Press Conference from Functional Viability Profiles of Breast Cancer

Summary Genomic screening is routinely used to guide the treatment of cancer patients in many countries. However, several multi-layered factors make this effort difficult to deliver within a clinically relevant timeframe. Here we share the learnings from the CRUK-funded Stratified Medicine Programme for advanced NSCLC patients, which could be useful to better plan future studies.

Supplementary Table 1 from High-Throughput RNAi Screening Reveals Novel Regulators of Telomerase

Supplementary Figures 1-7 from High-Throughput RNAi Screening Reveals Novel Regulators of Telomerase

Abstract The design of targeted therapeutic strategies for cancer has largely been driven by the identification of tumor-specific genetic changes. However, the large number of genetic alterations present in tumor cells means that it is difficult to discriminate between genes that are critical for maintaining the disease state and those that are merely coincidental. Even when critical genes can be identified, directly targeting these is often challenging, meaning that alternative strategies such as exploiting synthetic lethality may be beneficial. To address these issues, we have carried out a functional genetic screen in >30 commonly used models of breast cancer to identify genes critical to the growth of specific breast cancer subtypes. In particular, we describe potential new therapeutic targets for PTEN-mutated cancers and for estrogen receptor–positive breast cancers. We also show that large-scale functional profiling allows the classification of breast cancers into subgroups distinct from established subtypes. Significance: Despite the wealth of molecular profiling data that describe breast tumors and breast tumor cell models, our understanding of the fundamental genetic dependencies in this disease is relatively poor. Using high-throughput RNA interference screening of a series of pharmacologically tractable genes, we have generated comprehensive functional viability profiles for a wide panel of commonly used breast tumor cell models. Analysis of these profiles identifies a series of novel genetic dependencies, including that of PTEN-null breast tumor cells upon mitotic checkpoint kinases, and provides a framework upon which additional dependencies and candidate therapeutic targets may be identified. Cancer Discovery; 1(3); 260–73. © 2011 AACR. Read the Commentary on this article by Beijersbergen and Bernards, p. 205 This article is highlighted in the In This Issue feature, p. 189

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The majority of targeted therapies for non-small-cell lung cancer (NSCLC) are directed against oncogenic drivers that are more prevalent in patients with light exposure to tobacco smoke1–3. As this group represents around 20% of all patients with lung cancer, the discovery of stratified medicine options for tobacco-associated NSCLC is a high priority. Umbrella trials seek to streamline the investigation of genotype-based treatments by screening tumours for multiple genomic alterations and triaging patients to one of several genotype-matched therapeutic agents. Here we report the current outcomes of 19 drug–biomarker cohorts from the ongoing National Lung Matrix Trial, the largest umbrella trial in NSCLC. We use next-generation sequencing to match patients to appropriate targeted therapies on the basis of their tumour genotype. The Bayesian trial design enables outcome data from open cohorts that are still recruiting to be reported alongside data from closed cohorts. Of the 5,467 patients that were screened, 2,007 were molecularly eligible for entry into the trial, and 302 entered the trial to receive genotype-matched therapy—including 14 that re-registered to the trial for a sequential trial drug. Despite pre-clinical data supporting the drug–biomarker combinations, current evidence shows that a limited number of combinations demonstrate clinically relevant benefits, which remain concentrated in patients with lung cancers that are associated with minimal exposure to tobacco smoke. Current outcomes are reported from the ongoing National Lung Matrix Trial, an umbrella trial for the treatment of non-small-cell lung cancer in which patients are triaged according to their tumour genotype and matched with targeted therapeutic agents.

