HDAC6 deficiency or inhibition blocks FGFR3 accumulation and improves bone growth in a model of achondroplasia.

Mutations that cause increased and/or inappropriate activation of FGFR3 are responsible for a collection of short-limbed chondrodysplasias. These mutations can alter receptor trafficking and enhance receptor stability, leading to increased receptor accumulation and activity. Here, we show that wildtype and mutant activated forms of FGFR3 increase expression of the cytoplasmic deacetylase HDAC6 (Histone Deacetylase 6) and that FGFR3 accumulation is compromised in cells lacking HDAC6 or following treatment of fibroblasts or chondrocytes with small molecule inhibitors of HDAC6. The reduced accumulation of FGFR3 was linked to increased FGFR3 degradation that occurred through a lysosome-dependent mechanism. Using a mouse model of Thanatophoric Dysplasia Type II (TDII) we show that both HDAC6 deletion and treatment with the small molecule HDAC6 inhibitor tubacin reduced FGFR3 accumulation in the growth plate and improved endochondral bone growth. Defective endochondral growth in TDII is associated with reduced proliferation and poor hypertrophic differentiation and the improved bone growth was associated with increased chondrocyte proliferation and expansion of the differentiation compartment within the growth plate. These findings further define the mechanisms that control FGFR3 accumulation and contribute to skeletal pathology caused by mutations in FGFR3.

Mutant activated FGFR3 impairs endochondral bone growth by preventing SOX9 downregulation in differentiating chondrocytes.

Fibroblast growth factor receptor 3 (FGFR3) plays a critical role in the control of endochondral ossification, and bone growth and mutations that cause hyperactivation of FGFR3 are responsible for a collection of developmental disorders that feature poor endochondral bone growth. FGFR3 is expressed in proliferating chondrocytes of the cartilaginous growth plate but also in chondrocytes that have exited the cell cycle and entered the prehypertrophic phase of chondrocyte differentiation. Achondroplasia disorders feature defects in chondrocyte proliferation and differentiation, and the defects in differentiation have generally been considered to be a secondary manifestation of altered proliferation. By initiating a mutant activated knockin allele of FGFR3 (FGFR3K650E) that causes Thanatophoric Dysplasia Type II (TDII) specifically in prehypertrophic chondrocytes, we show that mutant FGFR3 induces a differentiation block at this stage independent of any changes in proliferation. The differentiation block coincided with persistent expression of SOX9, the master regulator of chondrogenesis, and reducing SOX9 dosage allowed chondrocyte differentiation to proceed and significantly improved endochondral bone growth in TDII. These findings suggest that a proliferation-independent and SOX9-dependent differentiation block is a key driving mechanism responsible for poor endochondral bone growth in achondroplasia disorders caused by mutations in FGFR3.

The activities of MYC, MNT and the MAX-interactome in lymphocyte proliferation and oncogenesis.

The MYC family of proteins plays essential roles in embryonic development and in oncogenesis. Efforts over the past 30 years to define the transcriptional activities of MYC and how MYC functions to promote proliferation have produced evolving models of MYC function. One picture that has emerged of MYC and its partner protein MAX is of a transcription factor complex with a seemingly unique ability to stimulate the transcription of genes that are epigenetically poised for transcription and to amplify the transcription of actively transcribed genes. During lymphocyte activation, MYC is upregulated and stimulates a pro-proliferative program in part through the upregulation of a wide variety of metabolic effector genes that facilitate cell growth and cell cycle progression. MYC upregulation simultaneously sensitizes cells to apoptosis and activated lymphocytes and lymphoma cells have pro-survival attributes that allow MYC-driven proliferation to prevail. For example, the MAX-interacting protein MNT is upregulated in activated lymphocytes and was found to protect lymphocytes from MYC-dependent apoptosis. Here we review the activities of MYC, MNT and other MAX interacting proteins in the setting of T and B cell activation and oncogenesis. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis.

