Daniel J. Treacy

Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

Single-cell expression profiles of melanoma Tumors harbor multiple cell types that are thought to play a role in the development of resistance to drug treatments. Tirosh et al. used single-cell sequencing to investigate the distribution of these differing genetic profiles within melanomas. Many cells harbored heterogeneous genetic programs that reflected two different states of genetic expression, one of which was linked to resistance development. Following drug treatment, the resistance-linked expression state was found at a much higher level. Furthermore, the environment of the melanoma cells affected their gene expression programs. Science, this issue p. 189 Melanoma cells show transcriptional heterogeneity. To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.

MAP Kinase Pathway Alterations in BRAF-Mutant Melanoma Patients with Acquired Resistance to Combined RAF/MEK Inhibition

Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.

ERK Mutations Confer Resistance to Mitogen-Activated Protein Kinase Pathway Inhibitors

The use of targeted therapeutics directed against BRAF(V600)-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAF(V600)-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor-resistant alleles were sensitive to RAF/MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents.

Systematic functional characterization of resistance to PI3K inhibition in breast cancer

PIK3CA (which encodes the PI3K alpha isoform) is the most frequently mutated oncogene in breast cancer. Small-molecule PI3K inhibitors have shown promise in clinical trials; however, intrinsic and acquired resistance limits their utility. We used a systematic gain-of-function approach to identify genes whose upregulation confers resistance to the PI3K inhibitor BYL719 in breast cancer cells. Among the validated resistance genes, Proviral Insertion site in Murine leukemia virus (PIM) kinases conferred resistance by maintaining downstream PI3K effector activation in an AKT-independent manner. Concurrent pharmacologic inhibition of PIM and PI3K overcame this resistance mechanism. We also observed increased PIM expression and activity in a subset of breast cancer biopsies with clinical resistance to PI3K inhibitors. PIM1 overexpression was mutually exclusive with PIK3CA mutation in treatment-naïve breast cancers, suggesting downstream functional redundancy. Together, these results offer new insights into resistance to PI3K inhibitors and support clinical studies of combined PIM/PI3K inhibition in a subset of PIK3CA-mutant cancers. SIGNIFICANCE PIM kinase overexpression confers resistance to small-molecule PI3K inhibitors. Combined inhibition of PIM and PI3K may therefore be warranted in a subset of breast cancers. Cancer Discov; 6(10); 1134-47. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069.

Assigning clinical meaning to somatic and germ-line whole-exome sequencing data in a prospective cancer precision medicine study

Purpose:Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care.Methods:One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences.Results:At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach.Conclusion:The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017

PLZF, a Tumor Suppressor Genetically Lost in Metastatic Castration-Resistant Prostate Cancer, Is a Mediator of Resistance to Androgen Deprivation Therapy

Whole-exome sequencing of metastatic castration-resistant prostate cancer (mCRPC) reveals that 5% to 7% of tumors harbor promyelocytic leukemia zinc finger (PLZF) protein homozygous deletions. PLZF is a canonical androgen-regulated putative tumor suppressor gene whose expression is inhibited by androgen deprivation therapy (ADT). Here, we demonstrate that knockdown of PLZF expression promotes a CRPC and enzalutamide-resistant phenotype in prostate cancer cells. Reintroduction of PLZF expression is sufficient to reverse androgen-independent growth mediated by PLZF depletion. PLZF loss enhances CRPC tumor growth in a xenograft model. Bioinformatic analysis of the PLZF cistrome shows that PLZF negatively regulates multiple pathways, including the MAPK pathway. Accordingly, our data support an oncogenic program activated by ADT. This acquired mechanism together with the finding of genetic loss in CRPC implicates PLZF inactivation as a mechanism promoting ADT resistance and the CRPC phenotype.

Systematic genomic and translational efficiency studies of uveal melanoma

To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches including viability and translational efficiency assays. Knockdown of both wild type and mutant EIF1AX was lethal to uveal melanoma cells. We probed the function of N-terminal tail EIF1AX mutations by performing RNA sequencing of polysome-associated transcripts in cells expressing endogenous wild type or mutant EIF1AX. Ribosome occupancy of the global translational apparatus was sensitive to suppression of wild type but not mutant EIF1AX. Together, these studies suggest that cells expressing mutant EIF1AX may exhibit aberrant translational regulation, which may provide clonal selective advantage in the subset of uveal melanoma that harbors this mutation.

