Mutant C-myc Research Articles

Computational modeling to understand the interaction of TMPyP4 with a G-quadruplex

The potential of small molecules to bind to G-quadruplex-forming sequences in oncogene promoter regions, thereby regulating their structural equilibrium, has been explored as a promising strategy for cancer chemotherapy. The model drug 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4) has been shown to have an affinity toward G-quadruplex DNA. However, the precise sites and modes of TMPyP4 binding to G-quadruplex DNA remain a subject of debate. In this study, we focus on identifying potential binding sites on a mutant c-MYC sequence known to fold into a single 1:2:1 loop isomer quadruplex. Our findings provide insights into the 4:1 stoichiometry reported for TMPyP4 binding to this G-quadruplex. Binding enthalpy and free energy calculations show that intercalation of a TMPyP4 molecule between the quadruplexes is thermodynamically favorable. Our calculations suggest that two of the binding sites are located at the top and bottom of the quadruplex, respectively, while the remaining two are likely intercalations.

A common druggable signature of oncogenic c-Myc, mutant KRAS and mutant p53 reveals functional redundancy and competition among oncogenes in cancer

The major driver oncogenes MYC, mutant KRAS, and mutant TP53 often coexist and cooperate to promote human neoplasia, which results in anticancer therapeutic opportunities within their downstream molecular programs. However, little research has been conducted on whether redundancy and competition among oncogenes affect their programs and ability to drive neoplasia. By CRISPR‒Cas9-mediated downregulation we evaluated the downstream proteomics and transcriptomics programs of MYC, mutant KRAS, and mutant TP53 in a panel of cell lines with either one or three of these oncogenes activated, in cancers of the lung, colon and pancreas. Using RNAi screening of the commonly activated molecular programs, we found a signature of three proteins - RUVBL1, HSPA9, and XPO1, which could be efficiently targeted by novel drug combinations in the studied cancer types. Interestingly, the signature was controlled by the oncoproteins in a redundant or competitive manner rather than by cooperation. Each oncoprotein individually upregulated the target genes, while upon oncogene co-expression each target was controlled preferably by a dominant oncoprotein which reduced the influence of the others. This interplay was mediated by redundant routes of target gene activation - as in the case of mutant KRAS signaling to c-Jun/GLI2 transcription factors bypassing c-Myc activation, and by competition - as in the case of mutant p53 and c-Myc competing for binding to target promoters. The global transcriptomics data from the cell lines and patient samples indicate that the redundancy and competition of oncogenic programs are broad phenomena, that may constitute even a majority of the genes dependent on oncoproteins, as shown for mutant p53 in colon and lung cancer cell lines. Nevertheless, we demonstrated that redundant oncogene programs harbor targets for efficient anticancer drug combinations, bypassing the limitations for direct oncoprotein inhibition.

Hepatitis C virus may accelerate breast cancer progression by increasing mutant p53 and c-Myc oncoproteins circulating levels

BackgroundHepatitis C virus (HCV) was reported to relate to polymorphous and frequent extrahepatic manifestation. Despite the limited studies, HCV viral oncoproteins may be implicated in breast cancer (BC) tumor aggressiveness. In a trial to elucidate a mechanistic link, this study aimed to investigate a mutant p53 and c-Myc oncoprotein expression levels in BC patients with and without HCV infection.MethodsA total of 215 BC patients (119 infected and 96 non-infected with HCV) were collected. ELISA was used for detection of anti-HCV antibodies, mutant p53, c-Myc, HCV-NS4, CEA, CA 125, and CA-15.3.ResultsHCV infection was related to BC late stages, lymph-node invasion, distant metastasis, high grades, and large size. HCV-infected patients had a significantly (P < 0.05) higher WBCs, ALT and AST activity, bilirubin CEA, CA125 and CA15.3 levels, and reduced hemoglobin, albumin, and RBCs count. Regardless of tumor severity, HCV infection was associated with significant elevated levels of mutant p53 (22.5 ± 3.5 µg/mL; 1.9-fold increase) and c-Myc (21.4 ± 1.8 µg/mL; 1.5-fold increase). Among HCV-infected patients, elevated levels of p53 and c-Myc were significantly correlated with elevated tumor markers (CEA, CA 125, and CA15.3) and HCV-NS4 levels.ConclusionsThis study concluded that HCV infection may be accompanied with BC severity behavior and this may be owing to elevated expression of mutant p53 and c-Myc oncoproteins.

Lipid accumulation-mediated histone hypoacetylation drives persistent NK cell dysfunction in anti-tumor immunity

Hyperlipidemia impairs anti-tumor immune responses and is closely associated with increased human cancer incidence and mortality. However, the underlying mechanisms are not well understood. In the present study, we show that natural killer (NK) cells isolated from high-fat-diet mice or treated with oleic acid (OA) invitro exhibit sustainable functional defects even after removal from hyperlipidemic milieu. This is accompanied by reduced chromatin accessibility in the promoter region of NK cell effector molecules. Mechanistically, OA exposure blunts P300-mediated c-Myc acetylation and shortens its protein half-life in NK cells, which in turn reduces P300 accumulation and H3K27 acetylation and leads to persistent NK cell dysfunction. NK cells engineered with hyperacetylated c-Myc mutants surmount the suppressive effect of hyperlipidemia and display superior anti-tumor activity. Our findings reveal the persistent dysfunction of NK cells in dyslipidemia milieu and extend engineered NK cells as a promising strategy for tumor immunotherapy.

