Inactivation of MYC reverses tumorigenesis.

The MYC proto-oncogene is an essential regulator of many normal biological programmes. MYC, when activated as an oncogene, has been implicated in the pathogenesis of most types of human cancers. MYC overexpression in normal cells is restrained from causing cancer through multiple genetically and epigenetically controlled checkpoint mechanisms, including proliferative arrest, apoptosis and cellular senescence. When pathologically activated in the correct epigenetic and genetic contexts, MYC bypasses these mechanisms and drives many of the 'hallmark' features of cancer, including uncontrolled tumour growth associated with DNA replication and transcription, cellular proliferation and growth, protein synthesis and altered cellular metabolism. MYC also dictates tumour cell fate by enforcing self-renewal and by abrogating cellular senescence and differentiation programmes. Moreover, MYC influences the tumour microenvironment, including activating angiogenesis and suppressing the host immune response. Provocatively, brief or even partial suppression of MYC back to its physiological levels of activation can lead to the restoration of intrinsic checkpoint mechanisms, resulting in acute and sustained tumour regression associated with tumour cells undergoing proliferative arrest, differentiation, senescence and apoptosis, as well as remodelling of the tumour microenvironment, recruitment of an immune response and shutdown of angiogenesis. Hence, tumours appear to be addicted to the MYC oncogene because of both tumour cell intrinsic and host-dependent mechanisms. MYC is important for the regulation of both the initiation and maintenance of tumorigenesis.

Immunology in the clinic review series; focus on cancer: multiple roles for the immune system in oncogene addiction.

Despite complex genomic and epigenetic abnormalities, many cancers are irrevocably dependent on an initiating oncogenic lesion whose restoration to a normal physiological activation can elicit a dramatic and sudden reversal of their neoplastic properties. This phenomenon of the reversal of tumorigenesis has been described as oncogene addiction. Oncogene addiction had been thought to occur largely through tumour cell-autonomous mechanisms such as proliferative arrest, apoptosis, differentiation and cellular senescence. However, the immune system plays an integral role in almost every aspect of tumorigenesis, including tumour initiation, prevention and progression as well as the response to therapeutics. Here we highlight more recent evidence suggesting that oncogene addiction may be integrally dependent upon host immune-mediated mechanisms, including specific immune effectors and cytokines that regulate tumour cell senescence and tumour-associated angiogenesis. Hence, the host immune system is essential to oncogene addiction.

c-Myc is a critical target for c/EBPalpha in granulopoiesis.

CCAAT/enhancer binding protein alpha (C/EBPalpha) is an integral factor in the granulocytic developmental pathway, as myeloblasts from C/EBPalpha-null mice exhibit an early block in differentiation. Since mice deficient for known C/EBPalpha target genes do not exhibit the same block in granulocyte maturation, we sought to identify additional C/EBPalpha target genes essential for myeloid cell development. To identify such genes, we used both representational difference analysis and oligonucleotide array analysis with RNA derived from a C/EBPalpha-inducible myeloid cell line. From each of these independent screens, we identified c-Myc as a C/EBPalpha negatively regulated gene. We mapped an E2F binding site in the c-Myc promoter as the cis-acting element critical for C/EBPalpha negative regulation. The identification of c-Myc as a C/EBPalpha target gene is intriguing, as it has been previously shown that down-regulation of c-Myc can induce myeloid differentiation. Here we show that stable expression of c-Myc from an exogenous promoter not responsive to C/EBPalpha-mediated down-regulation forces myeloblasts to remain in an undifferentiated state. Therefore, C/EBPalpha negative regulation of c-Myc is critical for allowing early myeloid precursors to enter a differentiation pathway. This is the first report to demonstrate that C/EBPalpha directly affects the level of c-Myc expression and, thus, the decision of myeloid blasts to enter into the granulocytic differentiation pathway.

A murine model for B-cell lymphomagenesis in immunocompromised hosts: natural killer cells are an important component of host resistance to premalignant B-cell lines.

The accompanying paper (D. W. Felsher et al., Cancer Res., 50:7042-7049, 1990) describes a new panel of cloned murine B-cell lines with a premalignant phenotype and in vivo-derived malignant variants. This paper assesses the contribution of immune mediated antitumor mechanisms which might account for host resistance to the tumorigenicity of these cell lines. Conventional T-cell-dependent responses did not appear to be critical to host resistance. In vivo elimination of T-helper cells with anti-L3T4 monoclonal antibody did not reduce host resistance to the tumorigenicity of these cell lines, nor did these cell lines elicit cytotoxic T-cell activity. However, a strong correlation was found between tumorigenicity and host natural killer (NK) activity. In vitro studies demonstrated that the cell lines were as NK sensitive as the prototypical NK target, YAC-1, whereas the malignant variants fully tumorigenic in normal hosts were greater than 20-fold less NK sensitive than were the parent cell lines. In vivo depletion of NK cells with anti-asialo-GM1 in BALB/c strongly diminished host resistance to cell line tumorigenicity, whereas polydeoxyinosinic-deoxycytidilic acid induction of NK cells enhanced host resistance. These findings indicate that NK function is a critical component to host resistance in this system and suggest that endogenous cellular mechanisms which overcome NK sensitivity could be a target for secondary transforming events in B-cell lymphomagenesis. They also raise the unexpected possibility that a non-antigen-dependent (versus immune cytotoxic T-lymphocytes) effector mechanism may be the key deficit promoting B-cell neoplasia in the setting of immunocompromised states.

