Myc-dependent Cell Research Articles

Abstract B004: An isogenic CRISPR screen identifies novel MYC-driven vulnerabilities

Abstract c-MYC (MYC) is a central regulatory protein that is dysregulated in >50% of all human cancers and is linked to aggressive disease. Developing MYC inhibitors would revolutionize cancer treatment; however, developing small molecules that directly target MYC is challenging. An alternative approach is to identify and inhibit critical MYC partner proteins to inactivate MYC and trigger cancer cell death. Inhibiting these targets therapeutically can result in synthetic lethality (MYC-SL), which can be exploited in MYC-dysregulated cancers. To identify MYC-SL targets, we performed a genome-wide CRISPR knock-out screen using an isogenic pair of normal and MYC-driven breast cancer cells. In contrast to other screens, this model is dependent on MYC and recapitulates human disease at both pathological and molecular levels in vivo. We identified high-priority hits to validate from the screen using two independent approaches: 1) a traditional gene-set enrichment analysis to identify highly represented biological pathways; and 2) analyzing the Cancer Dependency Map (DEPMAP) to select hits that are likely to be robust beyond the context of our screening conditions. Using a traditional gene-set enrichment analysis approach, we identified topoisomerase 1 (TOP1) as an actionable vulnerability that can be targeted with clinically approved inhibitors. Genetic and pharmacological inhibition of TOP1 resulted in MYC-driven cell death compared to that in control cells. Finally, drug response to TOP1 inhibitors significantly correlated with MYC levels and activity across panels of breast cancer cell lines and patient-derived organoids, highlighting TOP1 as a promising target for MYC-driven cancers. As a secondary approach to interpreting our CRISPR screen hits, we analyzed DEPMAP to identify MYC-SLs that are differentially essential in MYC-dependent cancer cells. Specifically, data from RNA interference screens in hundreds of cancer cell lines were used to describe the response of these cells to MYC knockdown. These data were used to define MYC-dependent and MYC-independent cell lines within the context of this analysis. These two groups were then investigated for their in silico response to the knockdown of each of our MYC-SL hits. MYC-SLs, which were also differentially essential in MYC-dependent cancer cells from DEPMAP, were prioritized for further investigation. Critical MYC cofactors that have been validated by us and others (e.g., CDK9) were identified, providing confidence in this approach, and rationalizing ongoing investigations. Together, this work features two successful strategies to prioritize hits from hundreds of synthetic-lethal genome-wide CRISPR screens to identify novel MYC-driven vulnerabilities in cancer. Citation Format: Peter Lin, Linda Penn. An isogenic CRISPR screen identifies novel MYC-driven vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B004.

Design, Synthesis, and Investigation of the Pharmacokinetics and Anticancer Activities of Indenoisoquinoline Derivatives That Stabilize the G-Quadruplex in the MYC Promoter and Inhibit Topoisomerase I.

G-quadruplexes are noncanonical four-stranded DNA secondary structures. MYC is a master oncogene and the G-quadruplex formed in the MYC promoter functions as a transcriptional silencer and can be stabilized by small molecules. We have previously revealed a novel mechanism of action for indenoisoquinoline anticancer drugs, dual-downregulation of MYC and inhibition of topoisomerase I. Herein, we report the design and synthesis of novel 7-aza-8,9-methylenedioxyindenoisoquinolines based on desirable substituents and π-π stacking interactions. These compounds stabilize the MYC promoter G-quadruplex, significantly lower MYC levels in cancer cells, and inhibit topoisomerase I. MYC targeting was demonstrated by differential activities in Raji vs CA-46 cells and cytotoxicity in MYC-dependent cell lines. Cytotoxicities in the NCI-60 panel of human cancer cell lines were investigated. Favorable pharmacokinetics were established, and in vivo anticancer activities were demonstrated in xenograft mouse models. Furthermore, favorable brain penetration, brain pharmacokinetics, and anticancer activity in an orthotopic glioblastoma mouse model were demonstrated.

