Inhibition Of MYC Research Articles

BET inhibitors synergize with venetoclax to induce apoptosis in MYC-driven lymphomas with high BCL-2 expression

AbstractAlthough the MYC oncogenic network represents an attractive therapeutic target for lymphoma, MYC inhibitors have been difficult to develop. Alternatively, inhibitors of epigenetic/ transcriptional regulators, particularly the bromodomain and extraterminal (BET) family, have been used to modulate MYC. However, current benzodiazepine-derivative BET inhibitors (BETi) elicit disappointing responses and dose-limiting toxicity in relapsed/refractory lymphoma, potentially because of enrichment of high-risk molecular features and chemical backbone-associated toxicities. Consequently, novel nonbenzodiazepine BETi and improved mechanistic understanding are required. Here we characterize the responses of aggressive MYC-driven lymphomas to 2 nonbenzodiazepine BETi: PLX51107 and PLX2853. Both invoked BIM-dependent apoptosis and in vivo therapy, associated with miR-17∼92 repression, in murine Eµ-myc lymphomas, with PLX2853 exhibiting enhanced potency. Accordingly, exogenous BCL-2 expression abrogated these effects. Because high BCL-2 expression is common in diffuse large B-cell lymphoma (DLBCL), BETi were ineffective in driving apoptosis and in vivo therapy of DLBCL cell lines, mirroring clinical results. However, BETi-mediated BIM upregulation and miR-17∼92 repression remained intact. Consequently, coadministration of BETi and ABT199/venetoclax restored cell death and in vivo therapy. Collectively, these data identify BIM-dependent apoptosis as a critical mechanism of action for this class of BETi that, via coadministration of BH3 mimetics, can deliver effective tumor control in DLBCL.

TLR2/6 signaling promotes the expansion of premalignant hematopoietic stem and progenitor cells in the NUP98–HOXD13 mouse model of MDS

Toll-like receptor 2 (TLR2) expression is increased on hematopoietic stem and progenitor cells (HSPCs) of patients with myelodysplastic syndromes (MDS), and enhanced TLR2 signaling is thought to contribute to MDS pathogenesis. Notably, TLR2 heterodimerizes with TLR1 or TLR6, and while high TLR2 is associated with lower-risk disease, high TLR6, but not TLR1, correlates with higher-risk disease. This raises the possibility of heterodimer-specific effects of TLR2 signaling in MDS, and in the work described here, we tested the effects of specific modulation of TLR1/2 versus TLR2/6 signaling on premalignant HSPCs. Indeed, chronic stimulation of TLR2/6, but not TLR1/2, accelerates leukemic transformation in the NHD13 mouse model of MDS, and conversely, loss of TLR6, but not TLR1, slows this process. TLR2/6 stimulation expands premalignant HSPCs, and chimeric mouse studies revealed that cell-autonomous signaling contributes to this expansion. Finally, TLR2/6 stimulation is associated with an enrichment of Myc and mTORC1 activities. While Myc inhibition partially suppressed the TLR2/6 agonist-mediated expansion of premalignant HSPCs, inhibition of mTORC1 exacerbated it, suggesting that these pathways play opposite roles in regulating the effects of TLR2/6 ligation on HSPCs. Together, these data reveal heterodimer-specific effects of TLR2 signaling on premalignant HSPCs, with TLR2/6 signaling promoting their expansion and leukemic transformation.

MYC Upregulation Confers Resistance to Everolimus and Establishes Vulnerability to Cyclin-Dependent Kinase Inhibitors in Pancreatic Neuroendocrine Neoplasm Cells