Abstract 4380: Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-sequencing

Tumors are heterogeneous ecosystems composed of genetically and epigenetically distinct cancer cell populations embedded in an intricate tumor microenvironment. The complexity and cell-to-cell interactions within this system pose a tremendous therapeutic challenge and opportunity. Due to technical constraints, current profiling technologies only provide average signals that do not reflect this intrinsic genetic and phenotypic variability. Here, we applied single-cell RNA-sequencing to examine 4,645 single cells isolated from 19 freshly procured melanomas, profiling malignant, immune and stromal cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, stem-like cells, spatial context, and a drug treatment resistance program. All tumors harbored malignant cells from two distinct transcriptional cell states, such that treatment-sensitive “MITF-high” tumors also contained drug-resistant “AXL-high” tumor cells; similar heterogeneity was present in 18 established melanoma cell lines. The frequency of AXL-high cells increased in post-relapse resistant tumors following treatment with BRAF/MEK inhibitors. Using multiplexed, quantitative single-cell immunofluorescence analysis and FACS, we validated these observations in melanoma cell lines treated with BRAF±MEK inhibitors. Signatures of cell types identified from single-cell analysis revealed distinct patterns of the tumor microenvironment. We inferred cell-to-cell interactions between stromal, immune and malignant cells, and identified factors, including known secreted gene products (e.g. CXCL12) and several complement factors. We validated the association between cancer-associated fibroblast (CAF)-expressed complement factor 3 (C3) and TIL infiltration in an independent set of 308 melanomas. Finally, analysis of TILs revealed T-cell activation dependent and independent exhaustion programs that varied among patients dependent on their exposure to treatment with immune checkpoint-inhibitors. In addition to co-expression of several known co-inhibitory receptors, including PD1, CTLA-4, and TIM-3, we identified common markers associated with cytotoxicity-independent T-cell exhaustion across patients. To identify potential T-cell clones, we classified single T-cells by their isoforms of the V and J segments of the alpha and beta TCR chains, allowing us to identify expanded T-cell clones. We found that clonally expanded T-cells expressed a strong exhaustion program, while non-expanded T-cells lacked this phenotype. This study represents the most comprehensive single-cell genomics analysis in humans to date and begins to unravel the cellular ecosystem of tumors. Single-cell genomics offer new insights with implications for both targeted and immune therapies by simultaneously profiling numerous aspects of a tumor with a single assay. Citation Format: Benjamin Izar, Itay Tirsh, Sanjay Prakadan, Marc Wadsworth, Daniel Treacy, John Trombetta, Asaf Rotem, Christine Lian, George Murphy, Mohammad Fallahi-Sichani, Ken Dutton-Regester, Jia-Ren Lin, Judit Jane-Valbuena, Orit Rozenblatt-Rosen, Charles Yoon, Alex Shalek, Aviv Regev, Levi Garraway. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-sequencing. [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 4380.

Abstract P3-06-11: Understanding resistance mechanisms of PIK3a inhibition in breast cancer

Breast cancer is the most common non-cutaneous cancer in women worldwide. PIK3CA is the most frequently mutated oncogene in all invasive breast cancers, with about one third of cases harboring activating mutations. Small molecule therapeutics targeting PI3K pathway has been developed and investigated for a decade. Everolimus (an mTOR inhibitor) is now successfully used in conjunction with hormonal therapy in patients with metastatic hormone-receptor positive breast cancers. Inhibitors of PI3Kα have shown promise as a single agent as well as in combination with hormonal therapies. The high rate of primary and acquired resistance; however, limits clinical usage of this class of drugs. There is an urgent need to understand the resistance mechanisms of PI3K inhibition and develop strategies to overcome the resistance in breast cancer. We took a genome-scale functional screening approach to identify resistance mechanisms to PI3K inhibition in breast cancer. We performed a whole-genome lentiviral open-reading frame (ORF) overexpression screen in a breast cancer cell line (T47D) in the presence of a PI3Kα inhibitor. We screened a total of 15,590 ORFs and identified 75 genes whose overexpression confers resistance to PI3K inhibition. Once we validate the hits, we plan to determine the mechanisms by which the genes confer resistance. We also plan to perform RNA-sequencing in patient samples with PI3Ka resistance to evaluate the clinical relevance of these genes and pathways. Citation Format: Le X, Luo F, Treacy D, Wulf G, Garraway L. Understanding resistance mechanisms of PIK3a inhibition in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-11.

Abstract CN07-04: Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

A single tumor is composed of malignant cells in diverse genetic and epigenetic states, and this diversity presents a significant barrier for targeted therapies. Furthermore, diverse non-malignant cells, such as immune, fibroblasts and endothelial cells shape the tumor microenvironment and are emerging as important drug targets. However, the diversity of cellular states among malignant and non-malignant cells within any tumor remains poorly understood. To begin to address these challenges we applied single-cell RNA-seq to profile >3,000 single cells isolated from 16 fresh human melanomas, and characterized distinct cell types and cell states. We found that malignant cells within the same tumor display transcriptional heterogeneity associated with multiple biological processes. In particular, subpopulations of cells in treatment-naive tumors expressed a transcriptional program associated with resistance to RAF/MEK inhibition, and these were enriched in post-relapsed samples. Among several non-malignant cell types that were identified we focused on tumor-infiltrating T-cells and identified multiple profiles of exhaustion which differed among patients and could be linked to prior immunotherapies. Finally, we used our single cell–derived profiles of cell types within melanoma to deconvolve publicly available bulk tumor profiles and infer interactions between cells in the tumor microenvironment. This work demonstrates the capacity of single cell transcriptomics to offer new insights with implications for both targeted and immune therapies and will be broadly applicable to other tumor types. Citation Format: Itay Tirosh, Benjamin Izar, Sanjay M. Prakadan, Marc H. Wadsworth II, Daniel Treacy, John J. Trombetta, Diana Lu, Asaf Rotem, Christine Lian, George Murphy, Ofir Cohen, Eli van Allen, Monica Bertagnolli, Alex Genshaft, Travis K. Hughes, Carly G. K. Ziegler, Samuel W. Kazer, Aleth Gaillard, Kellie E. Kolb, Judit Valbuena1, Charles Yoon, Orit Rozenblatt-Rosen, Alex K. Shalek, Aviv Regev and Levi Garraway. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. [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 CN07-04.