Targeting MYC and exploring the role of mitochondrial metabolism in childhood neuroblastoma

MYCN is a member of the MYC family of proto-oncogenes, encoding transcription factors (c-MYC, MYCN and L-MYC) that play crucial roles for normal cellular functions and during development. However, the expression of MYC (here referring to c-MYC and MYCN) is found elevated in a large number of human cancers where it is implicated in most aspects of tumorigenesis and correlates to poor clinical outcome. Neuroblastoma is a heterogenous childhood cancer of the sympathetic nervous system. Tumors harboring amplification of the MYCN gene are highly aggressive and these patients have a poor prognosis. Consequently, new treatments directed against high MYC expressing tumors could help to improve the survival rates of these children. In Paper I, we screened 80 chemotherapeutic drugs and small chemical compounds to assess their selectivity against MYC-overexpression, using cancer cells with conditional c-MYC or MYCN expression. Positive hits belonged to distinct classes of chemical agents acting on selective cellular processes, including RNA, DNA and protein synthesis and turnover, and those inhibiting microtubules and topoisomerases. These results may provide indications for future drug development and treatment optimization towards MYC. One important goal in cancer research is to identify small molecules, which can interfere with MYC’s function, since today, no therapeutically relevant therapy acting directly against MYC exists. In Paper II we demonstrated that a previously identified c-MYC binding molecule, 10058-F4, showed selectivity towards high MYCN expressing neuroblastoma cells and resulted in prolonged survival in a MYCN-driven transgenic mouse model of neuroblastoma. In Paper IV, we further demonstrated that 10058-F4 and a few additional c-MYC-binding small molecules bind directly to the corresponding region of MYCN, and that their binding affinities correlated with the level of growth suppression in cells. Metabolic rewiring is an important feature in aggressive tumors. In Paper II we showed that downregulation of MYCN in neuroblastoma cells leads to accumulation of cytoplasmic lipid droplets caused by mitochondrial dysfunction. In this regard, MYCN was found to be linked with an overall elevated mitochondrial metabolism important for mediating tumor aggressiveness in neuroblastoma. In Paper III, we carried out a systematic investigation of metabolic alterations associated with MYCN in neuroblastoma, using patient gene expression data, quantitative proteomics and functional studies of metabolic pathway fluxes. MYCN was found to positively regulate glycolysis, respiration as well as oxidation of exogenous fatty acids in neuroblastoma cells, suggesting that MYCN mediates metabolic plasticity, which could account for an important survival mechanism during neuroblastoma tumor progression. Together the work comprised in this thesis support the development of targeted therapy against MYCN and identified MYCN-induced metabolic signals as a potential approach to target high risk neuroblastoma

Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine) and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide) as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C). Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity

Targeting of the MYCN protein with small molecule c-MYC inhibitors

This study was funded by grants from the Swedish Research Council and the Swedish Cancer Society. IM and HZ were recipients of graduate student grants from KI (KID), MAH was recipient of a Senior Investigator Award from the Swedish Cancer Society, and NJW was a Royal Society University Research Fellow when this work began.Members of the MYC family are the most frequently deregulated oncogenes in human cancer and are often correlated with aggressive disease and/or poorly differentiated tumors. Since patients with MYCN-amplified neuroblastoma have a poor prognosis, targeting MYCN using small molecule inhibitors could represent a promising therapeutic approach. We have previously demonstrated that the small molecule 10058-F4, known to bind to the c-MYC bHLHZip dimerization domain and inhibiting the c-MYC/MAX interaction, also interferes with the MYCN/MAX dimerization in vitro and imparts anti-tumorigenic effects in neuroblastoma tumor models with MYCN overexpression. Our previous work also revealed that MYCN-inhibition leads to mitochondrial dysfunction resulting in accumulation of lipid droplets in neuroblastoma cells. To expand our understanding of how small molecules interfere with MYCN, we have now analyzed the direct binding of 10058-F4, as well as three of its analogs; #474, #764 and 10058-F4(7RH), one metabolite C-m/z 232, and a structurally unrelated c-MYC inhibitor 10074-G5, to the bHLHZip domain of MYCN. We also assessed their ability to induce apoptosis, neurite outgrowth and lipid accumulation in neuroblastoma cells. Interestingly, all c-MYC binding molecules tested also bind MYCN as assayed by surface plasmon resonance. Using a proximity ligation assay, we found reduced interaction between MYCN and MAX after treatment with all molecules except for the 10058-F4 metabolite C-m/z 232 and the non-binder 10058-F4(7RH). Importantly, 10074-G5 and 10058-F4 were the most efficient in inducing neuronal differentiation and lipid accumulation in MYCN-amplified neuroblastoma cells. Together our data demonstrate MYCN-binding properties for a selection of small molecules, and provide functional information that could be of importance for future development of targeted therapies against MYCN-amplified neuroblastoma.Publisher PDFPeer reviewe

