High Levels Of c-Myc Research Articles

Mechanical stress during confined migration causes aberrant mitoses and c-MYC amplification

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.

Abstract 6799: The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy with rapidly acquired chemotherapy resistance. While the inflamed SCLC subtype is associated with a significantly improved response to immunotherapy, the non-inflamed SCLC subtypes, encompassing most SCLC patients, experience only modest survival gains with chemo-immunotherapy. Wnt signaling pathway activation is associated with resistance to chemotherapy and an immune checkpoint inhibitor in several tumors. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers, but the efficacy of TNIK inhibition in SCLC has not previously been researched. We previously demonstrated that single agent TNIK inhibitors are effective in a subset of SCLC. Here, we hypothesize that targeting TNIK will show an anti-tumor effect based on specific biomarkers and also modify the tumor immune environment in SCLC. Methods: We evaluated susceptibility to a small molecule TNIK inhibitor, NCB-0846, in 29 human-derived SCLC cell lines using 96-hour proliferation assays. To investigate efficacy biomarkers, we correlated NCB-0846 IC50 values with proteomic expression (profiled using reverse phase protein array). cMYC and FOXK1 expression was reduced by siRNA. Chemokine concentrations were determined by ELISA. Results: SCLC cell lines with high cMYC levels, were more sensitive to the TNIK inhibitor, NCB-0846 (F.C.=-2.07, p=0.01), while SCLC cell lines with high expression of TTF-1 were more resistant (F.C.=2.48, p<0.01). Knockdown of cMYC conferred resistance to NCB-0846 in cMYC-high cells, while NCB-0846 treatment resulted in decreased cMYC expression in a concentration-dependent manner. Additionally, NCB-0846 treatment decreased the immunosuppressive chemokine CCL2 levels in the supernatant of human-derived SCLC cell lines and GEMM-derived cell lines. The expression of FOXK1, a transcription factor of CCL2, was also reduced in cell lines treated with NCB-0846. Conclusions: These findings show that TNIK inhibition is more effective in cMYC-high SCLC, acting through downregulating cMYC levels. TNIK inhibition also decreases the production of CCL2, an immunosuppressive chemokine implicated in resistance to anti-PD-L1 immunotherapy. Together, these findings highlight the promising potential of TNIK inhibitors in cMYC-high SCLC. Additionally, suppressing CCL2 by TNIK inhibition supports the rationale for combining the TNIK inhibitor and an anti-PD-L1 antibody in the non-inflamed SCLC subtypes. Citation Format: Azusa Tanimoto, Kavya Ramkumar, Allison Stewart, Bingnan Zhang, Robert Cardnell, Shen Li, Qi Wang, Jing Wang, Carl Gay, Lauren Averett Byers. The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer [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 6799.

Abstract A110: Identifying sensitive patient populations for CDK7 inhibitors using cell panel screens and bioinformatic approaches

Abstract Background Dysregulation of cell cycle and transcriptional processes promote tumorigenesis and tumor growth. Due to its dual role in regulating both cellular processes, CDK7 is an attractive therapeutic target, with several CDK7 inhibitors in clinical trials. Preclinical data suggests that such agents could have utility across a range of tumor types particularly those driven by defects in the regulation of cell cycle and transcriptional processes. Consequently, potential patient populations are broad and often lack well-defined patient stratification. To identify specific cancers with greater dependence on CDK7 activity, we employed the use of a cell line panel screen coupled with bioinformatic analysis of association of drug sensitivity to molecular features. Materials and Methods To explore tumor sensitivities to CDK7 inhibition we profiled two CDK7 inhibitors (THZ1 and LY3405105; Eli Lilly) and the non-selective kinase inhibitor staurosporine as a control across a panel of 468 human cancer cell lines with varied genetic backgrounds. Activity area was used to define drug responses whilst multi-omics features of cell lines obtained from DepMap were used to define potential molecular features associated with drug response. Differential gene expression between sensitive and resistant cell lines was used to identify expression signatures associated with sensitivity to CDK7 inhibition. Results were validated in vitro by characterisation of cell line models in viability assays, western blots and RT-qPCR. Our findings were further validated in vivo in a NSCLC mouse xenograft model. Results Cell panel data showed a wide range of sensitives to selective CDK7 inhibition (LY3405105) compared to the profile of the less selective THZ1 which had a similar distribution of sensitivity as the control staurosporine. Bioinformatics analysis identified a global c-MYC signature with a significant correlation between CDK7 expression and c-MYC expression across the entire cell panel which confers sensitivity to CDK7 inhibition and was highly correlated in lung cancer. Further analysis of CDK7 inhibitor sensitivity in lung cancer as a function of c-MYC expression found that SCLC and NSCLC had the highest correlation. Using an independent set of cell lines with varying c-MYC levels, we confirmed these findings and the results were further validated in vivo where we observed partial tumor regressions. Conclusions Cell panel data coupled to bioinformatic analysis was used to define potentially sensitive patient populations for CDK7 inhibitors. This work demonstrates in vitro and in vivo activity of selective CDK7 inhibition in SCLC and NSCLC cancer models with high c-MYC levels. Citation Format: Keisha Hearn, Maria Ahn, Luke Bevan, Jessica Brothwood, Charlotte East, Anna Esteve-Arenys, Lynsey Fazal, Christopher Hindley, Sabrina James, Justyna Kucia-Tran, John Lyons, Joanne Munck, Harpreet Saini, Mathieu Unbekandt, Dhaval Varshney, Steve Wagner, Andrew Woodhead, Nicola Wallis. Identifying sensitive patient populations for CDK7 inhibitors using cell panel screens and bioinformatic approaches [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 A110.

Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias.

The tumor suppressor TP53 is frequently inactivated in a mutation-independent manner in cancers and is reactivated by inhibiting its negative regulators. We here cotarget MDM2 and the nuclear exporter XPO1 to maximize transcriptional activity of p53. MDM2/XPO1 inhibition accumulated nuclear p53 and elicited a 25- to 60-fold increase of its transcriptional targets. TP53 regulates MYC, and MDM2/XPO1 inhibition disrupted the c-MYC-regulated transcriptome, resulting in the synergistic induction of apoptosis in acute myeloid leukemia (AML). Unexpectedly, venetoclax-resistant AMLs express high levels of c-MYC and are vulnerable to MDM2/XPO1 inhibition in vivo. However, AML cells persisting after MDM2/XPO1 inhibition exhibit a quiescence- and stress response-associated phenotype. Venetoclax overcomes that resistance, as shown by single-cell mass cytometry. The triple inhibition of MDM2, XPO1, and BCL2 was highly effective against venetoclax-resistant AML in vivo. Our results propose a novel, highly translatable therapeutic approach leveraging p53 reactivation to overcome nongenetic, stress-adapted venetoclax resistance.

Asparagine Bioavailability Regulates the Translation of MYC oncogene in Lymphoid Malignancies

Non-essential amino acids are becoming increasingly important in cancer metabolism because of their roles beyond protein translation. These include providing precursors for macromolecule biosynthesis, serving as substrates for epigenetic modifications, regulating redox states, and modulating cell signaling. Acute lymphoblastic leukemia (ALL) is one such example where asparagine is shown to be conditionally essential for cancer growth and proliferation. Exploiting this metabolic vulnerability, L-Asparaginase (ASNase) has been used in the clinic to successfully treat ALL patients through depleting the circulating asparagine. Our investigation into the mechanism of resistance to ASNase in ALL cells yielded a striking correlation between asparagine bioavailability and c-MYC protein levels. c-MYC is a key transcription factor and has been shown to regulate the transcription of more than 10% of genes, including many metabolic genes, to meet the demands of tumor growth and progression. In addition, it has a well-documented role in normal hematopoiesis and hematological malignancies, including ALL, where high c-MYC levels have been shown to be a marker for leukemic initiation and growth. Using asparagine auxotrophic ALL cells that do not express asparagine synthetase (ASNS), the asparagine biosynthesis enzyme, we found that c-MYC protein levels were acutely depleted when asparagine was withdrawn from the tissue culture medium. However, c-MYC protein levels were sustained when ASNS cDNA was re-introduced into these cells. These results indicate the possibility of a reciprocal association between metabolism and a critical oncogene MYC. We also discovered that asparagine depletion inhibited the translation of c-MYC without changing its mRNA expression or altering the rate of protein degradation. Mechanistic Target of Rapamycin Complex 1 (mTORC1) and General Controlled Nonderepressible 2 (GCN2) are the two central molecular components involved in amino acid sensing and translation control. Asparagine starvation notably caused a decrease in global protein synthesis, mediated by suppression of mTORC1 and simultaneous activation of GCN2. Our studies using small molecule inhibitors and polysome profiling showed that repression of c-MYC mRNA translation is not due to the activation of the GCN2 pathway or a consequence of global translation suppression. In addition, both the '5' and '3' untranslated regions (UTRs) were not required for this inhibitory effect on c-MYC mRNA translation during asparagine depletion. Our results suggest that while asparagine depletion causes a defect in global translation at the initiation step, its effect on MYC mRNA translation is possibly due to elongation stalling. While ASNase can efficiently deplete circulating asparagine, its clinical applications are limited to ALL due to its low-level expression of ASNS. However, aspartate, a critical substrate for asparagine biosynthesis, can be pharmacologically targeted using electron transport chain (ETC) inhibitors. Our studies in ASNase-resistant ALL and lymphomas showed that while asparagine depletion alone did not affect c-MYC protein expression, co-treatment with ETC inhibitors (phenformin) showed a significant decrease in c-MYC protein levels. We extended this observation to test the synergy of ASNase and phenformin to treat an orthotopic MYC-driven B-cell lymphoma model. While either of the single treatments only modestly reduced the tumor burden, the double treatment showed the most significant reduction in the tumor burden, which correlated with a significant reduction of c-MYC protein expression. These results suggest that perturbing the asparagine biosynthesis pathway can be clinically employed to inhibit c-MYC protein expression and improve therapeutic outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genomic screening reveals ubiquitin-like modifier activating enzyme 1 as a potent and druggable target in c-MYC-high triple negative breast cancer models.

