Proteasomal Degradation Of c-Myc Research Articles

SET facilitates immune escape of microsatellite stability colorectal cancer by inhibiting c-Myc degradation.

Microsatellite stability (MSS) colorectal cancer (CRC) exhibits a low mutation load and poor immunogenicity, contributing to immune escape of tumor cells and less benefit from immune checkpoint blockade (ICB) treatment. The mechanisms underlying immunotherapeutic resistance in MSS CRC remain to be elucidated. Here, we identified that nuclear proto-oncogene SET is significantly higher expressed in MSS CRC compared to microsatellite instability (MSI) CRC and facilitates immune escape of MSS CRC. Mechanistically, SET represses the expression of C-C motif chemokine ligand 5 (CCL5) and upregulates mismatch repair (MMR) proteins expression in a c-Myc-dependent manner, which inhibits infiltration and migration of CD8+ T cells to tumor tissues and results in low immunogenicity in MSS CRC. In addition, we found that SET impairs ubiquitination and proteasomal degradation of c-Myc by disrupting the interaction between E3 ligase FBXW7 and c-Myc. Moreover, SET inhibition enhances the response to immunotherapy in MSS CRC invivo. Overall, this study reveals the critical roles and posttranslational regulatory mechanism of SET in immune escape and highlights the SET/c-Myc axis as a potential target for immunotherapy of MSS CRC that have implications for targeting a unique aspect of this disease.

ELP3 stabilizes c-Myc to promote tumorigenesis.

ELP3, the catalytic subunit of the Elongator complex, is an acetyltransferase and associated with tumor progression. However, the detail of ELP3 oncogenic function remains largely unclear. Here, we found that ELP3 stabilizes c-Myc to promote tumorigenesis in an acetyltransferase-independent manner. Mechanistically, ELP3 competes with the E3-ligase FBXW7β for c-Myc binding, resulting in the inhibition of FBXW7β-mediated ubiquitination and proteasomal degradation of c-Myc. ELP3 knockdown diminishes glycolysis and glutaminolysis and dramatically retards cell proliferation and xenograft growth by downregulating c-Myc, and such effects are rescued by the reconstitution of c-Myc expression. Moreover, ELP3 and c-Myc were found overexpressed with a positive correlation in colorectal cancer and hepatocellular carcinoma. Taken together, we elucidate a new function of ELP3 in promoting tumorigenesis by stabilizing c-Myc, suggesting that inhibition of ELP3 is a potential strategy for treating c-Myc-driven carcinomas.

PL02.1.A DELINEATING AND TARGETING A NOVEL METABOLISM-BASED POST-TRANSLATIONAL MECHANISM REGULATING THE ABUNDANCE OF THE ‘UNDRUGGABLE’ ONCOPROTEIN C-MYC IN MEDULLOBLASTOMA

Abstract BACKGROUND Brain tumors are the leading cause of cancer death in children, and medulloblastoma (MB) is one of the most common pediatric central nervous system malignancies. Amplification of the c-MYC oncogene is frequently observed in the most aggressive and lethal subgroup of this disease, group 3 (G3), but not in other subgroups. Patients that have G3 MB tumors with high c-MYC abundance are more likely to present as metastatic and are prone to develop fatal recurrent tumors. Unfortunately, the functional ubiquity and disordered structure of c-MYC makes it difficult to target for cancer treatment. Therefore, it is critical to identify novel, out-of-the-box strategies to suppress oncogenic c-MYC in highly aggressive G3 MB brain tumors. Recently, metabolism has emerged as a major regulator of overall cellular signaling processes through post-translational and epigenetic mechanisms. While c-MYC is known to regulate cellular metabolism, whether metabolism plays a role in reciprocally supporting enhanced c-MYC abundance in cancer is unknown. We hypothesize that an intrinsic feedback mechanism may exist where metabolic activity modulates c-MYC abundance that could be exploited as a therapeutic strategy to improve outcomes for G3 MB patients. MATERIAL AND METHODS Using various well-characterized G3 MB cells, patient-derived G3 MB cells, orthotopic intracerebellar xenograft models, patient tumor bioinformatics, detailed biochemical characterization, and point-mutation analyses, we have identified a novel metabolism-dependent post-translational modification that regulates c-MYC stability in G3 MB. RESULTS In-depth molecular analyses unveiled that c-MYC is susceptible to oxidation and proteasomal degradation under conditions of metabolic stress. Targeting mitochondrial respiration via inhibition of complex-I promotes the accumulation of reactive oxygen species (ROS) and leads to rapid cysteine oxidation and proteasomal degradation of c-MYC in G3 MB cells, but not normal human brain astrocytes or neural stem cells (NSCs). Point mutation analysis combined with biochemical oxidation assays identified the specific cysteine residues of c-MYC that are susceptible to oxidation and ultimately responsible for enhanced c-MYC degradation following complex-I inhibition. Importantly, oral administration of a blood-brain barrier permeable complex-I inhibitor impaired the growth of intracerebellar MB xenograft tumors in mice, significantly prolonging animal survival. CONCLUSION Altogether, these findings unveil a novel mechanism through which metabolism regulates the post-translational stability of c-MYC and provides insights for designing rationale therapeutic strategies for the treatment of MB patients. SUPPORT This work is supported by a project grant from the Canadian Institutes of Health Research (CIHR).