Abstract 5964: Design and anticancer biological evaluation of novel 7-aza-8,9-methylenedioxyindenoisoquinoline derivatives that inhibit MYC and topoisomerase I

Abstract MYC is one of the most validated driver oncogenes and is overexpressed in most human cancers. However, the MYC protein is considered “undruggable” due to its disordered structure and difficulties associated with targeting transcription factors. The MYC gene promoter forms G- quadruplex, a four-stranded non-canonical DNA secondary structure. The MYC G-quadruplex functions as a transcription silencer and further inhibits MYC expression upon drug binding, representing an exciting approach for MYC-targeting cancer therapy. Indenoisoquinolines are topoisomerase I inhibitors with better physicochemical and biological properties than the clinical camptothecin drugs. We previously revealed a novel mechanism of action for active anticancer indenoisoquinolines by dual inhibition of MYC and topoisomerase I, where potent MYC inhibition was achieved through targeting its promoter G-quadruplex. Herein, we report the design, cellular activity, and in vivo efficacy of novel anticancer 7-aza-8,9-methylenedioxyindenoisoquinoline derivatives based on optimized substituents and π-π stacking interactions. Using biophysical, biochemical, and cellular assays, we demonstrated that the new indenoisoquinoline derivatives greatly stabilized the MYC promoter G-quadruplex, significantly lowered MYC protein and mRNA levels in cancer cells, and inhibited topoisomerase I activity. A MYC G-quadruplex mediated mechanism of action was evident by differential activities in Raji vs CA-46 cells. The importance of MYC targeting was corroborated by potent cytotoxicity against a panel of MYC-dependent cancer cell lines as well as the NCI-60 panel of human cancer cell lines. The top three candidates were evaluated for pharmacokinetics and in vivo anticancer properties. Excellent metabolic stability, bioavailability, and tumor accumulation were observed in xenograft mouse models. Potent and significant tumor growth inhibition was demonstrated in the aggressive RD-ES Ewing Sarcoma xenograft mouse model while the drugs were well-tolerated. In addition, favorable brain penetration and pharmacokinetics were established, which encourage future evaluations in difficult-to-treat brain tumors. In conclusion, we designed and developed 7-aza-8,9-methylenedioxyindenoisoquinoline derivatives that are dual inhibitors of MYC and topoisomerase I. Our drug candidates demonstrated excellent pharmacokinetics and in vivo anticancer activities with promising indications in MYC-dependent cancers. Citation Format: Yichen Han, Luying Chen, Adam Buric, Venkat Chintareddy, Pooran Chand, Randall Riggs, Mark Cushman, Danzhou Yang. Design and anticancer biological evaluation of novel 7-aza-8,9-methylenedioxyindenoisoquinoline derivatives that inhibit MYC and topoisomerase I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5964.