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.

Biosimulation Using the Cellworks Computational Omics Biology Model (CBM) Identifies Genomic and Molecular Markers for Decitabine (DAC) Plus Valproic-Acid (VPA) Treatment Response in Patients with Myelodysplastic Syndromes (MDS)

Biosimulation Using the Cellworks Computational Omics Biology Model (CBM) Identifies Genomic and Molecular Markers for Decitabine (DAC) Plus Valproic-Acid (VPA) Treatment Response in Patients with Myelodysplastic Syndromes (MDS)

CRY2 missense mutations suppress P53 and enhance cell growth

Disruption of circadian rhythms increases the risk of several types of cancer. Mammalian cryptochromes (CRY1 and CRY2) are circadian transcriptional repressors that are related to DNA-repair enzymes. While CRYs lack DNA-repair activity, they modulate the transcriptional response to DNA damage, and CRY2 can promote SKP1 cullin 1-F-box (SCF)FBXL3-mediated ubiquitination of c-MYC and other targets. Here, we characterize five mutations in CRY2 observed in human cancers in The Cancer Genome Atlas. We demonstrate that two orthologous mutations of mouse CRY2 (D325H and S510L) accelerate the growth of primary mouse fibroblasts expressing high levels of c-MYC. Neither mutant affects steady-state levels of overexpressed c-MYC, and they have divergent impacts on circadian rhythms and on the ability of CRY2 to interact with SCFFBXL3 Unexpectedly, stable expression of either CRY2 D325H or of CRY2 S510L robustly suppresses P53 target-gene expression, suggesting that this may be a primary mechanism by which they influence cell growth.

G9a/GLP targeting in MM promotes autophagy-associated apoptosis and boosts proteasome inhibitor–mediated cell death

AbstractMultiple myeloma (MM) is an (epi)genetic highly heterogeneous plasma cell malignancy that remains mostly incurable. Deregulated expression and/or genetic defects in epigenetic-modifying enzymes contribute to high-risk disease and MM progression. Overexpression of the histone methyltransferase G9a was reported in several cancers, including MM, correlating with disease progression, metastasis, and poor prognosis. However, the exact role of G9a and its interaction partner G9a-like protein (GLP) in MM biology and the underlying mechanisms of action remain poorly understood. Here, we report that high G9a RNA levels are associated with a worse disease outcome in newly diagnosed and relapsed MM patients. G9a/GLP targeting using the specific G9a/GLP inhibitors BIX01294 and UNC0638 induces a G1-phase arrest and apoptosis in MM cell lines and reduces primary MM cell viability. Mechanistic studies revealed that G9a/GLP targeting promotes autophagy-associated apoptosis by inactivating the mTOR/4EBP1 pathway and reducing c-MYC levels. Moreover, genes deregulated by G9a/GLP targeting are associated with repressive histone marks. G9a/GLP targeting sensitizes MM cells to the proteasome inhibitors (PIs) bortezomib and carfilzomib, by (further) reducing mTOR signaling and c-MYC levels and activating p-38 and SAPK/JNK signaling. Therapeutic treatment of 5TGM1 mice with BIX01294 delayed in vivo MM tumor growth, and cotreatment with bortezomib resulted in a further reduction in tumor burden and a significantly prolonged survival. In conclusion, we provide evidence that the histone methyltransferases G9a/GLP support MM cell growth and survival by blocking basal autophagy and sustaining high c-MYC levels. G9a/GLP targeting represents a promising strategy to improve PI-based treatment in patients with high G9a/GLP levels.

