Methyl lysine readers, specifically PHD fingers, are emerging epigenetic targets in human diseases. For example, several PHD finger fusions are implicated in clinical cases of acute myeloid leukemia, highlighting the potential for PHD... more
Methyl lysine readers, specifically PHD fingers, are emerging epigenetic targets in human diseases. For example, several PHD finger fusions are implicated in clinical cases of acute myeloid leukemia, highlighting the potential for PHD inhibitors in disease regulation. However, limited chemical matter exists targeting PHD fingers. Here we report the first fragment-based screen against the BPTF PHD to identify several of the first reported BPTF PHD-targeting small molecule ligands. We used ligand-observed NMR to first screen a fragment library, followed by biophysical validation to prioritize two scaffolds, pyr-rolidine- and pyridazine-containing fragments. Structural predictions show these respective scaffolds may engage two distinct subpockets on the protein. The demonstrated ligandability of the BPTF PHD supports the future development of methyl lysine reader chemical probes to study their oncogenic functions.
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Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis‐targeting chimera (PROTAC) approaches, owing to favorable drug‐like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs... more
Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis‐targeting chimera (PROTAC) approaches, owing to favorable drug‐like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD‐based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra‐terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4‐11 cells at low picomolar concentrations (IC50=3 pM; BRD4 DC50=0.87 nM). These findings strongly support the utility of PG deriv...
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N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their... more
N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-te...
Research Interests: Biochemistry, Biology, Enzyme Inhibitors, Medicine, Screening, and 15 moreProtein-Protein Interaction, Ubiquitin, Humans, Proteins, Post Translational Modifications, Acetylation, Ubiquitin ligase, DNA ligase, Cell Molecular Biology and Developmental Biology, Pharmacy and Pharmaceutical Sciences, Structure activity Relationship, X Ray Crystallography, Biochemistry and cell biology, Molecular Structure, and binding sites
While the PROTAC approach to targeted protein degradation greatly benefits from rational design, the discovery of molecular glue degraders currently relies on screening strategies. Here, we describe screening of a library containing 3,630... more
While the PROTAC approach to targeted protein degradation greatly benefits from rational design, the discovery of molecular glue degraders currently relies on screening strategies. Here, we describe screening of a library containing 3,630 cereblon binders against a panel of 9 patient-derived cancer cell lines. This led to the discovery of SJ7590, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-guided optimization strategy we developed SJ3149, a uniquely potent and selective CK1α degrader. The crystal structure of the CK1α+CRBN+DDB1+SJ3149 quaternary complex provided a rationale for the improved degradation properties via direct contacts between SJ3149 and CK1α. In a panel of 115 human cancer cell lines SJ3149 displayed broad antiproliferative activity. This activity profile, which showed statistically significant correlation with MDM2 inhibitor Nutlin-3a, suggests potential for the development of treatments for hematological cancers, as well as solid tumors.
High-risk neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Despite extensive study, treatments remain highly toxic and are often insufficient to cure. Thus, there is an urgent need to develop more effective and... more
High-risk neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Despite extensive study, treatments remain highly toxic and are often insufficient to cure. Thus, there is an urgent need to develop more effective and less toxic treatments for children with NB. NB commonly displays reliance on a relatively small cohort of transcription factors (TFs) and epigenetic proteins to drive cell growth, however, many of these targets are inaccessible with conventional small molecules. In recent years, new modalities such as targeted protein degradation (TPD) have emerged. TPD may permit small molecule degradation of traditionally “undruggable” targets, such as TFs. This makes TPD a particularly attractive modality for defining new therapeutic strategies for high-risk NB. One category of TPD agents are molecular glues (MGs). MGs are monovalent small molecules capable of simultaneously binding to an E3 ligase receptor such as CRBN (cereblon), and a target protein of intere...
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Supplementary Methods
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mouse models of supratentorial ependymoma and CPC and the number of cells implanted as allografts
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Tumor proliferation measured as Ki67 index in (A) mSEP-CR(-)RTBDNb and (B) mCPC treated with the indicated dose and route of administration of gemcitabine.