Abstract 518: The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation

Abstract Richter transformation (RT), characterized by the transformation of chronic lymphocytic leukemia (CLL) to an aggressive large-cell lymphoma, remains a challenge for the therapy. Genetic aberrations, such as Myc activation, TP53 abnormality, NOTCH1 gene mutation or loss of CDKN2A are associated with uncontrolled RT cell proliferation and escaping of apoptosis. Among those changes, gain of Myc activity, found in around 70% of RT, plays a nexus role in the pathogenesis of RT. As a transcription factor, Myc activation has a profound effect on cell survival, proliferation, adhesion and metabolism. Because of its essential role in lymphoma transformation and aggressiveness, Myc is a vital target for therapy toward RT. Fadraciclib (CYC065) is a second-generation CDK inhibitor with selective potency toward CDK2 and CDK9. We have shown previously that by inhibiting CDK9-mediated transcription, fadraciclib reduced expression of the short-lived anti-apoptotic protein Mcl-1 to initiate apoptosis in primary CLL cells. Like Mcl-1, Myc is a quintessential example of an oncogene with rapid turnover in both its mRNA and protein, therefore a promising target of CDK9 inhibition. Thus, we proposed that fadraciclib would target both the survival and proliferation pathways of RT pathogenesis through collaborative inhibition of Mcl-1, Myc and CDK2. First, our data showed that fadraciclib effectively induced apoptosis in the RT cell line HPRT1, as well as the lymphoma cell lines with Myc amplification, including Raji, Ramos and SU-DHL-2. The IC50 for apoptosis was 0.3 µM at 24h in the HPRT1 cells, which is less than the average IC50 for the primary CLL cells (0.8 µM). The IC50 for the lymphoma lines ranged from 0.8 µM to 1.3 µM. Immunoblots showed reduction of RNA Pol2 phosphorylation by fadraciclib, consistent with the inhibition of CDK9. As a result of loss of RNA Pol2 activity and pausing of transcription, there was a clear reduction of both Mcl-1 and Myc mRNA, measured by real-time RT-PCR, that was associated with the reduction of their protein levels. CDK2 was also inhibited by fadraciclib, shown by the reduction of the phosphorylation of both RB and NPM. Second, Fadraciclib inhibited cell proliferation of the HPRT1 cells at an IC50 of 0.25 µM measured by the CellTiter-Glo assay. The IC50 for growth inhibition in the lymphoma lines ranged from 0.5 to 0.7 µM. CFSE tracing demonstrated halting of cell division upon fadraciclib incubation. Cell cycle analysis by Click-iT EdU showed reduction of S-phase population and arresting of cells in both G1 and G2. We are currently working on dissecting the contributions of inhibiting Myc, Mcl-1 and CDK2 individually and together, in apoptosis induction and proliferation blockade. Taken together, these data suggest that fadraciclib is active in RT cells and Myc-dependent lymphoma cell lines and support expansion of its clinical testing in patients with RT. Citation Format: Rong Chen, Yuling Chen, Ping Xiong, William Plunkett. The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 518.

Abstract A059: Small molecule microarray lysate screen identifies bromodomain ligands that target the MYC transcription regulatory network

Abstract MYC is the most frequently amplified gene in human cancers and one of the most sought-after drug targets for cancer therapy. Its function as a transcription factor is essential for its oncogenic potential. However, development of small molecules that target oncogenic MYC function in cells has been intractable due to its lack of known ligand binding sites. To overcome these structural challenges, we leveraged Kronos Bio’s small molecule microarray (SMM) screening platform to identify small molecules that can bind to MYC transcriptional complexes in cell lysates from cancer cells with deregulated MYC function. Unbiased transcriptional signature-based profiling identified multiple SMM hits that modulated the MYC transcription regulatory network (TRN) in MYC-dependent cancer cell lines. One of the SMM hits mimicked signature changes due to MYC loss of function, which were also similar to signature changes effected by BET bromodomain inhibitors. Biochemical, biophysical, and structural analyses found that the SMM hit could directly engage with BET bromodomains in purified systems. Cell-based proximity labeling demonstrated binding to BET proteins in live cells, and comparative pharmacology approaches suggested that the BET protein engagement was likely driving the observed gene expression changes. These results demonstrate the utility of the SMM platform in identifying ligands that modulate transcription factor TRNs through binding to critical cofactors. Citation Format: Emily B Cohen, Zhihua Ma, Wayne L Glore, Oleg A Volkov, Tong Liang, Nikolaus D Obholzer, Andrew C Clay, Mulini Reddy Pingili, Yupeng Zheng, Jun Li, Shi Yun Wang, Tom Chen, David B Freeman, Hua Gao, Minyun Zhou, Priscilla Cheung, Tamara D Hopkins, Marius S Pop, Christopher J Dinsmore, B. Wesley Trotter, Charles Y Lin, Peter B Rahl. Small molecule microarray lysate screen identifies bromodomain ligands that target the MYC transcription regulatory network [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A059.