TORC1/2 kinase inhibition depletes glutathione and synergizes with carboplatin to suppress the growth of MYC-driven medulloblastoma.

Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. An in silico analysis of these cells identified mTOR inhibitors as potential therapeutic agents. We hypothesized that the orally bioavailable TORC1/2 kinase inhibitor TAK228 would have activity against MYC-driven medulloblastoma. TAK228 inhibited mTORC1/2, decreased cell growth and caused apoptosis in high-MYC medulloblastoma cell lines. Comprehensive metabolic profiling of medulloblastoma orthotopic xenografts showed upregulation of glutathione compared to matched normal brain. TAK228 suppressed glutathione production. Because glutathione is required to detoxify platinum-containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin in medulloblastoma. TAK228 synergized with carboplatin to inhibit cell growth and induce apoptosis and extended survival in orthotopic xenografts of high-MYC medulloblastoma. Brain-penetrant TORC1/2 inhibitors and carboplatin may be an effective combination therapy for high-risk medulloblastoma.

Wnt/β-catenin signaling pathway inhibits porcine reproductive and respiratory syndrome virus replication by enhancing the nuclear factor-κB-dependent innate immune response

The Wnt/β-catenin signaling pathway is an evolutionarily highly conserved signaling pathway related to the replication of various viruses. However, the interaction between the Wnt/β-catenin pathway and porcine reproductive and respiratory syndrome virus (PRRSV) is unknown. In the present study, we showed that PRRSV-infected Marc-145 and PAM cells expressed high levels of c-myc and cyclinD1 mRNA and accumulation of β-catenin in the nucleus. PRRSV nonstructural proteins (Nsps) 1α, 1β, 3, 4, 7, 10, and 12, and proteins encoded by open reading frames (ORFs) 2b, 3, and 5 induced the activation of the Wnt pathway according to TOP/FOP luciferase reporter assay. But, Nsp5 inhibited the activation of the Wnt pathway. Pre-treatment with Wnt3a inhibited PRRSV replication in Marc-145 cells in a dose-dependent manner. Over-expression of β-catenin also inhibited PRRSV replication, while silencing of β-catenin by small hairpin RNA increased its replication in Marc-145 cells. Over-expression of β-catenin increased interferon regulatory factor (IRF)-3 expression and nuclear factor (NF)-κB phosphorylation, NF-κB and interferon-stimulated response element promoter activities, and interferon-β, DExD/H-box helicase 58 (DDX58), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-8 mRNA expression. Conversely, silencing β-catenin decreased phosphorylated IRF-3 and NF-κB, NF-κB and IFIT1 promoter activities, and IFN-β, DDX58, IFIT1, IL-1β, TNF-α, and IL-8 mRNA levels in Marc-145 cells. Co-immunoprecipitation and immunofluorescence colocalization analyses confirmed that β-catenin interacted with NF-κB in Marc-145 cells. In conclusion, PRRSV infection activates the Wnt/β-catenin signaling pathway via Nsps 1α, 1β, 3, 4, 7, 10, and 12, and proteins encoded by ORFs 2b, 3, and 5. The Wnt/β-catenin pathway then inhibits PRRSV replication by enhancing the NF-κB-dependent innate immune response. These findings further our understanding of the role of the Wnt/β-catenin signaling pathway in regulating PRRSV replication and provide new insights into virus–host interactions.

The requirement for cyclin E in c-Myc overexpressing breast cancers

ABSTRACT Basal-like triple-negative breast cancers frequently express high levels of c-Myc. This oncoprotein signals to the core cell cycle machinery by impinging on cyclin E. High levels of E-type cyclins (E1 and E2) are often seen in human triple-negative breast tumors. In the current study, we examined the requirement for E-type cyclins in the c-Myc-driven mouse model of breast cancer (MMTV-c-Myc mice). To do so, we crossed cyclin E1- (E1-/-) and E2- (E2-/-) deficient mice with MMTV-c-Myc animals, and observed the resulting cyclin E1-/-/MMTV-c-Myc and cyclin E2-/-/MMTV-c-Myc females for breast cancer incidence. We found that mice lacking cyclins E1 or E2 developed breast cancers like their cyclin Ewild-type counterparts. In contrast, further reduction of the dosage of E-cyclins in cyclin E1-/-E2+/-/MMTV-c-Myc and cyclin E1+/-E2-/-/MMTV-c-Myc animals significantly decreased the incidence of mammary carcinomas, revealing arole for E-cyclins in tumor initiation. We also observed that depletion of E-cyclins in human triple-negative breast cancer cell lines halted cell cycle progression, indicating that E-cyclins are essential for tumor cell proliferation. In contrast, we found that the catalytic partner of E-cyclins, the cyclin-dependent kinase 2 (CDK2), is dispensable for the proliferation of these cells. These results indicate that E-cyclins, but not CDK2, play essential and rate-limiting roles in driving the proliferation of c-Myc overexpressing breast cancer cells.