Abstract 2411: Di-ganglioside GD2 expression and role in promoting tumorigenicity in prostate cancer

Abstract Background &amp; Significance: Prostate cancer (PCa) is the second leading cause of cancer deaths (~34,000 in 2021 (ACS)) in American men. Castration resistance and resistance to the next-gen androgen receptor (AR) targeted drugs are major challenges. Castration resistance involves multiple mechanisms, including androgen-independent signaling by androgen receptor (AR)- or its variants, and lineage plasticity (LP) with AR-indifferent neuroendocrine (NE) differentiation. Identifying new vulnerabilities across these multitude mechanisms could provide new therapeutic avenues against castration-resistant PCa (CRPC). The cell-surface di-ganglioside GD2 is overexpressed in neural crest cell tumors such as neuroblastoma &amp; melanoma and chimeric (Dinutuximab) or humanized (Naxitamab) anti-GD2 antibodies are now FDA-approved for high-risk neuroblastoma therapy. GD2 expression is reported in other cancers such as breast cancer and glioma and is linked to cancer stem cell behavior. While limited prior studies have detected GD2 expression in PCa cell lines or tumor tissues, nothing is known about the functional role of GD2 in PCa. Objectives: We hypothesized that GD2 overexpression in PCa could play a pro-tumorigenic role and that linkage of GD2 overexpression with CRPC progression may reveal the potential of targeting GD2 for CRPC therapy. Study Design &amp; Results: Immunohistochemical analysis of PCa patient and patient-derived xenograft tissue microarrays (TMAs) revealed GD2 expression in a subset of tumor cells. Fluorescence-activated cell sorter analysis of PCa cell lines showed strong constitutive GD2 expression on murine CRPC cell line RM-1 (derived from mutant Ras and c-Myc overexpressing prostatic epithelial cells) and human PCa line 22Rv1 (overexpresses wild-type AR and ARv7 splice variant). GD2 expression was induced de novo upon induction of lineage plasticity in GD2-negative LNCaP C4-2 prostate adenocarcinoma cell line by shRNA knockdown (KD) of RB1 or TP53. High GD2 expression was also induced when C4-2B cells were made enzalutamide resistant (C4-2BER). Induction of GD2 expression correlated with increased expression of rate-limiting GD2 biosynthetic pathway enzyme GD3 synthase (GD3S). CRISPR-Cas9 mediated stable GD3S knockout (KO) in the RM1 cell line led to the loss of GD2 expression. The GD3S-KO RM1 cells exhibited reduced proliferation, migration, invasion, and tumor sphere forming ability compared to the control cells. Intratibial injections in castrated male C57BL/6 mice showed a significant reduction in tumor development by GD3S KO RM1 cells compared to control cells. Conclusions: Our studies demonstrate that GD2 is expressed in a subset of prostate cancers. Cell line-based studies show that GD2 expression promotes pro-tumorigenic traits. Future studies will assess the biological roles of GD2 in PCa and the potential of targeting GD2+ CRPC with antibody-based approved therapeutic agents. Citation Format: Aaqib M. Bhat, Bhopal C. Mohapatra, Insha Mushtaq, Sukanya Chakraborty, Samikshan Dutta, Sameer Mirza, Matthew D. Storck, Subodh M. Lele, Ming-Fong Lin, Bruce J. Trock, Karen S. Sfanos, Colm Morrissey, Eva Corey, Jonathan Melamed, Leah Cook, Kaustubh Datta, Jane Meza, Jawed Siddiqui, Surinder K. Batra, Vimla Band, Hamid Band. Di-ganglioside GD2 expression and role in promoting tumorigenicity in prostate cancer [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 2411.

The Role of Oncogenes and Redox Signaling in the Regulation of PD-L1 in Cancer.

Simple SummaryPD-L1 is an important immune checkpoint molecule that is expressed in cancer tissues at various levels and plays a major role in tumor evasion from immune system. Therefore, understanding the mechanisms by which PD-L1 expression is regulated in tumor environment is important for the design of new therapeutic strategies to overcome tumor immune escape and improve the outcome of cancer treatment. Recent studies suggest that genetic, epigenetic, and transcriptional factors as well as posttranscriptional and posttranslational processes are crucial regulators of PD-L1 expression in tumor cells. This review focuses on two newly described regulations of PD-L1 mediated by oncogenes, and redox homeostasis. In this context, a recent research work describes the important role of oncogenic K-ras, growth factor receptors (FGFR1 and EGFR) and ROS in regulating PD-L1 expression in pancreatic cancer cells and aids significant new mechanistic insights in this important area.Tumor cells can evade the immune system via multiple mechanisms, including the dysregulation of the immune checkpoint signaling. These signaling molecules are important factors that can either stimulate or inhibit tumor immune response. Under normal physiological conditions, the interaction between programmed cell death ligand 1 (PD-L1) and its receptor, programmed cell death 1 (PD-1), negatively regulates T cell function. In cancer cells, high expression of PD-L1 plays a key role in cancer evasion of the immune surveillance and seems to be correlated with clinical response to immunotherapy. As such, it is important to understand various mechanisms by which PD-L1 is regulated. In this review article, we provide an up-to-date review of the different mechanisms that regulate PD-L1 expression in cancer. We will focus on the roles of oncogenic signals (c-Myc, EML4-ALK, K-ras and p53 mutants), growth factor receptors (EGFR and FGFR), and redox signaling in the regulation of PD-L1 expression and discuss their clinical relevance and therapeutic implications. These oncogenic signalings have common and distinct regulatory mechanisms and can also cooperatively control tumor PD-L1 expression. Finally, strategies to target PD-L1 expression in tumor microenvironment including combination therapies will be also discussed.

