Devan Murphy

Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape

An oncohistone deranges inhibitory chromatin Missense mutations (that change one amino acid for another) in histone H3 can produce a so-called oncohistone and are found in a number of pediatric cancers. For example, the lysine-36–to-methionine (K36M) mutation is seen in almost all chondroblastomas. Lu et al. show that K36M mutant histones are oncogenic, and they inhibit the normal methylation of this same residue in wild-type H3 histones. The mutant histones also interfere with the normal development of bone-related cells and the deposition of inhibitory chromatin marks. Science, this issue p. 844 The lysine-36–to–methionine mutation in histone H3 is oncogenic and interferes with inhibitory chromatin marks. Several types of pediatric cancers reportedly contain high-frequency missense mutations in histone H3, yet the underlying oncogenic mechanism remains poorly characterized. Here we report that the H3 lysine 36–to–methionine (H3K36M) mutation impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases. Depleting H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy the H3K36M mutation. After the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of polycomb repressive complex 1 and de-repression of its target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas in which novel K36M/I mutations in H3.1 are identified.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Combined Inhibition of MAP Kinase and KIT Signaling Synergistically Destabilizes ETV1 and Suppresses GIST Tumor Growth

UNLABELLED Gastrointestinal stromal tumor (GIST), originating from the interstitial cells of Cajal (ICC), is characterized by frequent activating mutations of the KIT receptor tyrosine kinase. Despite the clinical success of imatinib, which targets KIT, most patients with advanced GIST develop resistance and eventually die of the disease. The ETS family transcription factor ETV1 is a master regulator of the ICC lineage. Using mouse models of Kit activation and Etv1 ablation, we demonstrate that ETV1 is required for GIST initiation and proliferation in vivo, validating it as a therapeutic target. We further uncover a positive feedback circuit where MAP kinase activation downstream of KIT stabilizes the ETV1 protein, and ETV1 positively regulates KIT expression. Combined targeting of ETV1 stability by imatinib and MEK162 resulted in increased growth suppression in vitro and complete tumor regression in vivo. The combination strategy to target ETV1 may provide an effective therapeutic strategy in GIST clinical management. SIGNIFICANCE ETV1 is a lineage-specific oncogenic transcription factor required for the growth and survival of GIST. We describe a novel strategy of targeting ETV1 protein stability by the combination of MEK and KIT inhibitors that synergistically suppress tumor growth. This strategy has the potential to change first-line therapy in GIST clinical management.

Aberrant Activation of a Gastrointestinal Transcriptional Circuit in Prostate Cancer Mediates Castration Resistance

Prostate cancer exhibits a lineage-specific dependence on androgen signaling. Castration resistance involves reactivation of androgen signaling or activation of alternative lineage programs to bypass androgen requirement. We describe an aberrant gastrointestinal-lineage transcriptome expressed in ∼5% of primary prostate cancer that is characterized by abbreviated response to androgen-deprivation therapy and in ∼30% of castration-resistant prostate cancer. This program is governed by a transcriptional circuit consisting of HNF4G and HNF1A. Cistrome and chromatin analyses revealed that HNF4G is a pioneer factor that generates and maintains enhancer landscape at gastrointestinal-lineage genes, independent of androgen-receptor signaling. In HNF4G/HNF1A-double-negative prostate cancer, exogenous expression of HNF4G at physiologic levels recapitulates the gastrointestinal transcriptome, chromatin landscape, and leads to relative castration resistance.