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Purpose: Curing all children with brain tumors will require an understanding of how each subtype responds to conventional treatments and how best to combine existing and novel therapies. It is extremely challenging to acquire this... more
Purpose: Curing all children with brain tumors will require an understanding of how each subtype responds to conventional treatments and how best to combine existing and novel therapies. It is extremely challenging to acquire this knowledge in the clinic alone, especially among patients with rare tumors. Therefore, we developed a preclinical brain tumor platform to test combinations of conventional and novel therapies in a manner that closely recapitulates clinic trials.Experimental Design: A multidisciplinary team was established to design and conduct neurosurgical, fractionated radiotherapy and chemotherapy studies, alone or in combination, in accurate mouse models of supratentorial ependymoma (SEP) subtypes and choroid plexus carcinoma (CPC). Extensive drug repurposing screens, pharmacokinetic, pharmacodynamic, and efficacy studies were used to triage active compounds for combination preclinical trials with “standard-of-care” surgery and radiotherapy.Results: Mouse models display...
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Figure S1 shows siRNA screening in neuroblastoma cells. Figure S2 shows the dentification of ciclopirox that targets KDM4B. Figure S3 shows the CPX effect on other histone methyl marks. Figure S4 shows that CPX targets the Myc pathway.... more
Figure S1 shows siRNA screening in neuroblastoma cells. Figure S2 shows the dentification of ciclopirox that targets KDM4B. Figure S3 shows the CPX effect on other histone methyl marks. Figure S4 shows that CPX targets the Myc pathway. Figure S5 shows that CPX inhibits oxidative phosphorylation. Figure S6 shows the CPX effect on BE2C xenografts with KDM4B knockdown. Table S1 shows the sequences of the focused KDM siRNA library. Table S2 shows the primers for real time PCR.
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Background Pediatric high-grade glioma (pHGG) is largely incurable and accounts for most brain tumor-related deaths in children. Radiation is a standard therapy, yet the benefit from this treatment modality is transient, and most children... more
Background Pediatric high-grade glioma (pHGG) is largely incurable and accounts for most brain tumor-related deaths in children. Radiation is a standard therapy, yet the benefit from this treatment modality is transient, and most children succumb to disease within 2 years. Recent large-scale genomic studies suggest that pHGG has alterations in DNA damage response (DDR) pathways that induce resistance to DNA damaging agents. The aim of this study was to evaluate the therapeutic potential and molecular consequences of combining radiation with selective DDR inhibition in pHGG. Methods We conducted an unbiased screen in pHGG cells that combined radiation with clinical candidates targeting the DDR and identified the ATM inhibitor AZD1390. Subsequently, we profiled AZD1390 + radiation in an extensive panel of early passage pHGG cell lines, mechanistically characterized response to the combination in vitro in sensitive and resistant cells and evaluated the combination in vivo using TP53 wi...
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Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA-damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress and has been implicated... more
Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA-damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress and has been implicated as a potential biomarker to predict sensitivity to DNA-damaging agents (DDA). SLFN11 expression was quantified in 220 children with solid tumors using IHC. Sensitivity to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN) was assessed in sarcoma cell lines, including SLFN11 knock-out (KO) and overexpression models, and a patient-derived orthotopic xenograft model (PDOX). SLFN11 was expressed in 69% of pediatric sarcoma sampled, including 90% and 100% of Ewing sarcoma and desmoplastic small round-cell tumors, respectively, although the magnitude of expression varied widely. In sarcoma cell lines, protein expression strongly correlated with response to TAL and IRN, with SLFN11 KO resulting in significant loss of s...
