Distinct Transcriptional Programming Drive Response to MAPK Inhibition in BRAF -Mutant Melanoma Patient-Derived Xenografts
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Published OnlineFirst September 16, 2019; DOI: 10.1158/1535-7163.MCT-19-0028 Cancer Biology and Translational Studies Molecular Cancer Therapeutics Distinct Transcriptional Programming Drive Response to MAPK Inhibition in BRAFV600-Mutant Melanoma Patient-Derived Xenografts Tianshu Feng, Javad Golji, Ailing Li, Xiamei Zhang, David A. Ruddy, Daniel P. Rakiec, Felipe C. Geyer, Jane Gu, Hui Gao, Juliet A.Williams, Darrin D. Stuart, and Matthew J. Meyer Abstract Inhibitors targeting BRAF and its downstream kinase MEK are preferentially sensitive to MAPK inhibition. These gene- produce robust response in patients with advanced BRAFV600- expression programs are somewhat similar to the MITF-high mutant melanoma. However, the duration and depth of and -low phenotypes described in cancer cell lines, but response vary significantly between patients; therefore, pre- demonstrate an inverse relationship with drug response. dicting response a priori remains a significant challenge. Here, This suggests a discrepancy between in vitro and in vivo we utilized the Novartis collection of patient-derived xeno- experimental systems that warrants future investigations. grafts to characterize transcriptional alterations elicited by Finally, BRAFV600-mutant melanoma relies on either MAPK BRAF and MEK inhibitors in vivo, in an effort to identify or alternative pathways for survival under BRAF and MEK mechanisms governing differential response to MAPK inhibi- inhibition in vivo, which in turn predicts their response to tion. We show that the expression of an MITF-high, "epithelial- further pathway suppression using a combination of BRAF, like" transcriptional program is associated with reduced MEK, and ERK inhibitors. Our findings highlight the inter- sensitivity and adaptive response to BRAF and MEK inhibitor tumor heterogeneity in BRAFV600-mutant melanoma, and treatment. On the other hand, xenograft models that express the need for precision medicine strategies to target this an MAPK-driven "mesenchymal-like" transcriptional program aggressive cancer. Introduction A subset of BRAFV600-mutant melanoma patients progress rapidly upon treatment using targeted therapy (3–6). In contrast, Approximately 50% of cutaneous melanoma harbor oncogenic approximately 20% of patients benefit from durable response to mutations of BRAF. The majority of BRAF mutations occur in the BRAF and MEK inhibition, as measured by progression-free V600 position, resulting in constitutive activation of the MAPK survival at 3 years (10, 11). The disparity between these 2 patient signaling pathway (1, 2). The inhibition of BRAF, either alone or groups is not well understood. Clinical factors related to disease in combination with MEK, provides remarkable clinical benefits burden, including lactate dehydrogenase levels and the number for patients with advanced BRAFV600-mutant melanoma (3–6). of metastases, are important prognostic markers (10, 11). At the Nevertheless, despite the robust initial response, most patients molecular level, melanoma cell line sensitivity to BRAF-targeted eventually develop resistance. Acquired resistance to MAPK inhi- therapy is linked to the melanoma cell state (12, 13). bition involves heterogeneous genetic and nongenetic mechan- Gene-expression profiling revealed that melanoma falls into isms that activate MAPK or other compensatory signaling two distinct transcriptional states irrespective of their mutation pathways (7–9). As such, there is a need for patient stratification status, characterized by proliferative or invasive features and and treatment strategies in order to improve response and delay MITF expression levels (14–16). The invasive, or MITF-low and the onset of disease progression. AXL-high phenotype, confers intrinsic resistance to MAPK inhi- bition in BRAFV600-mutant melanoma in vitro (12, 13). However, Oncology Drug Discovery, Novartis Institutes for BioMedical Research (NIBR), both high and low MITF expression levels are implicated in Cambridge, Massachusetts. therapeutic resistance against MAPK inhibition in melano- Note: Supplementary data for this article are available at Molecular Cancer ma (17). It remains to be determined whether the melanoma Therapeutics Online (http://mct.aacrjournals.org/). cell state predicts response to BRAF inhibitor–based therapy in the Current address for T. Feng: Tango Therapeutics, Cambridge, MA; current clinical setting. address for A. Li and M.J. Meyer: Discovery Oncology, Bristol-Myers Squibb, In this study, we sought to elucidate mechanisms governing Cambridge, MA; and current address for H. Gao: AstraZeneca, Waltham, MA. differential sensitivity to BRAF and MEK