Huabo Wang

Improved low molecular weight Myc-Max inhibitors

Compounds that selectively prevent or disrupt the association between the c-Myc oncoprotein and its obligate heterodimeric partner Max (Myc-Max compounds) have been identified previously by high-throughput screening of chemical libraries. Although these agents specifically inhibit the growth of c-Myc–expressing cells, their clinical applicability is limited by their low potency. We describe here several chemical modifications of one of these original compounds, 10058-F4, which result in significant improvements in efficacy. Compared with the parent structure, these analogues show enhanced growth inhibition of c-Myc–expressing cells in a manner that generally correlates with their ability to disrupt c-Myc-Max association and DNA binding. Furthermore, we show by use of a sensitive fluorescence polarization assay that both 10058-F4 and its active analogues bind specifically to monomeric c-Myc. These studies show that improved Myc-Max compounds can be generated by a directed approach involving deliberate modification of an index compound. They further show that the compounds specifically target c-Myc, which exists in a dynamic and relatively unstructured state with only partial and transient α-helical content. [Mol Cancer Ther 2007;6(9):2399–408]

Structural Rationale for the Coupled Binding and Unfolding of the c-Myc Oncoprotein by Small Molecules

The basic-helix-loop-helix-leucine-zipper domains of the c-Myc oncoprotein and its obligate partner Max are intrinsically disordered (ID) monomers that undergo coupled folding and binding upon heterodimerization. We have identified the binding sites and determined the structural means by which two unrelated small molecules, 10058-F4 and 10074-G5, bind c-Myc and stabilize the ID monomer over the highly ordered c-Myc-Max heterodimer. In solution, the molecules bind to distinct regions of c-Myc and thus limit its ability to interact with Max and assume a more rigid and defined conformation. The identification of multiple, specific binding sites on an ID domain suggests that small molecules may provide a general means for manipulating the structure and function of ID proteins, such as c-Myc.

Direct inhibition of c-Myc-Max heterodimers by celastrol and celastrol-inspired triterpenoids

Many oncogenic signals originate from abnormal protein-protein interactions that are potential targets for small molecule inhibitors. However, the therapeutic disruption of these interactions has proved elusive. We report here that the naturally-occurring triterpenoid celastrol is an inhibitor of the c-Myc (Myc) oncoprotein, which is over-expressed in many human cancers. Most Myc inhibitors prevent the association between Myc and its obligate heterodimerization partner Max via their respective bHLH-ZIP domains. In contrast, we show that celastrol binds to and alters the quaternary structure of the pre-formed dimer and abrogates its DNA binding. Celastrol contains a reactive quinone methide group that promiscuously forms Michael adducts with numerous target proteins and other free sulfhydryl-containing molecules. Interestingly, triterpenoid derivatives lacking the quinone methide showed enhanced specificity and potency against Myc. As with other Myc inhibitors, these analogs rapidly reduced the abundance of Myc protein and provoked a global energy crisis marked by ATP depletion, neutral lipid accumulation, AMP-activated protein kinase activation, cell cycle arrest and apoptosis. They also inhibited the proliferation of numerous established human cancer cell lines as well as primary myeloma explants that were otherwise resistant to JQ1, a potent indirect Myc inhibitor. N-Myc amplified neuroblastoma cells showed similar responses and, in additional, underwent neuronal differentiation. These studies indicate that certain pharmacologically undesirable properties of celastrol such as Michael adduct formation can be eliminated while increasing selectivity and potency toward Myc and N-Myc. This, together with their low in vivo toxicity, provides a strong rationale for pursuing the development of additional Myc-specific triterpenoid derivatives.

Coordinated Activities of Multiple Myc-dependent and Myc-independent Biosynthetic Pathways in Hepatoblastoma.

Hepatoblastoma (HB) is associated with aberrant activation of the β-catenin and Hippo/YAP signaling pathways. Overexpression of mutant β-catenin and YAP in mice induces HBs that express high levels of c-Myc (Myc). In light of recent observations that Myc is unnecessary for long-term hepatocyte proliferation, we have now examined its role in HB pathogenesis using the above model. Although Myc was found to be dispensable for in vivo HB initiation, it was necessary to sustain rapid tumor growth. Gene expression profiling identified key molecular differences between myc+/+ (WT) and myc−/− (KO) hepatocytes and HBs that explain these behaviors. In HBs, these included both Myc-dependent and Myc-independent increases in families of transcripts encoding ribosomal proteins, non-structural factors affecting ribosome assembly and function, and enzymes catalyzing glycolysis and lipid bio-synthesis. In contrast, transcripts encoding enzymes involved in fatty acid β-oxidation were mostly down-regulated. Myc-independent metabolic changes associated with HBs included dramatic reductions in mitochondrial mass and oxidative function, increases in ATP content and pyruvate dehydrogenase activity, and marked inhibition of fatty acid β-oxidation (FAO). Myc-dependent metabolic changes included higher levels of neutral lipid and acetyl-CoA in WT tumors. The latter correlated with higher histone H3 acetylation. Collectively, our results indicate that the role of Myc in HB pathogenesis is to impose mutually dependent changes in gene expression and metabolic reprogramming that are unattainable in non-transformed cells and that cooperate to maximize tumor growth.

