Margaret M. Chou - US grants

The underlying hypothesis of the proposed studies is that the ability of activated Cdc42 to induce anchorage-independent growth derives from its effects on cyclin A. Thus, the proposal focuses on identifying the mechanisms by which Cdc42 induces anchorage-independent growth and how it stimulates cyclin A expression in the absence of mitogens. The application contains three specific aims. The first will be to determine the mechanism by which Cdc42 activates the cyclin A promoter. Here the goal will be to identify and characterize the cis- and trans-acting factors that confer Cdc42-responsiveness to the cyclin A promoter. The second aim is to identify Cdc42 effectors that are responsible for mediating cyclin A induction and anchorage-independent growth. The third aim will be to establish whether Cdc42V12 effectors are critical mediators of adhesion-dependent signals during the normal proliferative response. The importance of these experiments is to establish whether similar pathways are used by a transforming Cdc42 mutant and wild type Cdc42 under normal physiological conditions.

DESCRIPTION (provided by applicant): Acquisition of a motile, invasive phenotype is a key step in malignant transformation. The Rho family GTPases Cdc42 and Rac1 play a central role in this process by regulating dynamic actin remodeling and vesicular trafficking, coordination of which is essential for cell movement and invasiveness. Cdc42 and Rac1 act by promoting formation of filopodia and lamellapodia, respectively, at the leading edge of a motile cell, and invadopodia at the ventral surface. My laboratory has identified a novel effector of Cdc42 and Rac1, the TRE17 oncogene (a.k.a. USP6), which we hypothesize coordinates actin remodeling and targeted vesicular trafficking. TRE17 has been implicated in tumorigenesis in both humans and mice, yet little is known of its molecular functions. We have identified two direct targets of TRE17, IQGAP1 and the Arf6 GTPase, both of which have previously been linked to motile and invasive behavior. Indeed, high IQGAP1 expression has been linked to invasiveness in carcinomas. IQGAP1 directly regulates actin remodeling, while Arf6 controls endocytosis and plasma membrane recycling. Our preliminary data further identifies calcium/calmodulin (Ca2+/CaM) as an allosteric regulator of TRE17. We hypothesize that TRE17 induces motile, invasive behavior through simultaneous regulation of actin remodeling through IQGAP1 and vesicular trafficking through Arf6, in a manner that is regulated by Ca2+. Through this work we hope to understand how these functions are coordinated during cell movement, and to elucidate the mechanism of TRE17 transformation.

DESCRIPTION (provided by applicant): Bone and soft tissue tumors (BSTTs) are important yet understudied entities in human oncology. Our incomplete understanding of their pathogenesis has limited development of effective therapies. This proposal focuses on the role of TRE17/ubiquitin-specific protease 6 (USP6), in BSTT pathogenesis. Recurrent translocations of TRE17 occur in two BSTTs, aneurysmal bone cyst (ABC) and nodular fasciitis (NF), resulting in overexpression of wild type TRE17. TRE17 overexpression also arises selectively in other BSTTs, particularly the highly lethal alveolar rhabdomyosarcoma (ARMS). TRE17 potently activates the transcription factors NFkB and STAT3, in a manner dependent on its USP activity. Both NFkB and STAT3 are frequently dysregulated in cancer, often coordinately, to promote tumor cell survival and proliferation, inflammation, and angiogenesis. This proposal will test whether NFkB and STAT3 are critical cellular effectors of TRE17 in the pathogenesis of ARMS and ABC/NF. Aim 1: Determine role of Jak1/STAT and NFkB pathways in TRE17- mediated proliferation and survival. TRE17's USP activity is essential for tumorigenesis, and preliminary data implicate Jak1, the STAT3 kinase, as its first substrate. Exciting new results show that Jak1 can be modified by ubiquitin and the Ub-like molecule, ISG15, and suggest TRE17 may counteract both of these modifications to induce its stabilization. Cell culture-based studies will be used to test this, and to determine whether Jak1/STAT3 and NFkB are essential for TRE17-mediated proliferation, survival, and induction of modulators of the tumor microenvironment. Aim 2: Determine requirement for Jak1/STAT and NFkB in TRE17-mediated tumorigenesis in murine models of ABC/NF. We have developed murine xenograft models of ABC and NF, tumors driven by TRE17 translocation. We will examine whether NFkB/Jak1/STAT3 are required cell- autonomous requirement for tumor cell survival and proliferation, and determine their role in modulating the inflammatory and angiogenic microenvironment using immunohistochemistry and in vivo bioluminescence imaging. Aim 3: Determine role of TRE17 and NFkB/STAT3 in ARMS pathogenesis. ARMS is a highly malignant BSTT with dismal survival rates. Through analysis of patient-derived ARMS cell lines and primary tumor samples, we will determine whether TRE17 is an essential mediator of ARMS pathogenesis, and whether it functions through Jak1/STAT3 and NFkB. We will examine the requirement of these factors in growth and proliferation in vitro, including their role within ARMS cancer stem cells. Transcriptome analysis will be performed to define a TRE17-response signature. Finally, we will determine the roles of TRE17, Jak1/STAT3 and NFkB in ARMS tumor formation, maintenance, and metastasis in mice. These studies may ultimately lead to novel approaches (TRE17-specific USP inhibitors, and NFkB and Jak1/STAT inhibitors) for the treatment of lethal cancers such as ARMS, and other BSTTs in which TRE17 is overexpressed.