A murine model for B-cell lymphomagenesis in immunocompromised hosts: c-myc-rearranged B-cell lines with a premalignant phenotype.

Activation of c-myc or bcl-2 protooncogene is a common event in B-cell lymphomagenesis. Alone, each is insufficient to produce lymphoma, prompting the search for the additional steps required to complete the malignant phenotype. Among the existing systems of murine or human B-cell neoplasia, no commonly occurring complementary oncogenic activation has been found. This study introduces a new series of murine B-cell lines with a phenotype suggesting that such additional events might not involve intrinsic growth control, but instead host immune mechanisms which normally suppress tumorigenicity of premalignant B-cells. Four murine B-cell lines were isolated from the long-term culture of normal lymphoid tissue bearing a premalignant phenotype. (a) Their phenotype resembled naturally occurring lymphoid tumors of immunocompromised hosts with regard to c-myc activation, aberrant or absent immunoglobulin expression, preferential rearrangement of the lambda light chain locus, and a distinctive pattern of tissue invasion and tumor histology. (b) Their tumorigenicity was strictly dependent on host permissiveness correlated with immunodeficient status: C.B-17-scid greater than BALB/c-nu/nu greater than normal BALB/c much greater than other H-2d strains (NZB x NZW F1, NZB, DBA/2). (c) Host passage selected for malignant variants distinguished by a 10(4)-fold increase in tumorigenicity (as judged by limiting cell dose) and by novel tumorigenicity in nonpermissive syngeneic hosts. These features are analogous to properties of human lymphomas arising in immunocompromised states and, to our knowledge, unique among previously reported murine B-cell lines.

STK38 is a critical upstream regulator of MYC's oncogenic activity in human B-cell lymphoma.

The MYC protooncogene is associated with the pathogenesis of most human neoplasia. Conversely, its experimental inactivation elicits oncogene addiction. Besides constituting a formidable therapeutic target, MYC also has an essential function in normal physiology, thus creating the need for context-specific targeting strategies. The analysis of post-translational MYC activity modulation yields novel targets for MYC inactivation. Specifically, following regulatory network analysis in human B-cells, we identify a novel role of the STK38 kinase as a regulator of MYC activity and a candidate target for abrogating tumorigenesis in MYC-addicted lymphoma. We found that STK38 regulates MYC protein stability and turnover in a kinase activity-dependent manner. STK38 kinase inactivation abrogates apoptosis following B-cell receptor activation, whereas its silencing significantly decreases MYC levels and increases apoptosis. Moreover, STK38 knockdown suppresses growth of MYC-addicted tumors in vivo, thus providing a novel viable target for treating these malignancies.

Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas.

The anaplastic lymphoma kinase (ALK), tyrosine kinase oncogene is implicated in a wide variety of cancers. In this study we used conditional onco-ALK (NPM-ALK and TPM3-ALK) mouse MEF cell lines (ALK+ fibroblasts) and transgenic models (ALK+ B-lymphoma) to investigate the involvement and regulation of angiogenesis in ALK tumor development. First, we observed that ALK expression leads to downregulation of miR-16 and increased Vascular Endothelial Growth Factor (VEGF) levels. Second, we found that modification of miR-16 levels in TPM3-ALK MEF cells greatly affected VEGF levels. Third, we demonstrated that miR-16 directly interacts with VEGF mRNA at the 3'-untranslated region and that the regulation of VEGF by miR-16 occurs at the translational level. Fourth, we showed that expression of both the ALK oncogene and hypoxia-induced factor 1α (HIF1α) is a prerequisite for miR-16 downregulation. Fifth, in vivo, miR-16 gain resulted in reduced angiogenesis and tumor growth. Finally, we highlighted an inverse correlation between the levels of miR-16 and VEGF in human NPM-ALK+ Anaplastic Large Cell Lymphomas (ALCL). Altogether, our results demonstrate, for the first time, the involvement of angiogenesis in ALK+ ALCL and strongly suggest an important role for hypoxia-miR-16 in regulating VEGF translation.

Reversible tumorigenesis by MYC in hematopoietic lineages.

The targeted repair of mutant protooncogenes or the inactivation of their gene products may be a specific and effective therapy for human neoplasia. To examine this possibility, we have used the tetracycline regulatory system to generate transgenic mice that conditionally express the MYC protooncogene in hematopoietic cells. Sustained expression of the MYC transgene culminated in the formation of malignant T cell lymphomas and acute myleoid leukemias. The subsequent inactivation of the transgene caused regression of established tumors. Tumor regression was associated with rapid proliferative arrest, differentiation and apoptosis of tumor cells, and resumption of normal host hematopoiesis. We conclude that even though tumorigenesis is a multistep process, remediation of a single genetic lesion may be sufficient to reverse malignancy.