c-Myc and AMPK Control Cellular Energy Levels by Cooperatively Regulating Mitochondrial Structure and Function

The c-Myc (Myc) oncoprotein and AMP-activated protein kinase (AMPK) regulate glycolysis and oxidative phosphorylation (Oxphos) although often for different purposes. Because Myc over-expression depletes ATP with the resultant activation of AMPK, we explored the potential co-dependency of and cross-talk between these proteins by comparing the consequences of acute Myc induction in ampk+/+ (WT) and ampk-/- (KO) murine embryo fibroblasts (MEFs). KO MEFs showed a higher basal rate of glycolysis than WT MEFs and an appropriate increase in response to activation of a Myc-estrogen receptor (MycER) fusion protein. However, KO MEFs had a diminished ability to increase Oxphos, mitochondrial mass and reactive oxygen species in response to MycER activation. Other differences between WT and KO MEFs, either in the basal state or following MycER induction, included abnormalities in electron transport chain function, levels of TCA cycle-related oxidoreductases and cytoplasmic and mitochondrial redox states. Transcriptional profiling of pathways pertinent to glycolysis, Oxphos and mitochondrial structure and function also uncovered significant differences between WT and KO MEFs and their response to MycER activation. Finally, an unbiased mass-spectrometry (MS)-based survey capable of quantifying ~40% of all mitochondrial proteins, showed about 15% of them to be AMPK- and/or Myc-dependent in their steady state. Significant differences in the activities of the rate-limiting enzymes pyruvate kinase and pyruvate dehydrogenase, which dictate pyruvate and acetyl coenzyme A abundance, were also differentially responsive to Myc and AMPK and could account for some of the differences in basal metabolite levels that were also detected by MS. Thus, Myc and AMPK are highly co-dependent and appear to engage in significant cross-talk across numerous pathways which support metabolic and ATP-generating functions.

Discovery of methyl 4'-methyl-5-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-[1,1'-biphenyl]-3-carboxylate, an improved small-molecule inhibitor of c-Myc-max dimerization.

c‐Myc is a basic helix‐loop‐helix‐leucine zipper (bHLH‐ZIP) transcription factor that is responsible for the transcription of a wide range of target genes involved in many cancer‐related cellular processes. Over‐expression of c‐Myc has been observed in, and directly contributes to, a variety of human cancers including those of the hematopoietic system, lung, prostate and colon. To become transcriptionally active, c‐Myc must first dimerize with Myc‐associated factor X (Max) via its own bHLH‐ZIP domain. A proven strategy towards the inhibition of c‐Myc oncogenic activity is to interfere with the structural integrity of the c‐Myc–Max heterodimer. The small molecule 10074‐G5 is an inhibitor of c‐Myc–Max dimerization (IC50=146 μM) that operates by binding and stabilizing c‐Myc in its monomeric form. We have identified a congener of 10074‐G5, termed 3jc48‐3 (methyl 4′‐methyl‐5‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)‐[1,1′‐biphenyl]‐3‐carboxylate), that is about five times as potent (IC50=34 μM) at inhibiting c‐Myc–Max dimerization as the parent compound. 3jc48‐3 exhibited an approximate twofold selectivity for c‐Myc–Max heterodimers over Max–Max homodimers, suggesting that its mode of action is through binding c‐Myc. 3jc48‐3 inhibited the proliferation of c‐Myc‐over‐expressing HL60 and Daudi cells with single‐digit micromolar IC50 values by causing growth arrest at the G0/G1 phase. Co‐immunoprecipitation studies indicated that 3jc48‐3 inhibits c‐Myc–Max dimerization in cells, which was further substantiated by the specific silencing of a c‐Myc‐driven luciferase reporter gene. Finally, 3jc48‐3′s intracellular half‐life was >17 h. Collectively, these data demonstrate 3jc48‐3 to be one of the most potent, cellularly active and stable c‐Myc inhibitors reported to date.

