Reducing MYC's transcriptional footprint unveils a good prognostic gene signature in melanoma.

MYC's key role in oncogenesis and tumor progression has long been established for most human cancers. In melanoma, its deregulated activity by amplification of 8q24 chromosome or by upstream signaling coming from activating mutations in the RAS/RAF/MAPK pathway-the most predominantly mutated pathway in this disease-turns MYC into not only a driver but also a facilitator of melanoma progression, with documented effects leading to an aggressive clinical course and resistance to targeted therapy. Here, by making use of Omomyc, the most characterized MYC inhibitor to date that has just successfully completed a phase I clinical trial, we show for the first time that MYC inhibition in melanoma induces remarkable transcriptional modulation, resulting in severely compromised tumor growth and a clear abrogation of metastatic capacity independently of the driver mutation. By reducing MYC's transcriptional footprint in melanoma, Omomyc elicits gene expression profiles remarkably similar to those of patients with good prognosis, underlining the therapeutic potential that such an approach could eventually have in the clinic in this dismal disease.

Bacterial delivery of therapeutic proteins to the nuclei of cancer cells.

Targeting nucleic targets with therapeutic proteins would enhance the treatment of hard-to-treat cancers. However, exogenous proteins are excluded from the nucleus by both the cellular and nuclear membranes. We have recently developed Salmonella that deliver active proteins into the cytoplasm of cancer cells. Here, we hypothesized that bacterially delivered proteins accumulate within nuclei, nuclear localization sequences (NLSs) increase delivery, and bacterially delivered proteins kill cancer cells. To test this hypothesis, we developed intranuclear delivering (IND) Salmonella and quantified the delivery of three model proteins. IND Salmonella delivered both ovalbumin and green fluorescent protein to nuclei of MCF7 cancer cells. The amount of protein in nuclei was linearly dependent on the amount delivered to the cytoplasm. The addition of a NLSs increased both the amount of protein in each nucleus and the number of nuclei that received protein. Delivery of Omomyc, a protein inhibitor of the nuclear transcript factor, Myc, altered cell physiology, and significantly induced cell death. These results show that IND Salmonella deliver functional proteins to the nucleus of cancerous cells. Extending this method to other transcription factors will increase the number of accessible targets for cancer therapy.

EV20/Omomyc: A novel dual MYC/HER3 targeting immunoconjugate.

MYC is one of the most important therapeutic targets in human cancer. Many attempts have been made to develop small molecules that could be used to curb its activity in patients, but most failed to identify a suitable direct inhibitor. After years of preclinical characterization, a tissue-penetrating peptide MYC inhibitor, called Omomyc, has been recently successfully used in a Phase I dose escalation study in late-stage, all-comers solid tumour patients. The study showed drug safety and positive signs of clinical activity, prompting the beginning of a new Phase Ib combination study currently ongoing in metastatic pancreatic adenocarcinoma patients. In this manuscript, we have explored the possibility to improve Omomyc targeting to specific cancer subtypes by linking it to a therapeutic antibody. The new immunoconjugate, called EV20/Omomyc, was developed by linking a humanised anti-HER3 antibody, named EV20, to Omomyc using a bifunctional linker. EV20/Omomyc shows antigen-dependent penetrating activity and therapeutic efficacy in a metastatic model of neuroblastoma. This study suggests that directing Omomyc into specific cell types using antibodies recognising tumour antigens could improve its therapeutic activity in specific indications, like in the paediatric setting.

A big step for MYC-targeted therapies.

The MYC proto-oncogene encodes a master transcriptional regulator that is frequently dysregulated in human cancer. Decades of efforts have failed to identify a MYC-targeted therapeutic, and this is still considered to be a holy grail in drug development. We highlight a recent report by Garralda et al. of a Phase 1 clinical trial of OMO-103 in patients with solid malignancies.

Targeting c-MYC: a potential non-hormonal therapeutic approach for endometriosis treatment.