Small Molecule Activators of Protein Phosphatase 2A (SMAPs) Cause c-Myc Degradation and Tumor Growth Suppression in Preclinical Models of Lymphoid Malignancies

Introduction: c-myc is an oncogene implicated in the initiation and progression of multiple hematologic malignancies. Characteristic translocations of the c-myc gene with the immunoglobulin heavy chain gene (in Burkitt lymphoma and “double-hit” non-Hodgkin lymphoma) and with the T cell receptor gene (in T cell acute lymphoblastic leukemia, T-ALL) lead to constitutive expression of the c-myc transcription factor, ultimately driving dysregulated proliferation (Boxer & Dang, Oncogene 2001; 20(40):5595-5610). Strategies for inhibition of c-myc remain an area of active research; however, to date there are no approved myc-targeted agents within the cancer armamentarium, and cytotoxic chemotherapy remains the standard of care for c-myc-driven lymphoid malignancies. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase tumor suppressor protein that contributes to c-myc degradation through both direct dephosphorylation of an identified phosphodegron as well as by indirect mechanisms (Farrell & Sears, Cold Spring Harbor Perspect Med 2014; 4(3):1-15). Our laboratory has developed a series of first in class, orally bioavailable small molecule activators of PP2A (SMAPs) by derivatizing tricyclic antipsychotics to maximize antiproliferative activity and minimize psychotropic properties (Sangodkar et al., J Clin Invest 2017;127(6):2081-2090). We hypothesized that activation of PP2A with SMAPs would lead to c-myc degradation and tumor growth inhibition in models of c-myc-driven lymphoid malignancies.Methods: We investigated the effects of SMAP treatment on c-myc content, c-myc half-life (t1/2), cell viability, and xenograft tumor growth in Burkitt lymphoma (BL) and T-ALL cell lines with wild type and mutant c-myc phosphodegrons.Results: SMAP treatment induced apoptosis in Daudi (BL) cells, as evidenced by decreased viability and increased PARP and caspase cleavage. In Daudi (BL) and CCRF-CEM (T-ALL) cell lines, SMAP treatment led to rapid loss of phosphorylated and total c-myc protein. Cycloheximide (CHX)-chase experiments performed in CCRF-CEM cells confirmed that SMAP treatment affects c-myc degradation without affecting protein synthesis. This effect was abrogated by Ser to Glu mutation of residue 62 within the c-myc phosphodegron, suggesting that SMAP-mediated c-myc degradation is PP2A-dependent. Treatment of NOD/SCID mice bearing Daudi xenografts with twice daily oral SMAP decreased tumor c-myc content and inhibited tumor growth without obvious toxicity. Xenografts overexpressing c-myc with phosphodegron mutations (S62D, T58I) were resistant to SMAP treatment.Conclusions: These in vitro and in vivo observations establish PP2A activation as a potential pharmacologic strategy to target c-myc in cancers such as BL, T-ALL, and “double hit” non-Hodgkin lymphoma. SMAPs are well-tolerated, orally bioavailable activators of PP2A poised for further exploration in the treatment of c-myc-driven malignancies, either alone or in combination with other agents. Sequencing of the c-myc phosphodegron prior to initiation of treatment may facilitate patient selection in future clinical trials of PP2A activators. [Display omitted] DisclosuresNarla:Mount Sinai School of Medicine: Patents & Royalties: patents on the small molecule PP2A activators (GN).

A Driver Never Works Alone-Interplay Networks of Mutant p53, MYC, RAS, and Other Universal Oncogenic Drivers in Human Cancer.

The knowledge accumulating on the occurrence and mechanisms of the activation of oncogenes in human neoplasia necessitates an increasingly detailed understanding of their systemic interactions. None of the known oncogenic drivers work in isolation from the other oncogenic pathways. The cooperation between these pathways is an indispensable element of a multistep carcinogenesis, which apart from inactivation of tumor suppressors, always includes the activation of two or more proto-oncogenes. In this review we focus on representative examples of the interaction of major oncogenic drivers with one another. The drivers are selected according to the following criteria: (1) the highest frequency of known activation in human neoplasia (by mutations or otherwise), (2) activation in a wide range of neoplasia types (universality) and (3) as a part of a distinguishable pathway, (4) being a known cause of phenotypic addiction of neoplastic cells and thus a promising therapeutic target. Each of these universal oncogenic factors—mutant p53, KRAS and CMYC proteins, telomerase ribonucleoprotein, proteasome machinery, HSP molecular chaperones, NF-κB and WNT pathways, AP-1 and YAP/TAZ transcription factors and non-coding RNAs—has a vast network of molecular interrelations and common partners. Understanding this network allows for the hunt for novel therapeutic targets and protocols to counteract drug resistance in a clinical neoplasia treatment.