Abstract 3338: Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1

Abstract BACKGROUND: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients, with poor patient outcomes. FLT3 inhibitors are in clinical use, but with incomplete responses and onset of resistance. New targeting strategies are needed. The oncogenic serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD, directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop. We previously showed that concurrent treatment of FLT3-ITD cells with Pim kinase and FLT3 inhibitors enhanced apoptosis induction through post-translational downregulation of the master transcription factor c-Myc and the anti-apoptotic protein Mcl-1. The serine/threonine kinase GSK-3β regulates c-Myc and Mcl-1 protein via phosphorylation at T58 and S159, respectively; we hypothesized downregulation of both proteins by combination treatment through phosphorylation by GSK-3β. METHODS: FLT3-ITD cells lines (Ba/F3-ITD, MV4-11, and MOLM-14) and primary FLT3-ITD AML patient blasts were cultured with the pan-Pim kinase inhibitor AZD1208, the FLT3 inhibitors gilteritinib or quizartinib and the GSK-3β inhibitor TCG-24. Protein expression was measured by immunoblotting. To measure protein half-lives, cells were pretreated with cycloheximide with and without the proteasome inhibitor MG-132. Ba/F3-ITD cells were infected with Myc-T58A plasmid, preventing c-Myc T58 phosphorylation, or Mcl-1 S159A plasmid, preventing Mcl-1 S159 phosphorylation, or empty vector controls. Apoptosis was measured by Annexin V labeling, measured by flow cytometry. RESULTS: Combined treatment of FLT3-ITD-expressing cell lines and AML patient blasts with Pim and FLT3 inhibitors caused rapid downregulation of c-Myc, and then Mcl-1, protein expression, relative to single drugs. Combined Pim and FLT3 inhibitor treatment caused marked decrease in half-lives of both proteins, which was rescued by proteasome inhibition, as the mechanism of protein downregulation. Combined Pim and FLT3 inhibitor treatment rapidly activated (dephosphorylated) GSK-3β and culture with GSK-3β inhibitor prevented induction of c-Myc and Mcl-1 downregulation, increased c-Myc and Mcl-1 protein turnover by concurrent Pim and FLT3 inhibitor treatment. Finally, combination treatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids did not downregulate these proteins or increase their turnover, with reduced apoptosis induction. CONCLUSION: Pim inhibitors enhance efficacy of FLT3 inhibitors via GSK-3β activation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1, highlighting GSK-3β as a key target in cells with FLT3-ITD. Citation Format: Jonelle Lee, Mario Scarpa, Aditi Chatterjee, Mooath Mustafa Ali, Prerna Singh, Shivani Kapoor, Rossana Trotta, Maria Baer. Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3338.

