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

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.

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.

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.

The cooperating mutation or "second hit" determines the immunologic visibility toward MYC-induced murine lymphomas.

In Eµ-myc transgenic animals lymphoma formation requires additional genetic alterations, which frequently comprise loss of p53 or overexpression of BCL-2. We describe that the nature of the "second hit" affects the ability of the immune system to contain lymphoma development. Tumors with disrupted p53 signaling killed the host more rapidly than BCL-2 overexpressing ones. Relaxing immunologic control, using Tyk2(-/-) mice or by Ab-mediated depletion of CD8(+) T or natural killer (NK) cells accelerated formation of BCL-2-overexpressing lymphomas but not of those lacking p53. Most strikingly, enforced expression of BCL-2 prolonged disease latency in the absence of p53, whereas blocking p53 function in BCL-2-overexpressing tumors failed to accelerate disease. This shows that blocking apoptosis in p53-deficient cells by enforcing BCL-2 expression can mitigate disease progression increasing the "immunologic visibility." In vitro cytotoxicity assays confirmed that high expression of BCL-2 protein facilitates NK and T cell-mediated killing. Moreover, we found that high BCL-2 expression is accompanied by significantly increased levels of the NKG2D ligand MULT1, which may account for the enhanced killing. Our findings provide first evidence that the nature of the second hit affects tumor immunosurveillance in c-MYC-driven lymphomas and define a potential shortcoming of antitumor therapies targeting BCL-2.

The MYCN oncogene and differentiation in neuroblastoma.

Childhood neuroblastoma exhibits a heterogeneous clinical behavior ranging from low-risk tumors with the ability to spontaneously differentiate and regress, to high-risk tumors causing the highest number of cancer related deaths in infants. Amplification of the MYCN oncogene is one of the few prediction markers for adverse outcome. This gene encodes the MYCN transcriptional regulator predominantly expressed in the developing peripheral neural crest. MYCN is vital for proliferation, migration and stem cell homeostasis while decreased levels are associated with terminal neuronal differentiation. Interestingly, high-risk tumors without MYCN amplification frequently display increased c-MYC expression and/or activation of MYC signaling pathways. On the other hand, downregulation of MYCN leads to decreased proliferation and differentiation, emphasizing the importance of MYC signaling in neuroblastoma biology. Furthermore, expression of the neurotrophin receptor TrkA is associated with good prognosis, the ability to differentiate and spontaneous regression while expression of the related TrkB receptor is correlated with bad prognosis and MYCN amplification. Here we discuss the role of MYCN in neuroblastoma with a special focus on the contribution of elevated MYCN signaling for an aggressive and undifferentiated phenotype as well as the potential of using MYCN as a therapeutic target.

Deciphering the molecular events necessary for synergistic tumor cell apoptosis mediated by the histone deacetylase inhibitor vorinostat and the BH3 mimetic ABT-737.

The concept of personalized anticancer therapy is based on the use of targeted therapeutics through in-depth knowledge of the molecular mechanisms of action of these agents when used alone and in combination. We have identified the apoptotic proteins and pathways necessary for synergistic tumor cell apoptosis and in vivo antitumor responses seen when the HDAC inhibitor vorinostat is combined with the BH3-mimetic ABT-737 in lymphomas overexpressing Bcl-2. Vorinostat "primes" tumors overexpressing Bcl-2 for rapid ABT-737-mediated apoptosis by inducing expression of the BH3-only gene bmf. Moreover, these synergistic effects of vorinostat/ABT-737 were blunted in cells with an inactive p53 pathway or in cells lacking expression of the p53 target gene, noxa. These studies show the important and complex functional interaction between specific proapoptotic BH3-only proteins and the BH3-mimetic compound ABT-737 and provide the most comprehensive functional link between tumor genotype and the apoptotic and therapeutic effects of HDACi combined with ABT-737.

Targeting MYC-Regulated miRNAs to Combat Cancer.

