Anahita Dastur

Systematic identification of genomic markers of drug sensitivity in cancer cells

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines—which represent much of the tissue-type and genetic diversity of human cancers—with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing’s sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

mTOR Inhibition Specifically Sensitizes Colorectal Cancers with KRAS or BRAF Mutations to BCL-2/BCL-XL Inhibition by Suppressing MCL-1

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.

Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer

Significance Small-cell lung cancer (SCLC) is an aggressive carcinoma with few effective treatment options beyond first-line chemotherapy. BH3 mimetics, such as ABT-263, promote apoptosis in SCLC cell lines, but early phase clinical trials demonstrated no significant clinical benefit. Here, we examine the sensitivity of a large panel of cancer cell lines, including SCLC, to ABT-263 and find that high Bcl2-interacting mediator of cell death (BIM) and low myeloid cell leukemia 1 (MCL-1) expression together predict sensitivity. SCLC cells relatively resistant to ABT-263 are sensitized by TORC1/2 inhibition via MCL-1 reduction. Combination of ABT-263 and TORC1/2 inhibition stabilizes or shrinks tumors in xenograft models, in autochthonous SCLC tumors in a genetically engineered mouse model, and in a patient-derived xenograft SCLC model. Collectively, these data support a compelling new therapeutic strategy for treating SCLC. BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.

Exploitation of the Apoptosis-Primed State of MYCN-Amplified Neuroblastoma to Develop a Potent and Specific Targeted Therapy Combination

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.

Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

BackgroundThe development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance.MethodsHere we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost.ResultsUsing this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches.ConclusionWe have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens.

Primary Patient-Derived Cancer Cells and Their Potential for Personalized Cancer Patient Care

Summary Personalized cancer therapy is based on a patient’s tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient’s living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care.

Abstract A27: Transposon mutagenesis screen identifies genes conferring resistance to BRAF inhibition in melanoma

Drug resistance states can be affected by altered gene expression levels or functions. We sought to systematically identify genes that could confer resistance to BRAF inhibition in BRAF mutant melanoma by applying a novel transposon mutagenesis screening approach. Resistant genes identified in this study include both well-characterized molecules such as BRAF, CRAF, PTEN and many other genes whose functions have not been previously associated with drug resistance, and encompass functional groups such as signaling pathway components, transcription factors, ubiquitination and sumoylation enzymes, and G-protein coupled receptors. Overexpression of these genes also confers resistance to the MEK inhibitor AZD6244 demonstrating a pathway specific activity. The resistance mechanisms are diverse: some demonstrate elevated ERK or PI3K signaling, while others do not appear to be signaling-related. Two genes identified in this study elicit both ERK and AKT signals and respectively increase the EGFR baseline expression and PLX4720-induced phosphorylation. Combined treatment with EGFR and BRAF inhibitors in these two clones demonstrated marked drug synergy. Taken together with recently published studies implicating EGFR in resistance to BRAF our study indicates that multiple mechanisms of resistance to BRAF inhibitors can converge on EGFR signaling. Our study identifies additional genes that could be used to select patients most likely to benefit from treatments combining EGFR inhibitors with BRAF/MEK inhibitors to overcome resistance. Citation Format: Li Chen, Anahita Dastur, Xunqin Yin, Cyril Benes. Transposon mutagenesis screen identifies genes conferring resistance to BRAF inhibition in melanoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A27.

Abstract 3846: Sensitivity of NOTCH1 mutant T-ALL to ABT-263

Effective targeted therapies are lacking for refractory and relapsed T-cell Acute Lymphoblastic Leukemia (T-ALL). Analysis of a large cell line screen revealed that NOTCH1 mutant T-ALL cells are sensitive to the Bcl-2/xL inhibitor ABT-263. We show that NOTCH1 inhibits the mTOR complex 1, resulting in low MCL-1 levels and sensitization to ABT-263. Further suppression of mTORC1 using small molecule inhibitors in combination with ABT-263 results in very high levels of apoptosis and tumor regressions in vivo. This suggests new therapeutic opportunities for NOTCH1 mutant T-ALL, including in the setting of gamma-secretase inhibitor (GSI) resistance. Citation Format: Anahita Dastur, Carlotta Costa, Anthony Faber, Cyril Benes. Sensitivity of NOTCH1 mutant T-ALL to ABT-263. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3846.

