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IN THE SPOTLIGHT

What’s the fOX Got to Do with the KITten? Regulating the Lineage-specifi c Transcriptional Landscape in GIsT Donna M. Lee 1 and Anette Duensing 1 , 2

1 Cancer Therapeutics Program, UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania. 2 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Corresponding Author: Anette Duensing, University of Pittsburgh School of Medicine, UPMC Hillman Cancer Center , Research Pavilion, Suite G.17, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-5870; Fax: 412-623-1010; E-mail: aduensin@pitt.edu doi: 10.1158/2159-8290.CD-17-1370 ©2018 American Association for Cancer Research.

summary: Transcriptional regulation of the KIT receptor tyrosine kinase, a master regulator in gastrointestinal stromal tumors (GIST) and their precursors, the interstitial cells of Cajal (ICC), is part of a positive feedback loop involving the transcription factor ETV1. A new study now shows that the forkhead box (FOX) family transcription factor FOXF1 not only is an upstream regulator of ETV1 and hence ICC/GIST lineage-specifi c gene transcription, but also functions as lineage-specifi c pioneer factor with an active role in chromatin rearrangement to facilitate ETV1 binding and transcriptional activity. Cancer Discov; 8(2); 146–9. ©2018 AACR .

See related article by Ran et al., p. 234 (5).

The majority of cancers are defi ned by specifi c genomic alterations, such as gene mutations or chromosomal trans-locations, or distinct epigenetic changes. Oftentimes, these changes affect genes with important physiologic functions in the respective cellular lineage, but how these events might contribute to retaining the lineage specifi city of cancer cells is still largely unknown.

Things seemed to be simple in gastrointestinal stromal tumors (GIST), the most common type of soft-tissue sarcoma. GISTs are driven in more than 80% of cases by oncogenic muta-tions in the KIT receptor tyrosine kinase (whose viral homolog, v-kit , causes sarcoma in kittens; refs. 1, 2 ). KIT expression is a common denominator of GISTs and interstitial cells of Cajal (ICC), the pacemaker cells of the gut, both of whom presum-ably share a common, lineage-specifi c progenitor. Physiologic KIT signaling driven by its ligand stem cell factor is crucial for the unperturbed development and differentiation of ICCs. In contrast, oncogenically activated KIT leads to constitutive, ligand-independent activation of the receptor, resulting in constant activation of intracellular signaling cascades that promote proliferation, survival, and tumor growth.

A deeper understanding of the molecular wiring of GISTs came from a study published by Chi and colleagues in 2010 ( 3 ). The authors describe the discovery of ETV1, a member of the ETS family of transcription factors, as a lineage-specifi c transcriptional regulator in GIST. They not only found that ETV1 is highly expressed in GIST and in ICC subgroups that give rise to GIST, but could also show that ETV1 regulates the expression of ICC/GIST gene signatures and is crucial for ICC/GIST cell survival. Importantly, ETV1 and KIT are part of

a positive feedback loop, in which ETV1 stimulates KIT gene transcription, and oncogenic KIT signaling enhances ETV1 expression levels through the MEK–MAPK axis ( Fig. 1A , left panel). A follow-up study by Chi’s group positioned ETV1 as a therapeutic target in GIST by combining the KIT inhibitor imatinib mesylate (the current fi rst-line therapy for inoper-able and/or metastatic GIST) with a MEK inhibitor ( Fig. 1A , right panel; ref. 4 ). Promising preliminary results of a Phase Ib/II clinical trial were reported recently (clinicaltrials.gov; NCT01991379).

However, ETV1 has been implicated not only in GIST, but also in a number of other malignancies, including prostate cancer and melanoma. So, the question of how ETV1 can provide ICC/GIST lineage-specifi c signals remained open. Ran and colleagues now provide the answer in a further follow-up, published in this issue of Cancer Discovery ( 5 ). In this study, the authors start out by comparing the ETV1 cistrome between GIST and ETV1-driven prostate cancer cells. Interestingly, they found that although ETV1 promoter binding was shared among the two lineages, nonpromoter (“enhancer”) binding was largely different. In fact, GIST-specifi c, prostate-specifi c, and a minority of shared enhancer sites could be identifi ed. In a way, these fi ndings did not come as a surprise: The enhancer landscape has been postulated to be more lineage-specifi c than the promoter landscape. How-ever, the results indicate that additional factors than ETV1 itself are likely involved in lineage-specifi c enhancer binding. Could, for example, another transcription factor colocalize with ETV1? De novo motif analysis indeed revealed an enrich-ment for the forkhead box (FOX) binding motif (besides the ETV-binding motif) at both GIST- and prostate-specifi c enhancer binding sites. Interestingly, the FOX family tran-scription factor FOXA1 is a well-characterized transcriptional regulator in prostate cancer. Although the idea was tempting, FOXA1 did not turn out to be similarly important in GIST. However, another member of the family, FOXF1, fi t the bill. FOXF1 was highly and specifi cally expressed in GIST cell lines and primary tumors on the mRNA and protein level. Importantly, chromatin immunoprecipitation–sequencing experiments proved that FOXF1 colocalizes with ETV1 at GIST enhancers ( 5 ).