Pim Kinase Inhibitor Enhances FLT3 Inhibitor Efficacy through GSK-3β Activation and GSK-3β-Mediated Proteasomal Degradation of c-Myc

BACKGROUNDInternal tandem duplication in the fms-like tyrosine kinase 3 receptor tyrosine kinase (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients, associated with poor treatment outcomes due to rapid relapse. FLT3 inhibitors are used in the clinic, but with incomplete efficacy and development of resistance. Further treatment options are needed. The serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD; it directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual targeting of Pim-1 and FLT3 is a promising treatment strategy. The c-Myc transcription factor contributes to dysregulation of cell growth and apoptosis in cancers, including AML. In addition to transcriptional regulation, c-Myc is regulated post-translationally by T58 phosphorylation by the serine/threonine kinase glycogen synthase kinase-3- β (GSK-3β). Here we show that concurrent treatment of cells with FLT3-ITD with Pim and FLT3 inhibitors activates GSK-3β, which phosphorylates and post-translationally downregulates c-Myc.METHODSBa/F3-ITD and MV4-11 cells, with FLT3-ITD, and FLT3-ITD AML patient blasts were cultured with the pan-Pim inhibitor AZD1208 (1 μM) and/or the FLT3 inhibitors gilteritinib or quizartinib (15 nM, 1 nM), with and without the GSK-3β inhibitor TCG-24 (20 μM). c-Myc, p-GSK3-α/β (S21/9) and GSK3-α/β protein expression was measured by immunoblotting. c-Myc mRNA was measured by qPCR. Cells were also cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM) to measure protein half-life and proteasomal degradation. To study the role of c-Myc overexpression and activation, Ba/F3-ITD cells were infected with retroviral estrogen receptor (ER)-Myc plasmid, causing c-Myc nuclear translocation when activated by 4-hydroxytamoxifen (4-OHT; 300 nM). To study the role of c-Myc phosphorylation at T58, Ba/F3-ITD cells were infected with MycT58A plasmid, preventing c-Myc phosphorylation at T58. Apoptosis was detected by Annexin V and propidium iodide staining, measured by flow cytometry.RESULTSTreatment with Pim inhibitor AZD1208 and FLT3 inhibitor gilteritinib or quizartinib combination rapidly downregulated c-Myc protein expression in Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and in primary FLT3-ITD AML patient blasts, compared to quizartinib or gilteritinib alone. Pim inhibitor and FLT3 inhibitor combination treatment did not decrease c-Myc mRNA levels, but markedly decreased c-Myc protein half-life, from 36 mins without drugs and 24 mins with gilteritinib to 18 mins with combination. Half-life did not decrease when cells were pre-treated with the proteasome inhibitor MG-132, consistent with post-translational downregulation through proteasomal degradation. Apoptosis induction by Pim inhibitor and FLT3 inhibitor combination decreased by more than 50% in Ba/F3-ITD cells infected with ER-Myc plasmid and treated with 4-OHT, demonstrating the major role of c-Myc downregulation in apoptosis induction by combination treatment. GSK-3b is inactivated by phosphorylation, and combination treatment rapidly decreased p-GSK-3b levels, while total GSK-3b levels were unchanged, indicating activation of GSK-3b. Treatment of cells with FLT3-ITD with the GSK-3b inhibitor TCG-24 in addition to Pim and FLT3 inhibitors abrogated c-Myc protein downregulation, demonstrating that Pim and FLT3 inhibitor combination downregulates c-Myc through activation of GSK-3b. Finally, Pim and FLT3 inhibitor combination treatment did not downregulate c-Myc in Ba/F3-ITD cells transfected with c-Myc T58A, preventing c-Myc phosphorylation at T58, showing that c-Myc phosphorylation at T58 is necessary for its downregulation by combination treatment.CONCLUSIONSConcurrent treatment of cells with FLT3-ITD with Pim kinase inhibitor enhances the efficacy of FLT3 inhibitors through activation of GSK-3β and GSK-3β-mediated phosphorylation of c-Myc at T58, with resulting c-Myc downregulation through increased proteasomal degradation. This work and previous work in our laboratory on PP2A activating drugs and FLT3 inhibitor combination (Mol Cancer Ther 20:676, 2021) support GSK-3β activation as a mechanism for enhancing efficacy of FLT3 inhibitors in AML with FLT3-ITD. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1225: LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of cancer

