Sergio Nasi

Modelling Myc inhibition as a cancer therapy

Myc is a pleiotropic basic helix–loop–helix leucine zipper transcription factor that coordinates expression of the diverse intracellular and extracellular programs that together are necessary for growth and expansion of somatic cells. In principle, this makes inhibition of Myc an attractive pharmacological approach for treating diverse types of cancer. However, enthusiasm has been muted by lack of direct evidence that Myc inhibition would be therapeutically efficacious, concerns that it would induce serious side effects by inhibiting proliferation of normal tissues, and practical difficulties in designing Myc inhibitory drugs. We have modelled genetically both the therapeutic impact and the side effects of systemic Myc inhibition in a preclinical mouse model of Ras-induced lung adenocarcinoma by reversible, systemic expression of a dominant-interfering Myc mutant. We show that Myc inhibition triggers rapid regression of incipient and established lung tumours, defining an unexpected role for endogenous Myc function in the maintenance of Ras-dependent tumours in vivo. Systemic Myc inhibition also exerts profound effects on normal regenerating tissues. However, these effects are well tolerated over extended periods and rapidly and completely reversible. Our data demonstrate the feasibility of targeting Myc, a common downstream conduit for many oncogenic signals, as an effective, efficient and tumour-specific cancer therapy.

Making decisions through Myc

c‐Myc is a transcriptional regulator involved in carcinogenesis through its role in growth control and cell cycle progression. Here we attempt to relate its role in stimulating the G1–S transition to the ability to affect functioning of key cell cycle regulators, and we focus on how its property of modulating transcription of a wide range of target genes could explain its capacity to affect multiple pathways leading to proliferation, apoptosis, growth, and transformation.

Design and properties of a Myc derivative that efficiently homodimerizes

bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and specific protein-protein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and off cell proliferation, differentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization specificity. We report that Myc recognition specificity is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes efficiently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding efficiency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.

The Action Mechanism of the Myc Inhibitor Termed Omomyc May Give Clues on How to Target Myc for Cancer Therapy

Recent evidence points to Myc – a multifaceted bHLHZip transcription factor deregulated in the majority of human cancers – as a priority target for therapy. How to target Myc is less clear, given its involvement in a variety of key functions in healthy cells. Here we report on the action mechanism of the Myc interfering molecule termed Omomyc, which demonstrated astounding therapeutic efficacy in transgenic mouse cancer models in vivo. Omomyc action is different from the one that can be obtained by gene knockout or RNA interference, approaches designed to block all functions of a gene product. This molecule – instead – appears to cause an edge-specific perturbation that destroys some protein interactions of the Myc node and keeps others intact, with the result of reshaping the Myc transcriptome. Omomyc selectively targets Myc protein interactions: it binds c- and N-Myc, Max and Miz-1, but does not bind Mad or select HLH proteins. Specifically, it prevents Myc binding to promoter E-boxes and transactivation of target genes while retaining Miz-1 dependent binding to promoters and transrepression. This is accompanied by broad epigenetic changes such as decreased acetylation and increased methylation at H3 lysine 9. In the presence of Omomyc, the Myc interactome is channeled to repression and its activity appears to switch from a pro-oncogenic to a tumor suppressive one. Given the extraordinary therapeutic impact of Omomyc in animal models, these data suggest that successfully targeting Myc for cancer therapy might require a similar twofold action, in order to prevent Myc/Max binding to E-boxes and, at the same time, keep repressing genes that would be repressed by Myc.

Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis

Gliomas are the most common primary tumours affecting the adult central nervous system and respond poorly to standard therapy. Myc is causally implicated in most human tumours and the majority of glioblastomas have elevated Myc levels. Using the Myc dominant negative Omomyc, we previously showed that Myc inhibition is a promising strategy for cancer therapy. Here, we preclinically validate Myc inhibition as a therapeutic strategy in mouse and human glioma, using a mouse model of spontaneous multifocal invasive astrocytoma and its derived neuroprogenitors, human glioblastoma cell lines, and patient-derived tumours both in vitro and in orthotopic xenografts. Across all these experimental models we find that Myc inhibition reduces proliferation, increases apoptosis and remarkably, elicits the formation of multinucleated cells that then arrest or die by mitotic catastrophe, revealing a new role for Myc in the proficient division of glioma cells.

Non coding RNA and brain

Small non coding RNAs are a group of very different RNA molecules, present in virtually all cells, with a wide spectrum of regulatory functions which include RNA modification and regulation of protein synthesis. They have been isolated and characterized in all organisms and tissues, from Archaeobacteria to mammals. In mammalian brain there are a number of these small molecules, which are involved in neuronal differentiation as well as, possibly, in learning and memory. In this manuscript, we analyze the present knowledge about the function of the most important groups of small non-coding RNA present in brain: small nucleolar RNAs, small cytoplasmic RNAs, and microRNAs. The last ones, in particular, appear to be critical for dictating neuronal cell identity during development and to play an important role in neurite growth, synaptic development and neuronal plasticity.

