The Combination of Trametinib and Ganitumab is Effective in RAS-Mutated PAX-Fusion Negative Rhabdomyosarcoma Models.

PURPOSE: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. EXPERIMENTAL DESIGN: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. RESULTS: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. CONCLUSIONS: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

A Knock-In Mouse Model of Thymoma With the GTF2I L424H Mutation.

INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.

The thymoproteasome hardwires the TCR repertoire of CD8+ T cells in the cortex independent of negative selection.

The thymoproteasome expressed specifically in thymic cortical epithelium optimizes the generation of CD8+ T cells; however, how the thymoproteasome contributes to CD8+ T cell development is unclear. Here, we show that the thymoproteasome shapes the TCR repertoire directly in cortical thymocytes before migration to the thymic medulla. We further show that the thymoproteasome optimizes CD8+ T cell production independent of the thymic medulla; independent of additional antigen-presenting cells, including medullary thymic epithelial cells and dendritic cells; and independent of apoptosis-mediated negative selection. These results indicate that the thymoproteasome hardwires the TCR repertoire of CD8+ T cells with cortical positive selection independent of negative selection in the thymus.

A Shared Regulatory Element Controls the Initiation of Tcf7 Expression During Early T Cell and Innate Lymphoid Cell Developments.

The transcription factor TCF-1 (encoded by Tcf7) plays critical roles in several lineages of hematopoietic cells. In this study, we examined the molecular basis for Tcf7 regulation in T cells, innate lymphoid cells, and migratory conventional dendritic cells that we find express Tcf7. We identified a 1 kb regulatory element crucial for the initiation of Tcf7 expression in T cells and innate lymphoid cells, but dispensable for Tcf7 expression in Tcf7-expressing dendritic cells. Within this region, we identified a Notch binding site important for the initiation of Tcf7 expression in T cells but not in innate lymphoid cells. Our work establishes that the same regulatory element is used by distinct transcriptional controllers to initiate Tcf7 expression in T cells and ILCs.

YAP1/TAZ-TEAD transcriptional networks maintain skin homeostasis by regulating cell proliferation and limiting KLF4 activity.

The Hippo TEAD-transcriptional regulators YAP1 and TAZ are central for cell renewal and cancer growth; however, the specific downstream gene networks involved in their activity are not completely understood. Here we introduce TEADi, a genetically encoded inhibitor of the interaction of YAP1 and TAZ with TEAD, as a tool to characterize the transcriptional networks and biological effects regulated by TEAD transcription factors. Blockage of TEAD activity by TEADi in human keratinocytes and mouse skin leads to reduced proliferation and rapid activation of differentiation programs. Analysis of gene networks affected by TEADi and YAP1/TAZ knockdown identifies KLF4 as a central transcriptional node regulated by YAP1/TAZ-TEAD in keratinocyte differentiation. Moreover, we show that TEAD and KLF4 can regulate the activity of each other, indicating that these factors are part of a transcriptional regulatory loop. Our study establishes TEADi as a resource for studying YAP1/TAZ-TEAD dependent effects.

Identification of an Intronic Regulatory Element Necessary for Tissue-Specific Expression of Foxn1 in Thymic Epithelial Cells.

The thymus is critical for the establishment of the adaptive immune system and the development of a diverse T cell repertoire. T cell development depends upon cell-cell interactions with epithelial cells in the thymus. The thymus is composed of two different types of epithelial cells: cortical and medullary epithelial cells. Both of these express and critically depend on the transcription factor Foxn1 Foxn1 is also expressed in the hair follicle, and disruption of Foxn1 function in mice results in severe thymic developmental defects and the hairless (nude) phenotype. Despite its importance, little is known about the direct regulation of Foxn1 expression. In this study, we identify a cis-regulatory element (RE) critical for expression of Foxn1 in mouse thymic epithelial cells but dispensable for expression in hair follicles. Analysis of chromatin accessibility, histone modifications, and sequence conservation identified regions within the first intron of Foxn1 that possessed the characteristics of REs. Systematic knockout of candidate regions lead us to identify a 1.6 kb region that, when deleted, results in a near total disruption of thymus development. Interestingly, Foxn1 expression and function in the hair follicle were unaffected. RNA fluorescent in situ hybridization showed a near complete loss of Foxn1 mRNA expression in the embryonic thymic bud. Our studies have identified a genomic RE with thymic-specific control of Foxn1 gene expression.

MEK inhibition induces MYOG and remodels super-enhancers in RAS-driven rhabdomyosarcoma.

The RAS isoforms are frequently mutated in many types of human cancers, including PAX3/PAX7 fusion-negative rhabdomyosarcoma. Pediatric RMS arises from skeletal muscle progenitor cells that have failed to differentiate normally. The role of mutant RAS in this differentiation blockade is incompletely understood. We demonstrate that oncogenic RAS, acting through the RAF-MEK [mitogen-activated protein kinase (MAPK) kinase]-ERK (extracellular signal-regulated kinase) MAPK effector pathway, inhibits myogenic differentiation in rhabdomyosarcoma by repressing the expression of the prodifferentiation myogenic transcription factor, MYOG. This repression is mediated by ERK2-dependent promoter-proximal stalling of RNA polymerase II at the MYOG locus. Small-molecule screening with a library of mechanistically defined inhibitors showed that RAS-driven RMS is vulnerable to MEK inhibition. MEK inhibition with trametinib leads to the loss of ERK2 at the MYOG promoter and releases the transcriptional stalling of MYOG expression. MYOG subsequently opens chromatin and establishes super-enhancers at genes required for late myogenic differentiation. Furthermore, trametinib, in combination with an inhibitor of IGF1R, potently decreases rhabdomyosarcoma cell viability and slows tumor growth in xenograft models. Therefore, this combination represents a potential therapeutic for RAS-mutated rhabdomyosarcoma.

B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity.

The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.

Global regulation of promoter melting in naive lymphocytes.

Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, ∼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens.

Protection against human papillomavirus type 16-E7 oncogene-induced tumorigenesis by in vivo expression of dominant-negative c-jun.

Expression of the human papillomavirus (HPV) type 16 E6 and E7 gene products is a risk factor for human cervical carcinogenesis as well as skin and oral carcinogenesis. Expression of the HPV-16 E7 gene in mouse skin induces hyperplasia and enhances tumor promotion. Expression of dominant-negative c-jun (TAM67) in the mouse skin protects mice from 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papillomagenesis without blocking mitogen-induced hyperproliferation. To determine the role of activator protein-1 (AP-1) in HPV-induced cancer, we crossed HPV-16 E7 mice with TAM67 mice and analyzed the effects of DMBA/TPA on tumor promotion. We showed that expression of TAM67 protected mice from HPV-16 E7-enhanced tumorigenesis, suggesting AP-1 as a target for prevention of HPV-induced cancer.