Introduction: Dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1)-dependent pathways in pancreatic neuroendocrine neoplasms (PanNENs) underlies the introduction of the mTORC1 inhibitor everolimus as treatment of advanced progressive PanNENs. Although everolimus significantly increases progression-free survival, most patients acquire secondary resistance to the drug. This study aimed at identifying mechanisms involved in acquisition of resistance to everolimus. Methods: BON-1 and everolimus-resistant (ER) BON-1 cells were used as in vitro system of sensitivity and acquired resistance. Transcriptome changes occurring in BON-1 and ER-BON-1 were investigated by RNA sequencing and validated by quantitative PCR analysis. RNA extracted from patients’ biopsies was used to validate MYC upregulation. Drug screening and functional assays were performed using ER-BON-1 cells. Cell cycle progression was evaluated by FACS analysis. Results: Our results show that MYC overexpression is a key event in the development of secondary resistance to everolimus in PanNEN cell lines and in metastatic lesions from neuroendocrine neoplasm patients. MYC knockdown restored ER-BON-1 sensitivity to everolimus. Pharmacological inhibition of MYC mediated by the cyclin-dependent kinase inhibitor dinaciclib strongly reduced viability of ER-BON-1. Dinaciclib synergized with everolimus and inhibited ER-BON-1 cell cycle progression. Discussion: Our findings suggest that MYC upregulation drives the development of secondary resistance to everolimus in PanNENs and that its inhibition is an exploitable vulnerability. Indeed, our results indicate that combined treatments with cyclin-dependent kinase and mTOR inhibitors may counteract secondary resistance to everolimus in PanNENs and may pave the ground for new therapeutic regimens for these tumors.

ID1 overexpression promotes HCC progression by amplifying the AURKA/Myc signaling pathway.

Hepatocellular carcinoma(HCC) is the fourth most common cancer type worldwide, with a poor prognosis and high mortality rate. The aim of the present study was to investigate the mechanisms underlying HCC progression to potentially benefit the development of effective therapies for advanced HCC. The present study reported a novel mechanism that promotes the HCC malignant phenotype, in which the inhibitor of differentiation 1(ID1) activates the aurora kinase A(AURKA)/Myc signaling pathway. Specifically, a high expression of ID1 promoted a highly malignant phenotype of HCC cells that exhibit enhanced metastatic ability and drug resistance. However, the effects of ID1 were markedly reversed by the AURKA inhibitor VX689 and the Myc inhibitor 10058‑F4. Further studies demonstrated that ID1 competitively binds with anaphase‑promoting complex/cyclosome Cdh1(APC/CCdh1), which is responsible for ubiquitination‑mediated AURKA protein degradation, resulting in upregulation of AURKA. Increased AURKA expression subsequently enhanced Myc expression at both transcriptional and post‑transcriptional level, leading to amplification of the Myc oncogenic signaling pathway. This novel ID1/AURKA/Myc axis could be a promising therapeutic target for the development of effective therapeutic strategies for advanced HCC.

Combined Targeting of the BRD4-NUT-p300 Axis in NUT Midline Carcinoma by Dual Selective Bromodomain Inhibitor, NEO2734.

NUT midline carcinoma (NMC) is a rare, aggressive subtype of squamous carcinoma that is driven by the BRD4-NUT fusion oncoprotein. BRD4, a BET protein, binds to chromatin through its two bromodomains, and NUT recruits the p300 histone acetyltransferse (HAT) to activate transcription of oncogenic target genes. BET-selective bromodomain inhibitors have demonstrated on-target activity in patients with NMC, but with limited efficacy. P300, like BRD4, contains a bromodomain. We show that combining selective p300/CBP and BET bromodomain inhibitors, GNE-781 and OTX015, respectively, induces cooperative depletion of MYC and synergistic inhibition of NMC growth. Treatment of NMC cells with the novel dual p300/CBP and BET bromodomain-selective inhibitor, NEO2734, potently inhibits growth and induces differentiation of NMC cells in vitro; findings that correspond with potentiated transcriptional effects from combined BET and p300 bromodomain inhibition. In three disseminated NMC xenograft models, NEO2734 provided greater growth inhibition, with tumor regression and significant survival benefit seen in two of three models, compared with a lead clinical BET inhibitor or "standard" chemotherapy. Our findings provide a strong rationale for clinical study of NEO2734 in patients with NMC. Moreover, the synergistic inhibition of NMC growth by CBP/p300 and BET bromodomain inhibition lays the groundwork for greater mechanistic understanding of the interplay between p300 and BRD4-NUT that drives this cancer.