Background The APOBEC3 family of cytidine deaminases mutate the cancer genome in a range of cancer types. Although many studies have documented the downstream effects of APOBEC3 activity through next-generation sequencing, less is known about their upstream regulation. In this study, we sought to identify a molecular basis for APOBEC3 expression and activation. Results HER2 amplification and PTEN loss promote DNA replication stress and APOBEC3B activity in vitro and correlate with APOBEC3 mutagenesis in vivo . HER2-enriched breast carcinomas display evidence of elevated levels of replication stress-associated DNA damage in vivo . Chemical and cytotoxic induction of replication stress, through aphidicolin, gemcitabine, camptothecin or hydroxyurea exposure, activates transcription of APOBEC3B via an ATR/Chk1-dependent pathway in vitro . APOBEC3B activation can be attenuated through repression of oncogenic signalling, small molecule inhibition of receptor tyrosine kinase signalling and alleviation of replication stress through nucleoside supplementation. Conclusion These data link oncogene, loss of tumour suppressor gene and drug-induced replication stress with APOBEC3B activity, providing new insights into how cytidine deaminase-induced mutagenesis might be activated in tumourigenesis and limited therapeutically.

Abstract Background: Vascular endothelial growth factor (VEGF) is the major angiogenic factor in human cancer; VEGF-A is the most studied cytokine; VEGF-C is also involved in lymphatic development and both can act through binding to VEGFR2. Bevacizumab (Bev) is a humanized antibody directed towards VEGF with a modest effect in unselected HER2-negative breast cancer (BC). Patients & Methods: These VEGFs were determined in the randomized GeparQuinto trial investigating Bev as neoadjuvant treatment for HER2-negative BC (von Minckwitz et al N Eng J Med 2012). Patients were randomized to neoadjuvant chemotherapy with ECx4 and docetaxelx4 with or without concomitant Bev. VEGF-A, VEGF-C and VEGFR2 (VEGFs) were determined in serum samples prior to any treatment by use of commercial enzyme-linked immuno assays (Quantikine, R & D Systems, Minneapolis, USA). Serum levels of VEGF-A; -C and R2 were analyzed as dichotomized variables and correlated with pathological complete response (pCR, ypT0 ypN0) at surgery. Results: The VEGFs were determined in 830 (43%) trial participants of which 289 had triple negative (TN) and 541 hormone receptor positive (HR+) BC. The median age was 48 years (range 42-56), 81% had a ductal cancer and 44% were grade III. Median levels were 295.0 range 160-497 pg/mL for VEGF-A; 2102 range 1576-2588 pg/mL for VEGF-C; and 2328 range 1887-2913 pg/mL for VEGFR2, respectively. STEPP (subpopulation treatment effect pattern plot) analyses revealed a potential predictive value for all 3 VEGF's. In dichotomized analyses, TNBC patients with high VEGF-C had a higher chance of pCR with Bev in univariate analysis [interaction OR=4.4; p= 0.010] and multivariate analysis (interaction OR=6.5; p= 0.005) adjusted for age (≥ 50y vs. < 50y), histological type (ductal vs. lobular vs. others), grade (III vs. I-II), tumor stage (stage 4 vs. 1-3) and nodal status (node-negative vs. node positive). High VEGFR2 was correlated with pCR in univariate analysis [interaction OR=7.9; p= 0.017] whilst no effect was seen in multivariate analysis. HR+BC patients with high VEGF-A had an increased chance of pCR in univariate analysis [interaction OR=25.7; p< 0.001] and multivariate analysis [OR=29.4; p< 0.001]. A total 37.7% of the TNBC were classified as having high VEGFC and 31.2% obtained a pCR; 44.8% with Bev vs. 15.7% without Bev. A total of 89.3% of the TN patients were classified as “high VEGFR2” with a pCR rate of 31.4%; 39.5% with Bev vs. 23.9% without Bev. In the HR+ group, 41.6% were classified as having high VEGFA with a pCR rate of 6.7%; 11% with Bev vs. 2.6% without Bev. Conclusions: Results from the prospective GeparQuinto study demonstrate that VEGFs determined in serum can function as predictive tests for chemotherapy+/-Bev in the neoadjuvant setting. Prominent findings were a predictive value for bevacizumab for VEGF-C in TNBC and for VEGF-A in HR+BC. Citation Format: Barbro K. Linderholm, Gϋnter von Minckwitz, Stefano Caramuta, Fabrice André, Christos Sotiriou, Maria A. Cerone, Matthias Schwenkglenks, Patricia Blank, Carsten Denkert, Stephan Gade, Sibylle Loibl, on behalf of the GBG neoadjuvant board and the RESPONSIFY consortium. Serum-vascular endothelial growth factors (sVEGF) A and C have a potential as predictive tests for neoadjuvant bevacizumab in primary breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 896. doi:10.1158/1538-7445.AM2014-896

