Myc Family Proteins Research Articles

Recent Progress in Modulation of WD40-Repeat Domain 5 Protein (WDR5): Inhibitors and Degraders.

WD40-repeat (WDR) domain proteins play a crucial role in mediating protein-protein interactions that sustain oncogenesis in human cancers. One prominent example is the interaction between the transcription factor MYC and its chromatin co-factor, WD40-repeat domain protein 5 (WDR5), which is essential for oncogenic processes. The MYC family of proteins is frequently overexpressed in various cancers and has been validated as a promising target for anticancer therapies. The recruitment of MYC to chromatin is facilitated by WDR5, highlighting the significance of their interaction. Consequently, inhibiting the MYC-WDR5 interaction has been shown to induce the regression of malignant tumors, offering an alternative approach to targeting MYC in the development of anticancer drugs. WDR5 has two protein interaction sites, the "WDR5-binding motif" (WBM) site for MYC interaction and the histone methyltransferases SET1 recognition motif "WDR5-interacting" (WIN) site forming MLL complex. Significant efforts have been dedicated to the discovery of inhibitors that target the WDR5 protein. More recently, the successful application of targeted protein degradation technology has enabled the removal of WDR5. This breakthrough has opened up new avenues for inhibiting the interaction between WDR5 and the binding partners. In this review, we address the recent progress made in targeting WDR5 to inhibit MDR5-MYC and MDR5-MLL1 interactions, including its targeted protein degradation and their potential impact on anticancer drug discovery.

MDB-24. A DATA-DRIVEN APPROACH FOR THE IDENTIFICATION OF NEW SYNERGISTIC DRUG COMBINATIONS AGAINST NEURAL CANCERS

Abstract High-risk medulloblastomas are tumours with a poor survival prognosis (≤ 50%), which are currently treated with radiation therapy and DNA-damaging chemotherapy. The severe side effects of these treatments, including developmental problems and secondary tumours, underscore the need for new treatments with reduced toxicity. An attractive target for such therapy is the Myc-family proteins since high-risk medulloblastomas amplify either N-Myc (SHH-MB, G4-MB) or c-Myc (G3-MB). While the proteins from the Myc family themselves are often referred to as pharmacologically intractable, their levels and activity can be modulated by indirect targeting, e.g. inhibition of proteins located upstream or downstream of c-Myc/N-Myc. The diverse cellular processes and pathways that c-Myc and N-Myc are involved in, however, suggest that several Myc pathways must be interrupted simultaneously to achieve a lasting effect. To identify such multimodal interventions against Myc, we performed a principal component analysis of RNA sequencing data from patient-derived medulloblastoma cells (MB002, G3, MYC-amplified) and DAOY cells (SHH-group, no MYC amplification) that were treated with 120 small molecules. In the subsequent analysis, we focused on a selection of ~ 40 genes that represent common upstream regulators and downstream targets of either c-Myc or N-Myc. Applying a principal component analysis to these particular markers, we expect that the drugs that affect different pathways associated with c-Myc activity and are located at the opposite ends of the PC1 and PC2 axis should have a synergistic effect. Additionally, we expected that the drugs located closer to each other, which affect closely related pathways, would either have no effect or would be antagonistic. Our PCA distribution method can be used to select synergistic combinations in a manner that is easily extended to new compounds. This approach may allow fast screening of compounds for estimation of the treatment effect.

The Myc Family and the Metastasis Suppressor NDRG1: Targeting Key Molecular Interactions with Innovative Therapeutics.

