Impact of targeting MYC in metabolic reprogramming and differentiation of cancer

Alterations in several metabolic pathways due to increased energy and biomass demand, as consequence of the uncontrolled proliferation of cancer cells, is known as metabolic reprogramming. Mutations in tumor suppressor genes and oncogenes that initiate cancer development, are responsible directly and indirectly of the changes in major cellular energy production processes, including glycolysis, glutaminolysis, and lipid metabolism. Neuroblastoma (NB) is a solid tumor that develops extracranially in the sympathetic nervous system, and the most diagnosed cancer during the first year of life. Among several genetic alterations, MYCN-amplification occurs in approximately 25% of all cases, associated with poor survival rate. Although the MYCN protein plays a crucial role in NB progression, no inhibitors have been approved for clinical use, as targeting MYCN has proven to be challenging. Thus, other indirect strategies, such as targeting downstream processes controlled by MYCN including metabolism and differentiation, represent alternatives to overcome the drawbacks of directly targeting this oncoprotein. In clear cell renal cell carcinoma (ccRCC), loss of the von Hippel-Lindau (VHL) gene provokes constitutive activation of hypoxia inducible factors (HIFs). Due to disruptions in a myriad of metabolic pathways, and in part, as consequence of the continuous stabilization of HIFs, ccRCC is considered a metabolic disease. Moreover, although MYC amplification is found only in 5-10 % of the cases, increased MYC signaling has been associated with development of aggressive forms of ccRCC. In paper I we investigated the metabolic changes induced by MYCN amplification in NB. By combining proteomics, transcriptome analysis and functional metabolic assays, we demonstrated that MYCN induced changes in several metabolic enzymes, increasing glycolysis and oxidative phosphorylation. We also found that fatty acids were the preferred mitochondrial fuel for energy production in MYCN-amplified cells. Moreover, data from tracing experiments with 13C-labeled glucose or glutamine indicated that MYCN-amplified NB cells synthetized glutamine de novo. Furthermore, targeting fatty acid oxidation resulted in reduction of viability in NB cells with MYCN-amplification in vitro and in reduction of tumor burden in vivo. Since we found that fatty acid oxidation was relevant for MYCN-amplified NB, we further studied the effects of inhibiting de novo fatty acids synthesis in paper II. Using five different inhibitors targeting two consecutive enzymes in the process, we described that inhibition of the synthesis of fatty acids resulted in striking neuronal differentiation associated with activation of ERK signaling, and reduction of MYCN and MYC levels. Moreover, lipid composition as well as mitochondrial function and morphology of NB cells was altered. In addition, fatty acid synthesis inhibition led to reduced tumor formation and increased differentiation markers in several NB xenograft models. Together, the results in paper I and II suggested that targeting lipid metabolism could be a potential therapeutic approach for NB patients. In paper III we further analyzed the potential differentiation of NB cells induced by activation of nuclear hormone receptors (NHRs). Our data showed that the simultaneous activation of glucocorticoid receptor (GR), estrogen receptor α (ERα) and retinoic acid receptor α (RARα) potentiated neurite outgrowth, induced changes in the glycolytic and mitochondrial functions, accompanied with lipid droplet accumulation, and reduced proliferation in vitro as well as tumor burden in vivo. In addition, single cell nuclei analysis revealed a sequential expression of the three NHRs during adrenal gland development. Notably, in silico analysis of patient cohorts demonstrated that high expression of these NHRs were correlated with better overall survival. Thus, combination therapy with the concurrent activation of GR, ERα and RARα represents a promising strategy to induce differentiation in NB patients. Paper IV describes the mechanism behind lipid droplet (LD) accumulation induced by MYC inhibition during hypoxia in clear cell renal cell carcinoma (ccRCC). We found that HIF expression together with MYC inhibition resulted in LD deposition. Our results showed that due to HIF stabilization, glutamine-derived carbons were directed for synthesis of fatty acids, further accumulating in LDs. Importantly, we identified that the hypoxia inducible lipid droplet associated (HILPDA) gene, was overexpressed upon HIF induction and MYC inhibition, controlling LD formation in ccRCC cells. Hence, our study characterizes the molecular mechanism of LD accumulation in relation to hypoxia and MYC signaling, providing new understanding of metabolic adaption in ccRCC. Altogether, the data compiled in this thesis describes the important role of the MYC family of proteins in differentiation and metabolism of NB and in the metabolic reprogramming of ccRCC providing new knowledge and potential targets for development of novel therapeutic approaches

Inhibition of fatty acid synthesis induces differentiation and reduces tumor burden in childhood neuroblastoma

