MYC-dependent Manner Research Articles

Functional screen for mediators of onco-mRNA translation specificity.

Oncogenic protein dosage is tightly regulated to enable cancer cells to adapt and survive. Whether this is regulated at the level of translational control and the key factors in cis and trans remain unknown. The Myc oncogene is a central paradigm of an exquisitely regulated oncogene and a major driver of pancreatic ductal adenocarcinoma (PDAC). Using a functional genome-wide CRISPRi screen in PDAC cells, we identified activators of selective MYC translation through its 5' untranslated region (5'UTR) and validated four RNA binding proteins (RBPs), including epitranscriptome modifiers. Among these RBPs, our top hit was RBM42, which is highly expressed in PDAC and predicts poor survival. Combining polysome sequencing and CLIP-seq analyses, we find that RBM42 binds and selectively regulates the translation of MYC and a precise, yet vital suite of pro-oncogenic transcripts, including JUN and EGFR . Mechanistically, employing IP-mass spectrometry analysis, we find that RMB42 is a novel ribosome-associated protein (RAP). Using DMS-Seq and mutagenesis analysis, we show that RBM42 directly binds and remodels the MYC 5'UTR RNA structure, facilitating the formation of the translation pre-initiation complex. Importantly, RBM42 is necessary for human PDAC cell growth and fitness and PDAC tumorigenesis in xenograft mouse models in a Myc-dependent manner in vivo . In PDAC patient samples, RBM42 expression is correlated with Myc protein levels and transcriptional activity. This work transforms our understanding of the translational code in cancer and offers a new therapeutic opening to target the expression of oncogenes.

Abstract A078: Functional screen for mediators of onco-mRNA translation specificity in pancreatic cancer

Abstract Oncogenic protein dosage is tightly regulated to enable cancer cells to adapt and survive. In healthy cells, translation initiation of onco-mRNA transcripts encoding oncogenes and growth factors is tightly regulated to maintain accurate protein expression levels. However, this process is broken in cancer cells, which rely on the maintenance of high dosage of oncogenic proteins for their survival and to drive cancer development. Whether this is regulated at the level of translational control and the key factors in cis and trans remain unknown. The Myc oncogene is a central paradigm of an exquisitely regulated oncogene and a major driver of pancreatic ductal adenocarcinoma (PDAC). While there has been a tremendous effort to target Myc in cancer, clinically relevant Myc inhibition remains a challenge. Therefore, identifying selective promoters of MYC translation may present a novel therapeutic opportunity. Employing a genome-wide CRISPRi screen for modulators of MYC mRNA translation initiation in PDAC cells, we identified regulators of selective MYC translation and validated four RNA binding proteins (RBPs), including epitranscriptome modifiers. We focused on a top hit, a little studied RBP, RBM42, which is highly expressed in PDAC and predicts poor patient survival. We found that RBM42 binds and selectively regulates the translation of MYC and a precise, yet vital suite of pro-oncogenic transcripts, including JUN and EGFR. Mechanistically, employing immunoprecipitation-mass spectrometry analysis, we found that RMB42 is a novel ribosome-associated protein (RAP). Through RNA structure determination and mutagenesis analysis, we showed that RBM42 directly binds and remodels the MYC 5’UTR RNA structure, facilitating the formation of the translation pre-initiation complex and increasing MYC translation. Importantly, we demonstrated that RBM42 is necessary for human PDAC cell growth and fitness and PDAC tumorigenesis in xenograft mouse models in a Myc-dependent manner in vivo. In PDAC patient samples, RBM42 expression is correlated with Myc protein levels and transcriptional activity. This work transforms our understanding of the translational code in cancer and offers a new therapeutic opening to target the expression of oncogenes. Citation Format: Joanna R Kovalski, Goksu Sarioglu, Vishvak Subramanyam, Grace Hernandez, Gilles Rademaker, Juan A Oses-Prieto, Macey Slota, Kwun Wah Wen, Grace E Kim, Alma L Burlingame, Hani Goodarzi, Rushika M Perera, Davide Ruggero. Functional screen for mediators of onco-mRNA translation specificity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A078.

HYbrid DegRAding Copolymer (HYDRAC): Heterobifunctional polymers for targeted degradation of undruggable proteins.