Endometriosis is a benign gynecological disease in which eutopic endometrial tissue composed of glands and stroma grow within the pelvic cavity. The disease affects females of reproductive age and is characterized by pelvic pain, infertility and reduced quality of life. The majority of pharmacologic treatment modalities for endometriosis focus on suppression of estradiol production and/or action; an approach associated with adverse side effects. c-MYC is elevated in eutopic endometrium and endometriotic lesion tissue in patients with endometriosis and the disease shares many similar pathological characteristics with that of endometrial carcinoma. While targeting of c-MYC with Omomyc has recently gained substantial interest in the field of cancer research, there has been no recent attempt to evaluate the potential utility in targeting c-MYC for endometriosis treatment. The following perspective article compares the similarities between endometriosis and endometrial cancer and presents preliminary data suggesting that targeting c-MYC with Omomyc reduces endometriotic cell proliferation and viability in vitro. Future application of targeting c-MYC in endometriosis treatment and potential pros and cons are then discussed.

Pharmacokinetic Analysis of Omomyc Shows Lasting Structural Integrity and Long Terminal Half-Life in Tumor Tissue.

MYC is an oncoprotein causally involved in the majority of human cancers and a most wanted target for cancer treatment. Omomyc is the best-characterized MYC dominant negative to date. In the last years, it has been developed into a therapeutic miniprotein for solid tumor treatment and recently reached clinical stage. However, since the in vivo stability of therapeutic proteins, especially within the tumor vicinity, can be affected by proteolytic degradation, the perception of Omomyc as a valid therapeutic agent has been often questioned. In this study, we used a mass spectrometry approach to evaluate the stability of Omomyc in tumor biopsies from murine xenografts following its intravenous administration. Our data strongly support that the integrity of the functional domains of Omomyc (DNA binding and dimerization region) remains preserved in the tumor tissue for at least 72 hours following administration and that the protein shows superior pharmacokinetics in the tumor compartment compared with blood serum.

Inhibition of cMYC-MAX transcription factors hetero-dimerization with structurally engineered omoMYC to downregulate oncogenic pathways in renal carcinoma.

In the current study, we employed, structural informatics, and molecular simulation-based methods to engineer OmoMyc, a c-Myc dominant negative protein, to design novel mutants that could abrogate the c-MYC-MAX complex in Renal Carcinoma (RC). Among the total 472 mutations, only six mutations A61Q, Q64E, Q64K, N77R, Q64E-N77R, and Q64K-N77R were reported to increase the binding affinity and subjected to subsequent analysis such as protein-protein docking. The docking results revealed that the predicted mutants improve the functionality of the OmoMyc by not only increasing the binding affinity but also vdW and electrostatic energy in each complex that consequently increase the binding of the engineered OmoMyc by establishing extra hydrogen bonds, salt-bridges, and non-bonded contacts. Molecular simulation revealed a more stable behavior by the mutant complexes in contrast to the native OmoMyc however structural perturbations were reported in the regions, DBD (DNA-binding domain), loop region, and minor deviations at CTD (C terminal domain). Moreover, the hydrogen bonding and binding free energy results further validated the promising activity of our predicted mutants of OmoMyc. The results for TBE (total binding energy) revealed that the for each complex the TBE was calculated to be -87.88 ± 0.16 kcal/mol (WT OmoMyc-MAX), -91.89 ± 0.21 kcal/mol (A61Q OmoMyc-MAX), -91.55 ± 0.20 kcal/mol (Q64E OmoMyc-MAX), -95.17 ± 0.24 kcal/mol (Q64K OmoMyc-MAX), -96.49 ± 0.22 kcal/mol (N77R OmoMyc-MAX), -97.76 ± 0.22 kcal/mol (Q64E-N77R OmoMyc-MAX), and -95.31 ± 0.20 kcal/mol (Q64K-N77R OmoMyc-MAX) respectively. The results for TBE revealed promising results that allow the mutants to competitively inhibit the c-Myc-MAX complex more swiftly. Additionally, the internal motion and energy landscape were altered. These findings provide important insights into the potential of the mutants of OmoMyc as a therapeutic candidate for cancer treatment, particularly renal carcinoma, and could pave the way for the development of more effective clinical versions of OmoMyc.

Comparative analysis of compound NSC13728 as Omomyc homodimer stabilizer by molecular dynamics simulation and MM/GBSA free energy calculation.

