Inhibition Of MYC Expression Research Articles

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

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

MDB-05. HUMAN FETAL CEREBELLAR CELL ATLAS INFORMS MEDULLOBLASTOMA ORIGIN AND ONCOGENESIS

Abstract Medulloblastoma (MB) is the most common malignant childhood brain tumor, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatments. Previous analyses of mouse cerebella or human counterparts from frozen tissue nuclei5 have not fully defined the compositional heterogeneity of MBs. Here, we undertook an unprecedented single-cell profiling of freshly-isolated human fetal cerebella to establish a reference map delineating hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in developing fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group-3 and metastatic tumors. Single-cell multi-omics revealed underlying regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in group-3 MBs. Genomic and Hi-C profiling identified de novo long-range chromatin-loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group-3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group-3 MB growth. Our integrated single-cell atlases of human fetal cerebella and MBs reveal potential cell populations predisposed to transformation and regulatory circuitries underlying tumor cell states and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

KLF4-mediated upregulation of the NKG2D ligand MICA in acute myeloid leukemia: a novel therapeutic target identified by enChIP

The immunoreceptor NKG2D, which is expressed on NK cells and T cell subsets is critically involved in tumor immune surveillance. This applies in particular to acute myeloid leukemia (AML), which evades immune detection by downregulation of NKG2D ligands (NKG2D-L), including MICA. The absence of NKG2D-L on AML cells is moreover associated with leukemia stem cell characteristics. The NKG2D/NKG2D-L system thus qualifies as an interesting and promising therapeutic target.Here we aimed to identify transcription factors susceptible to pharmacological stimulation resulting in the expression of the NKG2D-L MICA in AML cells to restore anti-tumor activity. Using a CRISPR-based engineered ChIP (enChIP) assay for the MICA promoter region and readout by mass spectrometry-based proteomics, we identified the transcription factor krüppel-like factor 4 (KLF4) as associated with the promoter. We demonstrated that the MICA promoter comprises functional binding sites for KLF4 and genetic as well as pharmacological gain- and loss-of-function experiments revealed inducible MICA expression to be mediated by KLF4.Furthermore, induction in AML cells was achieved with the small compound APTO253, a KLF4 activator, which also inhibits MYC expression and causes DNA damage. This induction in turn yielded increased expression and cell surface presentation of MICA, thus rendering AML cells more susceptible to NK cell-mediated killing. These data unravel a novel link between APTO253 and the innate anti-tumor immune response providing a rationale for targeting AML cells via APTO253-dependent KFL4/MICA induction to allow elimination by endogenous or transplanted NK and T cells in vivo.D1rDmzAPriWUyYABRWPo9PVideo

Human fetal cerebellar cell atlas informs medulloblastoma origin and oncogenesis.

Medulloblastoma (MB) is the most common malignant childhood brain tumour1,2, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatments. Previous analyses of mouse cerebella3-5 have not fully defined the compositional heterogeneity of MBs. Here we undertook single-cell profiling of freshly isolated human fetal cerebella to establish a reference map delineating hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in developing fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group 3 and metastatic tumours. Single-cell multi-omics revealed underlying regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in group 3 MBs. Genomic and Hi-C profiling identified de novo long-range chromatin loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group 3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group 3 MB growth. Our integrated single-cell atlases of human fetal cerebella and MBs show potential cell populations predisposed to transformation and regulatory circuitries underlying tumour cell states and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

Activating a Non-Genetic MYC-Regulating Pathway Sensitizes Acute Myeloid Leukemia to Bromodomain Inhibition