22 Background: Of the ~20,000 proteins encoded by the human genome, only a small subset can be modulated via traditional pharmacological approaches. Included in the list of “undruggable” proteins are ones with pivotal roles in cancer. We aim to expand the arsenal of compounds able to affect the activity of these therapeutically relevant targets by introducing a modular proteomimetic platform technology designed to carry side chains consisting of targeting warheads and recruiters of cellular protein degradation machinery. We term these compounds HYbrid DegRAding Copolymers (HYDRACs) in reference to the multiplexing of different protein binders and degradation inducing sequences. Peptides arranged as HYDRACs exhibit favorable emergent properties including resistance to proteolysis, high levels of cell uptake, and payload-specific activity. Initial studies were conducted focusing on a undruggable transcription factor MYC, which is widely dysregulated in human cancers. Methods: A library of HYDRACs and control polymers were synthesized and were cell-penetrating with antiproliferative effects at sub-micromolar IC50s in a formulation- and MYC-dependent manner across multiple cell lines. Results: HYDRAC treatment resulted in significant decreases in MYC protein levels, which was rescued by proteasome inhibitor MG132 and neddylation inhibitor MLN4924, without changes in mRNA levels. RNAseq showed selective overexpression of MYC pathway genes only in HYDRAC treated cells, confirming on-target effects. Circular dichroism showed strong HYDRAC-MYC interactions, which was supported by the presence of MYC following pull-down of biotin-terminated HYDRACs. Removal of the RRRG degron, scrambling the H1 sequence, and disassociation of the two domains on separate polymers all abrogated MYC-degradation. Mice bearing transplanted Myc-CaP tumors showed delayed tumor growth and reduced tumor cell proliferation following HYDRAC treatment. Mice bearing luminescent Luc-MV-411 tumors treated with Cy5.5-labeled HDRACs showed selective tumor accumulation with detectable fluorescent signal in the tumor up to 72 hours following a single IP administration. The versatility of the platform was demonstrated by substituting the degron for recruiters of three different E3 ligases (VHL, KEAP1, and CRBN), which all maintained ability to degrade MYC. HYDRACs were also able to selectively degrade other protein targets including alpha-syn and RAS. Conclusions: We present a platform technology that addresses challenges inherent to small molecule drug design. HYDRACs have the potential to dramatically alter the drug discovery landscape, allowing for development of modulators of "undruggable" targets. We envision the HYDRAC platform as a generalizable approach to designing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for target protein degradation.

The effects of MYC on exosomes derived from cancer cells in the context of breast cancer.

MYC amplification and overexpression in breast cancer occur 16% and 22%, respectively, and MYC has a linchpin role in breast carcinogenesis. Emerging evidence has started to shed light on central role of MYC in breast cancer progression. On the contrary, tumor-derived exosomes and their cargo molecules are required for the modulation of the tumor environment and to promote carcinogenesis. Still, how MYC regulates tumor-derived exosomes is still a matter of investigation in the context of breast cancer. Here, we investigated for the first time how MYC affects the biological functions of normal breast cells cocultured with exosomes derived from MYC-expression manipulated breast cancer cells. Accordingly, exosomes were isolated from MCF-7 and MDA-MB-231 cells that MYC expression was manipulated through siRNAs or lentiviral vectors by using exosome isolation reagent. Then, normal breast epithelial MCF-10A cells were treated with breast cancer cell-derived exosomes. The cellular activity of MCF-10A was investigated by cell growth assay, wound healing assay, and transwell assay. Our results suggested that MCF-10A cells treated with exosomes derived from MYC-overexpressing breast cancer cells demonstrated higher proliferation and migration capability compared with nontreated cells. Likewise, MCF-10A cells treated with exosomes derived from MYC-silenced cancer cells did not show high proliferation and invasive capacity. Overall, MYC can drive the functions of exosomes secreted from breast cancer cells. This may allow exploring a new mechanism how tumor cells regulate cancer progression and modulate tumor environment. The present study clears the way for further researches as in vivo studies and multi-omics that clarify exosomal content in an MYC-dependent manner.