A Novel Combination Approach Targeting an Enhanced Protein Synthesis Pathway in MYC-driven (Group 3) Medulloblastoma

The MYC oncogene is frequently amplified in patients with medulloblastoma, particularly in group 3 patients, who have the worst prognosis. mTOR signaling-driven deregulated protein synthesis is very common in various cancers, including medulloblastoma, that can promote MYC stabilization. As a transcription factor, MYC itself is further known to regulate transcription of several components of protein synthesis machinery, leading to an enhanced protein synthesis rate and proliferation. Thus, inhibiting enhanced protein synthesis by targeting the MYC and mTOR pathways together may represent a highly relevant strategy for the treatment of MYC-driven medulloblastoma. Here, using siRNA and small-molecule inhibitor approaches, we evaluated the effects of combined inhibition of MYC transcription and mTOR signaling on medulloblastoma cell growth/survival and associated molecular mechanism(s) in MYC-amplified (group 3) medulloblastoma cell lines and xenografts. Combined inhibition of MYC and mTOR synergistically suppressed medulloblastoma cell growth and induced G1 cell-cycle arrest and apoptosis. Mechanistically, the combined inhibition significantly downregulated the expression levels of key target proteins of MYC and mTOR signaling. Our results with RNA-sequencing revealed that combined inhibition synergistically modulated global gene expression including MYC/mTOR components. In addition, the combination treatment significantly delayed tumor growth and prolonged survival of MYC-amplified medulloblastoma xenografted mice by downregulating expression of MYC and the key downstream components of mTOR signaling, compared with single-agent therapy. Together, our findings demonstrated that dual inhibition of MYC (transcription) and mTOR (translation) of the protein synthesis pathway can be a novel therapeutic approach against MYC-driven medulloblastoma.

DCE-MRI Evaluation of 10 patients with brain metastases treated with RRx-001, a Myc inhibitor and a CD47 and PD-L1 downregulator, in a phase I/II trial called BRAINSTORM.

e14509 Background: In a Phase 1/2 trial called BRAINSTORM (NCT02215512) for brain metastases from any histology, quantitative changes in perfusion MRI after administration of RRx-001, a mic inhibitor and CD47 and PD-L1 downregulator with vascular normalizing properties, were determined and correlated with response. Methods: Ten patients with 64 total lesions evaluable at baseline, 24 hours, and end of radiotherapy (RT) that participated in BRAINSTORM were subjected to a correlative dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) examination four days prior to the start of whole brain radiotherapy (WBRT) that evaluated Ktrans (capillary permeability) and Vp (plasma volume). The treatment comprised RRx-001 on Day -4, pre-WBRT then twice weekly during WBRT. Four dose levels were administered (5 mg/m2, 8.4 mg/m2, 16.5 mg/m2, and 27.5 mg/m2. Results: 10 patients underwent DCE-MRI scans and eight patients with 44 total evaluable lesions had available imaging at 1 month, and 6 patients with 29 total evaluable lesions had imaging at 4 months. On univariate analysis, only a decrease in 24-hour Vp from baseline after a single dose of RRx-001 was marginally associated with absolute tumor volume response 1 month after treatment (p-0.07). In a multivariate model, only Vp prior to therapy and 24-hour change in Vp were retained in the model after stepwise selection. A reduction in Vp 24 hours after RRx-001 (prior to WBRT) was associated with reduced tumor volume at 1 month (Estimate 0.88, 95% CI 0.37-1.40, p = 0.001) and 4 months (Estimate 1.51, 95% CI 0.58-2.43, p = 0.003). Likewise, a lower Vp prior to therapy was associated with reduced tumor volume at 1 month (Estimate 0.73, 95% CI 0.29-1.17, p = 0.002) and 4 months (Estimate 1.8, 95% CI 0.95-2.65, p = 0.0002), suggesting anti-angiogenic activity and early potential vascular normalization after a single dose of RRx-001 predictive of longer-term tumor response. Conclusions: RRx-001 induced a reduction in blood plasma volume, which was associated with tumor response and which suggests a vascular normalizing effect that merits further investigation in future planned studies. Clinical trial information: NCT02215512.