MYC (c-Myc) plays a central role in the oncogenic progression and is commonly overexpressed or amplified in more than 50% of human cancers, including acute myeloid leukemia (AML), which is an aggressive disease with over 70% of patients who cannot survive more than five years. Elevated MYC expression is associated with therapeutic resistance and predicts a poorer outcome in AML patients. While numerous lines of evidence have validated MYC as a therapeutic target of AML and many other cancer types, MYC protein itself has been a challenge for therapeutic targeting. Recently, MYC gene transcription has been linked to the bromodomain and extra-terminal domain (BET) family proteins. BET inhibitors (BETi) inhibit MYC expression and have shown promising antitumor activities in preclinical models and are being evaluated in clinical trials. Despite the therapeutic promise, BETi-associated drug resistance and dose-limiting side effects hinder the clinical applications. Therefore, there is a pressing need to understand the mechanisms controlling drug response and resistance to BET inhibitors. To understand the mechanisms that regulate BETi sensitivity, we leveraged a functional CRISPR screen focusing on about 1,700 human transcription factors. We discovered the aryl hydrocarbon receptor (AHR) as a novel regulator of BETi sensitivity in AML. Our results have further identified that a novel AHR-ELMSAN1 axis is critically involved in regulating MYC and BETi sensitivity. Specifically, we show that constitutive or ligand activation of AHR sensitizes leukemia cells to BETi. AHR signaling represses MYC and synergizes with BETi for maximum inhibition of MYC transcription. Ectopic expression of MYC could rescue the anti-leukemia effects induced by combined AHR activation and BET blockade. Mechanistically, our unbiased bioinformatic analysis unexpectedly identified that AHR functions through activating a non-canonical target, ELMSAN1, a less-studied epigenetic factor known as a member of the MiDAC histone deacetylase complex. Depletion of ELMSAN1 leads to upregulation of MYC and blunts AHR signaling-mediated MYC repression and BETi sensitization. Importantly, we demonstrate that AHR agonist and BETi synergistically inhibit MYC and leukemia cell growth in CD34+ primary AML patient samples in vitro and patient-derived xenografts in vivo. In summary, our data suggest that AHR-ELMSAN1 is a novel pathway for MYC repression, activation of which could sensitize AML to BET-targeting agents. As neither AHR nor ELMSAN1 is found mutated in AML, our research may open a new door to new MYC targeting paradigms by activating novel non-genetic MYC repressors.

STEM-24. SINGLE-CELL ATLAS OF HUMAN FETAL CEREBELLUM DECODING MEDULLOBLASTOMA ORIGIN AND ONCOGENESIS

Abstract Medulloblastomas (MBs) are the most common malignant childhood brain tumors, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatment strategies. Previous cell-type analyses of mouse cerebella or human counterparts from frozen tissue nuclei have not fully defined the compositional heterogeneity of MBs. Here, we undertook an unprecedented single-cell profiling of freshly-isolated human fetal cerebella at different developmental stages to establish a reference map for delineating the hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in human fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group-3 and metastatic tumors. Integrated single-cell multi-omic profiling revealed unique regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in aggressive group-3 MBs. Genomic profiling and Hi-C analyses identified de novo long-range chromatin loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group-3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group-3 MB growth. Together, our integrated single-cell atlases of human fetal cerebella and MBs reveal important cell populations predisposed to transformation and regulatory circuitries underlying tumor cell state evolution and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

Inhibition of MYC suppresses programmed cell death ligand-1 expression and enhances immunotherapy in triple-negative breast cancer.

Cancer immunotherapy has emerged as a promising strategy against triple-negative breast cancer (TNBC). One of the immunosuppressive pathways involves programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1), but many patients derived little benefit from PD-1/PD-L1 checkpoint blockades treatment. Prior research has shown that MYC, a master transcription amplifier highly expressed in TNBC cells, can regulate the tumor immune microenvironment and constrain the efficacy of immunotherapy. This study aims to investigate the regulatory relationship between MYC and PD-L1, and whether a cyclin-dependent kinase (CDK) inhibitor that inhibits MYC expression in combination with anti-PD-L1 antibodies can enhance the response to immunotherapy. Public databases and TNBC tissue microarrays were used to study the correlation between MYC and PD-L1. The expression of MYC and PD-L1 in TNBCs was examined by quantitative real-time polymerase chain reaction and Western blotting. A patient-derived tumor xenograft (PDTX) model was used to evaluate the influence of a CDK7 inhibitor THZ1 on PD-L1 expression. Cell proliferation and migration were detected by 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation and cell migration assays. Tumor xenograft models were established for in vivo verification. A high MYC expression level was associated with a poor prognosis and could alter the proportion of tumor-infiltrating immune cells (TIICs). The positive correlation between MYC and PD-L1 was confirmed by immunostaining samples from 165 TNBC patients. Suppression of MYC in TNBC caused a reduction in the levels of both PD-L1 messenger RNA and protein. In addition, antitumor immune response was enhanced in the TNBC cancer xenograft mouse model with suppression of MYC by CDK7 inhibitor THZ1. The combined therapy of CDK7 inhibitor THZ1 and anti-PD-L1 antibody appeared to have a synergistic effect, which might offer new insight for enhancing immunotherapy in TNBC.