Abstract 2714: Inhibiting Myc using heterofunctional polymeric degrading chimeras (HYDRACs): a novel class of compounds capable of targeted protein degradation

Abstract Background: The proto-oncogene Myc is known to play critical roles in tumorigenesis, being dysregulated in >70% of human cancers. Development of small molecule Myc drugs has been hampered due to the lack of suitable binding pockets. Myc inhibitory peptides H1, derived from the bHLH-LZ region, have been explored to address this. However, poor pharmacokinetic profiles have precluded clinical translation. Here, we use a novel modular platform technology referred to as the HYDRAC for the development of dual-functional Myc inhibitors/degraders. Methods: HYDRACs were generated using ROMP resulting in dense brush polymers where peptides occupy every side chain. Heterofunctional constructs were designed incorporating synergistic secondary sequences in addition to Myc-targeting H1. Specifically, a RRRG degron sequence was added to engage endogenous cellular machinery for targeted Myc degradation. Immunofluorescence and confocal microscopy were used to assess cellular penetration; cellular viability assays coupled with both cellular (isogenic pairs, Myc-dependent and independent lines) and biochemical (scrambled H1, monomeric peptides, etc) controls were used to assess anti-proliferative and Myc-specific responses. Myc engagement was assessed via biophysical assays and protein degradation monitored by Western blot. A CRISPR E3 ligase knockdown library and mass spec was used to identify pathways responsible for HYDRAC-induced protein degradation. Results: A library of HYDRACs with low PDI and predetermined DPs were generated. These compounds were shown to be cell-penetrating with antiproliferative effects at sub-micromolar IC50 values in a formulation- and Myc-dependent manner. HYDRAC treatment resulted in significant decreases in Myc protein levels, rescued by proteasomal inhibition with MG132. RNAseq showed selective overexpression of Myc pathway genes. Biophysical analysis showed strong HYDRAC-Myc interactions, in addtion to Myc protein present following pull-down of biotin-terminated HYDRACs. Selectivity studies revealed that HYDRAC activity requires an intact H1 targeting sequence. Identification of E3 ligases implicated in the degradation pathway are ongoing. Mice bearing transplanted Myc-CaP tumors showed delayed tumor growth following IP administration of Myc-degrading HYDRACs, with studies exploring the biodistribution and pharmacokinetic profile of the compound ongoing. Conclusion: We present a novel platform technology that addresses the challenges inherent to peptide delivery approaches. The presented work demonstrates the feasibility of HYDRACs for targeted Myc inhibition and degradation. This first-in-class compound also opens the door for the development of HYDRACs targeting other proteins of interest and multiplexing different E3 ligase recruiters for tissue or disease-specific activity. Citation Format: Max M. Wang, Mihai I. Truica, Brayley Gattis, Xiaoyu Zhang, Sarki Abdulkadir, Nathan C. Gianneschi. Inhibiting Myc using heterofunctional polymeric degrading chimeras (HYDRACs): a novel class of compounds capable of targeted protein degradation [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 2714.

Abstract 349: LncRNA Myca controls hepatocellular carcinoma growth and progression

Abstract Long non-coding RNAs (lncRNAs) have been shown to play critical roles in the tumorigenesis and progression of cancer. From a clinic and genomic perspective of evidence, the MYC signaling plays a crucial role in triggering hepatocellular carcinoma (HCC) and therefore represents an attractive target for HCC intervention. However, no specific drug is currently available to target MYC directly due to the lack of an enzymatic pocket. Therefore, deciphering the critical lncRNAs that functionally crosstalk with MYC protein levels may offer novel therapeutic strategies in HCC. Here we identify the involvement of a lncRNA Myca (Myc Activator) in HCC through controlling MYC protein levels. Overexpression of Myca triggers tumor growth in a MYC-dependent manner, while inactivation of Myca by antisense oligonucleotides (ASOs) profoundly reduces MYC protein levels, thus suppressing tumor progression in vitro. Mechanistically, we show that Myca attenuates MYC ubiquitination and leads to MYC accumulation. Clinically, overexpression of Myca is positively correlated with Hi-MYC transcriptional profile and HCC progression from the TCGA dataset. Together, our findings not only identify Myca in controlling MYC protein levels but also provide Myca as a new therapeutic lncRNA target for the HCC intervention. Citation Format: Hsin-Yi Chen, Yu-Yun Weng, Wei-Chien Yuan, Yu-Ru Lee. LncRNA Myca controls hepatocellular carcinoma growth and progression [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 349.

Sustained activation of EGFR-ERK1/2 signaling limits the response to tigecycline-induced mitochondrial respiratory deficiency in liver cancer.