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Combination therapy is increasingly central to modern medicine. Yet reliable analysis of combination studies remains an open challenge. Previous work suggests that common methods of combination analysis are too susceptible to noise to... more
Combination therapy is increasingly central to modern medicine. Yet reliable analysis of combination studies remains an open challenge. Previous work suggests that common methods of combination analysis are too susceptible to noise to support robust scientific conclusions. In this paper, we use simulated and real-world combination datasets to demonstrate that traditional index methods are unstable and biased by pharmacological and experimental conditions, whereas response-surface approaches such as the BRAID method are more consistent and unbiased. Using a publicly-available data set, we show that BRAID more accurately captures variations in compound mechanism of action, and is therefore better able to discriminate between synergistic, antagonistic, and additive interactions. Finally, we applied BRAID analysis to identify a clear pattern of consistently enhanced AKT sensitivity in a subset of cancer cell lines, and a far richer array of PARP inhibitor combination therapies for BRCA1...
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<p><b>(A)</b> Plot of phenotypic trajectory means for all eight test compounds in the “Cell Damage” and “Tubulin Intensity” dimensions. The extent of phenotypic trajectory means in the 154 test compounds has been... more
<p><b>(A)</b> Plot of phenotypic trajectory means for all eight test compounds in the “Cell Damage” and “Tubulin Intensity” dimensions. The extent of phenotypic trajectory means in the 154 test compounds has been included in grey for reference. Proteasome inhibitors are indicated by solid lines; unclassified compounds are indicated by dotted lines. <b>(B)</b> Plot of phenotypic trajectory means for all eight test compounds in the “Cell Damage” and “Blebbishness” dimensions. <b>(C)</b> The complete hierarchical clustering resulting from including the eight test compounds in the set. The eight test compounds have been indicated with black triangles. For more detail, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149439#pone.0149439.s002" target="_blank">S2 Fig</a>.</p
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<p><b>(A)</b> Dendrogram of similarity-based hierarchical clustering. For more detail, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149439#pone.0149439.s001"... more
<p><b>(A)</b> Dendrogram of similarity-based hierarchical clustering. For more detail, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149439#pone.0149439.s001" target="_blank">S1 Fig</a>. <b>(B)</b> Heatmap depicting phenotypic activity of all compounds in the dendrogram in Fig 5A. <b>(C)</b> Similarity matrix for all 154 training compounds (white indicates the minimum similarity of 0, black indicates the maximum similarity of 1).</p
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Medulloblastoma is the most common malignant pediatric brain tumor. Extensive molecular analysis by many groups around the world demonstrated four distinct subgroups, WNT, SHH, Group3 and Group4 that now all have been divided into 11... more
Medulloblastoma is the most common malignant pediatric brain tumor. Extensive molecular analysis by many groups around the world demonstrated four distinct subgroups, WNT, SHH, Group3 and Group4 that now all have been divided into 11 total subtypes, 8 for Grou3 and Group4 medulloblastoma. SHH with MYCN amplification and TP53 mutations and Group3 with MYC amplification are the most aggressive and the least curable with few to no therapeutic options for recurrent tumors. Over the last two decades, my group developed several murine models for Group3 with MYC and SHH with MYCN overexpression and p53 loss of function and patient-derived orthotopic xenografts that recapitulate the four tumor subgroups. In addition, several human Group3 medulloblastoma lines with MYC amplification have been established in 2D and 3D cultures. I will discuss how the development of these murine and human medulloblastoma models combined with high throughput drug screens and pre-clinical trials at St. Jude led ...
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The Bromodomain and Extra-Terminal domain (BET) family of proteins are involved in the regulation of gene transcription, and their dysregulation is implicated in several diseases including cancer. BET proteins contain two tandem... more
The Bromodomain and Extra-Terminal domain (BET) family of proteins are involved in the regulation of gene transcription, and their dysregulation is implicated in several diseases including cancer. BET proteins contain two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine residues and appear to have distinct biological roles. We compared several published co-crystal structures and found five positions near the substrate binding pocket that vary between BET bromodomains. One position located in the ZA loop has unique properties. In BRD2-4, this residue is glutamine in BD1 and lysine in BD2; in BRDT, this residue is arginine in BD1 and asparagine in BD2. Using molecular modeling, we identified differences in the water-mediated network at this position between bromodomains. Molecular dynamics simulations helped rationalize the observed bromodomain selectivity for exemplar BET inhibitors and a congeneric series of tetrahydroquinolines (THQ) that differed by...