DESCRIPTION (provided by applicant): Pediatric cancers represent an understudied area in oncology. The great majority of cancer research is dedicated to understanding adult malignancies, such as cancers of the breast, colon, lung and skin. However, pediatric cancers most commonly arise in distinct tissues, such as brain, bone, and blood. The biological landscape of such developing tissues is believed to confer a distinct susceptibility to oncogenic insults, but our understanding of the molecular basis of this phenomenon is still in its infancy. This application focuses on a novel agent implicated in the etiology of several bone and soft tissue tumors (BSTTs), a class of tumors that preferentially targets children and adolescents. TRE17 translocation occurs in two distinct BSTTs, aneurysmal bone cyst (ABC) and nodular fasciitis (NF), leading to its high level expression. Our screening of a wide panel of primary human tumors further revealed high expression specifically in a high percentage of alveolar rhabdomyosarcoma (ARMS) cases. Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma; of the various subtypes, ARMS carries the worst prognosis. My research is aimed at elucidating the pathogenic mechanisms of TRE17. Our recent work focusing on its functions in the context of ABC revealed that it functions cell-autonomously to promote tumor cell proliferation/survival, but also regulates the tumor microenvironment by inducing the production of multiple cytokines, chemokines, and growth factors, in a manner strictly dependent on its USP activity. We further identified NF?B and STAT3 as key effectors of TRE17 in ABC pathogenesis. Both of these transcription factors are widely dysregulated in cancer, often coordinately, where they function pleiotropically to promote multiple aspects of tumor growth and metastasis. We hypothesize that TRE17 plays a key role in ARMS pathogenesis, and that NF¿B and STAT3 function as critical effectors. This proposal will determine the requirement of these three factors both in vitro and in vivo. In vitro analyses will assess their rle in cell proliferation, survival, and cytokine/growth factor production, and will include examining their role within the cancer cell stem (CSC) subpopulation, which has been reported to be dependent on NF?B and STAT3 in other cancers. In vivo studies will examine their role in tumor formation, tumor maintenance, and metastasis. If successful, these studies would provide three novel targets for therapeutic intervention in ARMS. Notably, inhibitors for NF?B and STAT3 pathways are already being avidly developed for other cancers and immune disorders. Furthermore, TRE17-specific USP inhibitors might function as effective therapeutic agents for not only ARMS, but also other BSTTs driven by TRE17 overexpression. USP inhibitors are particularly appealing since TRE17 exhibits such limited expression, minimizing the likelihood of side effects.

PROJECT SUMMARY Bi-phenotypic sinonasal sarcoma (SNS) is a newly identified sarcoma that affects women three times more frequently than men. The only treatment for this highly invasive lesion is disfiguring facial surgery. We recently identified a novel fusion in SNS, which creates a novel chimera PAX3-MAML3 that fuses the DNA-binding domain of the PAX3 transcription factor with the Mastermind-like 3 (MAML3) transcriptional co-activator. The transcriptional program that PAX3-MAML3 triggers to elicit malignant transformation and invasive growth, and the basis of the gender dimorphism of SNS is completely uncharacterized. Preliminary data demonstrate that expression of PAX3-MAML3 is sufficient to drive formation of tumors that histologically mimic SNS, and to induce gene expression patterns observed in SNS. Notably, PAX3-MAML3 induced expression of estrogen receptor ? (ER?) and phosphorylation of ER?, and stimulated ER activity. Furthermore, tumors formed by xenografted PAX3-MAML3-expressing cells were significantly larger in female than male recipient mice. Transcriptome analysis of primary SNS tumors confirmed activation of an estrogen response signature in vivo. These results lead us to hypothesize that the ER plays an essential role in PAX3-MAML3's pathogenic activity in SNS, and that this underlies the gender dimorphic nature of this cancer. We further posit that anti- estrogens might serve as a complementary or even alternative approach to disfiguring surgery for the treatment of this highly invasive cancer. This will be tested through the following: Aim I: Determine role of ER in PAX3-MAML3 pathogenesis in vitro and in vivo We will examine the effects of ER inactivation, using CRISPR and anti-estrogenic agents (i.e. tamoxifen, fulvestrant, and aromatase inhibitor), on multiple aspects of PAX3-MAML3-induced transformation: (A) In vitro, we will monitor effects on cell proliferation and survival, anchorage-independent growth, and invasiveness. (B) In vivo, we will test the contribution of ER and estrogen signaling to PAX3-MAML3-mediated tumorigenesis. We will assess the effects of ER inactivation (by CRISPR- mediated depletion of ER?/ER? or by treatment of mice with anti-estrogenic agents), comparing tumor growth in male vs. female mice. Aim II: Determine how PAX3-MAML3 and ER coordinate gene expression to drive invasive growth We will: (A) define the transcriptional program induced by PAX3-MAML3 by RNA-seq, and determine how it is modulated by ER (through use of ER agonists, and CRISPR-mediated deletion of ER?/?); and (B) define the PAX3-MAML3 and ER chromatin occupancy landscapes to gain a mechanistic understanding of how PAX3-MAML3 and ER coordinate this transcriptional program. Transcriptome and ChIP- seq data sets will be intersected to distinguish direct from indirect target genes of PAX3-MAML3 and ER, and to gain insight into cooperative effects on transcription. A longterm goal of this integrated analysis is to facilitate identification of PAX3-MAML3/ER targets and their downstream pathways that are essential for the transformed, invasive phenotype, and may ultimately serve as additional therapeutic targets. !