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Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. Significance: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

Drug combinations are the basis of treatment for modern diseases but arriving at successful combination therapies is fraught with challenges. Decades ago, the limited number of drugs represented a tractable candidate list from which to design combination experiments. However, the current pool of single-agent drugs to potentially combine is far too large to brute-force screen, and purely computational predictions have performed poorly. Suitable screening methods are needed, but the design of experimental approaches has proven to be highly complex; researchers need to carefully balance many variables such as appropriate drug concentration ranges, number of doses, inclusion of replicates, and throughput. Perhaps the most significant obstacle facing these studies is the approach to data analysis, where conflicting definitions of synergy and unintuitive metrics serve to confuse researchers and render largely uninterpretable results. Collectively, these challenges hamper the progress of drug combination research and ultimately translational impact. To overcome these limitations, we have developed a fully self-contained framework to handle both the experimental design and analysis of drug combination experiments. Our method, called Combocat, provides a straightforward way to test and analyze any number of drug combinations and samples, and is suitable for high-throughput. Combocat provides a high-resolution of concentration combinations compared to most current approaches. This is automated by common instruments and uses scripts included within our protocol. Through careful template design, we were able to include 3 replicates of each 10x10 matrix, single-agent drugs, and controls - all within a single 384-well plate. We found our method to work robustly with varying sample types (Human cancer, bacteria, fungi), and readouts. After data generation, files can simply be dragged into our Combocat analysis tool directly. We provide a free, web-based software suite to fully automate the analysis after data collection. The Combocat web tool is intuitive and facilitates interactive exploration of synergy. It also provides a rich array of information such as dose-response curves, IC50 values, synergy matrices, ranked hit plots, and more. Data normalization, synergy algorithms, scoring functions, and other complex calculations are run swiftly and automatically in the background with no need for user input. Notably, we employ statistical testing by taking advantage of experimental replicates, which is a feature we found lacking in most methods. We use a well-documented synergy metric but also decided to formulate our own Combocat score which considers statistical measurements and assay quality. The Combocat score provides an easy interpretation of results and facilitates quick identification of top hits. Collectively, our platform will be used to enhance and expedite the selection of effective drug combinations. Citation Format: William C. Wright, Min Pan, Hyeong-Min Lee, Gregory A. Phelps, Jonathan Low, Duane Currier, Richard E. Lee, Taosheng Chen, Paul Geeleher. Combocat: A high-throughput framework for drug combination studies [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 1907.

The use of multikinase inhibitors (MKI) in oncology, such as sorafenib, is associated with a cutaneous adverse event called hand–foot skin reaction (HFSR), in which sites of pressure or friction become inflamed and painful, thus significantly impacting quality of life. The pathogenesis of MKI-induced HFSR is unknown, and the only available treatment options involve dose reduction or discontinuation of therapy, which have negative effects on primary disease management. To investigate the underlying mechanisms by which sorafenib promotes keratinocyte cytotoxicity and subsequent HFSR induction, we performed a transporter-directed RNAi screen in human epidermal keratinocytes and identified SLC22A20 (OAT6) as an uptake carrier of sorafenib. Further investigations into the intracellular mechanism of sorafenib activity through in situ kinome profiling identified the mitogen-activated protein kinase MAP3K7 (TAK1) as a target of sorafenib that induces cell death. Finally, we demonstrate that sorafenib induced keratinocyte injury in vivo and that this effect could be reversed by cotreatment with the OAT6 inhibitor probenecid. Collectively, our findings reveal a novel pathway that regulates the entry of some MKIs into keratinocytes and explains the basis underlying sorafenib-induced skin toxicity, with important implications for the therapeutic management of HFSR. Cancer Res; 76(1); 117–26. ©2015 AACR.