FOXF1 Defines the Core-Regulatory Circuitry in Gastrointestinal Stromal Tumor

The cellular context that integrates upstream signaling and downstream nuclear response dictates the oncogenic behavior and shapes treatment responses in distinct cancer types. Here, we uncover that in gastrointestinal stromal tumor (GIST), the forkhead family member FOXF1 directly controls the transcription of two master regulators, KIT and ETV1, both required for GIST precursor-interstitial cells of Cajal lineage specification and GIST tumorigenesis. Further, FOXF1 colocalizes with ETV1 at enhancers and functions as a pioneer factor that regulates the ETV1-dependent GIST lineage-specific transcriptome through modulation of the local chromatin context, including chromatin accessibility, enhancer maintenance, and ETV1 binding. Functionally, FOXF1 is required for human GIST cell growth in vitro and murine GIST tumor growth and maintenance in vivo The simultaneous control of the upstream signaling and nuclear response sets up a unique regulatory paradigm and highlights the critical role of FOXF1 in enforcing the GIST cellular context for highly lineage-restricted clinical behavior and treatment response.Significance: We uncover that FOXF1 defines the core-regulatory circuitry in GIST through both direct transcriptional regulation and pioneer factor function. The unique and simultaneous control of signaling and transcriptional circuitry by FOXF1 sets up an enforced transcriptional addiction to FOXF1 in GIST, which can be exploited diagnostically and therapeutically. Cancer Discov; 8(2); 234-51. ©2017 AACR.See related commentary by Lee and Duensing, p. 146This article is highlighted in the In This Issue feature, p. 127.

A Tmprss2-CreERT2 Knock-In Mouse Model for Cancer Genetic Studies on Prostate and Colon

Fusion between TMPRSS2 and ERG, placing ERG under the control of the TMPRSS2 promoter, is the most frequent genetic alteration in prostate cancer, present in 40–50% of cases. The fusion event is an early, if not initiating, event in prostate cancer, implicating the TMPRSS2-positive prostate epithelial cell as the cancer cell of origin in fusion-positive prostate cancer. To introduce genetic alterations into Tmprss2-positive cells in mice in a temporal-specific manner, we generated a Tmprss2-CreERT2 knock-in mouse. We found robust tamoxifen-dependent Cre activation in the prostate luminal cells but not basal epithelial cells, as well as epithelial cells of the bladder and gastrointestinal (GI) tract. The knock-in allele on the Tmprss2 locus does not noticeably impact prostate, bladder, or gastrointestinal function. Deletion of Pten in Tmprss2-positive cells of adult mice generated neoplasia only in the prostate, while deletion of Apc in these cells generated neoplasia only in the GI tract. These results suggest that this new Tmprss2-CreERT2 mouse model will be a useful resource for genetic studies on prostate and colon.

Abstract 3396: Dual lineage inhibition of ETV1 and KIT disrupts the ETV1-KIT feed forward circuit and potentiates imatinib antitumor effect in GIST oncogenesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Gastrointestinal stromal tumour (GIST) is one of the most common types of human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. Despite the initial clinical success of imatinib that specifically target mutant KIT and PDGFRA, imatinib resistance has become the biggest challenge in the management of advanced GIST patient. Novel therapeutics that can improve the efficacy of first line imatinib therapy and/or prevent and overcome imatinib resistance is imperative. The ETS family member, ETV1, has been previously identified as a lineage-specific survival factor that cooperates with mutant KIT by ETV1 protein stabilization by active MAP kinase signaling downstream of KIT signaling in GIST oncogenesis. Here we demonstrate that ETV1 is required for GIST initiation and maintenance in vivo using compound genetically engineered mouse models (GEMM). We further identified that ETV1 forms a feed forward circuit in GIST oncogenesis where the ETV1 protein is stabilized by active MAP kinase signaling downstream of KIT and stabilized ETV1enhances KIT expression through direct binding to the KIT enhancer regions in both GIST mouse models and human GIST cell lines. The dual lineage targeting of KIT by imatinib and ETV1 by MEK162 (an MEK inhibitor) disrupts the ETV1-KIT feed forward circuit and induces more apoptosis than single agent imatinib or MEK162 in human GIST cells. The combination therapy resulted in complete tumor regression whereas single agent imatinib or MEK162 resulted in stabilization of disease in human GIST xenograft studies. Moreover, the combination therapy also induced more tumor fibrosis than single agent imatinib or MEK162 in GIST GEMM. These observations demonstrate the in vivo role of ETV1 in GIST oncogenesis and the feasibility of targeting ETV1 protein stability by inhibiting MAP kinase signaling. They also suggest that the dual lineage targeting of ETV1 and KIT by the combination therapy may provide a more effective therapeutic strategy than imatinib alone in GIST management. Citation Format: Leili Ran, Inna Sirota, Zhen Cao, Devan Murphy, Shipra Shukla, Ferdinando Rossi, John Wongvipat, William D. Tap, Peter Besmer, Cristina R. Antonescu, Yu Chen, Ping Chi. Dual lineage inhibition of ETV1 and KIT disrupts the ETV1-KIT feed forward circuit and potentiates imatinib antitumor effect in GIST oncogenesis. [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 3396. doi:10.1158/1538-7445.AM2014-3396