Growth inhibition and apoptosis induction in <i>MYCN</i> amplified cells.

<p>(A) The amount of viable of cells was determined using crystal violet assay following 48 hours treatment of Kelly cells with increasing concentration of the indicated compound. Data are shown as percent of control (DMSO) treated cells and represent the mean of three independent experiments. Error bars indicate standard deviation. (B) Pearson's correlation between the IC₅₀ values in the growth inhibition assay (A, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097285#pone-0097285-t002" target="_blank">Table 2</a>) and the K_D values for binding to MYCN (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097285#pone-0097285-t001" target="_blank">Table 1</a>). (C–D) Quantification of apoptosis by propidium iodide staining for sub G1 DNA content of SK-N-BE(2) (C) and Kelly (D) cells. Data represent the means of at least three independent experiments. Error bars indicate standard deviation.</p

Reduction of MYCN/MAX interaction in NB cells after treatment with small molecules.

<p>A) Proximity ligation assay (PLA) for MYCN/MAX interaction after treatment of SMS-KCN69n cells with the different small molecules at the indicated concentrations for 6 hours. Green signals represent the proximity of the MYCN and MAX proteins, while the red signal shows the total MYCN signal in the cell. DNA is stained with DAPI. Scale bar: 10 µM. The pictures are representative from three independent experiments. B) Quantification of the PLA signals per cell after treatment with the different compounds. Error bars indicate standard error of the mean.</p

Reduction in MYCN protein levels in NB cells after treatment with small molecules.

<p>Western blot analysis of MYCN expression in SK-N-BE(2) cells treated for 48 hours with the indicated concentrations of 10058-F4, 10058-F4 metabolite C-<i>m/z</i> 232, 10058-F4 analogs #474 and #764 and 10074-G5. Membranes were probed with antibodies recognizing MYCN and GAPDH. The blots are representative from three independent experiments.</p

10074-G5 induces neuronal differentiation in NB cells.

<p>Morphological differentiation of SK-N-BE(2) cells in response to 15 days culture with 10058-F4 (60 µM) 10074-G5 (30 µM) or DMSO in the presence or absence of NGF (50 ng/ml). Phase contrast micrographs show representative pictures from one out of three independent experiments.</p

K_D values of small molecules binding to the bHLHZip of c-MYC and MYCN.

a<p>KD values are shown as mean ± SD</p

MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells

The MYC genes are the most frequently activated oncogenes in human tumors and are hence attractive therapeutic targets. MYCN amplification leads to poor clinical outcome in childhood neuroblastoma, yet strategies to modulate the function of MYCN do not exist. Here we show that 10058-F4, a characterized c-MYC/Max inhibitor, also targets the MYCN/Max interaction, leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-amplified neuroblastoma cells and to increased survival of MYCN transgenic mice. We also report the discovery that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction. This study expands on the current knowledge of how MYC proteins control the metabolic reprogramming of cancer cells, especially highlighting lipid metabolism and the respiratory chain as important pathways involved in neuroblastoma pathogenesis. Together our data support direct MYC inhibition as a promising strategy for the treatment of MYC-driven tumors