Genes encoding epigenetic regulators are commonly mutated at relapse in acute lymphoblastic leukemia, but the way in which such mutations promote drug resistance and their potential for therapeutic targeting is largely unknown. We previously identified mutations in CREBBP, encoding CREBBP binding protein, a histone and non-histone acetylase and transcriptional coregulator in relapsed ALL and non-Hodgkin’s lymphoma. In view of the known role of CREBBP mediation of the transcriptional response to glucocorticoids, we hypothesized that CREBBP mutations promote glucocorticoid resistance, and sought to comprehensively define the prevalence and spectrum of mutations in relapsed ALL, and elucidate their role in resistance. In studies of childhood ALL from St. Jude and the Children’s Oncology Group, CREBBP mutations were observed in 37 (21.2%) of patients, representing the most common target of sequence mutation at relapse. The mutations were either preserved from diagnosis to relapse, or evolved as new mutations or from minor clones to become clonal mutations at relapse. There were hotspots of mutation at R1446 and between P1488-P1494 in the histone acetyltransferase domain, residues critical for recognition of acetylation substrates, and are known or predicted to impair histone acetylation in biochemical assays and in Crebbp-/-;Ep300-/- mouse embryonic fibroblasts reconstituted with mutant CREBBP. To examine the role of CREBBP mutations in drug resistance, we generated isogenic CREBBP wild-type and mutant human ALL cell lines, and conventional and conditional Crebbp Q1501P (=human Q1500) and R1447H (=human R1446) mouse strains. Mice bearing germline Q1501P or R1447H alleles exhibited dysmorphology, reduced fertility, lymphoproliferation, and reduced lifespan. This phenotype was most pronounced in Crebbp R1447 mice, preventing establishment of a stable line; thus, we generated a conditional Crebbp-R1447H knockin strain (CREBBP-R1447H-CKI) that expresses CREBBP R1447H following Cre-mediated recombination. We generated BCR-ABL1 pre-B ALL models by transducing E13.5 fetal liver cells with BCR-ABL1 p185 retrovirus. Consistently, leukemias derived from heterozygous or homozygous CREBBP-Q1501P or heterozygous CREBBP-R1447H-CKI mice showed profound and selective resistance to prednisolone and dexamethasone. Similarly, NALM6 (DUX4/ERG) human ALL cells harboring CREBBP mutations induced by genome editing (NALM6-C001) also showed complete resistance to glucocorticoids, without change in expression of the glucocorticoid receptor (GR). Chromatin immunoprecipitation and sequencing showed that this was accompanied by marked attenuation of recruitment of GR to chromatin, attenuation of histone 3 lysine 18 (H3K18) and H3K27 acetylation, and reduced expression of H3K18Ac-marked genes following dexamethasone treatment. Global acetylome profiling showed that CREBBP mutations resulted in perturbed acetylation of multiple histone and non-histone targets, including CREBBP itself, IKZF1, and MYC, with reciprocal increase in EP300 acetylation. To identify agents that kill CREBBP mutant leukemic cells and that may reverse steroid resistance, we screened NALM6-C001 with a collection of 11,293 chemicals (5,832 unique compounds enriched for FDA-approved drugs) dosed at 10 mM in the presence or absence of dexamethasone for 48 or 96 hrs. 223 active compounds and 220 compounds targeting epigenetic modifications were tested in 10-point dose response screen. Seventeen compounds with IC50 These results demonstrate the high prevalence of CREBBP in relapsed ALL, and show that these mutations have a central role in driving glucocorticoid resistance. These data also suggest that these mutations create a therapeutic vulnerability through EP300 acetylation that may be exploited by bromodomain inhibition. Citation Format: Yunchao Chang, David W. Woessner, Wenwei Lin, Taosheng Chen, Beisi Xu, Yiping Fan, Haiyan Tan, Junmin Peng, Lawryn Kasper, Michelle L. Churchman, Daniela S. Gerhard, Mignon L. Loh, Stephen P. Hunger, Aman Seth, Karen Gascoigne, Charles G. Mullighan. Modeling and targeting CREBBP mutations in relapsed acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA12.

Ubiquitination of protein is well known mechanism for the regulation of cellular proteins by the ubiquitin-proteasome system (UPS). The ubiquitin-proteasome system (UPS) plays a crucial role in regulating cellular protein degradation and turnover. Dysregulation of the UPS has been associated with a wide variety of diseases including cancers, neurodegenerative disorders, and viral and bacterial infections. Recently, there are emerging evidences show that targeting this UPS by small molecule modulator is a useful anticancer strategy based on the previous successes of proteasome inhibitors, E3 inhibitors and the NEDD8 E1 inhibitor. In particular, we are targeting NEDD8 ligation to cullins via inhibition of DCN1 as a small molecule target. DCN1 was known to interacts with cullins and is required for neddylation in vivo. Furthermore, dysregulation of DCN1 is associated with a number of human cancers. DCN1 activity is controlled by its interaction with UBC12. Therefore, we have developed biochemical assay utilizing TR-FRET technology for the purpose of high-throughput screening (HTS) to identify the small molecule inhibitors that disrupt the DCN1-UBC12 protein-protein interaction and use them to elucidate the biological effects of DCN1 mediated UPS regulation. Herein, we report our progress including design, procedure and result of HTS with post-screen analysis to discover small molecule inhibitors of the DCN1 from our in-house library compounds (∼600K). Following hit identification and validation, we identified three novel scaffolds as novel DCN1 inhibitors by biochemical, biophysical, and crystallographic studies to follow for further hit optimization. To the best of our knowledge, this is the first work to report small molecule DCN1 inhibitors. Citation Format: Jaeki Min, Daniel C. Scott, Sergio C. Chai, Asli N. Goktug, Jared T. Hammill, Guochang Huang, Michele C. Connelly, Amy Matheny, Anang Shelat, Bhuvanesh Singh, Taosheng Chen, Brenda A. Schulman, R. Kiplin Guy. Discovery of novel small molecule inhibitors of the DCN1-Ubc12 interaction by high throughput screening technology. [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 LB-056.