A phase II trial of the super-enhancer inhibitor Minnelide™ in advanced refractory adenosquamous carcinoma of the pancreas.

Adenosquamous carcinoma of the pancreas (ASCP) is a very rare and highly aggressive variant of pancreatic ductal adenocarcinoma, accounting for 0.5-4% of all pancreatic cancer cases in the USA. Current data indicate that epigenetic changes and MYC overexpression lead to squamous transdifferentiation of pancreatic tumor cells and development of ASCP. Minnelide™, an oral anti-super-enhancer drug that inhibits MYC expression in preclinical models of ASCP, has demonstrated safety in a phase I study. We describe the design for a phase II, open-label, single-arm trial of Minnelide in patients with advanced refractory ASCP.

MiR-138 is a potent regulator of the heterogenous MYC transcript population in cancers

3′UTR shortening in cancer has been shown to activate oncogenes, partly through the loss of microRNA-mediated repression. This suggests that many reported microRNA-oncogene target interactions may not be present in cancer cells. One of the most well-studied oncogenes is the transcription factor MYC, which is overexpressed in more than half of all cancers. MYC overexpression is not always accompanied by underlying genetic aberrations. In this study, we demonstrate that the MYC 3′UTR is shortened in colorectal cancer (CRC). Using unbiased computational and experimental approaches, we identify and validate microRNAs that target the MYC coding region. In particular, we show that miR-138 inhibits MYC expression and suppresses tumor growth of CRC and hepatocellular carcinoma (HCC) cell lines. Critically, the intravenous administration of miR-138 significantly impedes MYC-driven tumor growth in vivo. Taken together, our results highlight the previously uncharacterized shortening of the MYC 3′UTR in cancer, and identify miR-138 as a potent regulator of the heterogenous MYC transcript population.

Abstract PO-054: A phase II trial of the super-enhancer inhibitor Minnelide in advanced refractory adenosquamous carcinoma of the pancreas (ASCP)

Abstract Background: Adenosquamous carcinoma of the pancreas (ASCP) is a very rare and highly aggressive variant of pancreatic ductal adenocarcinoma (PDA), accounting for 0.5-4% of all pancreatic cancer cases in the US. Current data indicates that epigenetic changes and Myc overexpression lead to squamous transdifferentiation of pancreatic tumor cells and development of ASCP. Minnelide is an oral anti-super-enhancer drug that inhibits Myc expression in preclinical models of ASCP. Oral Minnelide has been previously tested in the clinic where a Phase I study determined a recommended phase 2 dose of 2 mg/day. Major toxicities included neutropenia and sepsis. We hypothesize that Minnelide will be safe and effective in patients with advanced ASCP. Methods: This Phase II open label, single arm study being conducted at the NIH Clinical Center in Bethesda, MD (NCT04896073) will enroll up to 25 evaluable participants with advanced ASCP that has been previously treated. Eligibility criteria include: histologically confirmed ASCP (≥30% malignant squamous component), presence of metastatic, recurrent, or locally advanced unresectable disease, and progression or intolerance ≥1 prior systemic treatment for advanced disease. All participants will self-administer Minnelide at 2mg PO daily on days 1-21 of each 28-day cycle, for up to 12 cycles in the absence of disease progression or unacceptable toxicity. Restaging scans will be performed every 8 weeks. Optional biopsies will be requested at baseline, mid-cycle 1 and at time of progression. The primary objective is to determine the antitumor activity of Minnelide by evaluating the disease control rate. Secondary objectives are determination of drug safety in this patient population, progression free survival, and overall survival. Exploratory objectives include assessment of Minnelide effect on tumor cell epigenetics, Myc expression and immune cell populations using on-treatment tumor biopsy tissues. Changes in circulating tumor DNA and circulating immune cell populations upon treatment will also be evaluated. A Simon optimal two-stage phase II trial design will be used. If 3 of the first 12 patients experience disease control, then enrollment will continue up to 25 evaluable participants. If ≥8 of 25 participants experience disease control this would rule out an unacceptably low clinical benefit of 20% in favor of 40% (α = 0.1, β = 0.2). This trial opened for accrual in July 2021. Citation Format: Nebojsa Skorupan, Mehwish I. Ahmad, Seth M. Steinberg, Jane B. Trepel, Derek Cridebring, Haiyong Han, Daniel D. Von Hoff, Christine Alewine. A phase II trial of the super-enhancer inhibitor Minnelide in advanced refractory adenosquamous carcinoma of the pancreas (ASCP) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-054.