Many metabolic pathways, including lipid metabolism, are rewired in tumors tosupport energy and biomass production and to allow adaptation to stressful en-vironments. Neuroblastoma is the second deadliest solid tumor in children. Ge-netic aberrations, as the amplification of theMYCN-oncogene, correlate stronglywith disease progression. Yet, there are only a few molecular targets successfullyexploited in the clinic. Here we show that inhibition of fatty acid synthesis led toincreased neural differentiation and reduced tumor burden in neuroblastomaxenograft experiments independently ofMYCN-status. This was accompaniedby reduced levels of the MYCN or c-MYC oncoproteins and activation of ERKsignaling. Importantly, the expression levels of genes involved inde novofattyacid synthesis showed prognostic value for neuroblastoma patients. Our findingsdemonstrate that inhibition ofde novofatty acid synthesis is a promising pharma-cological intervention strategy for the treatment of neuroblastoma indepen-dently ofMYCN-status

Photophysical Characteristics of Polarity-Sensitive and Lipid Droplet-Specific Phenylbenzothiadiazoles

In this study, we present a series of solvatochromic phenylbenzothiadiazoles that display dual emission from the locally excited (LE) and intramolecular charge transfer (ICT) excited states. The donor-acceptor derivatives are highly sensitive to polarity changes, which can be monitored by differences in emission efficiency, spectroscopic shifts and variations of the LE/ICT ratio. One of the compounds in the series, containing a thiomethyl substituent, emerged as an excellent blue emitting stain for intracellular lipid droplets, a biomarker for various types of cancer. In addition, a non-emissive nitro derivative becomes fluorescent upon bioreduction in hypoxic cancer cells and accumulates in lipid droplets with a high signal-to-background ratio

Metabolic rewiring in MYC-driven medulloblastoma by BET-bromodomain inhibition

Medulloblastoma (MB) is the most common malignant brain tumour in children. High-risk MB patients harbouring MYC amplification or overexpression exhibit a very poor prognosis. Aberrant activation of MYC markedly reprograms cell metabolism to sustain tumorigenesis, yet how metabolism is dysregulated in MYC-driven MB is not well understood. Growing evidence unveiled the potential of BET-bromodomain inhibitors (BETis) as next generation agents for treating MYC-driven MB, but whether and how BETis may affect tumour cell metabolism to exert their anticancer activities remains unknown. In this study, we explore the metabolic features characterising MYC-driven MB and examine how these are altered by BET-bromodomain inhibition. To this end, we employed an NMR-based metabolomics approach applied to the MYC-driven MB D283 and D458 cell lines before and after the treatment with the BETi OTX-015. We found that OTX-015 triggers a metabolic shift in both cell lines resulting in increased levels of myo-inositol, glycerophosphocholine, UDP-N-acetylglucosamine, glycine, serine, pantothenate and phosphocholine. Moreover, we show that OTX-015 alters ascorbate and aldarate metabolism, inositol phosphate metabolism, phosphatidylinositol signalling system, glycerophospholipid metabolism, ether lipid metabolism, aminoacyl-tRNA biosynthesis, and glycine, serine and threonine metabolism pathways in both cell lines. These insights provide a metabolic characterisation of MYC-driven childhood MB cell lines, which could pave the way for the discovery of novel druggable pathways. Importantly, these findings will also contribute to understand the downstream effects of BETis on MYC-driven MB, potentially aiding the development of new therapeutic strategies to combat medulloblastoma

Targeting MYC induces lipid droplet accumulation by upregulation of HILPDA in clear cell renal cell carcinoma

Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Importantly, the concurrent inhibition of MYC signaling and glutamine metabolism prevented LD accumulation and reduced tumor burden in vivo. Furthermore, we identified the hypoxia-inducible lipid droplet–associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples postulated HILPDA as a specific ccRCC biomarker. Together, these results provide an attractive approach for development of alternative therapeutic interventions for the treatment of this type of renal cancer

Inhibition of fatty acid synthesis induces differentiation and reduces tumor burden in childhood neuroblastoma

Many metabolic pathways, including lipid metabolism, are rewired in tumors to support energy and biomass production and to allow adaptation to stressful environments. Neuroblastoma is the second deadliest solid tumor in children. Genetic aberrations, as the amplification of the MYCN-oncogene, correlate strongly with disease progression. Yet, there are only a few molecular targets successfully exploited in the clinic. Here we show that inhibition of fatty acid synthesis led to increased neural differentiation and reduced tumor burden in neuroblastoma xenograft experiments independently of MYCN-status. This was accompanied by reduced levels of the MYCN or c-MYC oncoproteins and activation of ERK signaling. Importantly, the expression levels of genes involved in de novo fatty acid synthesis showed prognostic value for neuroblastoma patients. Our findings demonstrate that inhibition of de novo fatty acid synthesis is a promising pharmacological intervention strategy for the treatment of neuroblastoma independently of MYCN-status