TOX expression and role in CTCL.

BACKGROUND: Cutaneous T-cell lymphomas (CTCL) are skin malignancies including mycosis fungoides (MF) and CD30(+) lymphoproliferative disorders (LPD). In early disease, CTCL can be difficult to diagnose, especially in MF for which there is no reliable diagnostic marker. MF/CTCL have increased expression of thymocyte selection-associated HMG box protein (TOX). Although TOX has been proposed to be a diagnostic marker for MF, further validation studies are needed. Moreover, it is unclear what drives TOX expression or its role in MF/CTCL. OBJECTIVE: We hypothesize evaluation of TOX levels across a spectrum of CTCL, including MF precursor (large plaque parapsoriasis, LPP), will help elucidate the implications of altered TOX expression. MATERIALS AND METHODS: TOX staining was performed in MF, CD30(+) LPD, LPP as well as benign inflammatory dermatoses (BID) and normal skin (NS). CTCL cell lines were utilized to evaluate the regulation of TOX. RESULTS: Positive TOX expression was identified in 73.6% of MF cases and in 31.6% of BID/NS. TOX had a positive predictive value (PPV) for MF of 86.7% and a negative predictive value (NPV) of 48.1%. TOX expression in MF was detected more commonly in Black patients (P = 0.015) and less commonly in transformed MF (P = 0.045). LPP had positive TOX staining in 70.0%. In CTCL cells, GATA3 knockdown decreased TOX mRNA and protein expression. TOX expression also decreased in the presence of CTCL therapeutics. CONCLUSION: Our data indicate that TOX is useful as a diagnostic marker in MF. Moreover, TOX expression was evident in LPP, indicating it may have a previously unappreciated role in the development of MF. Finally, our data suggest that GATA3 regulates TOX, revealing insight into TOX regulation.

Genetic markers associated with progression in early mycosis fungoides.

BACKGROUND: Mycosis fungoides (MF) is a rare, but potentially devastating malignancy. It classically presents with cutaneous patches and plaques and can progress to tumours on the skin with lymph node, blood and visceral involvement. While most patients with MF have a relatively benign disease course, a subset of patients will develop progressive disease that is often fatal. OBJECTIVE: The aim of this study was to identify genetic markers in early MF limited to the skin (stages IA-IIA) that distinguish those patients who will have progressive disease from those who will not, so that early appropriate treatment may be instituted. METHODS: The study includes 18 patients who were diagnosed with early stage MF at the time of biopsy and had follow-up to determine which patients developed progressive disease. RNA was extracted from skin biopsy specimens and analysed for expression of CD3, FOXP3, IFNγ, Interleukin (IL)-4, IL-13, KIR3DL2, MICB, PLS3 and STAT4 by quantitative real-time polymerase chain reaction. RESULTS/CONCLUSIONS: Reduced expression of FOXP3 and STAT4 and increased expression of IL-4 relative to CD3 expression levels were significantly associated with MF progression. Further studies will be needed to fully assess the usefulness of these genetic markers to predict disease progression and guide treatment options in patients diagnosed with early MF.

C9orf72 poly(PR) mediated neurodegeneration is associated with nucleolar stress.

The ALS/FTD-linked intronic hexanucleotide repeat expansion in the C9orf72 gene is translated into dipeptide repeat proteins, among which poly-proline-arginine (PR) displays the most aggressive neurotoxicity in-vitro and in-vivo . PR partitions to the nucleus when expressed in neurons and other cell types. Using drosophila and primary rat cortical neurons as model systems, we show that by lessening the nuclear accumulation of PR, we can drastically reduce its neurotoxicity. PR accumulates in the nucleolus, a site of ribosome biogenesis that regulates the cell stress response. We examined the effect of nucleolar PR accumulation and its impact on nucleolar function and determined that PR caused nucleolar stress and increased levels of the transcription factor p53. Downregulating p53 levels, either genetically or by increasing its degradation, also prevented PR-mediated neurotoxic phenotypes both in in-vitro and in-vivo models. We also investigated whether PR could cause the senescence phenotype in neurons but observed none. Instead, we found induction of apoptosis via caspase-3 activation. In summary, we uncovered the central role of nucleolar dysfunction upon PR expression in the context of C9-ALS/FTD.