Mnt (Max's next tango) is a Max-interacting transcriptional repressor that can antagonize both the proproliferative and proapoptotic functions of Myc in vitro. To ascertain the physiologically relevant functions of Mnt and to help define the relationship between Mnt and Myc in vivo, we generated a series of mouse strains in which Mnt was deleted in T cells in the absence of endogenous c-Myc or in the presence of ectopic c-Myc. We found that apoptosis caused by loss of Mnt did not require Myc but that ectopic Myc expression dramatically decreased the survival of both Mnt-deficient T cells in vivo and Mnt-deficient MEFs in vitro. Consequently, Myc-driven proliferative expansion of T cells in vitro and thymoma formation in vivo were prevented by the absence of Mnt. Consistent with T-cell models, mouse embryo fibroblasts (MEFs) lacking Mnt were refractory to oncogenic transformation by Myc. Tumor suppression caused by loss of Mnt was linked to increased apoptosis mediated by reactive oxygen species (ROS). Thus, although theoretically and experimentally a Myc antagonist, the dominant physiological role of Mnt appears to be suppression of apoptosis. Our results redefine the physiological relationship between Mnt and Myc and requirements for Myc-driven oncogenesis.

Disruption of a Sox9-ß-catenin circuit by mutant Fgfr3 in thanatophoric dysplasia type II.

Mutations in fibroblast growth factor (FGF) receptors are responsible for a variety of skeletal birth defects, but the underlying mechanisms responsible remain unclear. Using a mouse model of thanatophoric dysplasia type II in which FGFR3(K650E) expression was directed to the appendicular skeleton, we show that the mutant receptor caused a block in chondrocyte differentiation specifically at the prehypertrophic stage. The differentiation block led to a severe reduction in hypertrophic chondrocytes that normally produce vascular endothelial growth factor, which in turn was associated with poor vascularization of primary ossification centers and disrupted endochondral ossification. We show that the differentiation block and defects in joint formation are associated with persistent expression of the chondrogenic factor Sox9 and down-regulation of ß-catenin levels and activity in growth plate chondrocytes. Consistent with these in vivo results, FGFR3(K650E) expression was found to increase Sox9 and decrease ß-catenin levels and transcriptional activity in cultured mesenchymal cells. Coexpression of Fgfr3(K650E) and Sox9 in cells resulted in very high levels of Sox9 and cooperative suppression of ß-catenin-dependent transcription. Fgfr3(K650E) had opposing effects on Sox9 and ß-catenin protein stability with it promoting Sox9 stabilization and ß-catenin degradation. Since both Sox9 overexpression and ß-catenin deletion independently blocks hypertrophic differentiation of chondrocytes and cause chondrodysplasias similar to those caused by mutations in FGFR3, our results suggest that dysregulation of Sox9 and ß-catenin levels and activity in growth plate chondrocytes is an important underlying mechanism in skeletal diseases caused by mutations in FGFR3.

Differential regulation of N-Myc and c-Myc synthesis, degradation, and transcriptional activity by the Ras/mitogen-activated protein kinase pathway.

Myc transcription factors are important regulators of proliferation and can promote oncogenesis when deregulated. Deregulated Myc expression in cancers can result from MYC gene amplification and translocation but also from alterations in mitogenic signaling pathways that affect Myc levels through both transcriptional and post-transcription mechanisms. For example, mutations in Ras family GTPase proteins that cause their constitutive activation can increase cellular levels of c-Myc by interfering with its rapid proteasomal degradation. Although enhanced protein stability is generally thought to be applicable to other Myc family members, here we show that c-Myc and its paralog N-Myc respond to oncogenic H-Ras (H-Ras(G12V)) in very different ways. H-Ras(G12V) promotes accumulation of both c-Myc and N-Myc, but although c-Myc accumulation is achieved by enhanced protein stability, N-Myc accumulation is associated with an accelerated rate of translation that overcomes a surprising H-Ras(G12V)-mediated destabilization of N-Myc. We show that H-Ras(G12V)-mediated degradation of N-Myc functions independently of key phosphorylation sites in the highly conserved Myc homology box I region that controls c-Myc protein stability by oncogenic Ras. Finally, we found that N-Myc and c-Myc transcriptional activity is associated with their proteasomal degradation but that N-Myc may be uniquely dependent on Ras-stimulated proteolysis for target gene expression. Taken together, these studies provide mechanistic insight into how oncogenic Ras augments N-Myc levels in cells and suggest that enhanced N-Myc translation and degradation-coupled transactivation may contribute to oncogenesis.