Phase I pilot study of RRx-001 + nivolumab in patients with traditionally non-checkpoint inhibitor-responsive cancers (PRIMETIME).

e15119 Background: RRx-001 is a minimally toxic small molecule that downregulates CD47 via Myc inhibition and repolarizes tumor-associated macrophages (TAMs). A phase 1 pilot study was undertaken to determine the safety and feasibility of RRx-001 and nivolumab in patients with advanced traditionally non-immunogenic cancers and no standard options. Methods: This single arm, open-label pilot study (NCT02518958) was designed to evaluate the safety profile of RRx-001 + nivolumab in patients with advanced malignancies. A 3+3 trial design was used to establish safety of the combination at each dose level and guide the decision to escalate dose. RRx-001 is infused once weekly while nivolumab is given at 3mg/kg once every 2 weeks. RRx-001 starting dose was 2 mg IV weekly with 4 dose level escalations up to 16 mg IV weekly. From January 2015 to November 2015, 12 patients received treatment for 4 cycles (total 12 weeks) with the combination due to unavailability of nivolumab, which was not supplied to the Sponsor. Treatment-emergent (all cause, TEAEs) and treatment-related (TRAEs) adverse events occurring within 16 weeks of the first dose of RRx-001 + nivolumab were characterized according to CTCAE v4.03. Results: 12 patients received > 1 dose of RRx-001 and nivolumab. One discontinuation occurred due to pneumonitis and one to voluntary withdrawal after a post-procedural infection. There were no dose-limiting toxicities. The main adverse event related to RRx-001 was pain on infusion (33.3%). The main adverse event related to the combination was pseudoprogression (larger tumors in symptomatically improved patients) (25%). The most common immune-related treatment-emergent AEs were pneumonitis (8.3%), and hypothyroidism (8.3%). Objective response rate at 12 weeks was 25% and the disease control rate (DCR) consisting of > SD was 67% by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. 25% of the patients progressed on the combination. Conclusions: RRx-001 + nivolumab was well-tolerated with preliminary evidence of anti-cancer activity in non-immunogenic cancers. Further analyses with a larger sample size are required to confirm activity. Clinical trial information: NCT02518958 .

Results of a completed first-in-human phase Ib dose-escalation study of oral CBL0137 in patients with advanced solid tumors.

3607 Background: Curaxin CBL0137 is a novel compound with broad anticancer activity in animal models. The drug is a non-genotoxic DNA intercalator that interferes with histone/DNA binding causing decondensation of chromatin in tumor cells, functional inactivation of histone chaperone FACT, activation of p53 and IFN responses, and inhibition of pro-cancer transcriptional factors, MYC, NF-kB, HSF1, and HIF1a. Methods: The study enrolled adults with advanced chemorefractory solid tumors, ECOG PS ≤2, and adequate organ function. The primary objective was to find the maximum tolerated dose (MTD) and recommended dosing regimen (RDR). Secondary objectives were to evaluate CBL0137 safety, pharmacokinetics, and efficacy. CBL1037 was given orally once daily (QD) for the first 14 days of repeated 28-day cycles. A 3+3 dose escalation determined the MTD, defined as the highest dose at which ≤1 of 6 pts had Cycle 1 dose-limiting toxicity (DLT). Pharmacokinetics were assessed on Days 1 and 13. Efficacy was evaluated every 8 weeks. Results: 60 pts were enrolled (females/males [n]: 42/18; median [range] age 56 [25-76] years; ECOG PS [n] 0/1/2: 8/49/3); cancer types [n]: ovarian cancer [15], colorectal cancer [14], breast cancer [11], others [20]) over 16 dose levels ranging from 4 mg to 200 mg QD. Durations of therapy ranged from 6 to 342 days. Three DLTs were observed: prolongation of QTc Gr 3 (88 mg QD), neutropenia/thrombocytopenia Gr 4 (200 mg QD), and LV dysfunction Gr 3 (200 mg QD). Dose-dependent nausea/vomiting was observed and was Gr 2-4 at 200 mg QD. Gr 1/2 photosensitization occurred in 11 subjects across doses from 48 to 200 mg QD but was successfully managed with sun protection and resulted in no dose modifications or discontinuations. On Day 1, mean (range) plasma CBL0137 Tmax values were 5.1 (1-10) hrs. Generally linear increases in AUC occurred with increasing CBL0137 dose. Mean (range) t1/2 values were 25.6 (0.3-166) hrs, with minor dose dependency. Mean (range) Day 13/Day 1 Ctrough ratios showed 3.6 (1.7-7.2)-fold accumulations. Disease control was registered in 11 pts who had stable disease (SD). Target lesion regressions up to 21% were documented in 4 patients with breast cancer (2), sarcoma (1), and ovarian cancer (1). Pts with breast cancer (1) and sarcoma (1) had SD for > 36 weeks. Conclusions: The Phase 2 RDR for oral CBL0137 was established as 180 mg QD x 14 days in 28-day cycles based on bone marrow and gastrointestinal DLTs at 200 mg QD. CBL0137 showed a manageable safety profile with efficacy signals. Further study as a component of combinations is planned. Clinical trial information: 847 .