A New Module in Neural Differentiation Control: Two MicroRNAs Upregulated by Retinoic Acid, miR-9 and -103, Target the Differentiation Inhibitor ID2

The transcription factor ID2 is an important repressor of neural differentiation strongly implicated in nervous system cancers. MicroRNAs (miRNAs) are increasingly involved in differentiation control and cancer development. Here we show that two miRNAs upregulated on differentiation of neuroblastoma cells – miR-9 and miR-103 – restrain ID2 expression by directly targeting the coding sequence and 3′ untranslated region of the ID2 encoding messenger RNA, respectively. Notably, the two miRNAs show an inverse correlation with ID2 during neuroblastoma cell differentiation induced by retinoic acid. Overexpression of miR-9 and miR-103 in neuroblastoma cells reduces proliferation and promotes differentiation, as it was shown to occur upon ID2 inhibition. Conversely, an ID2 mutant that cannot be targeted by either miRNA prevents retinoic acid-induced differentiation more efficient than wild-type ID2. These findings reveal a new regulatory module involving two microRNAs upregulated during neural differentiation that directly target expression of the key differentiation inhibitor ID2, suggesting that its alteration may be involved in neural cancer development.

MiR-21 is an Ngf-modulated MicroRNA That supports Ngf signaling and regulates neuronal degeneration in PC12 cells

The neurotrophins Ngf, Bdnf, NT-3, NT4–5 have key roles in development, survival, and plasticity of neuronal cells. Their action involves broad gene expression changes at the level of transcription and translation. MicroRNAs (miRs)—small RNA molecules that control gene expression post-transcriptionally—are increasingly implicated in regulating development and plasticity of neural cells. Using PC12 cells as a model system, we show that Ngf modulates changes in expression of a variety of microRNAs, including miRs known to be modulated by neurotrophins—such as the miR-212/132 cluster—and several others, such as miR-21, miR-29c, miR-30c, miR-93, miR-103, miR-207, miR-691, and miR-709. Pathway analysis indicates that Ngf-modulated miRs may regulate many protein components of signaling pathways involved in neuronal development and disease. In particular, we show that miR-21 enhances neurotrophin signaling and controls neuronal differentiation induced by Ngf. Notably, in a situation mimicking neurodegeneration—differentiated neurons deprived of Ngf—this microRNA is able to preserve the neurite network and to support viability of the neurons. These findings uncover a broad role of microRNAs in regulating neurotrophin signaling and suggest that aberrant expression of one or more Ngf-modulated miRs may be involved in neurodegenerative diseases.

Targeting Id protein interactions by an engineered HLH domain induces human neuroblastoma cell differentiation

Inhibitor of DNA-binding (Id) proteins prevent cell differentiation, promote growth and sustain tumour development. They do so by binding to E proteins and other transcription factors through the helix-loop-helix (HLH) domain, and inhibiting transcription. This makes HLH-mediated Id protein interactions an appealing therapeutic target. We have used the dominant interfering HLH dimerization mutant 13I to model the impact of Id inhibition in two human neuroblastoma cell lines: LA-N-5, similar to immature neuroblasts, and SH-EP, resembling more immature precursor cells. We have validated 13I as an Id inhibitor by showing that it selectively binds to Ids, impairs complex formation with RB, and relieves repression of E protein-activated transcription. Id inactivation by 13I enhances LA-N-5 neural features and causes SH-EP cells to acquire neuronal morphology, express neuronal proteins such as N-CAM and NF-160, proliferate more slowly, and become responsive to retinoic acid. Concomitantly, 13I augments the cell-cycle inhibitor p27Kip1 and reduces the angiogenic factor vascular endothelial growth factor. These effects are Id specific, being counteracted by Id overexpression. Furthermore, 13I strongly impairs tumorigenic properties in agar colony formation and cell invasion assays. Targeting Id dimerization may therefore be effective for triggering differentiation and restraining neuroblastoma cell tumorigenicity.

Interplay of the E box, the cyclic AMP response element, and HTF4/HEB in transcriptional regulation of the neurospecific, neurotrophin-inducible vgf gene.

vgf is a neurotrophin response-specific, developmentally regulated gene that codes for a neurosecretory polypeptide. Its transcription in neuronal cells is selectively activated by the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3, which induce survival and differentiation, and not by epidermal growth factor. We studied a short region of the rat vgf promoter which is essential for its regulated expression. A cyclic AMP response element (CRE) within this region is necessary for NGF induction of vgf transcription. Two sites upstream of CRE, an E box and a CCAAT sequence, bind nuclear protein complexes and are involved in transcriptional control. The E box has a dual role. It acts as an inhibitor in NIH 3T3 fibroblasts, together with a second E box located downstream, and as a stimulator in the NGF-responsive cell line PC12. By expression screening, we have isolated the cDNA for a basic helix-loop-helix transcription factor, a homolog of the HTF4/HEB E protein, that specifically binds the vgf promoter E box. The E protein was present in various cell lines, including PC12 cells, and was a component of a multiprotein nuclear complex that binds the promoter in vitro. The E box and CRE cooperate in binding to this complex, which may be an important determinant for neural cell-specific expression.