OX40 engagement stabilizes Mxd4 and Mnt protein levels in antigen-stimulated T cells leading to an increase in cell survival.

OX40 engagement on activated T cells leads to increased proliferation, expansion and survival of Ag-specific T cells. Direct ex vivo examination of Ag-stimulated murine T cells show that the Myc antagonists, Mxd4 and Mnt, are transiently upregulated and translocated to the nucleus following OX40 engagement and may be involved in suppressing cell death. Both Mxd4 and Mnt are upregulated following OX40 stimulation through increased protein stability and we identify a critical phosphorylation site in Mxd4 that controls Mxd4 stability. The upregulation of Mxd4 and Mnt contributes to OX40-mediated T-cell survival because siRNA knockdown of Mxd4 and Mnt led to increased cell death. We hypothesize the upregulation of c-Myc following OX40 engagement drives T-cell proliferation and that upregulation of Mxd4 and Mnt suppresses Myc-dependent cell death. Thus, Mxd4 and Mnt upregulation following OX40 engagement most likely increases T-cell survival.

The role of senescence and prosurvival signaling in controlling the oncogenic activity of FGFR2 mutants associated with cancer and birth defects.

Mutations in fibroblast growth factor receptors (FGFRs) cause human birth defect syndromes and are associated with a variety of cancers. Although forced expression of mutant activated FGFRs has been shown to oncogenically transform some immortal cell types, their activity in primary cells remains unclear. Here, we show that birth defect and cancer-associated FGFR2 mutants promote DNA-damage signaling and p53-dependent senescence in primary mouse and human cells. Senescence promoted by FGFR mutants was associated with downregulation of c-Myc and forced expression of c-Myc facilitated senescence escape. Whereas c-Myc expression facilitated senescence bypass, mutant FGFR2 signaling suppressed c-Myc-dependent apoptosis and led to oncogenic transformation. Cells transformed by coexpression of a constitutively activated FGFR2 mutant plus c-Myc appeared to be become highly addicted to FGFR-dependent prosurvival activities, as small molecule inhibition of FGFR signaling resulted in robust p53-dependent apoptosis. Our data suggest that senescence-promoting activities of mutant FGFRs may normally limit their oncogenic potential and may be relevant to their ability to disrupt morphogenesis and cause birth defects. Our results also raise the possibility that cancers originating through a combination of constitutive FGFR activation and deregulated Myc expression may be particularly sensitive to small molecule inhibitors of FGF receptors.

A novel role of MNT as a negative regulator of REL and the NF-κB pathway.

MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix-loop-helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT-REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT-REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.

Mnt-Max to Myc-Max complex switching regulates cell cycle entry.

The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

Inflammatory disease and lymphomagenesis caused by deletion of the Myc antagonist Mnt in T cells.

Mnt is a Max-interacting protein that can antagonize the activities of Myc oncoproteins in cultured cells. Mnt null mice die soon after birth, but conditional deletion of Mnt in breast epithelium leads to tumor formation. These and related data suggest that Mnt functions as a tumor suppressor. Here we show that conditional deletion of Mnt in T cells leads to tumor formation but also causes inflammatory disease. Deletion of Mnt caused increased apoptosis of thymic T cells and interfered with T-cell development yet led to spleen, liver, and lymph node enlargement. The proportion of T cells in the spleen and lymph nodes was reduced, and the numbers of cells in non-T-cell immune cell populations were elevated. The disruption of immune homeostasis is linked to a strong skewing toward production of T-helper 1 (Th1) cytokines and enhanced proliferation of activated Mnt-deficient CD4+ T cells. Consistent with Th1 polarization in vivo, extensive intestinal inflammation and liver necrosis developed. Finally, most mice lacking Mnt in T cells ultimately succumbed to T-cell lymphoma. These results strengthen the argument that Mnt functions as a tumor suppressor and reveal a critical and surprising role for Mnt in the regulation of T-cell development and in T-cell-dependent immune homeostasis.

Suppression of FGFR3- and MYC-dependent oncogenesis by tubacin: association with HDAC6-dependent and independent activities.