Abstract A05: Context-dependent transformation with activated Ras isoforms in human normal epithelial cells

Abstract The classical mammalian ras genes exist three isoforms K-, H- and N-Ras. Ras mutations are genetic events that have been detected in 30 % of all human cancers. Unfortunately, mutated Ras tumors have not responded to chemotherapy, an IgG1 chimeric monoclonal antibody and tyrosine-kinase inhibitors against EGFR. Therefore, there is need for development of new therapeutic strategies to mutated Ras tumors. Although the function of three Ras isoforms is considered to be almost the same, the occurrence of Ras mutation is largely different in human each tissue. For example, APC and BRACA1 genes are associated with a high risk of developing tissue specific cancer. APC mutation caused familial adenomatous polyposis. Affected people have a nearly 100% lifetime risk of developing colorectal cancer, whereas other tumor types are rare. Such genes are therefore considered as tissue-specific cancer genes. Whether Ras genes have tissue specificity has not been clarified. In this study, we thought to elucidate whether mutated Ras isoform has tissue specificity and there is a difference in the function among three mutated Ras isoforms by using human normal epithelial cell lines (prostate, small airway, mammary, skin and bronchus). In clinical data, skin and bronchus frequently occurred H-Ras mutation, others occurred K-Ras mutation. Firstly, we induced immortalization of these cell lines by introduction of human papillomavirus E6/E7 and mutant c-myc (T58A). These cell lines could stably culture and were expressed cytokeratin 5, which is basal cell marker of epidermis. We analyzed the carcinogenesis efficiency by soft-agar assay when the cells infected with retrovirus of three mutated Ras in five immortalized cell lines. As a result, carcinogenesis efficiency was different for each cell line. These results were mostly consistent with clinical data. Also, we identified downstream signaling of Ras (PI3K-AKT and MAPK signaling) involved in carcinogenesis by performing Western blot analysis when the cells infected with mutated Ras, which induced highest carcinogenesis efficiency in each cell lines. As a result, the protein expression level that associated with growth-related PI3K/AKT and MAPK signaling affected differently in each mutated Ras isoform. These results indicated that mutated Ras isoform have tissue specificity and specific functions involved in carcinogenesis. Taken together, we concluded that these events present the possibility of new therapeutic target for mutated Ras tumors. Citation Format: Minami Kumazaki, Yusuke Yamamoto. Context-dependent transformation with activated Ras isoforms in human normal epithelial cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A05.

Berenil Binds Tightly to Parallel and Mixed Parallel/Antiparallel G-Quadruplex Motifs with Varied Thermodynamic Signatures.

Diminazene, DMZ, (or berenil) has been reported as a tight binder of G-quadruplexes. G-Quadruplex structures are often located in the promotor regions of oncogenes and may play a regulatory role in gene expression based on the stability of the folding topology. In this study, attempts have been made to characterize the specificity of DMZ binding toward multiple G-quadruplex topologies or foldamers. Mutant sequences of the G-quadruplex forming promotor regions of several oncogenes were designed to exhibit restricted loop lengths and folding topologies. Circular dichroism was used to confirm the quadruplex topology of mutant BCL2, KRAS, and c-MYC sequences, human telomere (Na+ and K+) G-quadruplexes and their complexes with DMZ and analogs thereof. Isothermal titration calorimetry was used to generate a complete thermodynamic profile (ΔG, ΔH, −TΔS) for the formation of DMZ and analog complexes with the target G-quadruplexes. DMZ binds to parallel and/or mixed parallel/antiparallel quadruplex DNA motifs with stoichiometries up to 8:1 and via three binding modes with varying affinities. In the case of the parallel G-quadruplexes, with the exception of the long-looped c-MYC mutant, the highest affinity binding event (mode 1) is driven by enthalpy. DMZ binding to the long-looped c-MYC mutant exhibits a very favorable entropy change in addition to a moderately favorable enthalpy change. Mode 1 binding to the antiparallel and mixed parallel/antiparallel hTel quadruplexes is also driven by favorable enthalpy changes. In all cases, the intermediate DMZ affinity binding (mode 2) is driven almost entirely by entropy, with small or unfavorable enthalpic contributions. The weakest binding event (mode 3) is also entropically driven with small or moderate enthalpic contributions.

Pharmacokinetics and biodistribution of polymeric micelles containing miRNA and small-molecule drug in orthotopic pancreatic tumor-bearing mice.