Results of a completed phase I trial of CBL0137 administered intravenously (IV) to patients (Pts) with advanced solid tumors.

3583 Background: The novel curaxin CBL0137 intercalates into DNA, interfering with histone/DNA binding. Consequent trapping of histone chaperone FACT leads to MYC, NF-kB, and HSF1 inhibition, p53 activation, and an IFN response. CBL0137 shows broad nonclinical antitumor activity (Gasparian et al. Sci Transl Med. 2011; 3(95):95ra74). Methods: This dose-ranging study assessed the CBL0137 maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D) and CBL0137 safety, pharmacokinetics (PK), and efficacy in adults with advanced treatment-refractory solid tumors. CBL0137 was administered IV on Days 1, 8, and 15 of repeated 28-day cycles until progressive disease (PD) or unacceptable toxicity. Doses were escalated using a 3+3 design based on Cycle 1 dose-limiting toxicities (DLTs). PK was assessed through 168 hours after Day 1. Efficacy was evaluated every 8 weeks. Results: The study enrolled 83 pts (M/F [n] = 49/34; median [range] age = 64 [33-85] years; ECOG status [n] = 1/2 [32/51]), with cancer types (n) of colorectal (23), prostate (7), glioblastoma (6), liver (6), non-small-cell (5), and others (36) across 17 dose levels from 10 to 700 mg/m2/infusion. Durations of therapy ranged to 24 months. Cycle 1 DLTs (n type) were observed at 240 mg/m2 (1 Gr 3 photosensitivity), 400 mg/m2 (1 Gr 3 anemia), 700 mg/m2 (1 Gr 4 thrombocytopenia, 1 Gr 4 neutropenia/Gr 4 thrombocytopenia), and 650 mg/m2 (1 Gr 3 thrombocytopenia, 1 Gr 4 neutropenia/Gr 3 thrombocytopenia). Nausea and vomiting were successfully prevented with dexamethasone/serotonin antagonists. Photosensitization was effectively managed with sun protection. Peripheral venous thrombosis required central vein infusion in subjects with glioblastoma. PK showed dose-proportional increases in plasma CBL0137 area under the concentration-time curve (AUC), a high mean (range) volume of distribution (Vd) of 1,030 (655-1460) L/m2 consistent with extensive tissue distribution and DNA intercalation, and an average mean (range) half-life (t1/2) of 24.7 (10.3-40.7) hours without dose dependence. The best response was stable disease: 2 pts with liver cancer had tumor control for 9 and 24 months and a maximum tumor regression of 10%; 2 pts with prostate cancer had tumor regressions by 11% and 22%; 1 pt with uterine cancer had a 20% tumor regression. Conclusions: CBL0137 administered IV was generally well tolerated with manageable toxicities The MTD and RP2D were estimated at 540 mg/m2 due to myelosuppressive DLTs. PK were predictable. Preliminary evidence of antitumor activity supports Phase 2 testing. Clinical trial information: NCT01905228 .

Structural and Biophysical Insights into the Function of the Intrinsically Disordered Myc Oncoprotein.