Abstract A15: Modeling sporadic gastrointestinal stromal tumor with BRAFV600E mutation

Gastrointestinal stromal tumor (GIST) is the most common subtype of sarcoma, characterized by activating mutations in KIT , PDGFRA , BRAF, N/H/KRA S, and loss of function genetic/epigenetic alterations in NF1 , SDH -complex. Despite clinical advances for KIT/PDGFRA-mutant GIST, the scientific and clinical advances for KIT/PDGFRA-wild type GISTs have remained extremely limited due to the lack of model systems. Due to the lack of ICC-lineage specific cre system, modeling for sporadic GIST has not been feasible. ETV1 has recently been described as an ICC and GIST-lineage specific master regulator. Here, using the cre-activated Rosa26 CAGGS-LSL-EYFP system, we demonstrate that Etv1 CREERT2 preferentially activates cre in the Etv1 -expressing ICC lineage by tamoxifen. Using the Braf CA conditional allele for cre-activated Braf V600E , we observed that Braf V600E alone, while leading to development of ICC hyperplasia, is not sufficient to drive GIST tumorigenesis. Combination of Braf V600E activation and Trp53 loss not only gives rise to ICC hyperplasia, but also leads to multifocal GIST-like tumors in mouse GI tract with 100% penetrance. We further demonstrate that the Braf V600E ; Tp53-/- sporadic GIST mouse models are amenable for therapeutic intervention and recapitulate clinical responses to RAF inhibitors in human GIST. Our observations provide the first in vivo model for understanding the pathogenesis and therapeutic responses in BRAF-mutant GISTs. Importantly, these observations provide the proof of principle for modeling sporadic GIST in adult mice using the Etv1 CREERT2 system and establish the feasibility and utility of establishing model systems for human GISTs that do not have any model systems available for mechanistic studies and therapeutic development. Citation Format: Leili Ran, Devan Murphy, Jessica Sher, Zhen Cao, Shangqian Wang, Edward Walczak, Youxin Guan, Yuanyuan Xie, Shipra Shukla, Yu Zhan, Cristina R. Antonescu, Yu Chen, Ping Chi. Modeling sporadic gastrointestinal stromal tumor with BRAFV600E mutation [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A15.

COP1-DET1-ETS axis regulates ERK transcriptome and sensitivity to MAPK inhibitors

Aberrant activation of MAPK signaling leads to the activation of oncogenic transcriptomes. How MAPK signaling is coupled with the transcriptional response in cancer is not fully understood. In 2 MAPK-activated tumor types, gastrointestinal stromal tumor and melanoma, we found that ETV1 and other Pea3-ETS transcription factors are critical nuclear effectors of MAPK signaling that are regulated through protein stability. Expression of stabilized Pea3-ETS factors can partially rescue the MAPK transcriptome and cell viability after MAPK inhibition. To identify the players involved in this process, we performed a pooled genome-wide RNAi screen using a fluorescence-based ETV1 protein stability sensor and identified COP1, DET1, DDB1, UBE3C, PSMD4, and COP9 signalosome members. COP1 or DET1 loss led to decoupling between MAPK signaling and the downstream transcriptional response, where MAPK inhibition failed to destabilize Pea3 factors and fully inhibit the MAPK transcriptome, thus resulting in decreased sensitivity to MAPK pathway inhibitors. We identified multiple COP1 and DET1 mutations in human tumors that were defective in the degradation of Pea3-ETS factors. Two melanoma patients had de novo DET1 mutations arising after vemurafenib treatment. These observations indicate that MAPK signaling–dependent regulation of Pea3-ETS protein stability is a key signaling node in oncogenesis and therapeutic resistance to MAPK pathway inhibition.