Genetic Basis of Extramedullary Plasmablastic Transformation of Multiple Myeloma.

In patients with multiple myeloma, plasmablastic transformation in the bone marrow is rare and associated with poor outcomes. The significance of discordant extramedullary plasmablastic transformation in patients with small, mature clonal plasma cells in the bone marrow has not been well studied. Here, we report the clinicopathologic, cytogenetic, and molecular features of 10 such patients (male/female: 6/4, median age: 65 y, range: 48 to 76 y) with an established diagnosis of multiple myeloma in the bone marrow composed of small, mature plasma cells in parallel with a concurrent or subsequent extramedullary plasmablastic transformation. Eight patients with available survival data showed an overall aggressive clinical course with a median survival of 4.5 months after the diagnosis of extramedullary plasmablastic transformation, despite aggressive treatment and even in patients with low-level bone marrow involvement. Pathologically, the extramedullary plasmablastic myeloma were clonally related to the corresponding bone marrow plasma cells, showed high levels of CMYC and/or P53 expression with a high Ki-67 proliferation index by immunohistochemistry and harbored more complex genomic aberrations including frequent mutations in the RAS pathway and MYC rearrangements compared with their bone marrow counterparts. In summary, although genetic and immunohistochemical studies were not uniformly performed on all cases due to the retrospective nature of this study, our data suggest that discordant extramedullary plasmablastic transformation of multiple myeloma has an aggressive clinical course and is characterized by frequent mutations in the RAS pathway and more complex genomic abnormalities.

The Novel Berbamine Derivative, Cbbm, Downregulates c-Myc to Block Diffuse Large B-Cell Lymphoma Cell Maintenance

▪Diffuse large B-cell lymphoma (DLBCL), is one of the most common lymphoma subtypes worldwide, and represents 30% of non-Hodgkin lymphoma (NHL). The current DLBCL chemotherapy R-CHOP regimen is limited by lack of specificity and toxicity to normal hematopoietic cells. Identification of a targeted therapeutic approach is an unmet need for the disease. Through translocations, amplifications or overexpression, c-Myc is dysregulated in many human cancers, including DLBCL. Aberrant overexpression of c-Myc promotes tumorigenesis by activating target genes critical for cell proliferation/survival. In our preliminary study, c-Myc protein levels in 119 cases of newly diagnosed DLBCL were evaluated by immunohistochemistry. We found that c-Myc levels were evident (>30% positive cells) in 40.52% (47/116) and high (>80% positive cells) in 6.90% (8/116) of patients. We also observed that relative to c-Myc negative cases, c-Myc positive cases (>30% positive cells) were associated with adverse prognostic indicators including high international prognostic index (IPI) score (≥60 years old group) (p=0.033), high Ki-67 index (p=0.002), high central nervous system involvement (p=0.003) and low complete response rate (p=0.028), confirming that c-Myc plays a critical role in DLBCL pathogenesis.Currently, c-Myc is considered to be an “undrugable” oncoprotein. Chemotherapeutic agents do not affect c-Myc activity. Our previous work in T-cell lymphoma (Cancer Cell, 32:115-128) demonstrated that berbamine, a natural bis-benzylisoquinoline alkaloid, effectively reduced N-Methyl-N-Nitrosourea (MNU)-induced lymphoma burden by disrupting the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII)γ/c-Myc axis. We also showed that CaMKIIγ stabilizes c-Myc by phosphorylating it at the serine 62 residue (Ser62). We created the berbamine derivative 4-chlorobenzoyl berbamine (CBBM) (Fig.1), and evaluated its efficacy in vitro using DLBCL cell lines (n=5) which expressed high levels of c-Myc. The IC50 of CBBM was an order of magnitude lower than that of berbamine across a panel of DLBCL cell lines: the IC50 (μmol/L) of CBBM vs. berbamine in these cells were OCI-Ly3 (1.39±0.04 vs 12.20±0.71), OCI-Ly10 (2.56±0.04 vs 23.66±0.53), U2932 (3.89±0.07 vs 17.78±0.29), SU-DHL 16 (1.82±0.05 vs 17.56±0.71) and Pfeiffer (3.73±0.06 vs 26.16±0.56). CBBM also induced apoptosis in a dose-dependent manner in these cell lines while sparing normal hematopoietic cells from healthy donors (n=2). As an example, CBBM treatment dose-dependently induced apoptosis on OCI-Ly3 cells (vehicle control was 4.43±0.06%, 4 μmol/L CBBM was 24.43±0.72% and 6 μmol/L CBBM was 75.43±0.25%, vehicle control vs CBBM at 4 μmol/L, p<0.001). Conversely, more than 85% of normal hematopoietic cells from mobilized healthy donor peripheral blood were still viable after exposure of CBBM at 10 μmol/L, which is a lethal dose for DLBCL lines. Moreover, CBBM treatment significantly reduced cycling of DLBCL cells (SG2M fractions: vehicle control 68.11±0.40%, CBBM 4 μmol/L 38.36±0.34%, p<0.001). Mechanistically, exposure of DLBCL lines (n=5) to CBBM decreased c-Myc protein levels in a dose-dependent manner. We also validated the reduction of PD-L1 expression, a reported c-Myc target through immunoblots. The proteasome inhibitor MG132 prevented CBBM-induced c-Myc protein degradation, indicating that reduced c-Myc after CBBM treatment is related to downregulation of protein stability. Importantly, we also explored the effect of CBBM on the CaMKIIγ/c-Myc axis. CBBM treatment reduced phospho-c-Myc (Ser62) levels in all the lines. We observed CBBM-mediated inhibitory effects (IC50 of five lines) was correlated with the basal level of CaMKIIγ (R2 = -0.8269, p=0.0323) (Fig.2). Beyond targeting c-Myc, CBBM also exhibited immune modulator potency including downregulation of autocrine interleukin 10 (IL10) level assayed by ELISA, leading to reduction of the downstream JAK2/STAT3 levels. Currently, we are in the process of testing CBBM in vivo effects using an OCI-Ly3 xenograft model. Although the overall survival result is pending, following 4 weeks of CBBM treatment (1 g/kg, i.g.), tumors from animals receiving drug treatment show decreased c-Myc expression levels relative to that of vehicle treated animals. Collectively, our results support further evaluation of CBBM as a promising compound to treat c-Myc associated DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Metabolic Reprogramming by Dual AKT/ERK Inhibition through Imipridones Elicits Unique Vulnerabilities in Glioblastoma.