Myc is a master transcriptional regulator that controls almost all cellular processes, whose function is dependent on dimerization with its obligate partner Max. Stabilization of Max homodimer by small molecules (such as compound NSC13728) has proven an effective way to reduce the availability of Myc-Max dimer. Omomyc, a peptide inhibitor of Myc, is able to form Omomyc homodimer, which can competitively inhibit the binding of Myc-Max to the E-box of DNA. Considering the high amino acid sequence homology between Omomyc and Max, we put forward the hypothesis that Max-Max stabilizers could stabilize the Omomyc homodimer. Hence, through molecular dynamics (MD) simulation and molecular mechanics/generalized Born surface area (MM/GBSA) free energy calculation, we discovered that the stability of Omomyc-Omomyc is remarkably higher than that of Max-Max. Moreover, after adding the compound NSC13728 into the well-defined "Site 3," the binding affinity between two Omomyc monomers can be further increased. Compound NSC13728 has stronger binding interaction to Omomyc-Omomyc than to Max-Max. "Site 3" of Omomyc is more hydrophobic than that of Max, which enlightens us that the more potent Omomyc-Omomyc stabilizers may be hydrophobic in structure.

Computational insights into the differentiated binding affinities of Myc, Max, and Omomyc dimers to the E-boxes of DNA.

Myc is a bHLHZip protein involved in growth control and cancer, which does not form a homodimer. Myc operates in a network with its heterodimerization partner Max, the latter of which can form homodimer and heterodimer. Omomyc, a polypeptide, can block Myc to treat cancers because it can both homodimerize as efficiently as Max and heterodimerize with both Myc and Max. However, the binding efficiencies to DNA for the mentioned two homodimers (Omomyc-Omomyc and Max-Max) and three heterodimers (Myc-Max, Omomyc-Myc, and Omomyc-Max) are still controversial. By molecular dynamics simulations and MM/GBSA free energy calculation, we ranked the binding affinities of five dimers to DNA and analyzed the contribution of single amino acids to the molecular recognition of dimers to DNA. Our simulation showed that the Omomyc-Omomyc dimer exhibited the highest binding energy to DNA, followed by the Omomyc-Myc, Max-Max, Omomyc-Max, and Myc-Max dimers. Moreover, five Arg residues (i.e., 7, 8, 15, 17, and 18 numbered by Omomyc) and five Lys residues (i.e., 6, 22, 40, 43, and 48 numbered by Omomyc) dominated the binding of various dimers to DNA while the residues Asp23 and Asp37 weakened the affinities via repulsive interaction. Our simulation would provide worthy information for further development of the structure-based design of novel Omomyc-like peptide inhibitors against Myc in the future.

Identification and Functional Characterization of Novel MYC-Regulated Long Noncoding RNAs in Group 3 Medulloblastoma.

The impact of protein-coding genes on cancer onset and progression is a well-established paradigm in molecular oncology. Nevertheless, unveiling the contribution of the noncoding genes-including long noncoding RNAs (lncRNAs)-to tumorigenesis represents a great challenge for personalized medicine, since they (i) constitute the majority of the human genome, (ii) are essential and flexible regulators of gene expression and (iii) present all types of genomic alterations described for protein-coding genes. LncRNAs have been increasingly associated with cancer, their highly tissue- and cancer type-specific expression making them attractive candidates as both biomarkers and therapeutic targets. Medulloblastoma is one of the most common malignant pediatric brain tumors. Group 3 is the most aggressive subgroup, showing the highest rate of metastasis at diagnosis. Transcriptomics and reverse genetics approaches were combined to identify lncRNAs implicated in Group 3 Medulloblastoma biology. Here we present the first collection of lncRNAs dependent on the activity of the MYC oncogene, the major driver gene of Group 3 Medulloblastoma. We assessed the expression profile of selected lncRNAs in Group 3 primary tumors and functionally characterized these species. Overall, our data demonstrate the direct involvement of three lncRNAs in Medulloblastoma cancer cell phenotypes.

Inhibition of Myc transcriptional activity by a mini-protein based upon Mxd1.