C9orf72 poly(PR) mediated neurodegeneration is associated with nucleolar stress.

The ALS/FTD-linked intronic hexanucleotide repeat expansion in the C9orf72 gene is aberrantly translated in the sense and antisense directions into dipeptide repeat proteins, among which poly proline-arginine (PR) displays the most aggressive neurotoxicity in-vitro and in-vivo. PR partitions to the nucleus when heterologously expressed in neurons and other cell types. We show that by lessening the nuclear accumulation of PR, we can drastically reduce its neurotoxicity. PR strongly accumulates in the nucleolus, a nuclear structure critical in regulating the cell stress response. We determined that, in neurons, PR caused nucleolar stress and increased levels of the transcription factor p53. Downregulating p53 levels also prevented PR-mediated neurotoxicity both in in-vitro and in-vivo models. We investigated if PR could induce the senescence phenotype in neurons. However, we did not observe any indications of such an effect. Instead, we found evidence for the induction of programmed cell death via caspase-3 activation.

Decreased Mdm2 expression inhibits tumor development induced by loss of ARF.

The tumor suppressor p14/p19(ARF) regulates Mdm2, which is known for controlling the p53 tumor suppressor. Here we report that loss of one allele of Mdm2 in cells that lack ARF resulted in a decreased rate of proliferation, fewer chromosomal aberrations, and suppression of Ras-induced transformation. Moreover, a haploinsufficiency of Mdm2 inhibited spontaneous tumor development in ARF-null mice. Remarkably, Mdm2(+/-)ARF(-/-) mice survived an average of 6 months longer than Mdm2(+/+)ARF(-/-) mice. The spectrum of tumors that arose in Mdm2(+/-)ARF(-/-) mice did not significantly differ from those that developed in mice lacking only ARF. However, the extended tumor latency allowed for the emergence of multiple primary tumors in a third of the Mdm2(+/-)ARF(-/-) mice, as compared to the single tumor type that arose in ARF-null only mice. Therefore, a decrease in Mdm2 levels restored regulation of critical cellular processes that are altered during transformation and that occur in the absence of ARF. Our findings also indicate that Mdm2 can function independently from ARF and imply that targeting Mdm2 in tumors that lack ARF expression should be an effective therapeutic approach.

Bax loss impairs Myc-induced apoptosis and circumvents the selection of p53 mutations during Myc-mediated lymphomagenesis.

The ARF and p53 tumor suppressors mediate Myc-induced apoptosis and suppress lymphoma development in E mu-myc transgenic mice. Here we report that the proapoptotic Bcl-2 family member Bax also mediates apoptosis triggered by Myc and inhibits Myc-induced lymphomagenesis. Bax-deficient primary pre-B cells are resistant to the apoptotic effects of Myc, and Bax loss accelerates lymphoma development in E mu-myc transgenics in a dose-dependent fashion. Eighty percent of lymphomas arising in wild-type E mu-myc transgenics have alterations in the ARF-Mdm2-p53 tumor suppressor pathway characterized by deletions in ARF, mutations or deletions of p53, and overexpression of Mdm2. The absence of Bax did not alter the frequency of biallelic deletion of ARF in lymphomas arising in E mu-myc transgenic mice or the rate of tumorigenesis in ARF-null mice. Furthermore, Mdm2 was overexpressed at the same frequency in lymphomas irrespective of Bax status, suggesting that Bax resides in a pathway separate from ARF and Mdm2. Strikingly, lymphomas from Bax-null E mu-myc transgenics lacked p53 alterations, whereas 27% of the tumors in Bax(+/-) E mu-myc transgenic mice contained p53 mutations or deletions. Thus, the loss of Bax eliminates the selection of p53 mutations and deletions, but not ARF deletions or Mdm2 overexpression, during Myc-induced tumorigenesis, formally demonstrating that Myc-induced apoptotic signals through ARF/Mdm2 and p53 must bifurcate: p53 signals through Bax, whereas this is not necessarily the case for ARF and Mdm2.