Purpose: The goal of this study is to enhance the efficacy of imipridones, a novel class of AKT/ERK inhibitors that displayed limited therapeutic efficacy against glioblastoma (GBM).Experimental Design: Gene set enrichment, LC/MS, and extracellular flux analyses were used to determine the mechanism of action of novel imipridone compounds, ONC206 and ONC212. Orthotopic patient-derived xenografts were utilized to evaluate therapeutic potency.Results: Imipridones reduce the proliferation of patient-derived xenograft and stem-like glioblastoma cell cultures in vitro and in multiple xenograft models in vivo ONC212 displayed the highest potency. High levels of c-myc predict susceptibility to growth inhibition and apoptosis induction by imipridones and increased host survival in orthotopic patient-derived xenografts. As early as 1 hour, imipridones elicit on-target inhibition, followed by dephosphorylation of GSK3β at serine 9. GSK3β promotes phosphorylation of c-myc at threonine 58 and enhances its proteasomal degradation. Moreover, inhibition of c-myc by BRD4 antagonists sensitizes for imipridone-induced apoptosis in stem-like GBM cells in vitro and in vivo Imipridones affect energy metabolism by suppressing both glycolysis and oxidative phosphorylation, which is accompanied by a compensatory activation of the serine-one carbon-glycine (SOG) pathway, involving the transcription factor ATF4. Interference with the SOG pathway through novel inhibitors of PHGDH results in synergistic cell death induction in vitro and in vivo Conclusions: These results suggest that c-myc expression predicts therapeutic responses to imipridones and that imipridones lead to suppression of tumor cell energy metabolism, eliciting unique metabolic vulnerabilities that can be exploited for clinical relevant drug combination therapies. Clin Cancer Res; 24(21); 5392-406. ©2018 AACR.

C-MYC, HIF-1α, ERG, TKT, and GSTP1: an Axis in Prostate Cancer?