Abstract A77: Repression of mTORC1 activity in NOTCH1 mutant T-ALL results in sensitivity to the BCL-2 inhibitor ABT-263

There is no effective molecularly targeted therapy for T-cell Acute Lymphoblastic Leukemias (T-ALL) and prognosis for relapsed or chemotherapeutic refractory cases remains poor. A large unbiased drug screen performed across cell lines from a broad spectrum of cancers revealed that NOTCH1 mutant T-ALL cells, including lines resistant to gamma-secretase inhibitors are very sensitive to the Bcl-2/xL inhibitor ABT-263. We found that NOTCH1 up regulates the mTOR suppressor REDD1, leading to suppression of the mTOR Complex 1. This in turn results in low MCL-1 levels and ABT-263 sensitivity. Inhibition of NOTCH1 activity in this context leads to robust and sustained up regulation of mTOR signaling which could limit the activity of gamma-secretase inhibitors. Analysis of a small set of patient samples support the findings that NOTCH1 mutant T-ALL have low MCL-1 levels. Most importantly, we show that suppression of mTORC1 by a small molecule inhibitor further sensitizes NOTCH1 mutant T-ALL to ABT-263 resulting in massive apoptosis and in vivo efficacy. SIGNIFICANCE: This study uncovers a previously unappreciated link between two major oncogenic pathways, the NOTCH and mTOR pathways, with NOTCH1 repressing mTORC1 through REDD1. Further, we show that this suppression results in low MCL-1 levels and that combining mTORC inhibition with BCL-2 inhibition is a promising therapeutic strategy for GSI-resistantNOTCH1-mutant T-ALL. Citation Format: Anahita Dastur, Anthony Faber, Carlotta Costa, Cyril Benes. Repression of mTORC1 activity in NOTCH1 mutant T-ALL results in sensitivity to the BCL-2 inhibitor ABT-263. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A77.

Abstract 5523: Study of the response of melanoma lines to BRAF/MEK inhibitors and combination drug treatments

The serine-threonine protein kinase, BRAF, is a frequently mutated oncogene, found in cancers of the skin, thyroid and large intestine. It is especially prevalent in malignant melanoma, where it occurs in 40-60% of the cases. The most common BRAF mutation is the valine to glutamate substitution at position 600 of the kinase (V600E). This substitution, seen in ninety percent of the BRAF cases, leads to constitutive activation of the kinase, and the downstream MEK-ERK pathway. Appropriately, several drugs have been designed to inhibit BRAF, with a broad range of outcomes in clinical trials. Unfortunately, all patients, including those showing complete responses, develop resistance to single agent therapy. Our goal is to come up with effective drug combinations that would delay, or even prevent, the cancer from becoming resistant to treatment. We used a panel of 43 melanoma lines, comprising of 28 BRAF mutants, 12 NRAS mutants and 3 lines that were wild type for these two genes. Besides BRAF and NRAS, we have the mutational status of 65 other commonly mutated cancer genes for all the lines. We assembled a set of 25 drugs targeting a wide range of proteins, including PI3K/mTOR inhibitors, the apoptosis machinery, as well as some epigenetic modulators. The melanoma lines were treated with these drugs, either (i) alone or (ii) in combination with the B-RAF inhibitor vemurafenib or (iii) in combination with the MEK inhibitor Selumetinib, for 72 hrs over a nine point, 256-fold concentration range. Viability and apoptosis were measured using cell titer glo (Promega). The results from this pilot study are discussed here. Note: This abstract was not presented at the meeting. Citation Format: Anahita Dastur, Cyril Benes. Study of the response of melanoma lines to BRAF/MEK inhibitors and combination drug treatments. [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 5523. doi:10.1158/1538-7445.AM2014-5523