The MYC protein controls many cellular processes, including proliferation, cell cycle progression, cell growth, metabolism, angiogenesis, differentiation, cell adhesion, and motility. This is primarily achieved through transcriptional regulation of large gene networks that ultimately results in activation or repression of target genes. Given its broad regulatory scope, the expression of the MYC gene itself needs to be tightly controlled. Deregulation of MYC expression promotes tumorigenesis and, not surprisingly, MYC is frequently activated in many different human cancers. Furthermore, these tumors become highly dependent on sustained MYC expression, while MYC inactivation results in desirable anticancer effects, such as cell death, differentiation, and/or senescence. Thus, MYC has emerged as an attractive target for cancer therapy. In addition to regulating protein-coding genes, MYC also governs the expression of microRNAs, many of which have important regulatory roles in cancer development and progression. Here we will discuss how MYC-regulated miRNAs could be exploited for therapeutic development for cancer.

Suppression of B-cell lymphomagenesis by the BH3-only proteins Bmf and Bad.

Oncogenic c-Myc is known to balance excessive proliferation by apoptosis that can be triggered by p53-dependent and p53-independent signaling networks. Here, we provide evidence that the BH3-only proapoptotic Bcl-2 family members Bcl-2 modifying factor (Bmf) and Bcl-2 antagonist of cell death (Bad) are potent antagonists of c-Myc-driven B-cell lymphomagenesis. Tumor formation was preceded by the accumulation of preneoplastic pre-B and immature immunoglobulin M-positive (IgM(+)) B cells in hematopoietic organs of Emu-myc/bmf(-/-) mice, whereas Emu-myc/bad(-/-) mice showed an increase of pre-B cells limited to the spleen. Although the loss of Bad had no impact on the tumor immunophenotype, Bmf deficiency favored the development of IgM(+) B cell over pre-B cell tumors. This phenomenon was caused by a strong protection of immature IgM(+) B cells from oncogene-driven apoptosis caused by loss of bmf and c-Myc-induced repression of Bmf expression in premalignant pre-B cells. Steady-state levels of B-cell apoptosis also were reduced in the absence of Bad, in support of its role as a sentinel for trophic factor-deprivation. Loss of Bmf reduced the pressure to inactivate p53, whereas Bad deficiency did not, identifying Bmf as a novel component of the p53-independent tumor suppressor pathway triggered by c-Myc.

Bcl2 family proteins in carcinogenesis and the treatment of cancer.

Deregulation of Bcl2 family members is a frequent feature of human malignant diseases and causal for therapy resistance. A number of studies have recently shed light onto the role of pro- and anti-apoptotic Bcl2 family members in tumour-pathogenesis and in mediating the effects of classical as well as novel front-line anticancer agents, allowing the development of more efficient and more precisely targeted treatment regimens. Most excitingly, recent progress in our understanding of how Bcl2-like proteins maintain or perturb mitochondrial integrity has finally enabled the development of rational-design based anticancer therapies that directly target Bcl2 regulated events at the level of mitochondria. This review aims to give an overview on the most recent findings on the role of the Bcl2 family in tumour development in model systems of cancer, to relate these findings with observations made in human pathologies and drug-action.

Loss of the BH3-only protein Bmf impairs B cell homeostasis and accelerates gamma irradiation-induced thymic lymphoma development.

Members of the Bcl-2 protein family play crucial roles in the maintenance of tissue homeostasis by regulating apoptosis in response to developmental cues or exogenous stress. Proapoptotic BH3-only members of the Bcl-2 family are essential for initiation of cell death, and they function by activating the proapoptotic Bcl-2 family members Bax and/or Bak, either directly or indirectly through binding to prosurvival Bcl-2 family members. Bax and Bak then elicit the downstream events in apoptosis signaling. Mammals have at least eight BH3-only proteins and they are activated in a stimulus-specific, as well as a cell type-specific, manner. We have generated mice lacking the BH3-only protein Bcl-2-modifying factor (Bmf) to investigate its role in cell death signaling. Our studies reveal that Bmf is dispensable for embryonic development and certain forms of stress-induced apoptosis, including loss of cell attachment (anoikis) or UV irradiation. Remarkably, loss of Bmf protected lymphocytes against apoptosis induced by glucocorticoids or histone deacetylase inhibition. Moreover, bmf(-/-) mice develop a B cell-restricted lymphadenopathy caused by the abnormal resistance of these cells to a range of apoptotic stimuli. Finally, Bmf-deficiency accelerated the development of gamma irradiation-induced thymic lymphomas. Our results demonstrate that Bmf plays a critical role in apoptosis signaling and can function as a tumor suppressor.