Rationale: Successful treatment of pancreatic cancer remains a challenge due to desmoplasia and prevalence of KRAS mutation. While hedgehog (Hh) ligand levels are upregulated in pancreatic cancer cells and contribute to desmoplasia, there is significant downregulation of tumor suppressor let-7b, which targets mutant KRAS, C-MYC and several other genes involved in pancreatic cancer progression, invasion, and metastasis. We recently explored combination therapy of GDC-0449 (Hh inhibitor) and let-7b mimic using poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (PEG-b-PCC-g-DC-g-TEPA) micelles in pancreatic tumor mouse model. Here, our objective was to determine the biodistribution (BD), pharmacokinetics (PK), therapeutic efficacy and toxicity of this micellar formulation.Methods: We determined the PK of micelles encapsulating Cy5.5-let-7b and GDC-0449 following intravenous injection in orthotopic pancreatic tumor-bearing NSG mice at doses of 2 mg/kg and 10 mg/kg, respectively. Mice were scanned for fluorescence by IVIS to determine the biodistribution of Cy5.5-let-7b at the whole-body level, and its concentration in plasma and major organs was determined by measuring fluorescence using a fluorimeter and by real-time RT-PCR. GDC-0449 concentration was determined by LC/MS/MS. Therapeutic efficacy and toxicity of the micellar formulation of let-7b and GDC-0449 was also determined after two weeks of treatment.Results: The use of a micellar formulation markedly prolonged the elimination half-life (t1/2, e) of Cy5.5-let-7b in plasma from 0.49 ± 0.19 h to 2.65 ± 0.46 h and increased the area-under-the-curve (AUC0-∞) by 7-fold, while t1/2,e and AUC0-∞of GDC-0449 were increased by 1.78-fold and 3.2-fold, respectively. The micelles significantly decreased the clearance of both encapsulated let-7b mimic and GDC-0449 compared to the emulsion formulation. Compared to the emulsion counterpart, the micellar formulation elevated the delivery of Cy5.5-let-7b and GDC-0449 to the orthotopic pancreatic tumor tissue by 7.8- and 4.2-fold, respectively. Furthermore, there was a significant reduction in tumor volume and negligible systemic toxicity as evident by hematological parameters and histological evaluation.Conclusion: PEG-b-PCC-g-DC-g-TEPA micelles carrying GDC-0449 and let-7b mimic have great potential to improve drug delivery for pancreatic cancer treatment.

An orthotropic mouse model of combined hepatocellular-cholangiocarcinoma derived from oncogene transformed hepatoblasts

Objective To establish an orthotropic mouse model of combined hepatocellular-cholangiocarcinoma. Methods Hepatoblasts were isolated by E-cadherin conjugated microbeads from E13.5 fetal liver of C57BL/6J mice. After culture for several days, expended hepatoblasts were transfected with retrovirus encoding c-Myc mutant T58A. Then 1×106 modified hepatoblasts were delivered into portal system of retrosine pretreated mice by splenic injection (c-Myc mutant T58A group, n=6). Non-modified hepatoblasts served as control for injection (wildtype group, n=6). Eight weeks after surgery, all 12 recipient mice were sacrificed for pathological examination. Immunohistochemistry and double-fluorescence immunostaining were performed on 4 μm sections to determine the phenotype of tumors. Results Mice delivered with hepatoblasts constructively expressing c-Myc mutant T58A showed high incidence of hepatocarcinogenesis (4 in 6 mice, 66.7%), while those delivered with wildtype hepatoblasts showed no tumor (0 in 6 mice, 0.0%). The tumors pathologically mimicked human combined hepatocellular-cholangiocarcinoma. By immunohistochemistry and double-fluorescence immunostaining, the tumor cells were confirmed with colocalization of ALB and K19. Conclusion We generated an orthotropic mouse model of combined hepatocellular-cholangiocarcinoma derived from oncogene transformed hepatoblasts. Key words: Combined hepatocellular-cholangiocarcinoma; Hepatoblast; Retrovirus; Model, animal; Oncogene

Abstract A15: Downregulation of c-myc is synthetic lethal with PARP inhibitors in high MYC cancers independent of BRCA status

Abstract PARP inhibitors represent a novel class of cancer therapeutics that exploit a synthetic lethal mechanism specific to cancer cells with a defect within the Homologous Recombination (HR) DNA repair pathway. Patients with a BRCA1/2 germ-line mutation have the highest response rate to PARP inhibitors. However, early clinical trial data has indicated that only ~30-40% BRCA mutant patients respond to PARP inhibitors. Coincidentally, the oncogene c-myc is the most frequently amplified gene in BRCA mutant tumors (~40-60%) which typically present as Triple Negative Breast Cancer (TNBC). The oncogene c-myc transcriptionally regulates several DNA repair genes, and correlates with poor survival among breast cancer patients. Therefore we hypothesized that dual combination therapy targeting c-myc and PARP is an effective therapeutic strategy in BRCA mutant tumors. C-myc siRNA induced increased sensitivity to PARP inhibitors in BRCA wild-type (MB231) &amp; BRCA mutant (SUM149) TNBC cell lines and down regulated HR repair. Pan CDK inhibitor Dinaciclib demonstrated significant MYC inhibition across several TNBC cell lines. Additionally, Dinaciclib demonstrated synergistic growth inhibition in combination with PARP inhibitor Niraparib in both BRCA wild-type and mutant c-myc expressing TNBC cell lines. Combination therapy also leads to an increase in DNA damage (via γH2AX) with a decrease in HR repair (BRCA1/RAD51). TNBC cell line mouse xenografts demonstrated down regulation of c-myc and RAD51 in Dinaciclib + Niraparib treated tumors. Dual Dinaciclib + Niraparib therapy also induced synergistic growth inhibition in PARP inhibitor acquired resistant cell lines while demonstrating significant growth inhibition across a panel of c-myc overexpressing cancer cell lines (Ovarian, Prostate, Pancreatic, Colon &amp; Lung). In conclusion, we demonstrated that dual CDK + PARP inhibition is synthetic lethal in both BRCA wild-type and mutant TNBC cell lines and is dependent upon down regulation of c-myc. This study supports c-myc as predictor of response to PARP inhibitor therapy and may also serve as a biomarker of response to Dinaciclib + PARPi therapy in high MYC expressing tumors. Citation Format: Jason PW Carey, Smruthi Vjayaraghavan, Kelly Hunt, Khandan Keyomarsi. Downregulation of c-myc is synthetic lethal with PARP inhibitors in high MYC cancers independent of BRCA status [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A15.