Mitoribosomal synthetic lethality overcomes multidrug resistance in MYC-driven neuroblastoma

Mitochondria are central for cancer responses to therapy-induced stress signals. Refractory tumors often show attenuated sensitivity to apoptotic signaling, yet clinically relevant molecular actors to target mitochondria-mediated resistance remain elusive. Here, we show that MYC-driven neuroblastoma cells rely on intact mitochondrial ribosome (mitoribosome) processivity and undergo cell death following pharmacological inhibition of mitochondrial translation, regardless of their multidrug/mitochondrial resistance and stem-like phenotypes. Mechanistically, inhibiting mitoribosomes induced the mitochondrial stress-activated integrated stress response (ISR), leading to downregulation of c-MYC/N-MYC proteins prior to neuroblastoma cell death, which could be both rescued by the ISR inhibitor ISRIB. The ISR blocks global protein synthesis and shifted the c-MYC/N-MYC turnover toward proteasomal degradation. Comparing models of various neuroectodermal tumors and normal fibroblasts revealed overexpression of MYC proteins phosphorylated at the degradation-promoting site T58 as a factor that predetermines vulnerability of MYC-driven neuroblastoma to mitoribosome inhibition. Reducing N-MYC levels in a neuroblastoma model with tunable MYCN expression mitigated cell death induction upon inhibition of mitochondrial translation and functionally validated the propensity of neuroblastoma cells for MYC-dependent cell death in response to the mitochondrial ISR. Notably, neuroblastoma cells failed to develop significant resistance to the mitoribosomal inhibitor doxycycline over a long-term repeated (pulsed) selection. Collectively, we identify mitochondrial translation machinery as a novel synthetic lethality target for multidrug-resistant MYC-driven tumors.

Cooperative Networks between MYC and XPO1 Associated with Decreased T-Cell Presence and a Depleted Tumor Microenvironment May be Addressed By the Synergistic Combination of AZD4573 and Selinexor

Background: Genomically-targeted Diffuse Large B-cell (DLBCL) treatments for refractory or relapsing patients remain a clinical need alongside the growing success of Chimeric Antigen T-cells (CAR-T), which have ushered in durable responses in 40-70% of patients. However, recent studies have identified factors indicative of an inferior CAR-T response. Recent tumor Ecotype studies from Kotlov and Steen reveal that a Depleted or Cold tumor microenvironment (TME) is associated with poor CAR-T response. Notable features of these environments include a decreased presence of native T-cells alongside oncogenic alterations associated with their exhaustion, namely MYC and XPO1. Few precision treatments exist given the difficulty targeting MYC, but two avenues have shown recent success: inhibition of the downstream oncogene CDK9 via AZD4573 and upstream inhibition of a MYC propagator, the nuclear exportin XPO1 via Selinexor. Herein, we report the first observations of synergy between AZD4573 and Selinexor within DLBCL cell lines and data supporting that these genes may serve as accessible markers for inferior tumor immune microenvironments. Methods: We combined progression vs. response results from four previous CAR-T genomics studies, noting MYC status (Jaeger 2020, Jain 2021, Sworder 2021, and Shouval 2022). We next analyzed a 418-patient de-novo DLBCL patient cohort (Xu-Monette 2020) and 48-patient TCGA cohort after GEDIT immune deconvolution for differential expression and component enrichment between patients expressing high levels of MYC and XPO1 (+1 Standard Deviation) compared to normal counterparts. Significantly expressed genes were evaluated via gene set enrichment analyses. We next assayed AZD4573 and Selinexor vs. 7 DLBCL cell lines, 5 of which harbor MYC alterations, across a series of 9 concentrations (78nM-20µM) across 3-9 replicates. We followed these results by combining treatments in the MYC-altered Ly3, DHL6, and DHL4 cell lines, analyzing results using the BLISS synergy model. Results: Integrative analyses support that MYC alterations are associated with significantly reduced CAR-T outcomes. Of the 93 MYC-altered patients within the total 246-patient cohort, 65.59% of them experienced progression during CAR-T treatment compared to just 50.33% of WT CAR-T patients (p = 0.0242). Differential gene expression analyses between de-novo patients expressing high MYC or XPO1 revealed losses in Cytokine Signaling pathways (FDR = 6.96E-18) and gains in Cell Cycle modulators (FDR = 2.525E-20). Importantly, CD8A expression was significantly lower in these patients (FDR < 0.0001) alongside other key reductions of CDKN1A and TNFAIP3. After immune deconvolution, both High-MYC and High-XPO1 patients displayed significant losses of CD8 T-cell association, respectively (FDR = 0.0005, FDR = 0.0396). Notably, High-MYC patients without a CD8 T-cell presence experienced an inferior survival prognosis compared to High-MYC counterparts with a CD8 presence (p = 0.0272). High MYC or XPO1 patients that experienced a failure event before 24-months (EFS24) had significantly lower activated CD4 T-cell presence compared to normal counterparts as well (FDR = 0.0095, FDR = 0.0069). Next, AZD4573 and Selinexor assays vs. DLBCL cell lines demonstrated dose and MYC-dependent cell inhibition, with ED50 values ranging between 58.1 nM to 8.07 µM. Lastly, the molecules were combined vs. Ly3, DHL6 and DHL4 cell lines, resulting in synergistic reduction of cell viability and BLISS synergy scores of 33.76, and 11.86, and 5.04, respectively. Conclusions: Our results support that the cooperation between MYC and XPO1 is associated with T-cell reductions characteristic of a Depleted or Cold TME, a key issue for CAR-T success. We applied the targeted therapies AZD4573 and Selinexor vs. cell line models to address this pathway, with both displaying anti-tumor effects as single agents. Most importantly, we observed synergistic activity in the MYC-harboring Ly3 and DHL6 cell lines, and in the WT DHL4 cell line when these molecules were combined. These results are supported by integrative analyses that highlight the importance of targeting aberrant MYC signaling via CDK9 and XPO1 inhibition, revealing a potential avenue to address inferior response rates faced by patients harboring MYC-positive, Depleted tumor microenvironments that would otherwise face an inferior CAR-T prognosis.

CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells.

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines-K562, ST486, HepG2, and MCF7-which revealed several essential E-boxes and genes. Among them, we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression, and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.

Targeting MYC with modular synthetic transcriptional repressors derived from bHLH DNA-binding domains.

Despite unequivocal roles in disease, transcription factors (TFs) remain largely untapped as pharmacologic targets due to the challenges in targeting protein-protein and protein-DNA interactions. Here we report a chemical strategy to generate modular synthetic transcriptional repressors (STRs) derived from the bHLH domain of MAX. Our synthetic approach yields chemically stabilized tertiary domain mimetics that cooperatively bind the MYC/MAX consensus E-box motif with nanomolar affinity, exhibit specificity that is equivalent to or beyond that of full-length TFs and directly compete with MYC/MAX protein for DNA binding. A lead STR directly inhibits MYC binding in cells, downregulates MYC-dependent expression programs at the proteome level and inhibits MYC-dependent cell proliferation. Co-crystallization and structure determination of a STR:E-box DNA complex confirms retention of DNA recognition in a near identical manner as full-length bHLH TFs. We additionally demonstrate structure-blind design of STRs derived from alternative bHLH-TFs, confirming that STRs can be used to develop highly specific mimetics of TFs targeting other gene regulatory elements.

TRAF6 functions as a tumor suppressor in myeloid malignancies by directly targeting MYC oncogenic activity.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an invivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.

The Diarylheptanoid Curcumin Induces MYC Inhibition and Cross-Links This Oncoprotein to the Coactivator TRRAP.