To analyze putative biomarkers for prostate cancer (PCA) characterization, the second leading cause of cancer-associated mortality in men. Quantification of the expression level of c-myc and HIF-1α was performed in 72 prostate cancer specimens. A cohort of 497 prostate cancer patients from The Cancer Genome Atlas (TCGA) database was further analyzed, in order to test our hypothesis. We found that high c-myc level was significantly associated with HIF-1α elevated expression (p = 0.008) in our 72 samples. Statistical analysis of 497 TCGA prostate cancer specimens confirmed the strong association (p = 0.0005) of c-myc and HIF-1α expression levels, as we found in our series. Moreover, we found high c-myc levels significantly associated with low Glutatione S-transferase P1 (GSTP1) expression (p = 0.01), with high Transketolase(TKT) expression (p < 0.0001). High TKT levels were found in TCGA samples with low GSTP1 mRNA (p < 0.0001), as shown for c-myc, and with ERG increased expression (p = 0.02). Finally, samples with low GSTP1 expression displayed higher ERG mRNA levels than samples with high GSTP1 score (p < 0.0001), as above shown for c-myc. Our study emphasizes the notion of a potential value of HIF-1α and c-myc as putative biomarkers in prostate cancer; moreover TCGA data analysis showed a putative crosstalk between c-myc, HIF-1α, ERG, TKT, and GSTP1, suggesting a potential use of this axis in prostate cancer.

MiR-137 mediates the functional link between c-Myc and EZH2 that regulates cisplatin resistance in ovarian cancer.

Platinum drugs are used in first-line to treat ovarian cancer, but most of the patients eventually generate resistance after treatment with these drugs. Although both c-Myc and EZH2 have been implicated in regulating cisplatin resistance in ovarian cancer, the interplay between these two regulators is poorly understood. Using RNA sequence analysis (RNA-seq), for the first time we find that miR-137 level is extremely low in cisplatin resistant ovarian cancer cells, correlating with higher levels of c-Myc and EZH2 expression. Further analyses indicate that in resistant cells c-Myc enhances the expression of EZH2 by directly suppressing miR-137 that targets EZH2 mRNA, and increased expression of EZH2 activates cellular survival pathways, resulting in the resistance to cisplatin. Inhibition of c-Myc-miR-137-EZH2 pathway re-sensitizes resistant cells to cisplatin. Both in vivo and in vitro analyses indicate that cisplatin treatment activates c-Myc-miR-137-EZH2 pathway. Importantly, elevated c-Myc-miR-137-EZH2 pathway in resistant cells is sustained by dual oxidase maturation factor 1 (DUOXA1)-mediated production of reactive oxygen species (ROS). Significantly, clinical studies further confirm the activated c-Myc-miR-137-EZH2 pathway in platinum drug-resistant or recurrent ovarian cancer patients. Thus, our studies elucidate a novel role of miR-137 in regulating c-Myc-EZH2 axis that is crucial to the regulation of cisplatin resistance in ovarian cancer.

Abstract 3179: TORC1/2 inhibition sensitizes MYC-driven medulloblastoma cells to carboplatin chemotherapy

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of high medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. We performed an in silico analysis of these cells, which predicted that mTOR inhibitors would be potential therapeutic agents. The dual TORC1/2 inhibitor TAK228 (also known as MLN128 and INK128) is orally bioavailable and penetrates the brain. TAK228 is currently in early clinical trials for adults with glioblastoma. We hypothesized that TAK228 would have activity against MYC-driven medulloblastoma. We tested this hypothesis using the MYC amplified medulloblastoma cell lines D425 and D283 as well as our human neural stem cell models. We determined that TAK228 was effective in inhibiting both mTORC1/2 as measured by western blots showing decreased p-S6 and p-AKT473 expression. P-S6 expression is decreased by 90% and p-AKT expression is decreased by 70% in D425 cell lines treated with 20nM TAK228. We found that treatment with TAK228 decreases cell growth in D425 and D283 high-MYC medulloblatoma cell lines at concentrations of 20nM (D425 p&amp;lt;0.005 vs vehicle, D283 p&amp;lt;0.0005 vs vehicle). We also found that TAK228 induces apoptosis as measured by cleaved caspase 3 staining and an increased expression of cleaved PARP (D425 p&amp;lt;0.05 vs vehicle, D283 p&amp;lt;0.0005 vs vehicle). Because mTOR inhibition can suppress glutathione production, and glutathione is required to detoxify platinum containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin, which is part of the current standard treatment for high-risk medulloblastoma. TAK228 in combination with carboplatin inhibits cell growth as measured by MTS assay (D425 p&amp;lt;0.05 vs vehicle, D283 p&amp;lt;0.05 vs vehicle) and induces apoptosis as measured by cleaved PARP expression. Combination therapy of TAK228 and carboplatin treatment leads to a 7-fold increase in cleaved PARP compared to cells treated with the vehicle. In vivo testing in orthotopic xenografts is currently underway to determine the survival benefit of TAK228 in combination with carboplatin. TAK228 is effective at inhibiting growth and inducing apoptosis in high-MYC medulloblastoma cell lines and shows strong combinatorial efficacy with carboplatin. Citation Format: Rachael E. Maynard. TORC1/2 inhibition sensitizes MYC-driven medulloblastoma cells to carboplatin chemotherapy [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 3179.