inhibition, using our Corresponding Authors: Matthew J. Meyer, Bristol-Myers Squibb, 100 Binney internally established patient-derived xenograft (PDX) collec- Street, Cambridge, MA 02142. Phone: 617-494-7466; E-mail: tion (18). Work by our group and others demonstrated the [email protected]; and Darrin D. Stuart, Novartis Institutes for Bio- prognostic value of PDXs in modeling clinical response to tar- Medical Research, 250 Massachusetts Avenue, Cambridge, MA 02139. – Phone: 617-871-5311; E-mail: [email protected] geted therapies (18 20). In comparison with other preclinical experimental systems, PDX models conserve tumor architecture Mol Cancer Ther 2019;18:1–12 and heterogeneity, and faithfully recapitulate patient response to doi: 10.1158/1535-7163.MCT-19-0028 chemo- and targeted therapies (18, 19, 21–23). Moreover, Ó2019 American Association for Cancer Research. because obtaining high-quality research biopsies from the clinic www.aacrjournals.org OF1 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst September 16, 2019; DOI: 10.1158/1535-7163.MCT-19-0028 Feng et al. is challenging, PDXs enable profiling of treatment and time amplified RNA-seq library products were quantified using the matched tumors with statistically powered replicates. Here, we Fragment Analyzer Standard Sensitivity NGS Fragment Analysis characterized on-treatment PDX tumor samples to compare the Kit (Advanced Analytical Technologies). Samples were diluted to pharmacodynamic effects of BRAF and MEK inhibitors in 10 nmol/L in Elution Buffer (QIAGEN), denatured, and loaded at models that achieved complete regression versus partial a range of 2.5 to 4.0 pmol/L on an Illumina cBOT using the HiSeq response and stable disease. Transcriptome profiling revealed 4000 PE Cluster Kit (Illumina). RNA-seq libraries were sequenced that in vivo,aMITF-high "epithelial-like" phenotype is associ- on a HiSeq 4000 at 75 base pair paired-end with 8 base pair dual ated with adaptive and intrinsic resistance, whereas a MAPK- indexes using the HiSeq 4000 SBS Kit, 150 cycles (Illumina). The high "mesenchymal-like" phenotype is linked to sensitivity to sequence intensity files were generated on instrument using the BRAF and MEK inhibitor treatment. This is different from Illumina Real-Time Analysis software and resulting intensity files findings using in vitro cancer cell lines (12, 13, 17) and suggests were demultiplexed with the bcl2fastq2. an in vitro–in vivo discrepancy that warrants additional inves- tigation. In less sensitive models, the lack of tumor regression Gene-expression analysis in response to treatment was model dependent and mediated Gene-level read counts were normalized and voom transfor- by either MAPK-dependent or -independent mechanisms. Our mated (24) for differential expression using the limma frame- results suggested that response of BRAFV600-mutant melanoma work (25), and P values were corrected for multiple testing xenografts to BRAF and MEK inhibitors is heterogeneous, and using the Benjamini–Hochberg method. Genes were considered precision medicine–based combination strategies are required differentially expressed if the log2-fold change > 1.5 in absolute to target this aggressive cancer. Importantly, this study provided value and the adjusted P value < 0.05. Log-transformed and insights into determinants of therapeutic response in BRAFV600 TMM-normalized (26) expression values were used for cluster- melanoma in vivo. ing analysis with NbClust (27). K ¼ 5 was selected as the most informative partition. A hypergeometric distribution function was used to determine enrichment P values for overlap of each Materials and Methods cluster membership with gene sets from mSigDB (28), in Mouse xenograft studies addition to transcription factor and canonical gene sets Mice were maintained and handled in accordance with the exported from MetaCore (Clarivate Analytics). Gene set enrich- Novartis Institutes for BioMedical Research Animal Care and Use ment P values were adjusted using the Benjamini–Hochberg Committee protocols and regulations. PDX models were pro- method. Mutation and copy-number calls were made as cured and established as previously described (18). Surgical described in Gao et al. (18). The RNA-seq data have been tumor tissues were obtained from treatment-na€ve cancer submitted to the Sequence Read Archive database; submission patients, all of whom provided informed written consent for the number PRJNA562481. samples procured by Novartis Inc. PDX fragments were implanted subcutaneously into the right Clinical data set flanks of female nude mice (Charles River) using a trocar. Once Normalized and background corrected microarray data were the tumor volumes reached 300 to 500 mm3 in size, mice were downloaded from Gene-Expression Omnibus public database, randomized into respective