Cancer is a leading cause of death worldwide, resulting in ∼10 million deaths in 2020. Major oncogenic effectors are the Myc proto-oncogene family, which consists of three members including c-Myc, N-Myc, and L-Myc. As a pertinent example of the role of the Myc family in tumorigenesis, amplification of MYCN in childhood neuroblastoma strongly correlates with poor patient prognosis. Complexes between Myc oncoproteins and their partners such as hypoxia-inducible factor-1α and Myc-associated protein X (MAX) result in proliferation arrest and pro-proliferative effects, respectively. Interactions with other proteins are also important for N-Myc activity. For instance, the enhancer of zest homolog 2 (EZH2) binds directly to N-Myc to stabilize it by acting as a competitor against the ubiquitin ligase, SCFFBXW7, which prevents proteasomal degradation. Heat shock protein 90 may also be involved in N-Myc stabilization since it binds to EZH2 and prevents its degradation. N-Myc downstream-regulated gene 1 (NDRG1) is downregulated by N-Myc and participates in the regulation of cellular proliferation via associating with other proteins, such as glycogen synthase kinase-3β and low-density lipoprotein receptor-related protein 6. These molecular interactions provide a better understanding of the biologic roles of N-Myc and NDRG1, which can be potentially used as therapeutic targets. In addition to directly targeting these proteins, disrupting their key interactions may also be a promising strategy for anti-cancer drug development. This review examines the interactions between the Myc proteins and other molecules, with a special focus on the relationship between N-Myc and NDRG1 and possible therapeutic interventions. SIGNIFICANCE STATEMENT: Neuroblastoma is one of the most common childhood solid tumors, with a dismal five-year survival rate. This problem makes it imperative to discover new and more effective therapeutics. The molecular interactions between major oncogenic drivers of the Myc family and other key proteins; for example, the metastasis suppressor, NDRG1, may potentially be used as targets for anti-neuroblastoma drug development. In addition to directly targeting these proteins, disrupting their key molecular interactions may also be promising for drug discovery.

Abstract 3906: The mRNA translational plasticity of small cell lung carcinoma is associated with its phenotypic transdifferentiation

Abstract Small cell lung carcinoma(SCLC), classified as a recalcitrant cancer type, accounts for 13-15% of all lung cancers with a median overall survival less than 10 months in patients with extensive stage SCLC. SCLC was once considered as molecularly homogeneous due to nearly universal loss of TP53 and RB1, whereas subsets of MYC-driven, non-neuroendocrine (non-NE) SCLC variants, possess chemoresistance, suggesting its high plasticity and intertumoral heterogeneity related to rapid refractory to first-line chemotherapies. Current pathognomonic classification stratifies SCLC into two NE subtypes (ASCL1+, NEUROD1+) and two non-NE subtypes (POU2F3+ and triple negative), associated with different expression patterns of MYC family proteins. MYC is a master regulator of mRNA translation, whether translational regulation actively tunes SCLC plasticity and the associated chemosensitivity remains an enigma. Here, we delineate the translation landscape of NE and non-NE SCLC cells by using paired human NE SCLC cell line and constitutive or induced non-NE SCLC cells. As expected, non-NE SCLC cells are more resistant to the clinically relevant first-line chemotherapy than NE SCLC cells. With polysome profiling in combination with polysome RNA-seq, we showed differential expression at both transcription- and translation-level. However, transcription-level changes minimally overlapped with the translational changes between NE and non-NE SCLC cells, with a global reduction of translation activity observed in non-NE SCLC cells. Through analyzing the polysome profiles, we observed a striking accumulation of 60S monosomes and diminishment of 80S ribosomes derived from non-NE SCLC cells compared to those from NE SCLC cells. This unique translational pattern in non-NE SCLC cells was correlated with an increased expression of eIF6, a ‘sitting on the bench’ member of eukaryotic initiation factor (eIF) family. The eIF6 couples 60S monosome maturation and recycling, enrolling a quintessential role in functional 80S ribosome formation. Positive correlation between eIF6 expression and non-NE SCLC tumors was observed in 81 tumor samples derived from patients with limited stage SCLC and CCLE cell consortium, in which c-MYC expression was also positively correlated with eIF6 level. By using both ultracentrifugation and fraction analysis from polysome profiling, we found that less association of eIF6 with 60S may mitigate 80S ribosome assembly in non-NE SCLC cells. In addition, in vivo xenograft models showed that eIF6 expression was higher in residual tumors upon chemotherapy. Taken together, these data revealed an eIF6-dependent translational regulation in non-NE transdifferentiation of SCLC subtypes, suggesting an underappreciated function of translational regulation of SCLC plasticity and the potential therapeutic value of eIF6 for overcoming SCLC resistance to chemotherapy. Citation Format: Haoning Peng, Mengyao Wang, Lu Li, Lunxu Liu, Shensi Shen. The mRNA translational plasticity of small cell lung carcinoma is associated with its phenotypic transdifferentiation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3906.

