Bringing order to disorder : structural and functional analysis for development of strategies to target myc
Abstract: Several cellular processes such as cell cycle progression, apoptosis, cell adhesion and motility, cell growth, angiogenesis and differentiation are regulated by the MYC (here MYCN and c-MYC) proto-oncogenes. Elevated expression of MYC is present in a wide range of human tumors including Burkitt´s Lymphoma, neuroblastoma, medulloblastoma, colon cancer and glioblastoma. In cancer, high MYC expression levels have been correlated with fast tumor growth and poor prognosis. Neuroblastoma, a heterogeneous childhood cancer has a broad clinical spectrum ranging from spontaneously regressing tumors to those with fast progression and an unfavorable outcome. One of the markers for poor survival is MYCN-amplification. Importantly, down-regulation of MYCN results in differentiation. While better treatment options has increased the overall survival for low-risk tumors, the survival of children with high-risk tumors has not improved and new therapeutic strategies such as targeting MYC are needed. Currently, there are no drugs directly targeting MYC available in the clinic. In paper I we demonstrated that several known c-MYC inhibitors also bind directly to MYCN and prevented MYCN/MAX interaction. We found that the affinities of the molecules correlated with growth inhibition of neuroblastoma cells. We further showed that the compounds lead to increased apoptosis in MYCN-amplified cells compared to non-amplified cells. In addition, treatment resulted in differentiation and lipid accumulation processes previously correlated to down-regulation of MYCN. These data show the proof-of-principle of directly targeting MYC. However, there is a great need for more potent and specific molecules targeting MYC. In paper II we have screened for new molecules targeting both MYCN and c-MYC. Using a cell-based assay we identified six compounds that target both MYC proteins. The small molecule #2.7 was chosen for further characterization due to its ability to directly targeting MYC. Treatment of MYCN-amplified and MYCN-non-amplified neuroblastoma cells with compound #2.7 lead to decreased proliferation in a MYCN-dependent manner and to the inhibition of transformation by RAS and MYCN in vitro. Collectively, our results in paper I and II support the idea that direct targeting of MYC is a new strategy for cancer treatment. In paper III we demonstrated that MYCN upregulates the miR-17~92 cluster in neuroblastoma. Importantly, we showed that two members of the cluster miR-18a and miR-19a bind to and down-regulate the ESR1 gene encoding for the estrogen receptor-α (ER-α) thereby inhibiting differentiation of neuroblastoma cells. In contrast, ERoverexpression lead to neural differentiation. We further demonstrated ESR1 expression during normal development of the human sympathetic nervous system and showed a correlation between low ESR1 levels and poor survival. Based on these results, we suggest that targeting MYCN regulated miRNAs could be another strategy to treat MYCN-amplified neuroblastoma. The transactivation domain (TAD) of c-MYC is a highly flexible region, which is necessary for all of MYC’s functions. In order to gain more information about the c-MYC-TAD we have performed structural studies in paper IV. Circular Dichroism confirmed the presence of little secondary structure (about 9% α-helical and 30% β-sheet) in the mainly disordered (61%) c-MYC-TAD. Small-angle light scattering analysis suggested that the overall structure is most probably that of an elliptical cylinder. Our results show that small-angle light scattering together with NMR and CD might be used in future studies to gain even more insight in the structure of the c-MYC-TAD. Taken together, two approaches to target MYCN-amplified neuroblastoma can be suggested based on the data presented in this thesis. The MYCN protein could either be targeted directly with small molecules or indirectly by using anti-miRNAs. We have identified several novel small molecules that target MYC that will be characterized and may have potential for further development. Importantly, we have also aimed to increase the knowledge concerning the structure of the c-MYC transactivation domain. This new information may contribute to the development of future tailored and selective targeting of MYC and its interaction partners and may also give further insight to MYC function.
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