Abstract Women have a lower incidence of and improved clinical outcomes for most cancer types, but the mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that many cancer types lack classic nuclear estrogen receptors. The GPER agonist G-1, a racemate, has been demonstrated to have anticancer activities by many independent laboratories, though there are some contradictory findings in the literature. We resolved the enantiomers that compose G-1 and determined that LNS8801 is the sole enantiomer responsible for G-1's anticancer effects. Using CRISPR to deplete GPER, we determined that the anticancer activity of LNS8801 is dependent upon GPER expression within the tumor cells. Previously, we determined that the anticancer mechanism of GPER activation is through a rapid proteasomal degradation of c-Myc. The PRISM screen was performed on LNS8801 at the Broad Institute, and it was determined that this compound has universal anticancer activity across multiple cancer types, consistent with our inhouse data. Sensitivity to LNS8801 was highly correlated with expression of c-Myc target genes, which is consistent with the known mechanism of action. Lineage analysis identified two specific cancer types that were uniformly more sensitive to LNS8801 than other cancer types: diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL). DLBCL has historically been considered to be highly dependent on c-Myc, which is consistent with the known mechanism of action of LNS8801; however, the identification of ALCL was unexpected. Deeper analysis determined that high expression of anaplastic lymphoma kinase (ALK) fusion genes tightly correlated with sensitivity to LNS8801 in ALCL and other ALK+ cancer types. Using non-small cell lung cancer cell lines engineered to express EML4-ALK, we validated that ALK fusion expression results in increased sensitivity to LNS8801 relative to isogenic controls. This work has identified an enantiomerically pure GPER agonist that has potent activity across many cancer types, with c-Myc-dependent and ALK+ cancer types being enriched for heightened sensitivity. Citation Format: Chris Natale, Tina Garyantes, Todd Ridky. LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1225.

BIOM-60. BIOMARKER EVALUATION FOR IMIPRIDONE ONC206 REVEALS ClpP, ATF4, MYC, EGFR and HIF1 AS KEY PREDICTORS OF ANTI-CANCER EFFICACY

Abstract ONC206 is a DRD2 antagonist and ClpP agonist that is a chemical derivative of ONC201, which is in Phase II clinical trials for H3 K27M-mutant glioma. We have previously reported that dopamine receptor expression correlates with ONC201 and ONC206 efficacy. Here, we evaluated additional predictive biomarkers for both agents in the GDSC panel using RNA-seq expression data. ClpP emerged as a strong predictor of efficacy for ONC206 (IC50: p = 1.83E-4, AUC: 9.92E-13). ClpP activation enhances degradation of its substrates, including electron transport chain members responsible for oxidative phosphorylation (OXPHOS). Imipridones activate the integrated stress response involving ATF4 and cause proteasomal degradation of c-myc, which also reduces OXPHOS. Accordingly, high ATF4 (IC50: p = 4.92E-5, AUC: p=2.84E-9) and elevated c-myc correlated with ONC206 efficacy (IC50: p = 6.42E-6, AUC: 3.31E-12). EGFR expression is inversely correlate with DRD2 expression in glioma and its activation is associated with glycolysis. Low EGFR expression correlated with increased ONC206 efficacy (IC50: p = 4.32E-7, AUC: p = 6.44E-20). Loss of HIF1 shunts cellular metabolism toward OXPHOS. Low HIF1 expression correlated with increased anticancer efficacy for ONC206 (IC50: p = 1.96E-3, AUC: p = 1.56E-11). Expression of each of the five markers was significantly different in cell lines that achieved an IC90 with ONC206 (~10%) versus those that did not. A similar analysis for ONC201 revealed that ClpP, MYC and EGFR are more predictive of efficacy relative to ATF4 and HIF1. Combinatorial biomarker analyses revealed MYC/EGFR as the most significant predictor of IC50 for both agents. ClpP/MYC and ClpP/HIF1 were the most significant predictors of AUC for ONC201 and ONC206, respectively. Ongoing studies are further investigating tumor type enrichment of biomarkers. Prediction of innate imipridone sensitivity using biomarkers identified in this study may guide patient and tumor type selection in clinical trials.

Abstract 3173: Predictive biomarker evaluation for the anti-cancer imipridone ONC201