UNLABELLEDThe design of targeted therapeutic strategies for cancer has largely been driven by the identification of tumor-specific genetic changes. However, the large number of genetic alterations present in tumor cells means that it is difficult to discriminate between genes that are critical for maintaining the disease state and those that are merely coincidental. Even when critical genes can be identified, directly targeting these is often challenging, meaning that alternative strategies such as exploiting synthetic lethality may be beneficial. To address these issues, we have carried out a functional genetic screen in >30 commonly used models of breast cancer to identify genes critical to the growth of specific breast cancer subtypes. In particular, we describe potential new therapeutic targets for PTEN-mutated cancers and for estrogen receptor-positive breast cancers. We also show that large-scale functional profiling allows the classification of breast cancers into subgroups distinct from established subtypes.SIGNIFICANCEDespite the wealth of molecular profiling data that describe breast tumors and breast tumor cell models, our understanding of the fundamental genetic dependencies in this disease is relatively poor. Using high-throughput RNA interference screening of a series of pharmacologically tractable genes, we have generated comprehensive functional viability profiles for a wide panel of commonly used breast tumor cell models. Analysis of these profiles identifies a series of novel genetic dependencies, including that of PTEN-null breast tumor cells upon mitotic checkpoint kinases, and provides a framework upon which additional dependencies and candidate therapeutic targets may be identified.

Abstract Telomerase is considered an attractive anticancer target on the basis of its common and specific activation in most human cancers. While direct telomerase inhibition is being explored as a therapeutic strategy, alternative strategies to target regulators of telomerase that could disrupt telomere maintenance and cancer cell proliferation are not yet available. Here, we report the findings of a high-throughput functional RNA interference screen to globally profile the contribution of kinases to telomerase activity (TA). This analysis identified a number of novel telomerase modulators, including ERK8 kinase, whose inhibition reduces TA and elicited characteristics of telomere dysfunction. Given that kinases represent attractive drug targets, we addressed the therapeutic implications of our findings, such as demonstrating how limiting TA via kinase blockade could sensitize cells to inhibition of the telomere-associated protein tankyrase. Taken together, our findings suggest novel combinatorial approaches to targeting telomere maintenance as a strategy for cancer therapy. Cancer Res; 71(9); 3328–40. ©2011 AACR.

RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.

Upon treatment with retinoic acid, NTera‐2 (NT2) human teratocarcinoma and SK‐N‐SH neuroblastoma cells can be induced to terminally differentiate into postmitotic neuronal cells. The neuronal cell yield obtained from the NT‐2 cells is partially dependent on the time of differentiation (24–55 days). SK‐N‐SH cells differentiate into a mixed population of neuronal and epithelium‐like cells. Here we report modified protocols that increase the number of differentiated NT‐2 and SK‐N‐SH cells and that establish an enriched neuronal SK‐N‐SH‐derived cell population essentially devoid of nonneuronal cells. Differentiated cells express the cytoskeleton‐associated protein tau and other typical neuronal markers, such as Map2, Ngn1, NeuroD, Mash1, and GluR which are also expressed in primary human fetal neurons. Telomerase activity is down‐regulated in differentiated cells, which is consistent with the telomerase status of primary fetal human neurons. Thus, differentiated NT2 and SK‐N‐SH cells may represent an excellent source for studies investigating the role of telomerase or other survival‐promoting activities in protecting human neuronal cells from cell death‐mediating stresses associated with neurodegenerative diseases. © 2006 Wiley‐Liss, Inc.