The c-Myc protein (MYC) is a transcription factor with strong oncogenic potential controlling fundamental cellular processes. In most human tumors, MYC is overexpressed by enhanced transcriptional activation, gene amplification, chromosomal rearrangements, or increased protein stabilization. To pharmacologically suppress oncogenic MYC functions, multiple approaches have been applied either to inhibit transcriptional activation of the endogenous MYC gene, or to interfere with biochemical functions of aberrantly activated MYC. Other critical points of attack are targeted protein modification, or destabilization leading to a non-functional MYC oncoprotein. It has been claimed that the natural compound curcumin representing the principal curcumoid of turmeric (Curcuma longa) has anticancer properties although its specificity, efficacy, and the underlying molecular mechanisms have been controversially discussed. Here, we have tested curcumin’s effect on MYC-dependent cell transformation and transcriptional activation, and found that this natural compound interferes with both of these MYC activities. Furthermore, in curcumin-treated cells, the endogenous 60-kDa MYC protein is covalently and specifically cross-linked to one of its transcriptional interaction partners, namely the 434-kDa transformation/transcription domain associated protein (TRRAP). Thereby, endogenous MYC levels are strongly reduced and cells stop to proliferate. TRRAP is a multidomain adaptor protein of the phosphoinositide 3-kinase-related kinases (PIKK) family and represents an important component of many histone acetyltransferase (HAT) complexes. TRRAP is important to mediate transcriptional activation executed by the MYC oncoprotein, but on the other hand TRRAP also negatively regulates protein stability of the tumor suppressor p53 (TP53). Curcumin-mediated covalent binding of MYC to TRRAP reduces the protein amounts of both interaction partners but does not downregulate TP53, so that the growth-arresting effect of wild type TP53 could prevail. Our results elucidate a molecular mechanism of curcumin action that specifically and irreversibly targets two crucial multifunctional cellular players. With regard to their broad impact in cancer, our findings contribute to explain the pleiotropic functions of curcumin, and suggest that this natural spice, or more bioavailable derivatives thereof, may constitute useful adjuvants in the therapy of MYC-dependent and TRRAP-associated human tumors.

Multiple Synthetic Routes to the Mini-Protein Omomyc and Coiled-Coil Domain Truncations.

The Myc transcription factor represents an "undruggable" target of high biological interest due to its central role in various cancers. An abbreviated form of the c-Myc protein, called Omomyc, consists of the Myc DNA-binding domain and a coiled-coil region to facilitate dimerization of the 90 amino acid polypeptide. Here we present our results to evaluate the synthesis of Omomyc using three complementary strategies: linear Fmoc solid-phase peptide synthesis (SPPS) using several advancements for difficult sequences, native chemical ligation from smaller peptide fragments, and a high-throughput bacterial expression and assay platform for rapid mutagenesis. This multifaceted approach allowed access to up to gram quantities of the mini-protein and permitted in vitro and in vivo SAR exploration of this modality. DNA-binding results and cellular activity confirm that Omomyc and analogues presented here, are potent binders of the E-box DNA engaged by Myc for transcriptional activation and that this 90-amino acid mini-protein is cell permeable and can inhibit proliferation of Myc-dependent cell lines. We also present additional results on covalent homodimerization through disulfide formation of the full-length mini-protein and show the coiled-coil region can be truncated while preserving both DNA binding and cellular activity. Altogether, our results highlight the ability of advanced peptide synthesis to achieve SAR tractability in a challenging synthetic modality.

Interaction between the BAG1S isoform and HSP70 mediates the stability of anti-apoptotic proteins and the survival of osteosarcoma cells expressing oncogenic MYC