Abstract Small molecule imipridone ONC201 is a selective, mixed competitive and non-competitive antagonist of dopamine receptor D2 (DRD2), a GPCR that is overexpressed in a broad range of malignancies (Madhukar et al, Nature Communications, 2019). ONC201 was recently found to also act as an allosteric agonist of the mitochondrial protease ClpP that degrades members of the electron transport chain and other mitochondrial substrates (Ishizawa et al, Cancer Cell 2019). ONC201 is currently being tested in several Phase II clinical trials with evidence of clinical activity in DRD2-dysregulated tumor types, including high-grade glioma, endometrial cancer, mantle cell lymphoma and adrenal tumors. Previously, we reported that DRD2 and DRD5 expression correlates with ONC201 efficacy in the NCI60 and GDSC panels (>1000 cancer cell lines) (Prabhu et al, Clinical Cancer Research, 2019). In this study, we explored additional predictive biomarkers of response for ONC201, including ClpP and known downstream effects of ONC201 across the NCI60 and GDSC cancer cell line panels. Elevated ClpP expression emerged as a top predictive biomarker for ONC201 anti-cancer efficacy (low IC50) in the NCI60 and GDSC (p = 0.0066 and p = 0.00071, respectively) panels. Upregulation of HIF1 expression is known to drive cancer cells toward glycolysis; conversely, its loss shunts cellular energetics toward oxidative phosphorylation as the primary mechanism of ATP generation (Clark et al, Cell, 2019) that is inhibited by ONC201 via ClpP activation. Accordingly, decreased HIF1a expression correlated with increased anti-cancer efficacy in the NCI60 (p = 0.058) and GDSC (p = 0.0021) panels. ONC201 is known to cause downstream activation of the integrated stress response pathway, triggering induction of ATF4 (Ishizawa et al, Science signaling, 2016). High baseline ATF4 expression correlated with lower ONC201 IC50s in the GDSC panel (p = 0.027). Proteasomal degradation of c-myc by ONC201 has been reported in association with DRD2 antagonism and inhibition of oxidative phosphorylation in GBM (Ishida et al Clinical Cancer Research, 2018). High c-myc expression correlated with lower ONC201 IC50s in the GDSC panel (p = 2.5e-6). Ongoing studies are investigating biomarker combinations and identifying tumor types that are enriched for these predictive biomarkers. In conclusion, a series of target and downstream signaling biomarkers can be used to predict innate ONC201 tumor cell sensitivity that may provide guidance for patient and tumor type selection in future ONC201 clinical trials. Citation Format: Sara Morrow, Varun Vijay Prabhu, Joshua E. Allen, Wolfgang Oster. Predictive biomarker evaluation for the anti-cancer imipridone ONC201 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3173.

Abstract C052: FGF trapping impairs multiple myeloma growth through c-Myc degradation-induced mitochondrial oxidative stress

Abstract Multiple myeloma (MM) remains incurable because of chemotherapeutic resistance. Fibroblast growth factors (FGF) play a pivotal role in MM by acting as an autocrine/paracrine mitogen on plasma cells, bone marrow-derived endothelial cells and fibroblasts. Also, a recurrent chromosomal translocation t(4;14) is associated with FGFR3 upregulation in MM patients. Thus, agents able to hamper FGF signaling may represent a novel approach for MM treatment. Recently we have identified the PTX3-derived small molecule NSC12 as the first orally active antitumor pan-FGF trap. Here, the role of FGF/FGFR system in MM cell survival and the therapeutic potential of NSC12 were investigated. In vitro effects of NSC12 were tested on four human MM cell lines harboring (KMS-11 and OPM-2 cells) or not (U-266 and RPMI8226 cells) the t(4:14) translocation and on patient-derived MM cells (N = 26). In vivo effects of FGF blockade were investigated using subcutaneous (KMS-11 and RPMI8226 cells) and systemic (MM-1S cells) xenografts grown in NOD/SCID mice. In addition, gene expression profiling (GEP) and gene set enrichment analyses (GSEA) were performed in NSC12-treated MM cells. NSC12 blocked the proliferation of all human MM cell lines tested, causing inhibition of FGFR signaling, downregulation of the anti-apoptotic protein mcl-1 and caspase 3 activation. Cytofluorimetric analysis revealed a significant increase of apoptotic cells as early as 6 hrs after NSC12 treatment, with 100% of cell death after 24 hrs even when MM cells were co-cultured with patient-derived bone marrow stromal cells. In vivo, NSC12 blocked FGFR activation and reduced the growth of subcutaneous xenografts and MM cell dissemination in the BM. GEP and GSEA analyses of NSC12-treated cells revealed the upregulation of oxidative stress-induced genes and the downmodulation of c-Myc targets. Accordingly, NSC12 caused the rapid proteasomal degradation of c-Myc paralleled by GSH depletion, mitochondrial ROS production and depolarization, DNA damage and finally apoptosis in KMS-11 cells but not in KMS-11 cells expressing the undegradable mutated (T58A) form of c-Myc. Interestingly, these findings were confirmed on Bortezomib-resistant MM cells as well as on bone marrow-derived primary MM cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from high-risk MM patients. Our data reveal that FGF blockade triggers MM mitochondrial oxidative stress, DNA damage and apoptotic cell death via the proteasomal degradation of the c-Myc oncoprotein. Altogether, our findings dissect for the first time the mechanism by which the FGF/FGFR system plays a non-redundant role in MM cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for MM patients with poor prognosis and advanced disease stage. Citation Format: Roberto Ronca, Gaia Cristina Ghedini, Federica Maccarinelli, Antonio Sacco, Silvia Laura Locatelli, Eleonora Foglio, Sara Taranto, Elisabetta Grillo, Sara Matarazzo, Riccardo Castelli, Vanessa Desantis, Nadia Cattane, Annamaria Cattaneo, Marco Mor, Carmelo Carlo-Stella, Aldo Maria Roccaro, Marco Presta, Arianna Giacomini. FGF trapping impairs multiple myeloma growth through c-Myc degradation-induced mitochondrial oxidative stress [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C052. doi:10.1158/1535-7163.TARG-19-C052