Breast cancer is the most common malignancy among women. Current therapies for breast tumors are based on the use of chemotherapeutic drugs that are quite toxic for the patients and often result in resistance. Telomerase is up-regulated in 95% of breast carcinomas but not in adjacent normal tissues. Therefore, it represents a very promising target for anticancer therapies. Unfortunately, the antiproliferative effects of telomerase inhibition require extensive telomere shortening before they are fully present. Combining telomerase inhibition with common chemotherapeutic drugs can be used to reduce this lag phase and induce tumor cell death more effectively. Few studies have analyzed the effects of telomerase inhibition in combination with anticancer drugs in breast cancer cells. In this study, we inhibited telomerase activity in two breast cancer cell lines using a dominant-negative human telomerase reverse transcriptase and analyzed cell viability after treatment with different anticancer compounds. We found that dominant-negative human telomerase reverse transcriptase efficiently inhibits telomerase activity and causes telomere shortening over time. Moreover, cells in which telomerase was suppressed were more sensitive to anticancer agents independently of their mechanism of action and this sensitization was dependent on the presence of shorter telomeres. Altogether, our data show that blocking telomere length maintenance in combination with anticancer drugs can be used as an effective way to induce death of breast cancer cells. [Mol Cancer Ther 2006;5(7):1669–75]

: Telomerase is a ribonucleoprotein complex that maintains the stability of chromosome ends the telomeres and regulates cell replicative potential. The enzyme minimally contains a catalytic subunit with reverse transcriptase activity (hTERT) and a RNA subunit (hTR) with a region complementary to the telomeric repeats that is used as template. Telomerase is up-regulated in 05% of breast carcinoma but not in adjacent normal tissues and its activity increases with tumor aggressiveness. Therefore targeting telomerase may represent a promising approach for cancer therapy. Inhibition of telomerase would result in telomere shortening and cell death due to dysfunctional telomeres. The major limitation of this approach is the time necessary for the telomeres to shorten sufficiently to engage cell death. One possibility to overcome this lag phase is to target the telomeres by introducing hTRs with mutations in the template region which results in decreased cell viability and increased apoptosis. The aim of this study is to investigate the feasibility of a new anti-cancer approach based on the combination of telomere disturbances induced by mutant hTR and chemotherapeutic drugs. Our results show that interfering with telomere maintenance in breast cancer cells results in increased susceptibility to anti-cancer drugs independently of initial telomere length and mechanisms of telomere maintenance. These results suggest that this strategy could lead to the development of a general approach for the treatment of all human cancers.

Dyskeratosis congenita (DC) is a rare multi-system syndrome characterized by nail dystrophy, abnormal skin pigmentation and mucosal leukoplakia. The gene mutated in the X-linked form of human DC encodes for dyskerin, a nucleolar pseudourydilase that is involved in rRNA maturation. Dyskerin is also involved in telomerase function through its interaction with the telomerase RNA (hTR). Mutations in dyskerin result in low levels of hTR, decreased telomerase activity and telomere shortening. Autosomal dominant DC is characterized by mutations in hTR, supporting the hypothesis that the DC phenotype may be caused by impaired telomere maintenance. Several mutations have been identified in different regions of hTR in patients affected by autosomal dominant DC. Recent reports have shown that coexpression of wild-type hTR with hTR harboring mutations found in the pseudoknot domain does not affect telomerase activity in vitro. However, these studies did not assess the consequences of mutant hTR expression at the telomeres. Here we provide the first direct in vivo evidence that a mutant hTR carrying the GC to AG double substitution in the pseudoknot at nucleotides 107-108 found in patients affected by autosomal dominant DC does not behave as a dominant-negative for telomere maintenance. Rather it reconstitutes a weakly active telomerase enzyme, which is defective in telomere elongation.