Identification of tumor dependencies is important for developing therapeutic strategies for liver cancer. A genome-wide CRISPR screen was performed for finding critical vulnerabilities in liver cancer cells. Compounds screen, RNA sequencing, and human phospho-receptor tyrosine kinase arrays were applied to explore mechanisms and search for synergistic drugs. We identified mitochondrial translation-related genes associated with proliferation for liver cancer cells. Tigecycline induced deficiency of respiratory chain by disturbing mitochondrial translation process and showed therapeutic potential in liver cancer. For liver cancer cells extremely insensitive to tigecycline, a compounds screen was applied to identify MEK inhibitors as synergistic drugs to tigecycline-insensitive liver cancer cells. Mechanistically, sustained activation of EGFR-ERK1/2-MYC cascade conferred the insensitivity to tigecycline, which was mediated by enhanced secretion of EREG and AREG. Moreover, glycolytic enzymes, such as HK2 and PKM2 were upregulated to stimulate glycolysisin a MYC-dependent manner. Tigecycline induced respiratory chain deficiency in combination with cutting off EGFR-ERK1/2-MYC cascade by MEK inhibitors or EGFR inhibitors, resulting in decrease of both oxidative phosphorylation and glycolysis in liver cancer cells. Our study proved that blocking EGFR-ERK1/2-MYC cascade combined with tigecycline could be a potential therapeutic strategy for liver cancer. This work was funded by grants from the National Natural Science Foundation of China (82073039,82222047, 81920108025), Program of Shanghai Academic/Technology Research Leader (22XD1423100), Shanghai Municipal Science and Technology Project (20JC1411100), 111 Project (B21024), Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZDCX20212700, SHSMU-ZDCX20210802) and Shanghai Jiao Tong University School of Medicine (YG2019GD01).

STEM-26. ASTROCYTIC SOX2+ CELLS RELAY IN NON-CANONICAL GLI SIGNALING TO FACILITATE MEDULLOBLASTOMA RELAPSE

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Despite the fair efficacy of current treatment approaches, 30 % of patients with medulloblastoma relapse. Cells expressing the stemness biomarker SRY (sex determining region Y)-box 2 (SOX2) are known to play key roles in sustaining medulloblastoma growth, providing chemo-resistance, and driving tumor relapse. It is therefore critical to elucidate the underlying mechanisms that propagate these cells and ensure therapies to target them. Single cell sequencing analyses revealed the existence of a subset of astrocyte-like SOX2+ cells expressing biomarkers indicative of SHH signaling activation. Intriguingly, such SOX2+ cells were not affected by the upstream SHH inhibitor vismodegib. Using SOX2+ enriched cultures, we observed that astrocyte-like SOX2 cells not only express SHH effectors, but require GLI signaling to proliferate, and that GLI is activated in a non-canonical and MYC dependent manner. Importantly, in vivo inhibition of SHH signaling downstream of SMO depleted the vismodegib resistant SOX2+ cell pool, while reduced the ability of residual medulloblastoma tissues to engraft in vivo. Our data show that in medulloblastoma, a subset of SOX2+ tumor cells rely on non-canonical GLI signaling to propagate, and emphasizes the importance of using therapies that deplete SOX2+ cells to prevent tumor recurrence.

Noncanonical activation of GLI signaling in SOX2+ cells drives medulloblastoma relapse.

SRY (sex determining region Y)–box 2 (SOX2)–labeled cells play key roles in chemoresistance and tumor relapse; thus, it is critical to elucidate the mechanisms propagating them. Single-cell transcriptomic analyses of the most common malignant pediatric brain tumor, medulloblastoma (MB), revealed the existence of astrocytic Sox2+ cells expressing sonic hedgehog (SHH) signaling biomarkers. Treatment with vismodegib, an SHH inhibitor that acts on Smoothened (Smo), led to increases in astrocyte-like Sox2+ cells. Using SOX2-enriched MB cultures, we observed that SOX2+ cells required SHH signaling to propagate, and unlike in the proliferative tumor bulk, the SHH pathway was activated in these cells downstream of Smo in an MYC-dependent manner. Functionally different GLI inhibitors depleted vismodegib-resistant SOX2+ cells from MB tissues, reduced their ability to further engraft in vivo, and increased symptom-free survival. Our results emphasize the promise of therapies targeting GLI to deplete SOX2+ cells and provide stable tumor remission.