Abstract 3536: Role of Myc family proteins in fusion-positive rhabdomyosarcoma

Abstract Fusion-positive rhabdomyosarcoma (FP RMS) is an aggressive pediatric malignancy characterized by the presence of a PAX3-FOXO1 (P3F) or PAX7-FOXO1 fusion gene. Previous RNA expression studies have shown that these FP RMS tumors also express high levels of MYCN and lower levels of MYC. Our recent studies of FP RMS tumors revealed an inverse relationship between MYCN and MYC RNA expression in FP RMS tumors. Western blot studies of FP RMS cell lines and patient-derived xenograft (PDX) tumors identified tumors with high level MYCN and MYC protein expression as well as tumors with high level protein expression of either MYC or MYCN. To model the interaction of P3F with Myc family proteins in FP RMS, we generated human Dbt myoblasts with doxycycline-inducible P3F (iP3F) with or without constitutively expressed MYCN. We previously observed that doxycycline-treated human Dbt myoblasts engineered with iP3F and constitutive MYCN expression (Dbt-MYCN-iP3F) form foci in vitro and rapidly form tumors in vivo, while doxycycline-treated Dbt myoblasts engineered with only iP3F expression (Dbt-iP3F) do not form foci in vitro and form tumors in vivo at a slower rate. Western blot and qRT-PCR analysis of the parental and tumor-derived (TD) Dbt-MYCN-iP3F lines revealed high MYCN and low MYC expression. In contrast, Dbt-iP3F parental lines express moderate levels of MYC and very low levels of MYCN whereas iP3F TD lines show increased MYC and MYCN expression, though this level of MYCN expression is considerably less than that seen in Dbt-MYCN-iP3F lines. Using CRISPR-Cas9 technology to knockdown MYCN or MYC, we found a primary dependence on MYCN in both Dbt-MYCN-iP3F parental and TD lines as indicated by loss of focus formation following MYCN knockdown while MYC knockdown does not interfere with oncogenicity. In contrast, oncogenicity in Dbt-iP3F TD lines is primarily dependent upon MYC whereas MYCN knockdown results in a modest and variable effect on oncogenicity. To elucidate the role of Myc proteins on the expression of P3F target genes, we measured expression of several transcriptional targets in these parental and TD lines by RNA sequencing and qRT-PCR. Several target genes (such as FGFR4) showed comparable upregulation by P3F in both parental and TD lines with or without constitutive MYCN expression. In contrast, a few target genes (such as FGF8) were stimulated by P3F at low levels in Dbt-iP3F parental lines and were stimulated at much higher levels in Dbt-iP3F TD lines as well as in both Dbt-MYCN-iP3F parental and TD lines. This finding indicates that some P3F target genes do not require high MYCN or MYC expression whereas other P3F target genes can only be maximally stimulated by P3F in the presence of high levels of MYCN and/or MYC. We postulate that the dependence of FP RMS on high level expression of a Myc family protein may be explained by the need to stimulate expression of one or more of the P3F target genes in this latter category. Citation Format: Bishwanath Chatterjee, Salah Boudjadi, Puspa Raj Pandey, Hana Kim, Wenyue Sun, Frederic G. Barr. Role of Myc family proteins in fusion-positive rhabdomyosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3536.

Abstract 4871: Dysregulation of MYC-family proteins sensitizes cancers to NMT inhibition: identification of NMTi sensitivity and mechanism