MicroRNA‑27b inhibits the development of melanoma by targeting MYC

Cutaneous malignant melanoma is a malignancy with one of the fastest increasing incidence rates worldwide; however, the mechanism underlying the occurrence and development of melanoma remains unclear. The aim of the present study was to identify novel biomarkers for the occurrence and development of melanoma. The results of the present study demonstrated that the expression levels of microRNA (miR)-27b were decreased in melanoma tissue samples compared with those in adjacent noncancerous tissue samples and cells according to online and experimental data. By contrast, MYC expression levels were upregulated in melanoma compared with those in adjacent noncancerous tissue samples. miR-27b overexpression significantly inhibited A375 and A2085 melanoma cell DNA synthesis, viability and invasive ability. Dual-luciferase reporter assay results demonstrated that miR-27b inhibited MYC expression through binding to the 3′-untranslated region of MYC mRNA. MYC knockdown in melanoma cells exerted similar effects to those of miR-27b overexpression on DNA synthesis, cell viability and invasive ability; the effects of miR-27b inhibition were significantly reversed by MYC knockdown. In conclusion, the miR-27b/MYC axis may modulate malignant melanoma cell biological behaviors and may be a potential target for melanoma treatment.

FTO Inhibition Enhances the Antitumor Effect of Temozolomide by Targeting MYC-miR-155/23a Cluster-MXI1 Feedback Circuit in Glioma

Malignant glioma constitutes one of the fatal primary brain tumors in adults. Such poor prognosis calls for a better understanding of cancer-related signaling pathways of this disease. Here we elucidate a MYC-miRNA-MXI1 feedback loop that regulates proliferation and tumorigenesis in glioma. MYC suppressed MXI1 expression via microRNA-155 (miR-155) and the microRNA-23a∼27a∼24-2 cluster (miR-23a cluster), whereas MXI1, in turn, inhibited MYC expression by binding to its promoter. Overexpression of miR-155 and the miR-23a cluster promoted tumorigenesis in U87 glioma cells. Furthermore, fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) RNA demethylase, regulated the loop by targeting MYC. The ethyl ester form of meclofenamic acid (MA2) inhibited FTO and enhanced the effect of the chemotherapy drug temozolomide on suppressing proliferation of glioma cells and negatively regulated the loop. These data collectively highlight a key regulatory circuit in glioma and provide potential targets for clinical treatment. SIGNIFICANCE: These findings elucidate a novel feedback loop that regulates proliferation in glioma and can be targeted via inhibition of FTO to enhance the efficacy of temozolomide.

Protein phosphatase 2A activation as a therapeutic strategy for managing MYC-driven cancers.

The tumor suppressor protein phosphatase 2A (PP2A) is a serine/threonine phosphatase whose activity is inhibited in most human cancers. One of the best-characterized PP2A substrates is MYC proto-oncogene basic helix-loop-helix transcription factor (MYC), whose overexpression is commonly associated with aggressive forms of this disease. PP2A directly dephosphorylates MYC, resulting in its degradation. To explore the therapeutic potential of direct PP2A activation in a diverse set of MYC-driven cancers, here we used biochemical assays, recombinant cell lines, gene expression analyses, and immunohistochemistry to evaluate a series of first-in-class small-molecule activators of PP2A (SMAPs) in Burkitt lymphoma, KRAS-driven non-small cell lung cancer, and triple-negative breast cancer. In all tested models of MYC-driven cancer, the SMAP treatment rapidly and persistently inhibited MYC expression through proteasome-mediated degradation, inhibition of MYC transcriptional activity, decreased cancer cell proliferation, and tumor growth inhibition. Importantly, we generated a series of cell lines expressing PP2A-dependent phosphodegron variants of MYC and demonstrated that the antitumorigenic activity of SMAPs depends on MYC degradation. Collectively, the findings presented here indicate a pharmacologically tractable approach to drive MYC degradation by using SMAPs for the management of a broad range of MYC-driven cancers.

Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells

The transcriptional regulator BRD4 has been shown to be important for the expression of several oncogenes including MYC. Inhibiting of BRD4 has broad antiproliferative activity in different cancer cell types. The small molecule JQ1 blocks the interaction of BRD4 with acetylated histones leading to transcriptional modulation. Depleting BRD4 via engineered bifunctional small molecules named PROTACs (proteolysis targeting chimeras) represents the next-generation approach to JQ1-mediated BRD4 inhibition. PROTACs trigger BRD4 for proteasomale degradation by recruiting E3 ligases. The aim of this study was therefore to validate the importance of BRD4 as a relevant target in colorectal cancer (CRC) cells and to compare the efficacy of BRD4 inhibition with BRD4 degradation on downregulating MYC expression. JQ1 induced a downregulation of both MYC mRNA and MYC protein associated with an antiproliferative phenotype in CRC cells. dBET1 and MZ1 induced degradation of BRD4 followed by a reduction in MYC expression and CRC cell proliferation. In SW480 cells, where dBET1 failed, we found significantly lower levels of the E3 ligase cereblon, which is essential for dBET1-induced BRD4 degradation. To gain mechanistic insight into the unresponsiveness to dBET1, we generated dBET1-resistant LS174t cells and found a strong downregulation of cereblon protein. These findings suggest that inhibition of BRD4 by JQ1 and degradation of BRD4 by dBET1 and MZ1 are powerful tools for reducing MYC expression and CRC cell proliferation. In addition, downregulation of cereblon may be an important mechanism for developing dBET1 resistance, which can be evaded by incubating dBET1-resistant cells with JQ1 or MZ1.

Abstract 2096: Resistance to APTO-253 caused by internal deletion and alternate promoter usage of the MYC gene in malignant B cells

Abstract APTO-253 is a clinical stage small molecule that acts in part by down-regulation of MYC at mRNA and protein levels. MYC onco-protein dysregulation, which leads to increased proliferation and survival, is implicated in the etiology of numerous cancer subtypes. In the current study, we explored sensitivity of B-cell cancer cells to APTO-253 and sought to understand how changes in MYC can yield resistance to APTO-253. APTO-253 showed potency against B-cell cancer cell lines (IC50 30-600 nM) and bone marrow samples from patients. Sensitivity of cell lines correlated with MYC over-expression, amplification or translocation. APTO-253 resistant Raji cells (Raji253R) are characterized by upregulation of the ABCG2 transporter. Treatment with ABCG2 inhibitors only partially reversed the resistant phenotype suggesting additional contributing mechanisms. RNA-seq analysis revealed that while levels of MYC mRNA were unchanged in Raji253R, a truncation of exon 2 was present. This internal deletion occurred at the DNA level, not by alternative splicing, and resulted in a truncated MYC protein (45 kDa) lacking the conserved core sequence of MYC box III (MBIII). At early stages in the evolution of resistance residual full length MYC mRNA and protein was detectable; however, this expression was eventually lost in Raji253R cells as selection continued. Deletion of MBIII has been implicated in MYC protein stability, in vitro and in vivo transformation, gene repression, and MYC dependent apoptosis. Cycloheximide treatment of Raji and Raji253R cells demonstrated a modest increase in stability of truncated over full length MYC protein. In addition, Raji253R cells exhibited hypersensitivity to etoposide. The internal truncation is flanked by sites of microhomology suggesting a method for deletion. We hypothesize that DNA damage in response to APTO-253 treatment led to error prone repair by microhomology end joining and resulted in a MYC internal deletion. Interestingly, transcription from the canonical P1/P2 promoter, as seen in WT Raji cells, was systematically lost and transcription initiation shifted to a downstream promoter, P3. APTO-253 inhibits MYC expression via binding and stabilizing the G-quadruplex (G4) sequence in the P1/P2 promoter therefore switching of transcriptional initiation to P3 should relieve APTO-253 mediated repression. Indeed, APTO-253 treatment of Raji253R cells did not repress MYC expression, even at concentrations 50-fold higher than required for MYC repression in Raji WT cells. Presence of the active intra-cellular form of APTO-253, Fe(253)3, was confirmed in Raji253R cells. In summary, MYC driven Raji cells become resistant to APTO-253 by acquisition of a more stable MYC protein and by utilization of an alternate promoter not inhibited by G4 binding and stabilization, thereby confirming the essential role of MYC repression in the mechanism of action of APTO-253. Citation Format: Andrea Local, Cheng-Yu Tsai, Hongying Zhang, Susan Sheng, Khalid Benbatoul, Stephen B. Howell, William G. Rice. Resistance to APTO-253 caused by internal deletion and alternate promoter usage of the MYC gene in malignant B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2096.