C-Myc Alteration Determines the Therapeutic Response to FGFR Inhibitors.

Purpose: Lately, emerging evidence has suggested that oncogenic kinases are associated with specific downstream effectors to govern tumor growth, suggesting potential translational values in kinase-targeted cancer therapy. Tyrosine kinase FGFR, which is aberrant in various cancer types, is one of the most investigated kinases in molecularly targeted cancer therapy. Herein, we investigated whether there exists key downstream effector(s) that converges FGFR signaling and determines the therapeutic response of FGFR-targeted therapy.Experimental Design: A range of assays was used to assess the role of c-Myc in FGFR aberrant cancers and its translational relevance in FGFR-targeted therapy, including assessment of drug sensitivity using cell viability assay, signaling transduction profiling using immunoblotting, and in vivo antitumor efficacy using cancer cell line-based xenografts and patient-derived xenografts models.Results: We discovered that c-Myc functioned as the key downstream effector that preceded FGFR-MEK/ERK signaling in FGFR aberrant cancer. Disruption of c-Myc overrode the cell proliferation driven by constitutively active FGFR. FGFR inhibition in FGFR-addicted cancer facilitated c-Myc degradation via phosphorylating c-Myc at threonine 58. Ectopic expression of undegradable c-Myc mutant conferred resistance to FGFR inhibition both in vitro and in vivo c-Myc level alteration stringently determined the response to FGFR inhibitors, as demonstrated in FGFR-responsive cancer subset, as well as cancers bearing acquired or de novo resistance to FGFR inhibition.Conclusions: This study reveals a stringent association between FGFR and the downstream effector c-Myc in FGFR-dependent cancers, and suggests the potential therapeutic value of c-Myc in FGFR-targeted cancer therapy. Clin Cancer Res; 23(4); 974-84. ©2016 AACR.

Autocrine IGF1 Signaling Mediates Pancreatic Tumor Cell Dormancy in the Absence of Oncogenic Drivers

Mutant KRAS and c-MYC are oncogenic drivers and rational therapeutic targets for the treatment of pancreatic cancer. Although tumor growth and homeostasis are largely dependent on these oncogenes, a few residual cancer cells are able to survive the ablation of mutant KRAS and c-MYC. By performing a genome-wide gene expression analysis of invivo-derived bulk tumor cells and residual cancer cells lacking the expression of mutant KRAS or c-MYC, we have identified an increase in autocrine IGF1/AKT signaling as a common survival mechanism in dormant cancer cells. The pharmacological inhibition of IGF-1R reduces residual disease burden and cancer recurrence, suggesting that this molecular pathway is crucial for the survival of cancer cells in the absence of the primary oncogenic drivers.

Mutant p53 Enhances the Development and Sustained Growth of MYC-Driven Lymphoma and Exerts a Dominant Negative Effect Preferentially Deregulating Pathways for Metabolism and DNA Repair