Abstract N-myristoyltransferases (NMTs) catalyze the protein N-terminal modification N-myristoylation, a lipidation event which affects >150 proteins and is involved in protein localization, stability, and function. NMT has been suggested as a target in cancers, but there has been a lack of rationale to identify patients who may respond to NMT inhibition. Additionally, the mechanism of action of NMT inhibitors (NMTi) is difficult to dissect, as NMT substrates feature in multiple biological pathways, and many studies have focused only on a single substrate. Here, a combination of bioinformatic, biochemical, proteomic, and cellular biology techniques have been combined to show that deregulation of MYC-family proteins sensitizes cells to NMT inhibition. Cell lines sensitive to NMTi were identified through screening of hundreds of cancer cell lines, and the transcriptome of sensitive and insensitive lines compared to obtain a “Sensitive to NMTi” gene set. This gene set correlated well with MYC-related gene sets and mutation, amplification, or chromosomal rearrangement in MYC(N) were predictive for NMTi sensitivity, suggesting that MYC deregulated cancer cells are sensitive to NMTi. This was verified by cytotoxicity assays in B-cell lymphoma and neuroblastoma lines, where highly MYC(N) expressing lines exhibited increased NMTi sensitivity, including in the SHEP21N and P493-6 lines, in which MYC(N) levels can be regulated. Furthermore, proteomic profiling identified multiple pathways which are affected by NMTi, and these were further validated. In particular, an impact on Complex I formation was seen, and mitochondrial dysfunction in high MYC(N) cells upon NMTi was seen through the loss of basal and maximal respiration, ATP production and spare respiratory capacity. Loss of Complex I formation was shown to be caused by the loss of the NMT substrate NDUFAF4 upon NMTi treatment. Furthermore, application of an orally bioavailable NMTi eliminated tumors in mouse models of both diffuse large B-cell lymphoma and neuroblastoma and were well tolerated with no changed in body weight, suggesting that NMT inhibitors are well tolerated and efficacious in vivo. Citation Format: James Zhang, Gregor A. Lueg, Monica Faronato, Evon Poon, Andrii Gorelik, Andrea G. Grocin, Eva Caamano-Gutierrez, Francesco Falciani, Roberto Solari, Robin Carr, Andrew S. Bell, Edward Bartlett, Jennie Hutton, Miriam Llorian-Sopena, Probir Chakravarty, Bernadette Brzezicha, Martin Janz, Matther J. Garnett, Louis Chesler, Dinis P. Calado, Edward W. Tate. Dysregulation of MYC-family proteins sensitizes cancers to NMT inhibition: identification of NMTi sensitivity and mechanism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4871.

Clinical significance of MYC family protein expression in surgically resected high-grade neuroendocrine carcinoma of the lung.

MYC family genes including MYC, MYCN, and MYCL are amplified and overexpressed as oncogenic drivers in high-grade neuroendocrine carcinoma of the lung (HGNEC), but little is known about their clinical significance. This study evaluated the prognostic impact of MYC family protein expression in patients with surgically resected HGNEC. Immunohistochemical analyses were performed on 83 resected specimens of HGNEC using antibodies against MYC family proteins (c-MYC, n-MYC, and l-MYC). When nuclear staining of any intensity in ≥10% of tumor cells showed immunoreactivity with any one or more of c-MYC, n-MYC, or l-MYC, the specimens were defined as MYC family-positive. A total of 83 patients were analyzed. MYC family-positive status was observed in 33.7% (28 of 83 cases) and was not correlated with clinicopathological factors. The protein expression was mutually exclusive and no duplicate cases were observed. A log-rank test showed that MYC family-positive status was significantly associated with shorter overall survival (OS) (p=0.003) and recurrence-free survival (RFS) (p=0.039). According to Cox multivariate analysis, MYC family-positive status had a significant effect on shorter OS (hazard ratio [HR]=2.217, 95% confidence interval [CI] 1.179-4.169, p=0.014) and RFS (HR=1.802, 95% CI 1.014-3.202, p=0.045). In patients with pathological stage I, MYC family-positive status also showed significantly poor OS (HR=2.847, 95% CI 1.236-6.557, p=0.014) and RFS (HR=2.088, 95% CI 1.006-4.332, p=0.048) in the multivariate analysis. MYC family protein expression could be an independent unfavorable prognostic factor in patients with surgically resected HGNEC.

Recent advances in small cell lung cancer: the future is now?

Small cell lung cancer is a relevant clinical issue as it is a highly malignant cancer, often diagnosed in advanced stage. Similarly to non-small cell lung cancer, tobacco smoking is currently the main risk factor. Its incidence, at least in males, has declined over the past decades, due to the worldwide decreased percentage of active smokers. The typical small cells of this tumor type are characterized by a high Proliferation Index, chromosomal deletions such as 3p(14-23) involving the tumor-suppressor gene FHIT, alterations of the MYC or Notch family proteins and the frequent expression of neuroendocrine markers. The combination of thoracic radiotherapy and chemotherapy is the standard treatment for limited stage disease, while platinum-based chemotherapy is the most effective choice for extensive stage disease. Unfortunately, whatever chemotherapy is used, the results are disappointing. No regimen has proved to be effective in the long run, indeed small cell lung cancer rapidly progresses after a frequent initial strong response, and the mortality rate remains still high. The advent of immunotherapy is actually changing the landscape in oncology. As well as in other cancers, recent trials have demonstrated the efficacy of the combination of immune checkpoint inhibitors and chemotherapy, opening new perspectives for the future of our patients.