Apoptosis triggered by Myc-induced suppression of Bcl-X(L) or Bcl-2 is bypassed during lymphomagenesis.

Enforced Bcl-2 expression inhibits Myc-induced apoptosis and cooperates with Myc in transformation. Here we report that the synergy between Bcl-2 and Myc in transforming hematopoietic cells in fact reflects a Myc-induced pathway that selectively suppresses the expression of the Bcl-X(L) or Bcl-2 antiapoptotic protein. Myc activation suppresses Bcl-X(L) RNA and protein levels in cultures of primary myeloid and lymphoid progenitors, and Bcl-X(L) and Bcl-2 expression is inhibited by Myc in precancerous B cells from Emu-myc transgenic mice. The suppression of bcl-X RNA levels by Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression of Bcl-2 or Bcl-X(L) by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-X(L) levels are markedly elevated in over one-half of all lymphomas arising in Emicro-myc transgenic mice. Bcl-2 and/or Bcl-X(L) overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways are inactivated independently. Therefore, the suppression of Bcl-X(L) or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.

Myc enhances B-cell receptor signaling in precancerous B cells and confers resistance to Btk inhibition.

Dysregulation of the oncogenic transcription factor MYC induces B-cell transformation and is a driver for B-cell non-Hodgkin lymphoma (B-NHL). MYC overexpression in B-NHL is associated with more aggressive phenotypes and poor prognosis. Although genomic studies suggest a link between MYC overexpression and B-cell receptor (BCR) signaling molecules in B-NHL, signaling pathways essential to Myc-mediated B-cell transformation have not been fully elucidated. We utilized intracellular phospho-flow cytometry to investigate the relationship between Myc and BCR signaling in pre-malignant B cells. Utilizing the Eµ-myc mouse model, where Myc is overexpressed specifically in B cells, both basal and stimulated BCR signaling were increased in precancerous B lymphocytes from Eµ-myc mice compared with wild-type littermates. B cells overexpressing Myc displayed constitutively higher levels of activated CD79α, Btk, Plcγ2 and Erk1/2. Notably, Myc-overexpressing B cells maintained elevated BCR signaling despite treatment with ibrutinib, a Bruton's tyrosine kinase inhibitor. Furthermore, PI3K/Akt pathway signaling was also increased in Eµ-myc B cells, and this increase was partially suppressed with ibrutinib. In addition, experiments with Btk-null B cells revealed off-target effects of ibrutinib on BCR signaling. Our data show that in pre-malignant B cells, Myc overexpression is sufficient to activate BCR and PI3K/Akt signaling pathways and further enhances signaling following BCR ligation. Therefore, our results indicate that precancerous B cells have already acquired enhanced survival and growth capabilities before transformation, and that elevated MYC levels confer resistance to pharmacologic inhibitors of BCR signaling, which has significant implications for B-NHL treatment.

Defective replication stress response inhibits lymphomagenesis and impairs lymphocyte reconstitution.

DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.

Histone deacetylase inhibition reveals a tumor-suppressive function of MYC-regulated miRNA in breast and lung carcinoma.

Histone deacetylase (HDAC) inhibition leads to dynamic changes in the epigenetic landscape that is postulated to alter the expression of critical mediators of cellular proliferation and death. While current HDAC inhibitors have shown to be efficacious in the treatment of specific hematologic malignancies, their therapeutic utility in epithelial-based cancers warrants further evaluation. Moreover, the mechanisms of HDAC inhibition-induced cancer cell death are not completely understood. Therefore, elucidation of the underlying pathways engaged by HDAC inhibition may enable the development of more effective therapeutic strategies. Here, we report that HDAC inhibition in human breast and lung carcinoma cells activates an apoptotic mechanism mediated by microRNA (miRNA) and induced by the oncogene MYC. Specifically, following HDAC inhibition, MYC, which normally represses miR-15 and let-7 families, transcriptionally activated their expression and MYC was required for this miRNA upregulation. As a result, transcript levels of the tumor-suppressive miR-15 and let-7 families increased, which targeted and decreased the expression of the crucial prosurvival genes BCL-2 and BCL-XL, respectively. MYC was also required for the downregulation of BCL-2 and BCL-XL following HDAC inhibition. Blocking the binding sites of the miR-15 and let-7 families in the 3'-untranslated regions of BCL-2 and BCL-XL protected against HDAC inhibition-induced apoptosis. These results provide important insight into the molecular underpinnings of HDAC inhibition-induced cell death in breast and lung cancer and reveal a tumor-suppressive role for MYC-regulated miRNA that is activated with HDAC inhibition.