Myc is a transcription factor driving growth and proliferation of cells and involved in the majority of human tumors. Despite a huge body of literature on this critical oncogene, our understanding of the exact molecular determinants and mechanisms that underlie its function is still surprisingly limited. Indubitably though, its crucial and non-redundant role in cancer biology makes it an attractive target. However, achieving successful clinical Myc inhibition has proven challenging so far, as this nuclear protein is an intrinsically disordered polypeptide devoid of any classical ligand binding pockets. Indeed, Myc only adopts a (partially) folded structure in some contexts and upon interacting with some protein partners, for instance when dimerizing with MAX to bind DNA. Here, we review the cumulative knowledge on Myc structure and biophysics and discuss the implications for its biological function and the development of improved Myc inhibitors. We focus this biophysical walkthrough mainly on the basic region helix–loop–helix leucine zipper motif (bHLHLZ), as it has been the principal target for inhibitory approaches so far.

Blocking Myc to Treat Cancer: Reflecting on Two Decades of Omomyc.

First designed and published in 1998 as a laboratory tool to study Myc perturbation, Omomyc has come a long way in the past 22 years. This dominant negative has contributed to our understanding of Myc biology when expressed, first, in normal and cancer cells, and later in genetically-engineered mice, and has shown remarkable anti-cancer properties in a wide range of tumor types. The recently described therapeutic effect of purified Omomyc mini-protein—following the surprising discovery of its cell-penetrating capacity—constitutes a paradigm shift. Now, much more than a proof of concept, the most characterized Myc inhibitor to date is advancing in its drug development pipeline, pushing Myc inhibition into the clinic.

MYC Instructs and Maintains Pancreatic Adenocarcinoma Phenotype.

The signature features of pancreatic ductal adenocarcinoma (PDAC) are its fibroinflammatory stroma, poor immune activity, and dismal prognosis. We show that acute activation of Myc in indolent pancreatic intraepithelial neoplasm (PanIN) epithelial cells in vivo is, alone, sufficient to trigger immediate release of instructive signals that together coordinate changes in multiple stromal and immune-cell types and drive transition to pancreatic adenocarcinomas that share all the characteristic stromal features of their spontaneous human counterpart. We also demonstrate that this Myc-driven PDAC switch is completely and immediately reversible: Myc deactivation/inhibition triggers meticulous disassembly of advanced PDAC tumor and stroma and concomitant death of tumor cells. Hence, both the formation and deconstruction of the complex PDAC phenotype are continuously dependent on a single, reversible Myc switch. SIGNIFICANCE: We show that Myc activation in indolent Kras G12D-induced PanIN epithelium acts as an immediate pleiotropic switch, triggering tissue-specific signals that instruct all the diverse signature stromal features of spontaneous human PDAC. Subsequent Myc deactivation or inhibition immediately triggers a program that coordinately disassembles PDAC back to PanIN.See related commentary by English and Sears, p. 495.

Discovery of WD Repeat-Containing Protein 5 (WDR5)-MYC Inhibitors Using Fragment-Based Methods and Structure-Based Design.

The frequent deregulation of MYC and its elevated expression via multiple mechanisms drives cells to a tumorigenic state. Indeed, MYC is overexpressed in up to ∼50% of human cancers and is considered a highly validated anticancer target. Recently, we discovered that WD repeat-containing protein 5 (WDR5) binds to MYC and is a critical cofactor required for the recruitment of MYC to its target genes and reported the first small molecule inhibitors of the WDR5-MYC interaction using structure-based design. These compounds display high binding affinity, but have poor physicochemical properties and are hence not suitable for in vivo studies. Herein, we conducted an NMR-based fragment screening to identify additional chemical matter and, using a structure-based approach, we merged a fragment hit with the previously reported sulfonamide series. Compounds in this series can disrupt the WDR5-MYC interaction in cells, and as a consequence, we observed a reduction of MYC localization to chromatin.

Inhibition of Myc transcriptional activity by a mini-protein based upon Mxd1.