Abstract 5731: Structure-based development of a novel MYC inhibitor for neuroendocrine prostate cancer

Abstract Background: MYC oncoproteins are important drivers of human cancers. More specifically, NMYC is amplified and overexpressed in neuroendocrine prostate cancer (NEPC). NMYC elicits its oncogenic effects by forming a heterodimer with MAX. This complex binds to regulating elements and activates the transcription of MYC target genes that play roles in tumor growth and progression. It is well known that members of the MYC family of proteins make compelling targets for cancer treatments. However, to this date, no tangible drug candidates have been developed into the clinics for MYC. In previous studies utilizing a rational computer-aided drug discovery (CADD) approach, we identified VPC-70551 as our most active hit. Subsequent optimization with the VPC-70551 scaffold has led to the identification of a new series of compounds. Using in silico drug screening followed by functional validation, we identified a small molecule inhibitor, VPC-70619, that exhibits higher microsomal stability and is better absorbed and tolerated orally than most MYC inhibitors described in the literature. Method: We used a transcriptional reporter assay to determine the effect of VPC-70619 on MYC-mediated transcription. To validate on-target effect, we evaluated the effect of VPC-70619 treatment on MYC-positive (LASCPC-01, NCIH660, LNCaP N-MYC, 22Rv1 N-MYC) and MYC-negative (HO15.19) cancer cells. We also evaluated the effect of VPC-70619 on MYC/MAX interaction by PLA and on DNA binding by BLI. The direct binding between recombinant MYC/MAX protein complex and VPC-70619 was evaluated by MST. Results: VPC-70619 inhibited MYC transcriptional activity in dose dependent manner and the proliferation of MYC-positive cell lines. VPC-70619 did not interfere with MYC/MAX interaction however it blocked the complex interaction with DNA. We confirmed the direct binding of VPC-70619 to the purified MYC/MAX complex by using MST. Conclusion: This project presents the identification of a new MYC inhibitor that blocks the transcriptional activity of this oncogene and elucidates the molecular mechanism of action of this inhibitor. Our findings help prelude the development and discovery of more effective treatments for NEPC patients. Citation Format: Jane Foo, Anh-Tien Ton, Kriti Singh, Fuqiang Ban, Helene Morin, Joseph Lee, Eric LeBlanc, Nada Lallous, Artem Cherkasov. Structure-based development of a novel MYC inhibitor for neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5731.

Bromodomain Inhibitor JQ1 Provides Novel Insights and Perspectives in Rhabdomyosarcoma Treatment.

Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. It is classified into two main subtypes: embryonal (eRMS) and alveolar (aRMS). MYC family proteins are frequently highly expressed in RMS tumors, with the highest levels correlated with poor prognosis. A pharmacological approach to inhibit MYC in cancer cells is represented by Bromodomain and Extra-Terminal motif (BET) protein inhibitors. In this paper, we evaluated the effects of BET inhibitor (+)-JQ1 (JQ1) on the viability of aRMS and eRMS cells. Interestingly, we found that the drug sensitivity of RMS cell lines to JQ1 was directly proportional to the expression of MYC. JQ1 induces G1 arrest in cells with the highest steady-state levels of MYC, whereas apoptosis is associated with MYC downregulation. These findings suggest BET inhibition as an effective strategy for the treatment of RMS alone or in combination with other drugs.

EZH2 depletion potentiates MYC degradation inhibiting neuroblastoma and small cell carcinoma tumor formation

Efforts to therapeutically target EZH2 have generally focused on inhibition of its methyltransferase activity, although it remains less clear whether this is the central mechanism whereby EZH2 promotes cancer. In the current study, we show that EZH2 directly interacts with both MYC family oncoproteins, MYC and MYCN, and promotes their stabilization in a methyltransferase-independent manner. By competing against the SCFFBW7 ubiquitin ligase to bind MYC and MYCN, EZH2 counteracts FBW7-mediated MYC(N) polyubiquitination and proteasomal degradation. Depletion, but not enzymatic inhibition, of EZH2 induces robust MYC(N) degradation and inhibits tumor cell growth in MYC(N) driven neuroblastoma and small cell lung carcinoma. Here, we demonstrate the MYC family proteins as global EZH2 oncogenic effectors and EZH2 pharmacologic degraders as potential MYC(N) targeted cancer therapeutics, pointing out that MYC(N) driven cancers may develop inherent resistance to the canonical EZH2 enzymatic inhibitors currently in clinical development.