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Figure 1.  FOXF1 is a transcriptional master regulator and pioneer factor of the ICC/GIST lineage. A, The receptor tyrosine kinase KIT and the transcrip-tion factor ETV1 are part of a positive feedback loop, in which KIT signaling enhances ETV1 expression levels via the MEK–MAPK signaling cascade (left). This, in turn, leads to increased transcription of KIT as well as other ICC/GIST lineage-specific genes. The circuit can be disrupted by inhibition of either KIT or MEK signaling (or a combination of both), resulting in reduced proliferation and survival (right; ref. 4). B, FOXF1 is highly and differentially expressed in the ICC/GIST lineage and colocalizes with ETV1 at ICC/GIST–specific enhancers. Positioned upstream of ETV1, it positively regulates the transcriptional expres-sion of ICC/GIST lineage-specific genes, including KIT and ETV1. However, the signaling events upstream of FOXF1 activation and whether FOXF1 plays an additional role in regulating ETV1 expression beyond transcriptional control are currently unknown. C, FOXF1 is a pioneer factor in GIST with an active role in chromatin rearrangement. As shown by Ran and colleagues (5), it is highly likely that FOXF1 can bind to condensed chromatin to stimulate the addition of H3K4me1 enhancer chromatin marks (indicated by small red dots on histone tails). This, in turn, leads to nucleosomal depletion and enhanced chromatin accessibility, enabling the “effector” transcription factor ETV1 to bind and trigger active transcription of ICC/GIST-specific genes. MEKi, MEK inhibitor.

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FOXF1 is a rather poorly characterized member of the FOX transcription factor family. Mutations in FOXF1 have been reported in a rare lung developmental disorder [alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV)], but FOXF1’s role in cancer is still somewhat unclear. Although deletions and epigenetic silencing have been described in several cancer types, paradoxically, FOXF1 overexpression was shown to be associated with a mesenchy-mal phenotype and increased invasive potential (6).

After defining FOXF1 as a likely transcriptional coregu-latory factor of ETV1 in GIST, Ran and colleagues set out to prove that FOXF1 can indeed modulate the expression of ICC/GIST lineage-specific genes. A vast amount of data employing transcriptomics and gene set enrichment analy-ses proves this point. Importantly, though, FOXF1 seemed to be upstream of ETV1: Downregulation of FOXF1 led to downregulation not only of ICC/GIST lineage genes, but also of ETV1, whereas downregulation of ETV1 did not affect FOXF1 expression levels (Fig. 1B). In the same vein, downregulation of FOXF1 perturbed ETV1 binding at ETV1 promoters and enhancers, but ETV1 downregulation did not perturb FOXF1 binding. Interestingly, there was significantly more reduction of ETV1 binding at ETV1/FOXF1 colocalized enhancers than at ETV1-only enhancers after FOXF1 knock-down, indicating that FOXF1 regulates ETV1 chromatin binding beyond mere protein dosage. It seemed likely that FOXF1 somehow recruits ETV1 to ETV1/FOXF1 enhancers. However, FOXF1 and ETV1 did not coimmunoprecipitate. Instead, FOXF1 was shown to change the global chroma-tin landscape and to modulate chromatin accessibility in a more general fashion: Its presence enabled the addition of H3K4me1 enhancer chromatin marks, likely resulting in nucleosomal depletion and increased chromatin accessibility (Fig. 1C). Together, these findings indicate that FOXF1 acts as a “pioneer factor” in GIST.