Myc, a transcription factor with oncogenic activity, is upregulated by amplification, translocation, and mutation of the cellular pathways that regulate its stability. Inhibition of the Myc oncogene by various modalities has had limited success. One Myc inhibitor, Omomyc, has limited cellular and invivo activity. Here, we report a mini-protein, referred to as Mad, which is derived from the cellular Myc antagonist Mxd1. Mad localizes to the nucleus in cells and is 10-fold more potent than Omomyc in inhibiting Myc-driven cell proliferation. Similar to Mxd1, Mad also interacts with Max, the binding partner of Myc, and with the nucleolar upstream binding factor. Mad binds to E-Box DNA in the promoters of Myc target genes and represses Myc-mediated transcription to a greater extent than Omomyc. Overall, Mad appears to be more potent than Omomyc both invitro and in cells.

Myc-mediated SDHA acetylation triggers epigenetic regulation of gene expression and tumorigenesis.

The transcriptional role of cMyc (or Myc) in tumorigenesis is well appreciated; however, it remains to be fully established how extensively Myc is involved in the epigenetic regulation of gene expression. Here, we show that by deactivating succinate dehydrogenase complex subunit A (SDHA) via acetylation, Myc triggers a regulatory cascade in cancer cells that leads to H3K4me3 activation and gene expression. We find that Myc facilitates the acetylation-dependent deactivation of SDHA by activating the SKP2-mediated degradation of SIRT3 deacetylase. We further demonstrate that Myc inhibition of SDH-complex activity leads to cellular succinate accumulation, which triggers H3K4me3 activation and tumour-specific gene expression. We demonstrate that acetylated SDHA at Lys 335 contributes to tumour growth in vitro and in vivo, and we confirm increased tumorigenesis in clinical samples. This study illustrates a link between acetylation-dependent SDHA deactivation and Myc-driven epigenetic regulation of gene expression, which is critical for cancer progression.

Estrogen Induces Mammary Ductal Dysplasia via the Upregulation of Myc Expression in a DNA-Repair-Deficient Condition.

SummaryMammary ductal dysplasia is a phenotype observed in precancerous lesions and early-stage breast cancer. However, the mechanism of dysplasia formation remains elusive. Here we show, by establishing a novel dysplasia model system, that estrogen, a female hormone, has the potential to cause mammary ductal dysplasia. We injected estradiol (E2), the most active form of estrogen, daily into scid mice with a defect in non-homologous end joining repair and observed dysplasia formation with cell proliferation at day 30. The protooncogene Myc is a downstream target of estrogen signaling, and we found that its expression is augmented in mammary epithelial cells in this dysplasia model. Treatment with a Myc inhibitor reduced E2-induced dysplasia formation. Moreover, we found that isoflavones inhibited E2-induced dysplasia formation. Our dysplasia model system provides insights into the mechanistic understanding of breast tumorigenesis and the development of breast cancer prevention.

Nanotherapy delivery of c-myc inhibitor targets Protumor Macrophages and preserves Antitumor Macrophages in Breast Cancer.

Tumor-associated macrophages (TAMs) enhance tumor growth in mice and are correlated with a worse prognosis for breast cancer patients. While early therapies sought to deplete all macrophages, current therapeutics aim to reprogram pro-tumor macrophages (M2) and preserve those necessary for anti-tumor immune responses (M1). Recent studies have shown that c-MYC (MYC) is induced in M2 macrophages in vitro and in vivo where it regulates the expression of tumor-promoting genes. In a myeloid lineage MYC KO mouse model, MYC had important roles in macrophage maturation and function leading to reduced tumor growth. We therefore hypothesized that targeted delivery of a MYC inhibitor to established M2 TAMs could reduce polarization toward an M2 phenotype in breast cancer models.Methods: In this study, we developed a MYC inhibitor prodrug (MI3-PD) for encapsulation within perfluorocarbon nanoparticles, which can deliver drugs directly to the cytosol of the target cell through a phagocytosis independent mechanism. We have previously shown that M2-like TAMs express significant levels of the vitronectin receptor, integrin β3, and in vivo targeting and therapeutic potential was evaluated using αvβ3 integrin targeted rhodamine-labeled nanoparticles (NP) or integrin αvβ3-MI3-PD nanoparticles.Results: We observed that rhodamine, delivered by αvβ3-rhodamine NP, was incorporated into M2 tumor promoting macrophages through both phagocytosis-independent and dependent mechanisms, while NP uptake in tumor suppressing M1 macrophages was almost exclusively through phagocytosis. In a mouse model of breast cancer (4T1-GFP-FL), M2-like TAMs were significantly reduced with αvβ3-MI3-PD NP treatment. To validate this effect was independent of drug delivery to tumor cells and was specific to the MYC inhibitor, mice with integrin β3 knock out tumors (PyMT-Bo1 β3KO) were treated with αvβ3-NP or αvβ3-MI3-PD NP. M2 macrophages were significantly reduced with αvβ3-MI3-PD nanoparticle therapy but not αvβ3-NP treatment.Conclusion: These data suggest αvβ3-NP-mediated drug delivery of a c-MYC inhibitor can reduce protumor M2-like macrophages while preserving antitumor M1-like macrophages in breast cancer.