Genetic and Histopathological Heterogeneity of Neuroblastoma and Precision Therapeutic Approaches for Extremely Unfavorable Histology Subgroups.

Peripheral neuroblastic tumors (neuroblastoma, ganglioneuroblastoma and ganglioneuroma) are heterogeneous and their diverse and wide range of clinical behaviors (spontaneous regression, tumor maturation and aggressive progression) are closely associated with genetic/molecular properties of the individual tumors. The International Neuroblastoma Pathology Classification, a biologically relevant and prognostically significant morphology classification distinguishing the favorable histology (FH) and unfavorable histology (UH) groups in this disease, predicts survival probabilities of the patients with the highest hazard ratio. The recent advance of neuroblastoma research with precision medicine approaches demonstrates that tumors in the UH group are also heterogeneous and four distinct subgroups—MYC, TERT, ALT and null—are identified. Among them, the first three subgroups are collectively named extremely unfavorable histology (EUH) tumors because of their highly aggressive clinical behavior. As indicated by their names, these EUH tumors are individually defined by their potential targets detected molecularly and immunohistochemically, such as MYC-family protein overexpression, TERT overexpression and ATRX (or DAXX) loss. In the latter half on this paper, the current status of therapeutic targeting of these EUH tumors is discussed for the future development of effective treatments of the patients.

The regulatory function of tandem repeat VNTR2-1 in hTERT gene involves basic Helix-loop-helix family transcription factors.

Telomerase is a ribonucleoprotein enzyme that synthesizes telomere end sequence. The expression of hTERT gene, encoding the catalytic subunit of human telomerase, is restricted to highly proliferative tissues and is undetectable in most somatic cells. Abnormal activation of hTERT gene is found in 90% of human tumors. Previously, we identified tandem repeat of 42-bp/unit, VNTR2-1, in intron 2 of the hTERT gene, as a novel regulatory element important for hTERT transcription in cancer cells. In the current study, we found that multiple 42-bp repeats of VNTR2-1 activated luciferase gene in reporter plasmids. Mutation of the predicted cis-regulatory elements within the 42-bp repeats, including a E-box motif, resulted in a partial or complete loss of its enhancer activity. Moreover, MYC family proteins, c-MYC, MAX, and MNT, regulated hTERT gene transcription through both VNTR2-1 and E-boxes at the proximal hTERT promoter. Chromatin segmentation analysis of published ChIP-sequencing data from K562 cells indicated that VNTR2-1 was a bivalent enhancer. In telomerase-expressing human melanoma cell line MelJuSo, deletion of VNTR2-1 caused the hTERT promoter chromatin status to change from an active state to a repressed state, accompanied by increases of H3K27me3 and H3K9me3 marks. Therefore, we provided additional evidence for VNTR2-1 as a functional regulatory element that regulated hTERT expression by MYC family transcription factors. These results have improved our knowledge on the functions of repetitive genomic DNAs and the regulatory mechanisms of human telomerase gene.

Drugging the "Undruggable" MYCN Oncogenic Transcription Factor: Overcoming Previous Obstacles to Impact Childhood Cancers.

Effective treatment of pediatric solid tumors has been hampered by the predominance of currently "undruggable" driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance in vivo, the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein-protein or protein-DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this "undruggable" transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.

Reciprocal Induction of MDM2 and MYCN in Neural and Neuroendocrine Cancers.

MYC family oncoproteins MYC, MYCN, and MYCL are deregulated in diverse cancers and via diverse mechanisms. Recent studies established a novel form of MYCN regulation in MYCN-overexpressing retinoblastoma and neuroblastoma cells in which the MDM2 oncoprotein promotes MYCN translation and MYCN-dependent proliferation via a p53-independent mechanism. However, it is unclear if MDM2 also promotes expression of other MYC family members and has similar effects in other cancers. Conversely, MYCN has been shown to induce MDM2 expression in neuroblastoma cells, yet it is unclear if MYC shares this ability, if MYC family proteins upregulate MDM2 in other malignancies, and if this regulation occurs during tumorigenesis as well as in cancer cell lines. Here, we report that intrinsically high MDM2 expression is required for high-level expression of MYCN, but not for expression of MYC, in retinoblastoma, neuroblastoma, small cell lung cancer, and medulloblastoma cells. Conversely, ectopic overexpression of MYC as well as MYCN induced high-level MDM2 expression and gave rise to rapidly proliferating and MDM2-dependent cone-precursor-derived masses in a cultured retinoblastoma genesis model. These findings reveal a highly specific collaboration between the MDM2 and MYCN oncoproteins and demonstrate the origin of their oncogenic positive feedback circuit within a normal neuronal tissue.