Ribosomopathy-like properties of murine and human cancers

Ribosomopathies comprise a heterogeneous group of hematologic and developmental disorders, often characterized by bone marrow failure, skeletal and other developmental abnormalities and cancer predisposition. They are associated with mutations and/or haplo-insufficiencies of ribosomal proteins (RPs) and inefficient ribosomal RNA (rRNA) processing. The resulting ribosomal stress induces the canonical p19ARF/Mdm2/p53 tumor suppressor pathway leading to proliferative arrest and/or apoptosis. It has been proposed that this pathway is then inactivated during subsequent neoplastic evolution. We show here that two murine models of hepatoblastoma (HB) and hepatocellular carcinoma (HCC) unexpectedly possess features that mimic the ribosomopathies. These include loss of the normal stoichiometry of RP transcripts and proteins and the accumulation of unprocessed rRNA precursors. Silencing of p19ARF, cytoplasmic sequestration of p53, binding to and inactivation of Mdm2 by free RPs, and up-regulation of the pro-survival protein Bcl-2 may further cooperate to drive tumor growth and survival. Consistent with this notion, re-instatement of constitutive p19ARF expression in the HB model completely suppressed tumorigenesis. In >2000 cases of human HCC, colorectal, breast, and prostate cancer, RP transcript deregulation was a frequent finding. In HCC and breast cancer, the severity of this dysregulation was associated with inferior survival. In HCC, the presence of RP gene mutations, some of which were identical to those previously reported in ribosomopathies, were similarly negatively correlated with long-term survival. Taken together, our results indicate that many if not all cancers possess ribosomopathy-like features that may affect their biological behaviors.

Sequential Adaptive Changes in a c-Myc-Driven Model of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common cancer that frequently overexpresses the c-Myc (Myc) oncoprotein. Using a mouse model of Myc-induced HCC, we studied the metabolic, biochemical, and molecular changes accompanying HCC progression, regression, and recurrence. These involved altered rates of pyruvate and fatty acid β-oxidation and the likely re-directing of glutamine into biosynthetic rather than energy-generating pathways. Initial tumors also showed reduced mitochondrial mass and differential contributions of electron transport chain complexes I and II to respiration. The uncoupling of complex IIs electron transport function from its succinate dehydrogenase activity also suggested a mechanism by which Myc generates reactive oxygen species. RNA sequence studies revealed an orderly progression of transcriptional changes involving pathways pertinent to DNA damage repair, cell cycle progression, insulin-like growth factor signaling, innate immunity, and further metabolic re-programming. Only a subset of functions deregulated in initial tumors was similarly deregulated in recurrent tumors thereby indicating that the latter can “normalize” some behaviors to suit their needs. An interactive and freely available software tool was developed to allow continued analyses of these and other transcriptional profiles. Collectively, these studies define the metabolic, biochemical, and molecular events accompanyingHCCevolution, regression, and recurrence in the absence of any potentially confounding therapies.

Abstract 4760: Inhibition of c-myc by the triterpenoid celastrol

c-Myc is a bHLH-ZIP transcription factor that is deregulated in a variety of human cancers. More specifically, c-Myc heterodimerizes with another bHLH-ZIP partner protein, Max, to bind to specific sequences (E-Boxes: CAC/TGTG) located near the transcriptional start sites of its numerous target genes. Because c-Myc9s highly variable downstream effects are difficult to attack individually, directly inhibiting the association between c-Myc and Max association by interfering with their bHLH-ZIP-mediated heterodimerization has emerged as an attractive therapeutic strategy. Thus far, high throughput screens of chemical libraries have identified a number of small molecules that specifically inhibit the c-Myc-Max association. However, these synthetic compounds and their analogs are generally of low potency and therefore have limited clinical utility. Here we report identification of celastrol, a naturally occurring triterpenoid, as a potent c-Myc inhibitor. We sought to further assess celastrol9s ability to inhibit c-Myc-Max interaction and cell growth, and determine its structure activity relationship. Surface plasmon resonance and electrophoretic mobility shift studies revealed that celastrol specifically binds to c-Myc and prevents both its interaction with Max, and subsequent DNA binding with a Kd in the low micromolar range. Co-immunoprecipitation studies confirmed that the dose-dependent disruption of c-Myc-Max heterodimers by celastrol could also be achieved in c-Myc-overexpressing human HL60 promyelocytic leukemia cells. Moreover, in vitro data demonstrated that celastrol significantly inhibited both HL60 and Burkitt9s lymphoma cell growth with IC50s in the nanomolar range. However, modifications to the C-28 carboxylic acid group of celastrol eliminated c-Myc binding, suggesting that celastrol binds to c-Myc via this functional group. This structure activity relationship may provide a basis for the development of more pharmacologically suitable analogs. Together these results suggest that celastrol is among the most potent Myc-Max disruptors yet identified, and suggest a mechanism of action. Celastrol thus represents a promising new agent with which to target c-Myc and provides a new structural chemical platform for further pharmaceutical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4760. doi:1538-7445.AM2012-4760