BackgroundThe oncoprotein MYC has the dual capacity to drive cell cycle progression or induce apoptosis, depending on the cellular context. BAG1 was previously identified as a transcriptional target of MYC that functions as a critical determinant of this cell fate decision. The BAG1 protein is expressed as multiple isoforms, each having an array of distinct biochemical functions; however, the specific effector function of BAG1 that directs MYC-dependent cell survival has not been defined.MethodsIn our studies the human osteosarcoma line U2OS expressing a conditional MYC-ER allele was used to induce oncogenic levels of MYC. We interrogated MYC-driven survival processes by modifying BAG1 protein expression. The function of the separate BAG1 isoforms was investigated by depleting cells of endogenous BAG1 and reintroducing the distinct isoforms. Flow cytometry and immunoblot assays were performed to analyze the effect of specific BAG1 isoforms on MYC-dependent apoptosis. These experiments were repeated to determine the role of the HSP70 chaperone complex in BAG1 survival processes. Finally, a proteomic approach was used to identify a set of specific pro-survival proteins controlled by the HSP70/BAG1 complex.ResultsLoss of BAG1 resulted in robust MYC-induced apoptosis. Expression of the larger isoforms of BAG1, BAG1L and BAG1M, were insufficient to rescue survival in cells with oncogenic levels of MYC. Alternatively, reintroduction of BAG1S significantly reduced the level of apoptosis. Manipulation of the BAG1S interaction with HSP70 revealed that BAG1S provides its pro-survival function by serving as a cofactor for the HSP70 chaperone complex. Via a proteomic approach we identified and classified a set of pro-survival proteins controlled by this HSP70/BAG1 chaperone complex that contribute to the BAG1 anti-apoptotic phenotype.ConclusionsThe small isoform of BAG1, BAG1S, in cooperation with the HSP70 chaperone complex, selectively mediates cell survival in MYC overexpressing tumor cells. We identified a set of specific pro-survival clients controlled by the HSP70/BAG1S chaperone complex. These clients define new nodes that could be therapeutically targeted to disrupt the survival of tumor cells driven by MYC activation. With MYC overexpression occurring in most human cancers, this introduces new strategies for cancer treatment.

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.

Design, synthesis and biological evaluation of novel 4-phenylisoquinolinone BET bromodomain inhibitors

The bromodomain and extra-terminal (BET) family of epigenetic proteins has attracted considerable attention in drug discovery given its involvement in regulating gene transcription. Screening a focused small molecule library based on the bromodomain pharmacophore resulted in the identification of 2-methylisoquinoline-1-one as a novel BET bromodomain-binding motif. Structure guided SAR exploration resulted in >10,000-fold potency improvement for the BRD4-BD1 bromodomain. Lead compounds exhibited excellent potencies in both biochemical and cellular assays in MYC-dependent cell lines. Compound 36 demonstrated good physicochemical properties and promising exposure levels in exploratory PK studies.

A general strategy for synthesis of cyclophane-braced peptide macrocycles via palladium-catalysed intramolecular sp3 C-H arylation.

New methods capable of effecting cyclization, and forming novel three-dimensional structures while maintaining favourable physicochemical properties are needed to facilitate the development of cyclic peptide-based drugs that can engage challenging biological targets, such as protein-protein interactions. Here, we report a highly efficient and generally applicable strategy for constructing new types of peptide macrocycles using palladium-catalysed intramolecular C(sp3)-H arylation reactions. Easily accessible linear peptide precursors of simple and versatile design can be selectively cyclized at the side chains of either aromatic or modified non-aromatic amino acid units to form various cyclophane-braced peptide cycles. This strategy provides a powerful tool to address the long-standing challenge of size- and composition-dependence in peptide macrocyclization, and generates novel peptide macrocycles with uniquely buttressed backbones and distinct loop-type three-dimensional structures. Preliminary cell proliferation screening of the pilot library revealed a potent lead compound with selective cytotoxicity toward proliferative Myc-dependent cancer cell lines.

Targeted Disruption of Myc-Max Oncoprotein Complex by a Small Molecule.

Myc plays important roles in cell cycle progression, cell growth, and stem cell self-renewal. Although dysregulation of Myc expression is a hallmark of human cancers, there is no Myc targeted therapy yet. Here, we report sAJM589, a novel small molecule Myc inhibitor, identified from a PCA-based high-throughput screen. sAJM589 potently disrupts the Myc-Max heterodimer in a dose dependent manner with an IC50 of 1.8 ± 0.03 μM. sAJM589 preferentially inhibits transcription of Myc target genes in a Burkitt lymphoma cell model, P493-6. Genome-wide transcriptome analysis showed that sAJM589 treatment and Myc depletion induced similar gene expression profiles. Consistently, sAJM589 suppressed cellular proliferation in diverse Myc-dependent cancer cell lines and anchorage independent growth of Raji cells. Disruption of the Myc-Max interaction by sAJM589 reduced Myc protein levels, possibly by promoting ubiquitination and degradation of Myc. Collectively, these results suggest that sAJM589 may be a basis for the development of potential inhibitors of Myc-dependent cell growth.