Abstract 3340: Endogenous, CRISPR-mediated overexpression of MYC family members as a framework to discover MYC-specific vulnerabilities

Abstract MYC activation reshapes cellular transcription, promotes cell growth, cell cycle progression and transformation and therefore MYC is considered a strong driver of tumor development. The MYC family includes c-MYC, MYCN, and MYCL that are typically associated with distinct tumor types. Based on the structural properties of these transcription factors MYC family members are considered undruggable targets. However, potential vulnerabilities associated with the cellular MYC status may provide a basis for therapeutic intervention. Currently, there is a lack of comparative studies assessing the specific contribution of each MYC family member to tumor development. Established cell lines in which MYC family members are overexpressed differ greatly in their genetic background, thereby impeding the analysis of MYC family member specific effects. Furthermore, the complexity of MYC regulation and MYC biology is not adequately recapitulated by traditional, heterologous MYC overexpression approaches. Therefore, we set out to establish an endogenous overexpression system using a CRISPR activation (CRISPRa) approach to activate the individual MYC family members for a systematic comparison of their impact on target gene activation, cell growth and drug sensitivity. Using sgRNAs specific to the promoter regions of the particular MYC family members in combination with a nucleolytically inactive Cas9-VP64 fusion protein, we successfully achieved transcriptional upregulation of all three MYC family members in multiple cell lines including NIH3T3 and MEF cells. The increase in MYC mRNA resulted in markedly elevated MYC protein levels. In addition, MYC activation enhanced transcription of canonical MYC target genes like NPM1, FBL, and 45S pre-rRNA suggesting a functional impact of upregulated MYC. Moreover, activation of c-Myc led to increased anchor-independent growth of cells in a soft-agar colony formation assay indicating that high c-Myc levels induce transformation in contrast to N-Myc or L-Myc. In line with previous studies that implicated upregulated c-Myc as an initiator of genome instability, we observed elevated levels of DNA damage by monitoring phosphorylated histone variant H2AX (γH2AX) in cells with activated c-Myc whereas N-Myc or L-Myc failed to induce γH2AX. Activation of c-Myc further led to increased sensitivity to cisplatin and other chemotherapeutic agents. In conclusion, we established an endogenous MYC overexpression model system that enables a systematic comparison of the different MYC family members and their specific contribution to tumor initiation, growth and drug sensitivity. The system successfully recapitulated previous observations of MYC activation and can serve as a platform to probe for MYC family member specific vulnerabilities and thereby pave the way for novel, targeted therapy approaches in MYC-driven cancer. Citation Format: Marcel A. Dammert, Martin L. Sos. Endogenous, CRISPR-mediated overexpression of MYC family members as a framework to discover MYC-specific vulnerabilities [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 3340.

Multiple DNA damage-dependent and DNA damage-independent stress responses define the outcome of ATR/Chk1 targeting in medulloblastoma cells

Targeting of oncogene-driven replicative stress as therapeutic option for high-risk medullobastoma was assessed using a panel of medulloblastoma cells differing in their c-Myc expression [i.e. group SHH (c-Myc low) vs. group 3 (c-Myc high)]. High c-Myc levels were associated with hypersensitivity to pharmacological Chk1 and ATR inhibition but not to CDK inhibition nor to conventional (genotoxic) anticancer therapeutics. The enhanced sensitivity of group 3 medulloblastoma cells to Chk1 inhibitors likely results from enhanced damage to intracellular organelles, elevated replicative stress and DNA damage and activation of apoptosis/necrosis. Furthermore, Chk1 inhibition differentially affected c-Myc expression and functions. In c-Myc high cells, Chk1 blockage decreased c-Myc and p-GSK3α protein and increased p21 and GADD45A mRNA expression. By contrast, c-Myc low cells revealed increased p-GSK3β protein and CHOP and DUSP1 mRNA levels. Inhibition of Chk1 sensitized medulloblastoma cells to additional replication stress evoked by cisplatin independent of c-Myc. Importantly, Chk1 inhibition only caused minor toxicity in primary rat neurons in vitro. Collectively, targeting of ATR/Chk1 effectively triggers death in high-risk medulloblastoma, potentiates the anticancer efficacy of cisplatin and is well tolerated in non-cancerous neuronal cells.