Interaction of MYC with host cell factor-1 is mediated by the evolutionarily conserved Myc box IV motif.

The MYC family of oncogenes encodes a set of three related transcription factors that are overexpressed in many human tumors and contribute to the cancer-related deaths of more than 70,000 Americans every year. MYC proteins drive tumorigenesis by interacting with co-factors that enable them to regulate the expression of thousands of genes linked to cell growth, proliferation, metabolism and genome stability. One effective way to identify critical co-factors required for MYC function has been to focus on sequence motifs within MYC that are conserved throughout evolution, on the assumption that their conservation is driven by protein-protein interactions that are vital for MYC activity. In addition to their DNA-binding domains, MYC proteins carry five regions of high sequence conservation known as Myc boxes (Mb). To date, four of the Mb motifs (MbI, MbII, MbIIIa and MbIIIb) have had a molecular function assigned to them, but the precise role of the remaining Mb, MbIV, and the reason for its preservation in vertebrate Myc proteins, is unknown. Here, we show that MbIV is required for the association of MYC with the abundant transcriptional coregulator host cell factor-1 (HCF-1). We show that the invariant core of MbIV resembles the tetrapeptide HCF-binding motif (HBM) found in many HCF-interaction partners, and demonstrate that MYC interacts with HCF-1 in a manner indistinguishable from the prototypical HBM-containing protein VP16. Finally, we show that rationalized point mutations in MYC that disrupt interaction with HCF-1 attenuate the ability of MYC to drive tumorigenesis in mice. Together, these data expose a molecular function for MbIV and indicate that HCF-1 is an important co-factor for MYC.

E2F-1 induces the stabilization of p53 but blocks p53-mediated transactivation.

E2F-1 induces p53 accumulation and E2F-1 and p53 form a physical complex, which affects the ability of E2F-1 to activate transcription. We mapped the domains on E2F-1 that interact with p53 and found two p53-binding domains. To understand the functional consequences of the E2F-1/p53 association on p53 activities we identified the domains of E2F-1 that were responsible for the accumulation of p53. Unexpectedly, we found that the E2F-1 transactivation domain was dispensable for p53 induction. By contrast, further deletion of the DP-1 interaction/'marked' box domain eliminated p53 accumulation. Radiolabeling pulse/chase analysis demonstrated that E2F-1 caused post-translational stabilization of p53. Although E2F-1 caused the stabilization of p53, E2F-1 expression impaired p53-dependent transactivation. Thus, the E2F-1 : p53 interaction may provide a checkpoint function to inactivate overactive E2F-1, but the association may also inactivate p53 transactivation to allow cell cycle progression.

Bcl-2 is an apoptotic target suppressed by both c-Myc and E2F-1.

Malignant transformation occurs in cells that overexpress c-Myc or that inappropriately activate E2F-1. Transformation occurs after the selection of cells that have acquired resistance to apoptosis that is triggered by these oncogenes, and a key mediator of this cell death process is the p53 tumor suppressor. In IL-3-dependent immortal 32D.3 myeloid cells the ARF/p53 apoptotic pathway is inactivated, as these cells fail to express ARF. Nonetheless, both c-Myc and E2F-1 overexpression accelerated apoptosis when these cells were deprived of IL-3. Here we report that c-Myc or E2F-1 overexpression suppresses Bcl-2 protein and RNA levels, and that restoration of Bcl-2 protein effectively blocks the accelerated apoptosis that occurs when c-Myc- or E2F-1-overexpressing cells are deprived of IL-3. Blocking p53 activity with mutant p53 did not abrogate E2F-1-induced suppression of Bcl-2. Analysis of immortal myeloid cells engineered to overexpress c-Myc and E2F-1 DNA binding mutants revealed that DNA binding activity of these oncoproteins is required to suppress Bcl-2 expression. These results suggest that the targeting of Bcl-2 family members is an important mechanism of oncogene-induced apoptosis, and that this occurs independent of the ARF/p53 pathway.