Abstract 4845: Fraxini and mistletoe lectins inhibit the proliferation of hepatocellular carcinoma by promoting proteasomal degradation of c-Myc

Abstract Mistletoe extracts, including Fraxini, have been used to treat cancer, including hepatocellular carcinoma (HCC), in Europe. However, the mechanism(s) underlying the effect of Fraxini in HCC is(are) inconclusive which hampers its optimal use for the treatment of HCC. We previously reported that Fraxini, and two other mistletoe extracts Iscador Q and M exerted strong antiproliferative and pro-apoptotic activities in Hep3B cells through targeting c-Myc protein. In current study, we examined the efficacy of Fraxini in HCC bearing animals and further investigated the mechanism by which Fraxini down-regulates c-Myc protein. How c-Myc was regulated by Fraxini was investigated by measuring the half-life of c-Myc protein with cycloheximide chase assay, proteasomal degradation and phosphorylation of c-Myc. The antitumor efficacy of the Fraxini was tested in the Hep3B mouse xenograft model and Fraxini was administered subcutaneously. We found that Fraxini dose dependently suppressed the c-Myc protein expression, but did not affect c-Myc mRNA level in Hep3B cells, suggesting Fraxini down-regulates c-Myc through post-translational regulation. Fraxini reduced the half-life of c-Myc protein from control (40 minutes) to 27 minutes. Inhibition of 26S proteasome activity using MG-132 in Hep3B cells notably antagonized Fraxini induced down-regulation of c-Myc, suggesting that Fraxini down-regulated c-Myc by promoting its proteasome degradation. Fraxini decreased phosphorylation of S62 (stabilizing c-Myc) while slightly increased p-cMycT58 (destabilized c-Myc) in Hep3B cells. When Burkitt's lymphoma Raji cells, which are known to host T58 mutation on c-Myc and result in c-Myc stabilization, were treated with Fraxini, Fraxini (up to 20 μg/ml) exerted minimum activity in cell growth and c-Myc protein expression, augmenting that Fraxini is acting on c-Myc phosphorylation sites which resulted in down-regulation c-Myc and elicited anticancer activity in HCC. Additionally, Fraxini injected (s.c.) to mice carrying Hep3B cell xenograft tumors for 2 weeks significantly reduced the average tumor volume (71.8 ± 22.2 mm3) compared to that in the control treated mice (179.0 ± 36.3 mm3, p < 0.05) and lead to reduction of c-Myc protein in the tumor tissues. Intriguingly, mistletoe lectin, a bioactive component in Fraxini, appears to be most effective in inhibiting the proliferation of c-Myc expressing Hep3B cells compared to that of PLC/RF5 cells with limited expression of c-Myc. Mistletoe lectin (1 pg/ml) in the meantime reduced almost 90% c-Myc protein expression in Hep3B cells. In conclusion, since c-MYC gene is required for hepatocyte proliferation and liver tumorigenesis, Fraxini and mistletoe lectin through targeting c-Myc may have great therapeutic potential for patients with advanced HCC and warrant further investigation. The study is supported by the start-up fund of UT MDACC. Citation Format: Yan Jiang, Yong Pan, Patrea R. Rhea, Richard Lee, Zhimin Lu, Peiying Yang. Fraxini and mistletoe lectins inhibit the proliferation of hepatocellular carcinoma by promoting proteasomal degradation of c-Myc. [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 4845.