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<p>(A1.) Zebrafish embryos were treated from 2 to 50 hpf with DMSO as a negative control, (<b>A2.</b>) with 10 µM dorsomorphin to dorsalize the embryos, or (<b>A3.</b>) injected with a chordin morpholino to... more
<p>(A1.) Zebrafish embryos were treated from 2 to 50 hpf with DMSO as a negative control, (<b>A2.</b>) with 10 µM dorsomorphin to dorsalize the embryos, or (<b>A3.</b>) injected with a chordin morpholino to ventralize the embryos. (<b>B-E.</b>) treatment with 5 and 10 µM isoliquiritigenin (<b>B1,2</b>), 5 and 10 µM 4′-hydroxychalcone (<b>C1, 2</b>), 10 and 20 µM apigenin (<b>D1, 2</b>), and 5 and 10 µM diosmetin (<b>E1, 2</b>) ventralized the embryos. Embryos were imaged at 50 hpf. At least 15 animals were analyzed per concentration and per compound. Images represent the phenotype found in ≥33% of animals in the respective groups. Arrow indicates the loss of the ventral tail fin. Scale bar, 500 µm.</p
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Brain tumors are often chemoresistant and these diseases are heterogeneous complicating efforts to discover effective new therapies. We describe a powerful interspecies genomics approach that meticulously matches subgroup specific driver... more
Brain tumors are often chemoresistant and these diseases are heterogeneous complicating efforts to discover effective new therapies. We describe a powerful interspecies genomics approach that meticulously matches subgroup specific driver mutations with cellular compartments to model cancer subgroups for drug screening. First, we performed a comprehensive genomics analysis to identify disease subgroups among >200 ependymomas. Subgroup specific alterations included amplifications and homozygous deletions of genes not yet implicated in ependymoma. We then identified CNS cell compartments most likely to give rise to ependymoma subgroups by matching the transcriptomes of human tumors to those of distinct types of mouse neural stem cell (NSC). Remarkably, activation of oncogenes in appropriate NSCs generated ependymomas that modelled the histology and transcriptome of the human disease. Stem-like tumor cells isolated from these mouse ependymomas were then cultured under conditions that...
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Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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... more
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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
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Rhabdoid tumors (RTs) predominantly affect young children and are among the deadliest pediatric solid tumors. Despite multimodal therapy consisting of surgery, radiation, and chemotherapy, children with these tumors have median survival... more
Rhabdoid tumors (RTs) predominantly affect young children and are among the deadliest pediatric solid tumors. Despite multimodal therapy consisting of surgery, radiation, and chemotherapy, children with these tumors have median survival of less than one year. RTs can arise throughout the body, including the central nervous system (CNS) where they are called atypical teratoid rhabdoid tumors (AT/RTs), and in extra-CNS locations such as the kidneys and other soft tissues where they are designated malignant RTs (MRTs). We previously identified MDM2 and MDM4 as therapeutic vulnerabilities in RTs and showed that treatment with the MDM2 inhibitor idasanutlin increased survival in mice bearing MRT xenografts. However, the therapeutic potential of idasanutlin in CNS RT tumors is unknown. Moreover, single agent therapies are prone to resistance and often show limited clinical benefit on their own. Therefore, we sought to identify combination strategies incorporating idasanutlin that would be...
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Additional file 1. Specified UPLC conditions, resulting chromatograms and analytical data including NMR, UV and MS spectra for the purified natural products.
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This data set contains the data used in Twarog et al. (2021) to examine the robustness and utility of response surface models in drug combination analysis. It includes simulated experimental data for the evaluation of traditional index... more
This data set contains the data used in Twarog et al. (2021) to examine the robustness and utility of response surface models in drug combination analysis. It includes simulated experimental data for the evaluation of traditional index methods, as well as a processed library of interaction metrics evaluated on the Merck OncoPolyPharmacology Screen (O'Neil et al., 2016), the scripts used to implement those metrics on all tested combinations in that screen, and scripts to evaluate the performance of those metrics in comparison with real-world mechanistic classifications. Finally, the data set includes data from several published and unpublished drug combination experiments, and scripts which allow the analyses of those experiments to be replicated and applied to new data.