Development of heterobifunctional proteomimetic polymers for delivery of MYC inhibitory peptides and targeted MYC degradation.

e15049 Background: The c-Myc(Myc) oncogene plays critical roles in tumorigenesis and therapeutic resistance, being dysregulated in up to 70% of all human cancers. Heterodimerization with the closely related bHLH-LZ protein Max is required for oncogenic transformation, yet the development of small molecule inhibitors has been challenging since disordered Myc and Max monomers lack suitable binding pockets, while the Myc-Max interface is flat and extensive. Myc inhibitory peptides derived from the first helix (H1) of the bHLH-LZ region have overcome the limited binding affinity of small molecules. However, poor cellular penetrance and extremely limited bioavailability have precluded clinical translation of peptide-based approaches. Aim: To address and improve the limitations and low potential for clinical translation of Myc inhibitory peptides via a versatile peptide delivery platform. This novel platform technology is referred to as the Protein-Like Polymer (PLP) and it is based on packaging peptides together as high-density brush polymers. Methods: We generated H1-PLPs using ring-opening metathesis polymerization and characterized them using HPLC, LC-MS, NMR, and GPC. We also explored the potential of PLPs to be designed as heterobifunctional compounds by copolymerizing with secondary side chain peptides (nuclear localization sequences and degron sequence). For biological studies, immunofluorescence and confocal microscopy were used to assess cellular penetration; cellular viability assays coupled with use of both cellular (isogenic pairs, Myc-dependent and independent lines) and biochemical (scrambled H1 sequence, homopolymers, monomeric peptides) controls were used to assess tumor antiproliferative effects and Myc-based selectivity of response. Myc target engagement was assessed via immunoprecipitation assays and dual-reporter luciferase-based assays. Results: Our studies show that heterobifunctional PLPs employing H1 peptide and a degron sequence are cell-penetrating and co-localize with Myc in the nucleus. They display antiproliferative effects with submicromolar IC50 values in a Myc-dependent manner. The PLPs lead to Myc/Max complex disruption, promote significant levels of Myc degradation and prevent binding of Myc/Max heterodimers to the Myc/Max consensus canonical DNA binding site. Selectivity studies reveal that antiproliferative effects are dependent on intact H1 sequence. Animal studies using transplanted Myc-CaP model, which mimics the clinical course of human castration resistant prostate cancer are ongoing. Conclusions: We present a novel peptide delivery platform that addresses the challenges inherent to peptide delivery approaches. The presented work demonstrates the feasibility of this platform in delivering Myc inhibitory peptides and provides rationale for further in vivo studies in Myc-dependent tumor models.

Polarity protein SCRIB interacts with SLC3A2 to regulate proliferation and tamoxifen resistance in ER+ breast cancer

Estrogen receptor (ER) positive breast cancer represents 75% of all breast cancers in women. Although patients with ER+ cancers receive endocrine therapies, more than 30% develop resistance and succumb to the disease, highlighting the need to understand endocrine resistance. Here we show an unexpected role for the cell polarity protein SCRIB as a tumor-promoter and a regulator of endocrine resistance in ER-positive breast cancer cells. SCRIB expression is induced by estrogen signaling in a MYC-dependent manner. SCRIB interacts with SLC3A2, a heteromeric component of leucine amino acid transporter SLC7A5. SLC3A2 binds to the N-terminus of SCRIB to facilitate the formation of SCRIB/SLC3A2/LLGL2/SLC7A5 quaternary complex required for membrane localization of the amino acid transporter complex. Both SCRIB and SLC3A2 are required for cell proliferation and tamoxifen resistance in ER+ cells identifying a new role for the SCRIB/SLC3A2 complex in ER+ breast cancer.

MYCMI-7: A Small MYC-Binding Compound that Inhibits MYC: MAX Interaction and Tumor Growth in a MYC-Dependent Manner.

Our findings demonstrate that the small-molecule MYCMI-7 binds MYC and inhibits interaction between MYC and MAX, thereby hampering MYC-driven tumor cell growth in culture and in vivo while sparing normal cells.

Induction of serine hydroxymethyltransferase 2 promotes tumorigenesis and metastasis in neuroblastoma.