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

Abstract 4634: Novel dual HDAC & PI3K inhibitor, CUDC-907, for MYC-driven malignancies

Abstract MYC family genes are among the most frequently deregulated oncogenic drivers in human cancer. Pharmacologic inhibition of HDAC activity and blockade of the PI3K pathway have both been shown to suppress MYC-induced transcription. HDAC activity is critical for MYC gene regulation, as MYC represses transcription of target genes through recruitment of HDACs. HDAC inhibitors have been shown to restore expression of genes suppressed by MYC family members and to induce rapid downregulation of expression of MYC itself. The PI3K pathway plays a central role in regulating MYC at the post-transcriptional level. Activation of PI3K signaling leads to activation of AKT, which phosphorylates and inhibits GSK3β. As GSK3β normally phosphorylates MYC which facilitates the degradation of MYC, activation of PI3K signaling leads to increased stability of MYC, whereas PI3K inhibitors decrease MYC stability. A recent study has demonstrated addiction to MYC signaling and hypersensitivity to PI3K inhibition in PTEN-deficient diffuse large B-cell (DLBCL) cell lines, suggesting that MYC-driven cancers may be particularly sensitive to PI3K inhibition. As HDACs and PI3K regulate MYC protein levels and functions through nonoverlapping mechanisms, simultaneous HDAC and PI3K inhibition may further enhance MYC suppression. CUDC-907 is an orally bioavailable, small-molecule dual HDAC and PI3K inhibitor that primarily inhibits class I and II HDACs and the PI3Kα, β, and δ isoforms. CUDC-907 shows greater anti-tumor activity in vitro than single-target HDAC or PI3K inhibitors, especially in MYC-dependent cell types, such as DLBCL and NUT midline carcinoma (NMC). In preclinical testing, CUDC-907 treatment leads to a dose-dependent decrease in MYC protein levels, and is also more potent in decreasing MYC than the HDAC inhibitor panobinostat and the pan-PI3K inhibitor pictilisib alone or in combination. Significant antitumor effects have been consistently observed in MYC-driven DLBCL xenograft and genetically engineered mouse models exposed to CUDC907. In particular, certain MYC translocation (Daudi), double-hit (concurrent MYC and BLC2 translocation, WSUDLCL2 and DOHH2), double-expresser (expression of MYC and BCL2 proteins, U2932) xenograft models, and the Eμ-Myc transgenic mouse model achieve tumor growth inhibition of 100%, 69%, 56%, 97% and 72%, respectively. These findings raise the possibility that hematologic and solid tumors driven by aberrant overexpression of MYC family genes (e.g., MYC-altered DLBCL and NMC) might be more responsive to simultaneous HDAC and PI3K inhibition with CUDC-907 than they are to single-target therapy. Clinical Phase 1 studies are currently testing CUDC-907 in patients with relapsed/refractory (R/R) DLBCL and advanced MYC-aberrant solid tumors. Preliminary data are encouraging and support the planned Phase 2 study in R/R MYC-altered DLBCL, as well as further testing in other MYC-driven malignancies. Citation Format: Kaiming Sun, Ruzanna Atoyan, Mylissa A. Borek, Steven Dellarocca, Maria E. Samson, Anna W. Ma, Guangxin Xu, Troy Patterson, David P. Tuck, Jaye L. Viner, Ali Fattaey, Jing Wang. Novel dual HDAC & PI3K inhibitor, CUDC-907, for MYC-driven malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4634.