFR activation as an escape mechanism in FGFR3 mutant cancer

UNLABELLED Activation of fibroblast growth factor receptors (FGFR) is a common oncogenic event. Little is known about the determinants of sensitivity to FGFR inhibition and how these may vary between different oncogenic FGFRs. Using parallel RNA interference (RNAi) genetic screens, we show that the EGF receptor (EGFR) limits sensitivity to FGFR inhibition in FGFR3-mutant and -translocated cell lines, but not in other FGFR-driven cell lines. We also identify two distinct mechanisms through which EGFR limits sensitivity. In partially FGFR3-dependent lines, inhibition of FGFR3 results in transient downregulation of mitogen-activated protein kinase signaling that is rescued by rapid upregulation of EGFR signaling. In cell lines that are intrinsically resistant to FGFR inhibition, EGFR dominates signaling via repression of FGFR3, with EGFR inhibition rescued by delayed upregulation of FGFR3 expression. Importantly, combinations of FGFR and EGFR inhibitors overcome these resistance mechanisms in vitro and in vivo. Our results illustrate the power of parallel RNAi screens in identifying common resistance mechanisms to targeted therapies. SIGNIFICANCE Our data identify a novel therapeutic approach to the treatment of FGFR3-mutant cancer, emphasizing the potential of combination approaches targeting both FGFR3 and EGFR. Our data extend the role of EGFR in mediating resistance to inhibitors targeting a mutant oncogene, showing that EGFR signaling can repress mutant FGFR3 to induce intrinsic resistance to FGFR targeting.

Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer.

Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity. Experimental Design: A panel of cell lines representative of TNBC was tested for in vitro and in vivo sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging. Results: The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both in vitro (P < 0.001 LAR vs. basal-like) and in vivo in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in PIK3CA-mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes. Conclusions: Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC. Clin Cancer Res; 23(18); 5561–72. ©2017 AACR.

An siRNA screen identifies the GNAS locus as a driver in 20q amplified breast cancer.

Poor-prognosis oestrogen receptor-positive breast cancer is characterised by the presence of high-level focal amplifications. We utilised a focused small interfering RNA screen in 14 breast cancer cell lines to define genes that were pathogenic in three genomic regions focally amplified in oestrogen receptor-positive breast cancer, 8p11-12, 11q13 and 20q. Silencing the GNAS locus, that encodes the G protein alpha stimulatory subunit Gαs, specifically reduced the growth of 20q amplified breast cancer cell lines. Examination of a publically available small hairpin RNA data set confirmed GNAS silencing to be selective for 20q amplified cancer cell lines. Cell lines with 20q amplification were found to overexpress specifically the extra long Gαs splice variant (XLαs). Overexpression of XLαs induced cAMP levels to a greater extent than Gαs, suggesting that amplification of the GNAS locus, and overexpression of the XLαs variant in particular, enhanced cAMP signalling. GNAS silencing in amplified cell lines reduced extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and conversely overexpression of exogenous XLαs in a non-amplified cell line increased MEK–ERK1/2 phosphorylation, identifying one potential downstream consequence of enhanced cAMP signalling. Our data indicate that amplification of the GNAS locus may contribute to the pathogenesis of breast cancer, and highlight a previously unrecognised role for the GNAS XLαs variant in cancer.

Joint adolescent-adult early phase clinical trials to improve access to new drugs for adolescents with cancer: proposals from the multi-stakeholder platform-ACCELERATE

The impressive progress recently observed in adult cancers through the introduction of new drugs has not yet been translated to adolescents between 12 and 17 years of age. Currently adolescents are grouped with children, so their access to new, effective drugs already available for adults is delayed because paediatric drug development starts late relative to adult programmes. Moreover, specific early phase trials designed exclusively for adolescents in rare diseases recruit poorly, even if conducted internationally. Evidence has shown that adolescents demonstrate similar toxicity profiles, maximum tolerated doses and pharmacokinetic parameters to adults. Although they may have specific vulnerabilities and their interests should be protected, they are, in many countries in Europe, entitled to provide informed consent themselves. There are no insurmountable scientific, medical, or regulatory barriers to their participation in Phase-I to III adult trials. Based on a review of the literature, the multi-stakeholder platform ACCELERATE, with representatives from academia, patient/parent advocacy groups, regulatory agencies and pharmaceutical companies, proposes the inclusion of adolescents in adult Phase-I to III trials of cancer drugs targeting a relevant disease or mechanism of action, without requiring preceding specific paediatric trials. The trials, however, should be delivered in age-appropriate clinical care settings by clinicians with adolescent trial expertise. Joint adolescent-adult trials will not exclude adolescents from participating in paediatric trials, as these approaches are complementary. This strategy is considered to be safe, rational, efficient and would provide more clinical trial options and accelerate drug development for adolescents with cancer.