Myc is a prognostic biomarker and potential therapeutic target in osteosarcoma.

Background:Over the past four decades, outcomes for osteosarcoma patients have plateaued as there have been few emerging therapies showing clinical results. Thus, the identification of novel biomarkers and therapeutic strategies are urgently needed to address these primary obstacles in patient care. Although the Myc-oncogene has known roles in oncogenesis and cancer cell growth, its expression and function in osteosarcoma are largely unknown.Methods:Expression of Myc was determined by Western blotting of osteosarcoma cell lines and patient tissues, and by immunohistochemistry of a unique osteosarcoma tissue microarray (TMA) constructed from 70 patient samples with extensive follow-up data. Myc specific siRNA and inhibitor 10058-F4 were applied to examine the effect of Myc inhibition on osteosarcoma cell proliferation. The clonogenicity and migration activity was determined by clonogenic and wound-healing assays. A mimic in vivo assay, three-dimensional (3D) cell culture model, was performed to further validate the effect of Myc inhibition on osteosarcoma cell tumorigenic markers.Results:Myc was significantly overexpressed in human osteosarcoma cell lines compared with normal human osteoblasts, and also highly expressed in fresh osteosarcoma tissues. Higher Myc expression correlated significantly with metastasis and poor prognosis. Through the addition of Myc specific siRNA and inhibitor, we significantly reduced Myc protein expression, resulting in decreased osteosarcoma cell proliferation. Inhibition of Myc also suppressed the migration, clonogenicity, and spheroid growth of osteosarcoma cells.Conclusion:Our results support Myc as an emerging prognostic biomarker and therapeutic target in osteosarcoma therapy.

Prevention of Colorectal Carcinogenesis by DNA-Binding Small-Molecule Curaxin CBL0137 Involves Suppression of Wnt Signaling.

Chemoprevention is considered a valid approach to reduce the incidence of colorectal cancer, one of the most common malignancies worldwide. Here, we investigated the tumor-preventive activity of curaxin CBL0137. This compound represents a new class of nonmutagenic DNA-binding small molecules that alter chromatin stability and inhibit the function of the histone chaperone FACT. Among downstream effects of CBL0137 treatment are activation of p53 and type I interferons and inhibition of NFκB, HSF1, and MYC. In addition, our data show that in both human and mouse colorectal cancer cells in vitro, CBL0137 inhibits the APC/WNT/β-catenin signaling pathway, which plays a key role in colon carcinogenesis. Using quantitative RT-PCR and microarray hybridization, we have demonstrated decreased expression of multiple components and downstream targets of the WNT pathway in colon cancer cells treated with CBL0137. At the same time, CBL0137 induced expression of WNT antagonists. Inhibition of WNT signaling activity by CBL0137 was also confirmed by luciferase reporter assay. Tumor-preventive activity of CBL0137 in vivo was tested in a murine model of colorectal carcinogenesis induced by 1,2-dimethylhydrazine (DMH), which is known to involve WNT pathway dysregulation. After DMH subcutaneous treatment, mice were administered CBL0137 in drinking water. Efficacy of CBL0137 in suppressing development of colorectal cancer in this model was evidenced by reduced incidence of adenocarcinomas and adenomas in both males and females and decrease in tumor multiplicity. These data support the prospective use of CBL0137 in chemoprevention of colorectal cancer as well as of other malignances associated with activated WNT signaling.