Fibroblast growth factor receptor 3 (FGFR3) is amplified, translocated or mutated in a number of different human cancer types, but most commonly in bladder cancers. We previously found that the accumulation of FGFR3 is dependent on histone deacetylase 6 (HDAC6). Here we show that HDAC6 loss or inhibition reduces FGFR3 accumulation in cells made tumorigenic by ectopic expression of a mutant activated version of FGFR3 together with the MYC oncoprotein and in a bladder cancer cell line whose tumorigenicity is dependent on expression of a translocated version of FGFR3. In tumor xenoplant assays, HDAC6 deficiency or small molecule inhibition by the selective HDAC6 inhibitors tubacin or tubastatin A was found to significantly impede tumor growth. However, tubacin was more effective at inhibiting tumor growth than tubastatin A or HDAC6 deficiency. The superior anti-tumor activity of tubacin was linked to its ability to not only inhibit accumulation of mutant FGFR3, but also to cause robust downregulation of MYC and cyclin D1, and to induce a DNA damage response and apoptosis. Neither HDAC6 deficiency nor treatment with tubastatin A altered MYC or cyclin D1 levels, and neither induced a DNA damage response or apoptosis. Thus while tubacin and tubastatin A inhibit HDAC6 with similar selectivity and potency, our results reveal unique HDAC6-independent activities of tubacin that likely contribute to its potent anti-tumor activity.

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas.

Although the MYC oncogene has been implicated in cancer, a systematic assessment of alterations of MYC, related transcription factors, and co-regulatory proteins, forming the proximal MYC network (PMN), across human cancers is lacking. Using computational approaches, we define genomic and proteomic features associated with MYC and the PMN across the 33 cancers of The Cancer Genome Atlas. Pan-cancer, 28% of all samples had at least one of the MYC paralogs amplified. In contrast, the MYC antagonists MGA and MNT were the most frequently mutated or deleted members, proposing a role as tumor suppressors. MYC alterations were mutually exclusive with PIK3CA, PTEN, APC, or BRAF alterations, suggesting that MYC is a distinct oncogenic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such as immune response and growth factor signaling; chromatin, translation, and DNA replication/repair were conserved pan-cancer. This analysis reveals insights into MYC biology and is a reference for biomarkers and therapeutics for cancers with alterations of MYC or the PMN.

Development of human gene reporter cell lines using rAAV mediated homologous recombination.

Understanding mechanisms of gene regulation has broad therapeutic implications for human disease. Here we describe a novel method for generating human cell lines that serve as reporters of transcriptional activity. This method exploits the ability of recombinant adeno-associated virus to mediate the insertion of exogenous DNA sequences into specific genomic loci through homologous recombination. To overcome the severe size limitation of the rAAV for carrying exogenous DNA, an enhanced green fluorescent protein (EGFP)-Luciferase fusion gene was used as both a selectable marker and gene expression reporter. EGFP was used for selection of correctly targeted alleles by taking advantage of known regulatory conditions that activate transcription of specific genes. Using this method, we describe the generation of primary human fibroblasts that express EGFP-Luciferase under the control of the c-Myc oncogene.

MNT and Emerging Concepts of MNT-MYC Antagonism.

MYC family proteins play fundamental roles in stem and progenitor cell homeostasis, morphogenesis and cancer. As expected for proteins that profoundly affect the fate of cells, the activities of MYC are regulated at a multitude of levels. One mechanism with the potential to broadly affect the activities of MYC is transcriptional antagonism by a group of MYC-related transcriptional repressors. From this group, the protein MNT has emerged as having perhaps the most far-reaching impact on MYC activities. In this review, we discuss the current understanding of MNT, its regulation and how, as a MYC antagonist, it functions both as a tumor suppressor and facilitator of MYC-driven proliferation and oncogenesis.

Mnt modulates Myc-driven lymphomagenesis.