Stage 4S Neuroblastoma: Molecular, Histologic, and Immunohistochemical Characteristics and Presence of 2 Distinct Patterns of MYCN Protein Overexpression-A Report From the Children's Oncology Group.

Stage 4S neuroblastoma (4SNB) is associated with spontaneous tumor regression and an excellent prognosis. However, a small group of the patients have a poor prognosis. One hundred eighty-five stage 4SNB cases filed at the Children's Oncology Group Neuroblastoma Pathology Reference Laboratory were studied. MYCN oncogene status [non-amplified (NA) vs. Amplified (A)] determined by fluorescence in situ hybridization, MYC-family (MYCN/MYC) protein expression [no-overexpression(-)/(+/-) vs. overexpression(+)] by immunohistochemistry and histopathology by International Neuroblastoma Pathology Classification [Favorable Histology (FH) vs. Unfavorable Histology (UH)] with particular attention to nucleolar hypertrophy [NH(-) vs. (+)] were assessed with patient survival. One hundred forty-seven (79.5%) tumors were MYCN-NA, FH, MYC-family protein(-)/(+/-), and NH(-) with a good prognosis [88.5±3.1% 3-y event-free survival (EFS); 94.1±2.3% 3-y overall survival (OS)]. Among MYCN-NA tumors, 11 demonstrated MYCN protein(+) with a moderate and uniform (M/U) staining pattern: they were FH(10/11), NH(-), 1 showed MYC protein(+) simultaneously, and all patients are alive. Also found were 5 MYC protein(+) and MYCN(-)/(+/-) tumors; they were FH without NH (4/5), and all patients are alive. Among MYCN-A tumors, 18 had MYCN protein(+) with a strong and heterogeneous (S/H) staining pattern, 9 had UH (44.4±23.4% EFS/OS) and 9 had FH (68.6±19.2% EFS/OS), and 15 showed NH(+). Two tumors had MYCN protein(-)/(+/-) despite MYCN-A; both were FH and NH(-), and 1 patient died. S/H staining pattern of MYCN protein overexpression by immunohistochemistry was associated with MYCN amplification, NH(+) and a poor prognosis. In contrast, the M/U staining pattern was associated with MYCN nonamplification and NH(-), and had no adverse prognostic effects for the stage 4SNB patients.

Recent advances in small cell lung cancer: the future is now?

Small cell lung cancer is a relevant clinical issue as it is a highly malignant cancer, often diagnosed in advanced stage. Similarly to non-small cell lung cancer, tobacco smoking is currently the main risk factor. Its incidence, at least in males, has declined over the past decades, due to the worldwide decreased percentage of active smokers. The typical small cells of this tumor type are characterized by a high proliferation index, chromosomal deletions such as 3p(14-23) involving the tumor-suppressor gene FHIT, alterations of the MYC or Notch family proteins and the frequent expression of neuroendocrine markers. The combination of thoracic radiotherapy and chemotherapy is the standard treatment for limited stage disease, while platinum-based chemotherapy is the most effective choice for extensive stage disease. Unfortunately, whatever chemotherapy is used, the results are disappointing. No regimen has proved to be effective in the long run, indeed small cell lung cancer rapidly progresses after a frequent initial strong response, and the mortality rate remains still high. The advent of immunotherapy is actually changing the landscape in oncology. As well as in other cancers, recent trials have demonstrated the efficacy of the combination of immune checkpoint inhibitors and chemotherapy, opening new perspectives for the future of our patients.

The MDM2 inhibitor CGM097 combined with the BET inhibitor OTX015 induces cell death and inhibits tumor growth in models of neuroblastoma.