Mdmx promotes genomic instability independent of p53 and Mdm2.

The oncogene Mdmx is overexpressed in many human malignancies, and together with Mdm2, negatively regulates the p53 tumor suppressor. However, a p53-independent function of Mdmx that impacts genome stability has been described, but this function is not well understood. In the present study, we determined that of the 13 different cancer types evaluated, 6-90% of those that had elevated levels of Mdmx had concurrent inactivation (mutated or deleted) of p53. We show elevated levels of Mdmx-inhibited double-strand DNA break repair and induced chromosome and chromatid breaks independent of p53, leading to genome instability. Mdmx impaired early DNA damage-response signaling, such as phosphorylation of the serine/threonine-glutamine motif, mediated by the ATM kinase. Moreover, we identified Mdmx associated with Nbs1 of the Mre11-Rad50-Nbs1 (MRN) DNA repair complex, and this association increased upon DNA damage and was detected at chromatin. Elevated Mdmx levels also increased cellular transformation in a p53-independent manner. Unexpectedly, all Mdmx-mediated phenotypes also occurred in cells lacking Mdm2 and were independent of the Mdm2-binding domain (RING) of Mdmx. Therefore, Mdmx-mediated inhibition of the DNA damage response resulted in delayed DNA repair and increased genome instability and transformation independent of p53 and Mdm2. Our results reveal a novel p53- and Mdm2-independent oncogenic function of Mdmx that provides new insight into the many cancers that overexpress Mdmx.

Suppression of Ras/Mapk pathway signaling inhibits Myc-induced lymphomagenesis.

Although the Myc transcription factor has been shown necessary for the oncogenic function of Ras, the contribution of Ras pathway signaling to the oncogenic function of Myc remains unresolved. We report the novel findings that Myc alone induced Ras/Mapk pathway signaling, and increased signaling following growth factor stimulation. Deletion of the scaffold protein kinase suppressor of Ras 1 (Ksr1) attenuated signaling through the Ras/Mapk pathway, including activation following Myc induction. B cells that lacked Ksr1 exhibited reduced proliferation and increased cytokine deprivation-induced apoptosis. Overexpression of Myc rescued the proliferation defect of Ksr1-null B cells, but loss of Ksr1 increased sensitivity of B cells to Myc-induced apoptosis. Notably, there was a significant delay in lymphoma development in Ksr1-null mice overexpressing Myc in B cells (Eµ-myc transgenic mice). There was an elevated frequency of p53 inactivation, indicative of increased selective pressure to bypass the p53 tumor suppressor pathway, in Ksr1-null Eµ-myc lymphomas. Therefore, loss of Ksr1 inhibits Ras/Mapk pathway signaling leading to increased Myc-induced B-cell apoptosis, and this results in reduced B-cell transformation and lymphoma development. Our data indicate that suppression of Myc-induced Ras/Mapk pathway signaling significantly impairs Myc oncogenic function. These results fill a significant gap in knowledge about Myc and should open new avenues of therapeutic intervention for Myc-overexpressing malignancies.

Aging mice have increased chromosome instability that is exacerbated by elevated Mdm2 expression.