The transcriptional represser Mnt is a functional antagonist of the proto-oncoprotein Myc. Both Mnt and Myc utilise Max as an obligate partner for DNA binding, and Myc/Max and Mnt/Max complexes compete for occupancy at E-box DNA sequences in promoter regions. We have previously shown in transgenic mouse models that the phenotype and kinetics of onset of haemopoietic tumours varies with the level of Myc expression. We reasoned that a decrease in the level of Mnt would increase the functional level of Myc and accelerate Myc-driven tumorigenesis. We tested the impact of reduced Mnt in three models of myc transgenic mice and in p53+/- mice. To our surprise, mnt heterozygosity actually slowed Myc-driven tumorigenesis in vavP-MYC10 and Eµ-myc mice, suggesting that Mnt facilitates Myc-driven oncogenesis. To explore the underlying cause of the delay in tumour development, we enumerated Myc-driven cell populations in healthy young vavP-MYC10 and Eµ-myc mice, expecting that the reduced rate of leukaemogenesis in mnt heterozygous mice would be reflected in a reduced number of preleukaemic cells, due to increased apoptosis or reduced proliferation or both. However, no differences were apparent. Furthermore, when mnt+/+ and mnt+/- pre-B cells from healthy young Eµ-myc mice were compared in vitro, no differences were seen in their sensitivity to apoptosis or in cell size or cell cycling. Moreover, the frequencies of apoptotic, senescent and proliferating cells were comparable in vivo in mnt+/- and mnt+/+ Eµ-myc lymphomas. Thus, although mnt heterozygosity clearly slowed lymphomagenesis in vavP-MYC10 and Eµ-myc mice, the change(s) in cellular properties responsible for this effect remain to be identified.

Mnt transcriptional repressor is functionally regulated during cell cycle progression.

The Myc/Max/Mad network of transcription factors regulates cell proliferation, differentiation, and transformation. Similar to other proteins of the network, Mnt forms heterodimers with Max and binds CACGTG E-Box elements. Transcriptional repression by Mnt is mediated through association with mSin3, and deletion of the mSin3-interacting domain (SID) converts Mnt to a transcriptional activator. Mnt is coexpressed with Myc in proliferating cells and has been suggested to be a modulator of Myc function. We report that Mnt is expressed both in growth-arrested and proliferating mouse fibroblasts and is phosphorylated when resting cells are induced to re-enter the cell cycle. Importantly, the interaction between Mnt and mSin3 is disrupted upon serum stimulation resulting in decreased Mnt-associated HDAC activity. Furthermore, we demonstrate that Mnt binds and recruits mSin3 to the Myc target gene cyclin D2 in quiescent mouse fibroblasts. Interference with Mnt expression by RNAi resulted in upregulation of cyclin D2 expression in growth-arrested fibroblasts, supporting the view that Mnt represses cyclin D2 transcription in quiescent cells. Our data suggest a model in which phosphorylation of Mnt at cell cycle entry results in disruption of Mnt-mSin3-HDAC1 interaction, which allows induction of Myc target genes by release of Mnt-mediated transcriptional repression.

Tbx3 impinges on the p53 pathway to suppress apoptosis, facilitate cell transformation and block myogenic differentiation.

Tbx3 is a member of the T-box family of transcription factors. Mutations in Tbx3 cause ulnar-mammary syndrome, an autosomal dominant disorder characterized by upper limb defects, apocrine-gland defects including mammary hypoplasia, and tooth, hair and genital defects. In cell culture, Tbx3 and its close relative Tbx2 are capable of immortalizing mouse embryo fibroblasts. We show that expression of Tbx3 together with Myc or oncogenic Ras (H-Ras(Val17)) leads to efficient transformation of mouse embryo fibroblasts. Oncogene cooperation by Tbx3 correlates with an ability of Tbx3 to suppress the induction of p19ARF and p53 that is typically caused by overexpression Myc and Ras, and to protect against Myc-induced apoptosis. Whereas Tbx3 is capable of interfering with apoptosis caused by excessive Myc levels, a Tbx3 mutant lacking its C-terminal repression domain shows no anti-apoptotic activity and fails to repress levels of p19ARF or p53. Consistent with an ability to suppress p53 pathway function, we find that Tbx3, but not a Tbx3 C-terminal mutant, efficiently blocks myogenic differentiation of C2C12 myoblasts. Our results support the idea that deregulation and/or excessive levels of Tbx3 may have oncogenic potential in vivo.