Targeting translation initiation by synthetic rocaglates for treating MYC-driven lymphomas.

MYC-driven lymphomas, especially those with concurrent MYC and BCL2 dysregulation, are currently a challenge in clinical practice due to rapid disease progression, resistance to standard chemotherapy and high risk of refractory disease. MYC plays a central role by coordinating hyperactive protein synthesis with upregulated transcription in order to support rapid proliferation of tumor cells. Translation initiation inhibitor rocaglates have been identified as the most potent drugs in MYC-driven lymphomas as they efficiently inhibit MYC expression and tumor cell viability. We found that this class of compounds can overcome eIF4A abundance by stabilizing target mRNA-eIF4A interaction that directly prevents translation. Proteome-wide quantification demonstrated selective repression of multiple critical oncoproteins in addition to MYC in B cell lymphoma including NEK2, MCL1, AURKA, PLK1, and several transcription factors that are generally considered undruggable. Finally, (−)-SDS-1–021, the most promising synthetic rocaglate, was confirmed to be highly potent as a single agent, and displayed significant synergy with the BCL2 inhibitor ABT199 in inhibiting tumor growth and survival in primary lymphoma cells in vitro and in patient-derived xenograft mouse models. Overall, our findings support the strategy of using rocaglates to target oncoprotein synthesis in MYC-driven lymphomas.

Identification of G-Quadruplex-Binding Inhibitors of Myc Expression through Affinity Selection–Mass Spectrometry

The Myc oncogene is overexpressed in many cancers, yet targeting it for cancer therapy has remained elusive. One strategy for inhibition of Myc expression is through stabilization of the G-quadruplex (G4), a G-rich DNA secondary structure found within the Myc promoter; stabilization of G4s has been shown to halt transcription of downstream gene products. Here we used the Automated Ligand Identification System (ALIS), an affinity selection–mass spectrometry method, to identify compounds that bind to the Myc G4 out of a pool of compounds that had previously been shown to inhibit Myc expression in a reporter screen. Using an ALIS-based screen, we identified hits that bound to the Myc G4, a small subset of which bound preferentially relative to G4s from the promoters of five other genes. To determine functionality and specificity of the Myc G4-binding compounds in cell-based assays, we compared inhibition of Myc expression in cells with and without Myc G4 regulation. Several compounds inhibited Myc expression only in the Myc G4-containing line, and one compound was verified to function through Myc G4 binding. Our study demonstrates that ALIS can be used to identify selective nucleic acid-binding compounds from phenotypic screen hits, increasing the pool of drug targets beyond proteins.

The kinases IKBKE and TBK1 regulate MYC-dependent survival pathways through YB-1 in AML and are targets for therapy

AbstractTo identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.

A miR-26a/E2F7 feedback loop contributes to tamoxifen resistance in ER-positive breast cancer.