High-risk neuroblastoma (NB) remains an extremely difficult subgroup to cure and is associated with MYCN amplification. Serine hydroxymethyltransferase 2 (SHMT2) regulates serine metabolism in a myc-dependent manner; it is upregulated in several cancers and is associated with tumor aggressiveness. Akt-2, an important regulator of MYCN via the PI3K/Akt pathway, induces metastatic potential in NB. The association between SHMT2 and PI3K/Akt in hepatocyte regeneration has been well established but its mechanistic interaction in cancer has yet to be clearly elucidated. Herein, we evaluated the exact role of SHMT2 on the PI3K/Akt pathway, in addition to NB tumorigenesis and metastatic potential in vitro. SHMT2 gene expression and overall survival (OS) were assessed. Two human NB cell lines were examined. SHMT2 silencing and overexpression were performed. The downstream effects were analyzed with immunoblotting, RT-qPCR and functional assays were performed. We found SHMT2 gene expression is associated with decreased OS and MYCN amplification. SHMT2 protein and mRNA expression are increased in MYCN-amplified cells. SHMT2 expression has a direct interaction with Akt-2 and MYCN. Induction of SHMT2 increased cellular proliferation, colony formation and cellular migration and SHMT2 expression was increased in metastatic NB cells. We conclude that SHMT2 regulates N-Myc via phosphorylation of Akt-2 and plays an important role in NB tumorigenesis by contributing to cell growth, migration, colony formation and metastasis in vitro.

Nedd8-Activating Enzyme Inhibition Enhances Anti-Tumor Immunity and PD1 Blockade in In Vivo lymphoma Models

Nedd8-Activating Enzyme Inhibition Enhances Anti-Tumor Immunity and PD1 Blockade in In Vivo lymphoma Models

Myelocytomatosis-Protein Arginine N-Methyltransferase 5 Axis Defines the Tumorigenesis and Immune Response in Hepatocellular Carcinoma.

HCC is a leading cause of cancer-related deaths globally with poor outcome and limited therapeutic options. Although the myelocytomatosis (MYC) oncogene is frequently dysregulated in HCC, it is thought to be undruggable. Thus, the current study aimed to identify the critical downstream metabolic network of MYC and develop therapies for MYC-driven HCC. Liver cancer was induced in mice with hepatocyte-specific disruption of Myc and control mice by administration of diethylnitrosamine. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses revealed that urinary dimethylarginine, especially symmetric dimethylarginine (SDMA), was increased in the HCC mouse model in an MYC-dependent manner. Analyses of human samples demonstrated a similar induction of SDMA in the urines from patients with HCC. Mechanistically, Prmt5, encoding protein arginine N-methyltransferase 5, which catalyzes SDMA formation from arginine, was highly induced in HCC and identified as a direct MYC target gene. Moreover, GSK3326595, a PRMT5 inhibitor, suppressed the growth of liver tumors in human MYC-overexpressing transgenic mice that spontaneously develop HCC. Inhibition of PRMT5 exhibited antiproliferative activity through up-regulation of the tumor suppressor gene Cdkn1b/p27, encoding cyclin-dependent kinase inhibitor 1B. In addition, GSK3326595 induced lymphocyte infiltration and major histocompatibility complex class II expression, which might contribute to the enhanced antitumor immune response. Combination of GSK3326595 with anti-programed cell death protein 1 (PD-1) immune checkpoint therapy (ICT) improved therapeutic efficacy in HCC. This study reveals that PRMT5 is an epigenetic executer of MYC, leading to repression of the transcriptional regulation of downstream genes that promote hepatocellular carcinogenesis, highlights a mechanism-based therapeutic strategy for MYC-driven HCC by PRMT5 inhibition through synergistically suppressed proliferation and enhanced antitumor immunity, and finally provides an opportunity to mitigate the resistance of "immune-cold" tumor to ICT.

Hyperoxia Inhibits Proliferation of Retinal Endothelial Cells in a Myc-Dependent Manner.

Oxygen supplementation is necessary to prevent mortality in severely premature infants. However, the supraphysiological concentration of oxygen utilized in these infants simultaneously creates retinovascular growth attenuation and vasoobliteration that induces the retinopathy of prematurity. Here, we report that hyperoxia regulates the cell cycle and retinal endothelial cell proliferation in a previously unknown Myc-dependent manner, which contributes to oxygen-induced retinopathy.