Cisplatin and oxaliplatin are among the most widely used anticancer drugs in both children and adults. The clinical use of these agents is associated with dose-limiting damage to kidneys (nephrotoxicity) and peripheral nerves (neurotoxicity). Mechanistic understanding of the underlying etiology remains incomplete and preventive strategies are currently unavailable. We previously reported that cisplatin nephrotoxicity (Filipski et al, CPT 2009) and oxaliplatin neurotoxicity (Sprowl et al, PNAS 2013) are dependent on organic cation transporter-mediated uptake mechanisms and can be reduced by genetic or pharmacological inhibition of OCT2. With a goal to identify small molecule OCT2 inhibitors, that could be used to prevent these toxicities, we carried out a high-throughput chemical library screen. Multiple tyrosine kinase inhibitors (TKIs), including dasatinib and vandetanib, were identified as potent OCT2 inhibitors. Further studies showed that TKIs inhibit OCT2 through a non-competitive mechanism raising the possibility that tyrosine phosphorylation by an unknown TKI-sensitive protein kinase(s) may be essential for OCT2 function. Using in vitro cell culture models and proteomics, site-directed mutagenesis and functional assays we found that OCT2 is indeed tyrosine phosphorylated and this phosphorylation is essential for OCT2 function and sensitive to inhibition by TKIs. A subsequent RNAi kinase library screen identified the Src-family member YES1 as the OCT2-phosphorylating kinase. This was further supported by experiments where a TKI-resistant YES1 mutant was able to rescue OCT2 inhibition by dasatinib. Non-TKI inhibitors of YES1 were also found to suppress OCT2 function. Importantly, tyrosine phosphorylation of OCT2 was observed in vivo, in murine kidneys and dorsal root ganglions, major sites of OCT2 expression and toxicities of platinum-based drugs. siRNA mediated in vivo knockdown of YES1 reduced OCT2 phosphorylation and suppressed its function in the kidneys. Treatment with the YES1 targeting TKI, Dasatinib, suppressed OCT2 phosphorylation in dorsal root ganglions, reduced oxaliplatin uptake and significantly prevented oxaliplatin induced neurotoxicity in mice models. These findings have not only provided new mechanistic insights into the regulation of membrane transporters; but have also provided a rationale for the translational development of new targeted interventions using TKI pulse-exposure to mitigate the debilitating side effects associated with platinum-based chemotherapeutics. Note: This abstract was not presented at the meeting. Citation Format: Navjotsingh Pabla, Jason A. Sprowl, Su Sien Ong, Alice A. Gibson, Guoqing Du, Wenwei Lin, Shuiying Hu, Lie Li, Taosheng Chen, Alex Sparreboom. Tyrosine phosphorylation is essential for OCT2 function and a target of inhibition by TKIs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4415. doi:10.1158/1538-7445.AM2015-4415

The cellular response to DNA damage comprises of DNA repair, cell-cycle checkpoint control and DNA damage-induced apoptosis to promote genomic integrity and suppress tumorigenesis. However, cancer cells can modulate the DNA damage response by suppressing DNA repair, altering cell cycle checkpoint control and ultimately downregulating tumor suppressors that contribute to apoptosis promoting genomic instability and avoidance of apoptosis. Although PXR is well established as a xenobiotic nuclear receptor that plays a central role in xenobiotic metabolism and disposition, emerging evidence suggest PXR as a regulator of apoptosis, promoting a malignant phenotype, both in vitro and in vivo. The tumor suppressor p53 can be activated in the presence of DNA damage and can induce cell cycle arrest to allow for DNA repair, or ultimately apoptosis suppressing tumor formation. We hypothesized that the novel protein-protein interaction between PXR and p53 contributes to the oncogenic functions of PXR enhancing colon carcinogenesis. Using immunoprecipitation, we found that PXR forms a protein-protein interaction with p53, resulting in reduced p53 activation and expression of downstream pro-apoptotic genes. In addition, utilizing the soft agar assay, we found that PXR overexpression leads to increased malignant transformation in colon cancer cells. Our findings show for the first time that PXR can physically bind to p53, resulting in downregulation of p53 activity and increased malignant transformation in colon cancer cells. Thus, these results provide insight into the biological significance of novel protein-protein interactions with PXR and how the PXR-p53 protein-protein interaction influences p53-mediated mechanism of tumor suppression. Citation Format: Delira F. Robbins, Jing Wu, Taosheng Chen. Regulation of cellular apoptosis via a novel protein-protein interaction of tumor suppressor p53 with the xenobiotic pregnane X receptor (PXR) in colon cancer cells. [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 1556. doi:10.1158/1538-7445.AM2014-1556