Tamoxifen (TAM) resistance is a substantial challenge in the treatment of estrogen receptor (ER)-positive breast cancer. Previous studies have revealed an important role of microRNA (miRNA/miR)-26a in TAM resistance in breast cancer. However, the mechanism underlying the regulatory effects of miR-26a on TAM resistance remains to be elucidated. The expression levels of miR-26a in ER-positive breast cancer were detected by reverse transcription-quantitative polymerase chain reaction. E2F transcription factor7(E2F7) and MYC proto-oncogene, bHLH transcription factor(MYC) levels were detected by western blotting. The present study demonstrated that miR-26a expression was reduced in ER-positive breast cancer compared with in normal breast tissues, whereas E2F7 expression was significantly elevated. Furthermore, an inverse correlation between miR-26a and E2F7 expression was detected in ER-positive breast cancer. The results indicated that miR-26a directly inhibited E2F7 expression through translational inhibition and indirectly inhibited MYC expression partly via E2F7 repression. E2F7, in turn, decreased miR-26a expression via MYC-induced transcriptional inhibition of miRNAs. Furthermore, transfection with miR-26a mimics increased the expression of its host genes (CTD small phosphatase like and CTD small phosphatase2), whereas ectopic E2F7 expression abrogated the effects of miR-26a. These findings indicated that miR-26a and E2F7 may form a double-negative feedback loop, resulting in downregulation of miR-26a and upregulation of E2F7 in ER-positive breast cancer. Both miR-26a knockdown and E2F7 overexpression conferred resistance to TAM in MCF-7 cells. Conversely, miR-26a overexpression and E2F7 silencing resensitized MCF-7 resistant cells to TAM. These findings revealed that a feedback loop between miR-26a and E2F7 may promote TAM resistance in ER-positive breast cancer.

Abstract 5790: TG-1601 is a novel BET inhibitor with strong binding affinity and long-lasting effect in pre-clinical models

Abstract BET (bromodomain and extra-terminal) proteins bind to acetylated lysine residues on chromatin and participate in the regulation of gene transcription. Inhibition of BET protein binding to chromatin with small molecules selectively suppresses the transcription of a set of oncogenes, including MYC and BCL-2. TG-1601 is a novel, selective and potent small molecule inhibitor of BET bromodomains. TG-1601 binds to the first and second bromodomains (BD1, BD2) of the BET protein family, BRD2, BRD3, BRD4, and BRDT, with Kd values ranging from 0.5 nM to 9.1 nM. MYC protein expression is strongly inhibited in the MV4-11 cancer cell line with an EC50 of 5 nM, with GI50 comprised between 15 nM and 85 nM in a variety of leukemia and myeloma cancer cell lines, indicating potent inhibition of cell proliferation. Time course and dose-response studies conducted in mice bearing subcutaneous MV4-11 xenografts showed that MYC protein was undetectable 3 hours following a single 25 mg/kg oral dose, with a TG-1601 tumor concentration of 6uM achieved. Interestingly, at 24h post-dose, while TG-1601 is cleared from the tumor, MYC protein level remains below 40% of its initial level, indicating a long-lasting effect pharmacodynamic of TG-1601, potentially attributable to enhanced binding affinity compared to earlier generation molecules. In agreement with this long-lasting effect, efficacy studies in MV4-11 tumor-bearing mice, dosed with a 20 mg/kg/day PO regimen interrupted by increasing drug holiday periods, showed that drug holidays of 2, 3 and 4 days per week only modestly affected efficacy (3%, 15% and 12% TGI respectively), suggesting discontinuous dosing of TG-1601 in clinic may not significantly impact efficacy. Anti-cancer agents have been shown - in vitro or in vivo - to synergize with BET inhibitors. Here we show that TG-1601 and anti PD-1 antibody demonstrated synergistic anti-tumor activity when combined in the B16F10 model, an aggressive syngeneic model of melanoma. TG-1601 inhibition of MYC, CCR-2 and IL1RN gene expression was tested in a whole blood ex-vivo experiment, and the genes were validated as pharmacodynamic markers to monitor TG-1601 activity in clinic. In conclusion, TG-1601 is a novel BET inhibitor with remarkably strong affinity and a potent ability to inhibit MYC expression and cell growth, with favorable pharmacokinetic properties supporting clinical development. Its properties in vivo provide an opportunity for the rational development of TG-1601 as an anti-cancer agent, taken alone or in combination with other small molecules or antibodies. IND enabling studies are underway, with clinical evaluation expected to commence in the first half of 2018. Citation Format: Emmanuel Normant, Leonid Gorelik, Rama Shmeis, Henry Le, Robert Nisch, Karen TenHuisen, Teja Turpuseema, James Oliviero, Hari P. Miskin, Peter Sportelli, Michael S. Weiss. TG-1601 is a novel BET inhibitor with strong binding affinity and long-lasting effect in pre-clinical models [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 5790.