Stabilization of the Max Homodimer with a Small Molecule Attenuates Myc-Driven Transcription

The transcription factor Max is a basic-helix-loop-helix leucine zipper (bHLHLZ) protein that forms homodimers or interacts with other bHLHLZ proteins, including Myc and Mxd proteins. Among this dynamic network of interactions, the Myc/Max heterodimer has crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Despite this significance, the arsenal of high-quality chemical probes to interrogate these proteins remains limited. We used small molecule microarrays to identify compounds that bind Max in a mechanistically unbiased manner. We discovered the asymmetric polycyclic lactam, KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppressestumor growth invivo. This approach demonstrates the feasibility of modulating Max with small molecules and supports altering Max dimerization as an alternative approach to targeting Myc.

B-Cell Receptor Signaling Drives Glycolysis in Chronic Lymphocytic Leukemia Cells

B-Cell Receptor Signaling Drives Glycolysis in Chronic Lymphocytic Leukemia Cells

A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC-dependent tumor cell proliferation

MYC is a key player in tumor development, but unfortunately no specific MYC-targeting drugs are clinically available. MYC is strictly dependent on heterodimerization with MAX for transcription activation. Aiming at targeting this interaction, we identified MYCMI-6 in a cell-based protein interaction screen for small inhibitory molecules. MYCMI-6 exhibits strong selective inhibition of MYC:MAX interaction in cells and in vitro at single-digit micromolar concentrations, as validated by split Gaussia luciferase, in situ proximity ligation, microscale thermophoresis and surface plasmon resonance (SPR) assays. Further, MYCMI-6 blocks MYC-driven transcription and binds selectively to the MYC bHLHZip domain with a KD of 1.6 ± 0.5 μM as demonstrated by SPR. MYCMI-6 inhibits tumor cell growth in a MYC-dependent manner with IC50 concentrations as low as 0.5 μM, while sparing normal cells. The response to MYCMI-6 correlates with MYC expression based on data from 60 human tumor cell lines and is abrogated by MYC depletion. Further, it inhibits MYC:MAX interaction, reduces proliferation and induces massive apoptosis in tumor tissue from a MYC-driven xenograft tumor model without severe side effects. Since MYCMI-6 does not affect MYC expression, it is a unique molecular tool to specifically target MYC:MAX pharmacologically and it has good potential for drug development.

Abstract 3952: Selective high affinity MYC-binding compound inhibits MYC-MAX interaction and MYC-dependent tumor cell growth

Abstract The MYC family oncoproteins/transcription factors MYC, MYCN and MYCL (here referred to as MYC) are key players in tumor development and are particularly associated with aggressive disease and poor prognosis. Efficient and specific MYC-targeting drugs are therefore highly warranted, but no such drugs are available in the clinic at present. MYC is strictly dependent on heterodimerization with MAX for activation of transcription. In a cell-based Bimolecular Fluorescence Complementation protein-protein interaction screen for small molecule inhibitors we identified a molecule that exhibits strong selective inhibition of MYC-MAX interaction in cells as validated by Gaussia luciferase protein complementation assay, coimmunoprecipitation and in situ proximity ligation (isPLA) assay, reaching an IC50 at single-digit micromolar concentrations. The molecule was shown to inhibit MYC-MAX interactions in a biochemical FRET assay and binds selectively to the MYC bHLHZip domain with affinity in the single digit micromolar range as demonstrated by Microscale Thermophoresis and Surface Plasmon Resonance. Further, within the same concentration range, this molecule blocks MYC-driven transcription and efficiently inhibits tumor cell growth in a MYC-dependent manner, but spares normal cells. Moreover, the growth inhibitory responses to the molecule correlated significantly with MYC expression levels in a cohort of 60 human tumor cell lines. Importantly, utilizing a mouse tumor model of MYCN-amplified neuroblastoma, treatment with the molecule resulted in significant inhibition of the MYC-MAX interaction in tumor tissue, as shown by isPLA, and massive induction of apoptosis in the tumors. Since this molecule, unlike many experimental MYC inhibitors, is selective, has high affinity for MYC, has high efficacy in cells, reaches its target in vivo and does not affect MYC expression levels, it can be used as a chemical tool to specifically study the role of the MYC-MAX complex in MYC biology in normal and cancerous cells, and it has potential for drug development. Citation Format: Alina Castell, Qinzi Yan, Karin Fawkner, Per Hydbring, Fan Zhang, Vasiliki Verschut, Marcela Franco, Giovanna Zinzalla, Lars-Gunnar Larsson. Selective high affinity MYC-binding compound inhibits MYC-MAX interaction and MYC-dependent tumor cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3952.