YM155 Suppresses Proliferation and Survival of Multiple Myeloma Cells via Proteasomal Degradation of c-Myc

The overall survival of patients with multiple myeloma has significantly improved over the last decade, mainly due to the use of novel agents such as thalidomide, lenalidomide and bortezomib; however, multiple myeloma remains incurable due to its eventual relapse, necessitating urgent development of newer agents. YM155 is a novel small molecule that suppresses transactivation of survivin and induces apoptosis in various cancer cell types. However, the effects of YM155 on multiple myeloma cells have not been fully elucidated. We herein found that YM155 inhibited proliferation of and induced caspase-dependent apoptosis in myeloma cells, partially via the suppression of survivin, myeloid cell leukemia sequence-1, and X-linked inhibitor of apoptosis protein. Interestingly, YM155 suppressed c-Myc protein expression via the proteasomal degradation. Moreover, YM155 increased F-box and WD-40 domain protein 7α expression that is a component of the Skp, Cullin, F-box-like ubiquitin ligase complex, suggesting that YM155-induced c-Myc down-regulation may be due to the proteasomal degradation through ubiquitination by F-box and WD-40 domain protein 7α. These results warrant further clinical evaluation of YM155 in MM patients.

A Small-Molecule Suppressant of Survivin YM155 Induces Cell Death Via Proteasomal Degradation of c-Myc in Multiple Myeloma Cells

[Background] Survivin is a member of the inhibitor of apoptosis protein (IAP) family with its dual roles in mitosis and apoptosis, and emerges as an attractive target for cancer therapy. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. However, the effect of this agent on multiple myeloma (MM) cells remains unclear. [Materials & Methods] Five human MM cell lines, RPMI8226, U266, KMS20, KMS28PE, and KMS34 were used in this study. Cell proliferation and cell death were evaluated by MTT assay and by flow cytometric analysis with annexin V/PI staining. Gene and protein expressions were analyzed with quantitative PCR (qPCR) and immunoblot, respectively. For proteasome inhibitory assay, cells were treated with YM155 and/or proteasome inhibitor MG132 for 6 hours. [Results] YM155 inhibited cell proliferation of these cells in a dose- and time-dependent manner. Annexin V assay showed that YM155 induced apoptosis in these cells. To better understand these effects of YM155 on MM cells, we evaluated the intracellular signaling and apoptosis-associated protein status. Immunoblot analyses showed that YM155 reduced not only survivin but also Mcl-1 and XIAP expressions. We also observed the activation of caspase-3 and PARP in YM155-treated cells, indicating that YM155 induces caspase-dependent apoptosis. In contrast, YM155 did not affect phosphorylation status of Erk1/2 and STAT3. Interestingly, YM155 suppressed c-Myc and IRF4 protein expressions, both of which are recognized as an important transcription factor in the pathogenesis of MM. In addition, qPCR assay showed that YM155 treatment did not reduce c-Myc mRNA level. On the other hand, proteasome inhibitor prevented the suppression of c-Myc expression by YM155 treatment, indicating a proteasomal degradation of c-Myc by YM155. [Conclusion] YM155 suppresses cell proliferation and survival in MM cells in part via not only inhibiting anti-apoptotic proteins such as survivin, Mcl-1 and XIAP but also suppressing c-Myc oncoprotein expression. Further study is needed to clarify the molecular mechanism of c-Myc degradation induced by YM155. Our results may provide a new concept in c-Myc-targeting therapy for MM because c-Myc oncoprotein have been considered undruggable. DisclosuresIshida:Bristol-Myers Squibb: Honoraria.