Transient excess of MYC activity can elicit genomic instability and tumorigenesis.

Overexpression of the MYC protooncogene has been implicated in the genesis of diverse human tumors. Tumorigenesis induced by MYC has been attributed to sustained effects on proliferation and differentiation. Here we report that MYC may also contribute to tumorigenesis by destabilizing the cellular genome. A transient excess of MYC activity increased tumorigenicity of Rat1A cells by at least 50-fold. The increase persisted for >30 days after the return of MYC activity to normal levels. The brief surfeit of MYC activity was accompanied by evidence of genomic instability, including karyotypic abnormalities, gene amplification, and hypersensitivity to DNA-damaging agents. MYC also induced genomic destabilization in normal human fibroblasts, although these cells did not become tumorigenic. Stimulation of Rat1A cells with MYC accelerated their passage through G1/S. Moreover, MYC could force normal human fibroblasts to transit G1 and S after treatment with N-(phosphonoacetyl)-L-aspartate (PALA) at concentrations that normally lead to arrest in S phase by checkpoint mechanisms. Instead, the cells subsequently appeared to arrest in G2. We suggest that the accelerated passage through G1 was mutagenic but that the effect of MYC permitted a checkpoint response only after G2 had been reached. Thus, MYC may contribute to tumorigenesis through a dominant mutator effect.

Independent rearrangement of Ig lambda genes in tissue culture-derived murine B cell lines.

Rearrangement of the lambda light chain locus is considered a late event in pre-B cell differentiation which occurs after successful heavy chain and unsuccessful kappa light chain rearrangement. However, this view has recently been challenged by the observation of apparently independent lambda rearrangement in certain B cell lines and Ig transgenic populations. In this study we have examined the pattern of Ig rearrangements and expression in several tissue culture-derived murine B cell lines. One pre-B cell line (BDL-1) displayed germline heavy and kappa light chain genes despite the presence of a productive lambda 1 light chain rearrangement. Two other cell lines (DAC-2, BDL-2) had multiple lambda rearrangements despite the presence of productive kappa chain rearrangements. These cell lines provide new precedents for rearrangement of the lambda locus independent of the kappa locus. Their phenotype suggests that accessibility at the different Ig loci may be controlled by a non-sequential mechanism.

A novel octamer regulatory element in the VH11 leader exon of B-1 cells.

B-1 cells (CD5 B cells) represent an initial fetal wave of B cell lymphopoiesis. B-1 cells have fundamental properties that are unique from conventional B cells, including a restricted Ab repertoire. We investigated the mechanism for the overrepresentation of one such Ig H chain variable-region gene, VH11, by murine B-1 cells. We postulated that a cis-regulatory element contributed to the use of VH11. We observed that the DNA encoding the leader peptide of VH11 was atypically A/T rich and thus was a candidate for nuclear protein binding. By electrophoretic mobility shift analysis, we found that the VH11 leader DNA specifically bound to three protein complexes present in the nucleus of the B-1 cell line AJ9. Of these bands, one was ubiquitous for all cells examined (lymphoid and nonlymphoid); another band was present only in B cells, and the third band was specific for B-1 cells that expressed VH11 or VH12. In addition to its binding properties, the VH11 leader sequence also displayed modest tissue-specific enhancer activity. By DNA footprint analysis, all three protein complexes were found to bind to an octamer motif embedded within the VH11 leader DNA. To identify the octamer-binding proteins, a panel of octamer-specific Abs was used. We found that the ubiquitous band was Oct-1, and the B cell-specific band was Oct-2. The B-1 cell-specific nuclear binding protein was neither Oct-1 nor Oct-2, but may be a novel POU domain protein. We hypothesize that the VH11 leader octamer site may target this gene for preferential rearrangement and/or expression and therefore would be a contributing factor in the increased use of this gene by B-1 cells.

Overexpression of MYC causes p53-dependent G2 arrest of normal fibroblasts.

Overexpression of the proto-oncogene MYC has been implicated in the genesis of diverse human cancers. One explanation for the role of MYC in tumorigenesis has been that this gene might drive cells inappropriately through the division cycle, leading to the relentless proliferation characteristic of the neoplastic phenotype. Herein, we report that the overexpression of MYC alone cannot sustain the division cycle of normal cells but instead leads to their arrest in G(2). We used an inducible form of the MYC protein to stimulate normal human and rodent fibroblasts. The stimulated cells passed through G(1) and S but arrested in G(2) and frequently became aneuploid, presumably as a result of inappropriate reinitiation of DNA synthesis. Absence of the tumor suppressor gene p53 or its downstream effector p21 reduced the frequency of both G(2) arrest and aneuploidy, apparently by compromising the G(2) checkpoint control. Thus, relaxation of the G(2) checkpoint may be an essential early event in tumorigenesis by MYC. The loss of p53 function seems to be one mechanism by which this relaxation commonly occurs. These findings dramatize how multiple genetic events can collaborate to produce neoplastic cells.