Aging is thought to negatively affect multiple cellular processes including the ability to maintain chromosome stability. Chromosome instability (CIN) is a common property of cancer cells and may be a contributing factor to cellular transformation. The types of DNA aberrations that arise during aging before tumor development and that contribute to tumorigenesis are currently unclear. Mdm2, a key regulator of the p53 tumor suppressor and modulator of DNA break repair, is frequently overexpressed in malignancies and contributes to CIN. To determine the relationship between aging and CIN and the role of Mdm2, precancerous wild-type C57Bl/6 and littermate-matched Mdm2 transgenic mice at various ages were evaluated. Metaphase analyses of wild-type cells showed a direct correlation between age and increased chromosome and chromatid breaks, chromosome fusions and aneuploidy, but the frequency of polyploidy remained stable over time. Elevated levels of Mdm2 in precancerous mice increased both the numerical and the structural chromosomal abnormalities observed. Chromosome and chromatid breaks, chromosome fusions, aneuploidy and polyploidy were increased in older Mdm2 transgenic mice compared with wild-type littermates. Unexpectedly, chromosome fusions, aneuploidy and polyploidy rates in Mdm2 transgenic mice, but not chromosome and chromatid breaks, showed cooperation between Mdm2 overexpression and age. Notably, Mdm2 overexpression promoted gains in one or more chromosomes with age, while it did not affect the rate of chromosome loss. Therefore, aging increased specific forms of genomic instability, and elevated Mdm2 expression cooperated with aging to increase the likelihood of gaining certain chromosomal abnormalities of the kind thought to lead to cancer development.

p53-dependent senescence delays Emu-myc-induced B-cell lymphomagenesis.

The effect of p53-dependent cell-cycle arrest and senescence on Emu-myc-induced B-cell lymphoma development remains controversial. To address this question, we crossed Emu-myc mice with the p53(515C) mutant mouse, encoding the mutant p53R172P protein that retains the ability to activate the cell-cycle inhibitor and senescence activator p21. Importantly, this mutant lacks the ability to activate p53-dependent apoptotic genes. Hence, Emu-myc mice that harbor two p53(515C) alleles are completely defective for p53-dependent apoptosis. Both Emu-myc::p53(515C/515C) and Emu-myc::p53(515C/+) mice survive significantly longer than Emu-myc::p53(+/-) mice, indicating the importance of the p53-dependent non-apoptotic pathways in B-cell lymphomagenesis. In addition, the p53(515C) allele is deleted in several Emu-myc::p53(515C/+) lymphomas, further emphasizing the functionality of p53R172P in tumor inhibition. Lymphomas from both Emu-myc::p53(515C/515C) and Emu-myc::p53(515C/+) mice retain the ability to upregulate p21, resulting in cellular senescence. Senescence-associated beta-galactosidase (SA beta-gal) activity was observed in lymphomas from Emu-myc::p53(+/+), Emu-myc::p53(515C/515C) and Emu-myc::p53(515C /+) mice but not in lymphomas isolated from Emu-myc::p53(+/-) mice. Thus, in the absence of p53-dependent apoptosis, the ability of p53R172P to induce senescence leads to a significant delay in B-cell lymphoma development.

A deficiency in Mdm2 binding protein inhibits Myc-induced B-cell proliferation and lymphomagenesis.

Mdm2 binding protein (MTBP) has been implicated in cell-cycle arrest and the Mdm2/p53 tumor suppressor pathway through its interaction with Mdm2. To determine the function of MTBP in tumorigenesis and its potential role in the Mdm2/p53 pathway, we crossed Mtbp-deficient mice to Emu-myc transgenic mice, in which overexpression of the oncogene c-Myc induces B-cell lymphomas primarily through inactivation of the Mdm2/p53 pathway. We report that Myc-induced B-cell lymphoma development in Mtbp heterozygous mice was profoundly delayed. Surprisingly, reduced levels of Mtbp did not lead to an increase in B-cell apoptosis or affect Mdm2. Instead, an Mtbp deficiency inhibited Myc-induced proliferation and the upregulation of Myc target genes necessary for cell growth. Consistent with a role in proliferation, Mtbp expression was induced by Myc and other factors that promote cell-cycle progression and was elevated in lymphomas from humans and mice. Therefore, Mtbp functioned independent of Mdm2 and was a limiting factor for the proliferative and transforming functions of Myc. Thus, Mtbp is a previously unrecognized regulator of Myc-induced tumorigenesis.