Over-expression of the c-MYC oncogene and Trp53 gene mutations are among the most common genetic alterations in human cancer and, when combined, result in highly aggressive malignant disease. Trp53 gene mutations produce over-expressed mutant TRP53 proteins that drive cancer growth through both loss of wild-type Trp53 tumor suppressor function and gain-of-function oncogenic properties. The Eμ-Myc mouse model provides a setting to study the functional interplay between c-Myc over-expression and mutant TRP53 proteins. Eμ-Myc transgenic mice carry a c-Myc transgene under the control of the immunoglobulin heavy chain gene enhancer (Eμ), recapitulating the chromosomal translocation underlying human Burkitt Lymphoma, and develop aggressive pre-B/B cell lymphoma with a high (~20%) spontaneous rate of Trp53 mutation.The effect of five mouse mutant TRP53 proteins (V170M, I192S, G280, R246Q, R270H) was initially examined in three settings (Trp53-/-, Trp53+/- and Trp53+/+;Eμ-Myc) using a hematopoietic stem and progenitor cell (HSPC) reconstitution model. Each mutant TRP53 protein studied corresponds to a commonly re-occurring Tp53 mutation in human cancer. Retroviral over-expression enabled the comparison of mutant-specific and genotype-specific features for each mutant TRP53 protein. Mutant TRP53 expression did not accelerate lymphoma development in mice receiving Trp53-/- or Trp53+/- HSPCs. However, mice reconstituted with Trp53+/- HSPCs expressing the TRP53 mutants displayed an altered tumor spectrum compared to mice reconstituted with control Trp53+/- HSPCs. In contrast, mutant TRP53 markedly accelerated lymphoma development in mice receiving Trp53+/+;Eμ-Myc HSPCs, highlighting a synergy between c-Myc over-expression and Trp53 mutations in neoplastic transformation. Furthermore, inducible mutant TRP53 expression demonstrated a dependency on sustained expression of mutant TRP53 in established MYC-driven lymphomas. Notably, none of the c-MYC plus mutant TRP53 driven lymphomas exhibited spontaneous endogenous Trp53 mutations.Despite the enhanced tumorigenesis, most established lymphomas from this model displayed sensitivity to TRP53-activating drugs consistent with a weak dominant negative effect over wild-type Trp53-induced apoptosis. Consistent with this finding, pre-malignant Trp53+/+;Eμ-Myc primary B-cells expressing mutant TRP53 were not protected against Trp53-induced apoptosis. Pre-malignant B-cells displayed a small increase in cell cycling and an expansion of the tumor-initiating pre/pro-B cell population. Most significantly, functional assessment of DNA damage in pre-malignant cells, using single cell gel electrophoresis (comet assay) and γ-H2AX staining, revealed increased DNA damage, suggesting an important role for defects in DNA repair during mutant TRP53-driven lymphoma development.To investigate the nature of the dominant negative effect, mutant TRP53 protein was exogenously expressed in mouse Eµ-Myc Trp53+/+ lymphoma cell lines. The impact of mutant TRP53 on the transcriptional function of the endogenous wild-type TRP53 protein was then studied using the TRP53-activating compound, nutlin-3a. Surprisingly, in established lymphoma cell lines, mutant TRP53 impaired nutlin-3a-induced apoptosis despite substantial induction of the critical pro-apoptotic effector, PUMA. To explore this finding further, we globally characterized the dominant negative effect, and assessed for mutant TRP53-specific transcriptional targets, by performing whole transcriptome sequence (RNAseq) analysis after treatment with nutlin-3a. Analysis of known wild-type Trp53 target genes (n=283) demonstrated that the induction of these genes as a group was repressed in the presence of the mutant TRP53 protein (ROAST test, p=6.7e-04). Remarkably, however, mutant TRP53 significantly repressed only 57% of the nutlin-3a-induced Trp53 target genes. Analysis of these strongly repressed genes highlighted the importance of several pathways, including metabolism, DNA damage repair and negative feedback loops in TRP53 signaling. This suggests a previously unrecognized selectivity of the dominant-negative-effect for certain p53 pathways that may be important in cancer initiation. Additional mutant TRP53-specific transcriptional targets were also identified and are under further investigation. DisclosuresNo relevant conflicts of interest to declare.

Concomitant reduction of c-Myc expression and PI3K/AKT/mTOR signaling by quercetin induces a strong cytotoxic effect against Burkitt’s lymphoma

Burkitt’s lymphoma is an aggressive B cell lymphoma whose pathogenesis involves mainly c-Myc translocation and hyperexpression, in addition to antigen-independent BCR signaling and, in some cases, EBV infection. As result of BCR signaling activation, the PI3K/AKT/mTOR pathway results constitutively activated also in the absence of EBV, promoting cell survival and counterbalancing the pro-apoptotic function that c-Myc may also exert. In this study we found that quercetin, a bioflavonoid widely distributed in plant kingdom, reduced c-Myc expression and inhibited the PI3K/AKT/mTOR activity in BL, leading to an apoptotic cell death. We observed a higher cytotoxic effect against the EBV-negative BL cells in comparison with the positive ones, suggesting that this oncogenic gammaherpesvirus confers an additional resistance to the quercetin treatment. Besides cell survival, PI3K/AKT/mTOR pathway also regulates autophagy: we found that quercetin induced a complete autophagic flux in BL cells, that contributes to c-Myc reduction in some of these cells. Indeed, autophagy inhibition by chloroquine partially restored c-Myc expression in EBV-positive (Akata) and EBV-negative (2A8) cells that harbor c-Myc mutation. Interestingly, chloroquine did not affect the quercetin-mediated reduction of c-Myc expression in Ramos cells, that have no c-Myc mutation in the coding region, although autophagy was induced.These results suggest that mutant c-Myc could be partially degraded through autophagy in BL cells, as previously reported for other mutant oncogenic proteins.

Abstract 667: Tumor cell dormancy is mediated by an increase in RTK signaling in reversible pancreatic cancer models expressing oncogenic K-Ras and c-Myc