Myc, a transcription factor with oncogenic activity, is upregulated by amplification, translocation, and mutation of the cellular pathways that regulate its stability. Inhibition of the Myc oncogene by various modalities has had limited success. One Myc inhibitor, Omomyc, has limited cellular and in vivo activity. Here, we report a mini-protein, referred to as Mad, which is derived from the cellular Myc antagonist Mxd1. Mad localizes to the nucleus in cells and is 10-fold more potent than Omomyc in inhibiting Myc-driven cell proliferation. Similar to Mxd1, Mad also interacts with Max, the binding partner of Myc, and with the nucleolar upstream binding factor. Mad binds to E-Box DNA in the promoters of Myc target genes and represses Myc-mediated transcription to a greater extent than Omomyc. Overall, Mad appears to be more potent than Omomyc both in vitro and in cells.

Blocking Myc to Treat Cancer: Reflecting on Two Decades of Omomyc.

First designed and published in 1998 as a laboratory tool to study Myc perturbation, Omomyc has come a long way in the past 22 years. This dominant negative has contributed to our understanding of Myc biology when expressed, first, in normal and cancer cells, and later in genetically-engineered mice, and has shown remarkable anti-cancer properties in a wide range of tumor types. The recently described therapeutic effect of purified Omomyc mini-protein-following the surprising discovery of its cell-penetrating capacity-constitutes a paradigm shift. Now, much more than a proof of concept, the most characterized Myc inhibitor to date is advancing in its drug development pipeline, pushing Myc inhibition into the clinic.

MYC, MYCL, and MYCN as therapeutic targets in lung cancer.

Introduction: Lung cancer is the leading cause of cancer-related mortality globally. Despite recent advances with personalized therapies and immunotherapy, the prognosis remains dire and recurrence is frequent. Myc is an oncogene deregulated in human cancers, including lung cancer, where it supports tumorigenic processes and progression. Elevated Myc levels have also been associated with resistance to therapy.Areas covered: This article summarizes the genomic and transcriptomic studies that compile evidence for (i) MYC, MYCN, and MYCL amplification and overexpression in lung cancer patients, and (ii) their prognostic significance. We collected the most recent literature regarding the development of Myc inhibitors where the emphasis is on those inhibitors tested in lung cancer experimental models and their potential for future clinical application.Expert opinion: The targeting of Myc in lung cancer is potentially an unprecedented opportunity for inhibiting a key player in tumor progression and maintenance and therapeutic resistance. Myc inhibitory strategies are on the path to their clinical application but further work is necessary for the assessment of their use in combination with standard treatment approaches. Given the role of Myc in immune suppression, a significant opportunity may exist in the combination of Myc inhibitors with immunotherapies.

Finding MYCure.

Inhibiting the nuclear protein MYC involved in the majority of human cancers has long been considered an impossible mission and several technical challenges have discouraged the development of MYC inhibitory strategies. Nevertheless, in our recent publication in Science Translational Medicine "Intrinsic cell-penetrating activity propels Omomyc from proof of concept to viable anti-MYC therapy", we demonstrate for the first time the feasibility of pharmacological MYC inhibition in vitro and in vivo using an Omomyc-based mini-protein.

Omomyc Reveals New Mechanisms To Inhibit the MYC Oncogene.

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.

Strategies to Inhibit Myc and Their Clinical Applicability.

Myc is an oncogene deregulated in most-perhaps all-human cancers. Each Myc family member, c-, L-, and N-Myc, has been connected to tumor progression and maintenance. Myc is recognized as a "most wanted" target for cancer therapy, but has for many years been considered undruggable, mainly due to its nuclear localization, lack of a defined ligand binding site, and physiological function essential to the maintenance of normal tissues. The challenge of identifying a pharmacophore capable of overcoming these hurdles is reflected in the current absence of a clinically-viable Myc inhibitor. The first attempts to inhibit Myc used antisense technology some three decades ago, followed by small molecule inhibitors discovered through "classical" compound library screens. Notable breakthroughs proving the feasibility of systemic Myc inhibition were made with the Myc dominant negative mutant Omomyc, showing both the great promise in targeting this infamous oncogene for cancer treatment as well as allaying fears about the deleterious side effects that Myc inhibition might have on normal proliferating tissues. During this time many other strategies have appeared in an attempt to drug the undruggable, including direct and indirect targeting, knockdown, protein/protein and DNA interaction inhibitors, and translation and expression regulation. The inhibitors range from traditional small molecules to natural chemicals, to RNA and antisense, to peptides and miniproteins. Here, we briefly describe the many approaches taken so far, with a particular focus on their potential clinical applicability.