Pioneer factors are transcription factors that can prime genes for transcription and that are therefore upstream of other “effec-tor” transcription factors. Many pioneer factors are able to bind to condensed chromatin and modulate chromatin accessibil-ity. This active chromatin rearrangement is oftentimes accom-plished through epigenetic modifications, such as histone and DNA methylation. As a result, downstream transcription fac-tors can then bind to and actively transcribe DNA (Fig. 1C). As outlined above, these are exactly the properties of FOXF1 in GIST. Pioneer factors can also act as passive “bookmarks” that are more permanently bound to enhancers, flagging these sites to quickly recruit downstream transcription factors after signal activation. This is for example the case for the above-mentioned FOXA1 in prostate and mammary epithelial cells, where it marks androgen and estrogen receptor–specific enhancers, respectively. Upon androgen/estrogen binding to their receptor, enhancers are quickly identified and transcription is initiated. It will be the topic of future studies to elucidate which direct upstream signal-ing event leads to FOXF1 and ETV1 transcriptional activation in ICCs/GIST. Although one possible link could be downstream of the KIT–MEK–MAPK signaling cascade, which modulates ETV1 levels, neither KIT nor MEK inhibition resulted in changes of FOXF1 expression (Fig. 1B; ref. 5).

Taking into account the above-mentioned findings, it appears obvious that FOXF1 has a direct role in regulating

GIST cell growth. Indeed, Ran and colleagues show in several ways that this is the case (5). Inducible FOXF1 knockdown in GIST cells in vitro leads to downregulation of cell-surface and intracellular KIT expression, downstream KIT signaling, as well as ETV1 levels. At the same time, cells exit the cell division cycle. Equivalent results were seen in the GIST-like tumors that spontaneously develop in a transgenic KitV558Δ/+ mouse model when combined with an inducible Foxf1 knockout (KitV558Δ/+; Foxf1f-HA-Foxf1/f-HA-Foxf1f; Rosa26Cre-ERT2). Here, loss of FOXF1 results in a reduction of tumor weight, reduced cellular prolif-eration, and induction of apoptosis. Reduction of ICC hyper-plasia, a characteristic of the KitV558Δ/+ model, was also seen.

Targeting FOXF1 therapeutically seems to be the logical next step: taking out the master regulator of the lineage-specific circuitry in GIST. This highly effective strategy would elimi-nate not only FOXF1 itself, but also KIT and ETV1. However, this task will be even more difficult to accomplish than the (somewhat indirect) targeting of ETV1 by MEK inhibition, because the upstream regulator(s) of FOXF1 are as of yet unknown. Once again, the obstacle of how to therapeutically target a transcription factor will have to be overcome. Recent drug-development efforts that focus on targeting specific pro-teins for degradation could prove to be helpful here (5, 7). On the other end of the spectrum, broader, already FDA-approved transcriptional inhibitors, such as mithramycin A and newly developed analogues (8) or—paradoxically—inhibitors of the 26S proteasome, could be repurposed with relatively little effort. Some of these compounds have already shown preclini-cal activity in several GIST models (9, 10). Targeting the line-age-specific transcriptional circuitry in GIST will not only be promising for patients with tyrosine kinase inhibitor–refrac-tory GIST, whose tumors are typically still KIT-dependent due to the emergence of secondary mutations. More importantly, this strategy could be crucial for GISTs without targetable KIT mutations, such as SDH-deficient tumors. First, however, a similarly prominent role of FOXF1 will have to be proven for this entity. Another caveat is the fact that FOXF1 was shown to localize to all ICC subtypes in a mouse model, even to ETV1lo ICCs that do not give rise to GISTs (5). The signifi-cance of this finding is still unclear. Could it be ETV1 that pro-vides the main proliferative stimulus in ICCs to develop into a GIST, whereas FOXF1 has a different role as key maintenance factor of ICCs? In this case, it may not be needed to also target FOXF1 therapeutically. In fact, it could lead to a global loss of ICCs and substantial toxicity.

Nevertheless, the current work by Ran and colleagues pro-vides another important piece to the puzzle of the transcrip-tional circuitry in ICCs and GISTs, which will undoubtedly aid in the development of better, more specific and highly effective therapies for patients with GIST.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThe authors would like to thank all members of the Duensing

laboratory for helpful discussions and feedback. The Duensing labo-ratory is supported by the GIST Cancer Research Fund, the Sarcoma Foundation of America, the Shadyside Hospital Foundation, the Out of the Woods Foundation, and numerous private donors. A. Duensing

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is supported by UPMC Hillman Cancer Center and in part by a grant from the Pennsylvania Department of Health. The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions.

Published online February 5, 2018.

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