Abstract Recent genetic studies of pancreatic cancer expressing mutant Kras and c-Myc strongly favor targeted therapies against these oncogenic drivers for the successful treatment of advanced staged pancreatic cancer. The potential limitation of these treatments is that they likely leave residual cancer cells that can play a role in tumor recurrence. Our study is focused on understanding the molecular mechanism of the survival of pancreatic cancer cells in the absence of the tumor-driving oncogene in order to successfully eliminate cancer cells and prevent tumor relapse. Using a reversible c-Myc-induced pancreatic cancer model, we have demonstrated that expression of c-Myc in pancreatic progenitors is sufficient to induce invasive pancreatic cancer, and its expression is required for the maintenance of tumors at primary and metastatic sites. However, despite the complete macroscopic remission of invasive pancreatic tumors at primary and metastatic sites upon downregulation of c-Myc, a few cancer cells survived and remained dormant for a protracted period of time (Lin et al., 2013). In this current study, we have used a reversible Kras-induced pancreatic tumor model and found that cancer cell dormancy is a common attribute in pancreatic cancer following oncogene ablation. In an effort to identify common, cancer cell-intrinsic molecular pathways that mediate residual disease following the ablation of oncogenic drivers, we performed a genome-wide gene expression analysis (RNA-Seq) of in vivo-derived bulk tumor cells and dormant cancer cells that survived the ablation of c-Myc and oncogenic Kras. Comparison of the gene expression profile of bulk and residual cancer cells revealed that the residual cancer cells have increased receptor tyrosine kinase (RTK)/Akt signaling as a common mechanism for their survival in both c-Myc and Kras-induced tumors. We have experimentally validated the importance of this signaling pathway in cancer cell survival in the absence of oncogenic signaling in both tumor models. In comparison to control mice, we observed a significant slow tumor growth and delayed tumor relapse resulting in longer survival of the tumor bearing mice though a combination of this RTK/Akt pathway inhibition and ablation of Kras or c-Myc. In conclusion, our current study in reversible cancer models highlights the importance for the development of an adjuvant therapeutic strategy targeting this RTK/Akt pathway in addition to oncogenic drivers to effectively eradicate residual cancer cells and to prevent pancreatic cancer recurrence. Citation Format: Nirakar Rajbhandari. Tumor cell dormancy is mediated by an increase in RTK signaling in reversible pancreatic cancer models expressing oncogenic K-Ras and 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 667.

WT1 Enhances Proliferation and Impedes Apoptosis in KRAS Mutant NSCLC via Targeting cMyc

Background: A novel link between oncogenic KRAS signalling and WT1 was recently identified. We sought to investigate the role of WT1 and KRAS in proliferation and apoptosis. Methods: KRAS mutations and WT1 (cMyc) expression were detected using Sanger sequencing and real-time PCR in 77 patients with non-small cell lung cancer (NSCLC). Overexpression and knockdown of WT1 were generated with plasmid and siRNA via transient transfection technology in H1299 and H1568 cells. MTT assay for detection of cell proliferation, and TUNEL assay and proteomic profiler assay for apoptosis evaluation were carried out. Dual luciferase reporter assay and ChIP-PCR were performed to validate the effect of WT1 on the cMyc promoter. Results: KRAS mutations showed a negative impact on overall survival (OS). High expressions of WT1 and cMyc were associated with poor OS in KRAS mutant subgroup. The potential mechanisms that WT1 promotes proliferation and impedes apoptosis through affecting multiple apoptosis-related regulators in KRAS mutant NSCLC cells were identified. WT1 could activate cMyc promoter directly in KRAS mutant cells. Conclusion: The results suggest that WT1 and c-MYC expression is important for survival in KRAS mutant tumors as opposed to KRAS wild-type tumors. For treatment of KRAS mutant NSCLC, targeting WT1 and cMyc may provide alternative therapeutic strategies.

Abstract 3519: The c-Myc oncogene over-activates dormant replication origins and sensitizes cancer cells to chemotherapy drugs

Abstract Several clinical studies have shown a correlation between elevated c-Myc levels and a better response to chemotherapy. Although elevated c-Myc might indirectly cause such chemosensitivity, it is possible that a direct effect is occurring due to underlying molecular mechanisms. The purpose of the current study was to determine whether c-Myc overexpression could acutely cause chemosensitivity. The c-Myc protein is known to activate DNA replication origins, but the mechanism for this is unknown. Origins are regulated by the loading of MCM2-7 hexamers, which form the core of the helicase that starts the initiation of replication. Mammalian cells load more MCM complexes than are necessary to complete one round of replication. The excess MCM complexes function as backups to allow recovery of replication when forks stall during drug treatment. Consistent with this, we found that loss of Mcm4 or Mcm7 sensitizes PDAC cell lines to 5-FU and Gemcitabine, two S phase stress drugs that are used to treat PDAC patients but are not highly effective on their own. Similar to depletion of backup MCM complexes via loss of Mcm4 or Mcm7, elevated c-Myc expression over-activates backup MCMs, leaving fewer available to recover from drug treatment. Cells containing overexpressed c-Myc display increased chemosensitivity to Gemcitabine that is not seen in cells containing normal c-Myc levels or a non-functional c-Myc mutant. These results demonstrate that reduction in backup MCM complexes, either by direct downregulation or by oncogene driven over-activation (via c-Myc), render cells sensitive to chemotherapy drugs. These results suggest that development of drugs that target MCM functionality may be useful in combination with S-phase stress drugs currently used in the clinic. These results also demonstrate that c-Myc has the ability to acutely cause chemosensitivity and c-Myc elevated tumors may respond better to chemotherapy due to the c-Myc effects on MCM complexes. Citation Format: Victoria Bryant, Mark G. Alexandrow. The c-Myc oncogene over-activates dormant replication origins and sensitizes cancer cells to chemotherapy drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3519. doi:10.1158/1538-7445.AM2014-3519