Nuclear receptor (NR) subfamily 4 member 2 (NR4A2), also known as Nurr1, has been shown to have oncogenic properties, in which it can mediate cell transformation, migration and drug resistance. However, the manner in which Nurr1 mediates these cancer-promoting features is not completely understood, but research implicates p53 as a potential intermediary. In this study, we show that Nurr1 enhances the proliferative rate of colorectal cancer cells. This increase in cell growth is accompanied by an upregulation of cyclin D3, a putative target gene of Nurr1. To understand how Nurr1 itself is regulated, we performed a reporter screen to identify microRNA regulators of Nurr1's 3’UTR. We identified miR-34c as a putative regulator of Nurr1 and this direct regulation was confirmed through site-directed mutagenesis of the predicted seed region. We next determined if p53 activation could affect Nurr1 expression through transcriptional activation of miR-34. Using Nutlin-3a to activate p53, we found that Nurr1 expression decreased in a dose response manner. Lastly, we sought to determine if Nurr1 could attenuate p53 activation. We found that overexpression of Nurr1 in colorectal cancer cells caused an attenuation of p53 transactivation of target genes following Nutlin-3a treatment. Our results suggest that a p53-miR-34-Nurr1 regulatory network exists, which would allow p53 to maintain Nurr1 in a suppressed state through miR-34-mediated regulation and prevent tumorigenesis. In cases of p53 mutation or Nurr1 overexpression, this network would be compromised, allowing Nurr1 to enhance tumorigenesis or drug resistance. Citation Format: Jordan A. Beard, Justin L. Hills, Alexa Tenga, Apana A. Takwi, Taosheng Chen. Nuclear receptor Nurr1 mediates cell proliferation and is involved in a p53-microRNA-34 regulatory network. [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 3393. doi:10.1158/1538-7445.AM2014-3393

Alveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein's transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR-mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS. To identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells’ motility. We also found that when PAX3-FKHR was downregulated, the expression level of carnitine palmitoyltransferase 1A (CPT1A) decreased. Analysis of the CPT1A promoter identified 4 putative PAX3-FKHR binding sites. Results of mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays showed that PAX3-FKHR binds directly and specifically to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel direct transcriptional target of PAX3-FKHR. Downregulating CPT1A, either by gene knockdown or by using a pharmacologic modulator of CPT1A activity, decreased cell motility in ARMS cells. Taken together, we have identified CPT1A as a novel direct transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting cell motility. CPT1A may represent a novel therapeutic target for the treatment of ARMS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2482. doi:1538-7445.AM2012-2482

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is a cancer of the cerebellum that occurs in children between the ages of 3 and 7. Human MBs have been classified into four subgroups, two of which harbor mutations in pathways that regulate normal development of the brain: Sonic Hedgehog/Patched (Shh/Ptch) (25%) and Wnt (15%). Our laboratory found that the bone morphogenetic proteins (BMP-2, 4, 7) antagonize Shh signaling by inducing the irreversible differentiation of MB cells into neurons leaving normal post-mitotic neurons intact. We designed a cell-based luciferase assay in which a human carcinoma cell line, C33A, expressed a multimerized BMP-responsive element from the Id1 promoter linked to luciferase. We then screened 3,488 compounds with known bioactivity and 31,441 compounds from the St. Jude diversity library for small molecule agonists of the BMP-2, 4, 7 signaling cascade. To date, 14 candidate compounds induced luciferase activity in a dose-dependent manner. Secondary screens showed that these small molecules induce P-Smad1/5/8 and Id2 expression in a dose-dependent manner in C33A cells by western blotting analysis, and alkaline phosphatase (ALP) activity which is a quantitative measure for the differentiation of C2C12 mouse myoblasts into osteoblasts. One of the compounds ventralized zebrafish embryos. Ongoing efforts are focused on understanding the mechanism of activation of BMP-4 signaling by these small molecule agonists. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4522. doi:10.1158/1538-7445.AM2011-4522