Survivin suppressant YM155 induces cell death via proteasomal degradation of c-Myc in multiple myeloma cells

PO-272 Survivin suppressant YM155 induces cell death via proteasomal degradation of c-Myc in multiple myeloma cells M. Asahi, S. Ito, Y. Ishida Hematology & Oncology, Department of Internal Medicine, Department of Medical Oncology, Iwate Medical University School of

Recombinant Human Adenovirus-p53 Injection Induced Apoptosis in Hepatocellular Carcinoma Cell Lines Mediated by p53-Fbxw7 Pathway, Which Controls c-Myc and Cyclin E

F-box and WD repeat domain-containing 7 (Fbxw7/hAgo/hCdc4/Fbw7) is a p53-dependent tumor suppressor and leads to ubiquitination-mediated suppression of several oncoproteins including c-Myc, cyclin E, Notch, c-Jun and others. Our previous study has indicated that low expression of Fbxw7 was negatively correlated with c-Myc, cyclin E and mutant-p53 in hepatocellular carcinoma (HCC) tissues. But the role and mechanisms of Fbxw7 in HCC are still unknown. Here, we investigated the function of Fbxw7 in HCC cell lines and the anti-tumor activity of recombinant human adenovirus-p53 injection (rAd-p53, Gendicine) administration in vitro and in vivo. Fbxw7-specific siRNA enhanced expression of c-Myc and cyclin E proteins and increased proliferation in cell culture. rAd-p53 inhibited tumor cell growth with Fbxw7 upregulation and c-Myc and cyclin E downregulation in vitro and a murine HCC model. This effect could be partially reverted using Fbxw7-specific siRNA. Here, we suggest that the activation of Fbxw7 by adenoviral delivery of p53 leads to increased proteasomal degradation of c-Myc and cyclin E enabling growth arrest and apoptosis. Addressing this pathway, we identified that rAd-p53 could be a potential therapeutic agent for HCC.

The X protein of hepatitis B virus binds to the F box protein Skp2 and inhibits the ubiquitination and proteasomal degradation of c-Myc

The HBx protein of hepatitis B virus is involved in deregulation of cell cycle and development of hepatocellular carcinoma. Since c-Myc also plays an important role in cell proliferation and tumor development, we studied its regulation by HBx in a human hepatoma cell line. Co-expression of HBx and c-Myc resulted in increased stability of intracellular c-Myc. HBx blocked the ubiquitination of Myc through a direct interaction with the F box region of Skp2 and destabilization of the SCF Skp2 complex. We suggest that sustained presence of c-Myc combined with mitogenic activity inherent to HBx may be associated with cell cycle deregulation and transformation.

Glucocorticoids Inhibit the Transcriptional Activity of LEF/TCF in Differentiating Osteoblasts in a Glycogen Synthase Kinase-3β-dependent and -independent Manner

Glucocorticoids, widely used as immune suppressors, cause osteoporosis by inhibiting bone formation. In MC3T3-E1 osteoblast-like cultures, dexamethasone (DEX) activates glycogen synthase kinase-3beta (GSK3beta) and inhibits a differentiation-related cell cycle that occurs at a commitment stage immediately after confluence. Here we show that DEX inhibition of the differentiation-related cell cycle is associated with a decrease in beta-catenin levels and inhibition of LEF/TCF-mediated transcription. These inhibitory activities are no longer observed in the presence of lithium, a GSK3beta inhibitor. DEX decreased the serum-responsive phosphorylation of protein kinase B/Akt-Ser(473) within minutes, and this inhibition was also observed after 12 h. When the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was inhibited by wortmannin, DEX no longer inhibited beta-catenin levels. Furthermore, DEX-mediated inhibition of LEF/TCF transcriptional activity was attenuated in the presence of dominant negative forms of either PI3K or protein kinase B/Akt. These results suggest cross-talk between the PI3K/Akt and Wnt signaling pathways. Consistent with a role for Wnt signaling in the osteoblast differentiation-related cell cycle, wortmannin partially negated the DEX inhibition of this cell cycle. DEX also induced histone deacetylase (HDAC) 1, which is known to inhibit LEF/TCF transcriptional activity. Overexpression of HDAC1 negated the inhibitory effect of DEX on LEF/TCF transcriptional activity. In the presence of trichostatin A, a deacetylase inhibitor, DEX-mediated inhibition of the differentiation-related cell cycle was partially negated. When administered together, wortmannin and trichostatin A completely negated the inhibitory effect of DEX on the differentiation-related cell cycle. These results suggest that inhibition of a PI3K/Akt/GSK3beta/beta-catenin/LEF axis and stimulation of HDAC1 cooperate to mediate the inhibitory effect of DEX on Wnt signaling and the osteoblast differentiation-related cell cycle.