BackgroundNeuroblastoma (NB) is the most common extracranial solid tumor in infants and children, with amplification of the oncogene MYCN being a hallmark of high‐risk disease and poor prognosis. Although less frequent, overexpression of MYC is similarly an indicator of poor prognosis. Most NB tumors initially respond to chemotherapy, however, most will relapse, resulting in chemoresistant disease. After relapse, there is growing evidence of p53 inactivation. MYC/MYCN and MDM2 have been shown to interact and contribute to NB growth and disease progression. MDM2 inhibitors and Bromodomain and Extra‐Terminal domain (BET) inhibitors have both shown promise in treating NB by increasing the expression of p53 and decreasing MYC/MYCN expression, respectively. Our study focuses on the combined treatment of a MDM2 inhibitor (CGM097) with a BET inhibitor (OTX015) in neuroblastoma.MethodsTwo p53 wild‐type and two p53 mutant established neuroblastoma cells lines were used to test this combination. Ray design assays were used to test whether this combination was synergistically cytotoxic to NB cells. Western blots were performed to check signaling pathways of interest after drug treatment. IncuCyte imaging and flow cytometry were utilized to quantify the apoptotic and cytostatic effects of these drugs on NB cells. In vivo studies were carried out to test the antitumor effect of this combination in a living host.ResultsThe combination of CGM097 and OTX015 resulted in p53 activation, decreased expression of MYC family proteins and a subsequent synergistic increase in NB cell death.ConclusionThis study warrants further investigation into the combination of MDM2 inhibitors and BET inhibitors for the treatment in NB.

Abstract 1831: Mistletoe lectin induces apoptosis in MYC-driven cancers through post-translational downregulation of c-MYC protein

Abstract c-Myc is part of the Myc family proteins associated with cellular proliferation, differentiation, and apoptosis. Dysregulated c-Myc expression is associated with tumorigenesis, and cancers with c-Myc overexpression are often associated with poor prognosis, partly due to difficulty in targeting c-Myc. Mistletoe lectin (ML), derived from the European mistletoe plant, has shown variable efficacy in cancer patients. One case series following 12 patients with advanced hepatocellular carcinoma (HCC) showed 2 patients with &gt30% reduction in the tumor marker alpha-fetoprotein and 1 patient who had stable disease for 9 months. These cases indicate promising results. ML consists of a heteroprotein complex of an A- and B-chain connected by a disulfide bond. The A-chain was previously described to inhibit translation by 28s rRNA cleavage, while the B-chain binds cell surface glycoproteins to trigger internalization of the lectin. We found that purified mistletoe extract, consisting primarily of ML, downregulates c-Myc protein and induces apoptosis in in vitro and in vivo models of human-derived cancer, and that the efficacy of ML treatment is strongly correlated with MYC gene expression and c-Myc protein abundance in in vitro human models of HCC, ovarian cancer, small cell lung cancer, and lymphoma. MTS assay data demonstrated that exogenous MYC expression can sensitize low-MYC cell lines to ML treatment, suggesting that anti-cancer effects of ML are dependent on its ability to downregulate c-Myc. While ML may be able to downregulate many proteins via general translational inhibition, we found that of 296 proteins profiled by RPPA, c-Myc was one of 11 proteins decreased by ML with at least a 2-fold change (p&lt0.01). We also found that ML promotes c-Myc ubiquitylation, suggesting that ML regulates c-Myc protein stability, rather than synthesis. In summary, here we describe a novel mechanism of action for ML in downregulating c-Myc and promoting apoptotic cell death in various models of MYC-driven cancer, and provide evidence for MYC gene expression as a predictive marker for response. This research supports the development of ML-based therapies that may provide a c-Myc-targeting treatment in multiple cancer types. Citation Format: Gabrielle Kennelley, Eric Yuan, Mohammad Shatat, Betsy Gauthier, Peiying Yang, Analisa Difeo, Richard Lee. Mistletoe lectin induces apoptosis in MYC-driven cancers through post-translational downregulation of c-MYC protein [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1831.

MAX Functions as a Tumor Suppressor and Rewires Metabolism in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a highly aggressive and lethal neoplasm. To identify candidate tumor suppressors we applied CRISPR/Cas9 gene inactivation screens to a cellular model of early-stage SCLC. Among the top hits was MAX, the obligate heterodimerization partner for MYC family proteins that is mutated in human SCLC. Max deletion increases growth and transformation in cells and dramatically accelerates SCLC progression in an Rb1/Trp53-deleted mouse model. In contrast, deletion of Max abrogates tumorigenesis in MYCL-overexpressing SCLC. Max deletion in SCLC resulted in derepression of metabolic genes involved in serine and one-carbon metabolism. By increasing serine biosynthesis, Max-deleted cells exhibit resistance to serine depletion. Thus, Max loss results in metabolic rewiring and context-specific tumor suppression.