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BACKGROUND Talazoparib combined with irinotecan and temozolomide demonstrated efficacy in a murine Ewing sarcoma model. Based on these data, we conducted a phase I trial of talazoparib and irinotecan with/without temozolomide in... more
BACKGROUND Talazoparib combined with irinotecan and temozolomide demonstrated efficacy in a murine Ewing sarcoma model. Based on these data, we conducted a phase I trial of talazoparib and irinotecan with/without temozolomide in paediatric patients with recurrent/refractory solid malignancies. PATIENTS AND METHODS Cohorts of 3-6 patients with recurrent/refractory solid malignancies received escalating doses of oral talazoparib and intravenous irinotecan (arm A) and oral talazoparib, oral temozolomide and intravenous irinotecan (arm B) in a 3 + 3 design. Talazoparib was administered on days 1-6, and intravenous irinotecan and oral temozolomide were administered on days 2-6, of a 21-day course. Serum for talazoparib and irinotecan pharmacokinetics was obtained during course 1. UGT1A1 polymorphism and Schlafen family member 11 (SLFN11) immunohistochemical staining were performed. RESULTS Forty-one patients (20 males; median age, 14.6 years; 24 with recurrent disease) were evaluable for dose escalation. Twenty-nine and 12 patients were treated on arm A and arm B, respectively, for a total of 208 courses. The most common diagnosis was Ewing sarcoma (53%). The most common ≥grade III haematologic toxicities in arms A and B included neutropenia (78% and 31%, respectively) and thrombocytopenia (42% and 31%, respectively). In arms A and B, febrile neutropenia (24% and 14%, respectively) and diarrhoea (21% and 7%, respectively) were the most common ≥grade III non-hematologic toxicities. Six patients (Ewing sarcoma [5 patients] and synovial sarcoma [1 patient]) had a response (1 with a complete response, 5 with a partial response). The objective response rates were 10.3% (arm A) and 25% (arm B). Pharmacokinetic testing demonstrated no evidence of drug-drug interaction between talazoparib and irinotecan. UGT1A1 was not related to response. SLFN11 positivity was associated with best response to therapy. CONCLUSIONS The combination of talazoparib and irinotecan with/without temozolomide is feasible and active in Ewing sarcoma, and further investigation is warranted.
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Medulloblastoma (MB), the most common malignant pediatric brain tumor, is classified into four major molecularly and histopathologically distinct subgroups, among which MYC-driven Group 3 MBs confer a poor prognosis. The Cyclin... more
Medulloblastoma (MB), the most common malignant pediatric brain tumor, is classified into four major molecularly and histopathologically distinct subgroups, among which MYC-driven Group 3 MBs confer a poor prognosis. The Cyclin D/CDK4/CDK6/RB pathway is frequently deregulated in MB leading to uncontrolled cell proliferation, but tumors express an intact RB protein (Northcott et al., Nature, 2017). Therefore, CDK4/6 inhibitor drugs offer a possible therapeutic approach to treat MBs. Because single agent therapy ultimately leads to drug resistance, we initiated in vitro combination drug screens to identify drug classes potentiating CDK4/6 inhibitors. We used Group 3 MB patient-derived orthotopic xenografts (PDOXs), a human cell line (HDMB03), and freshly dissociated tumor cells propagated only in the mouse brain. The drug screen included 90 compounds comprising targeted and cytotoxic drugs that are FDA approved or under active clinical investigation. Using a bioluminescence-based assa...
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Introduction. Acute erythroid leukemia (AEL) is a high-risk leukemia subtype of poorly understood genetic basis. In integrated comparative genomic analysis of 159 AEL and 1509 non AEL myeloid tumors, we identified 5 age-related AEL... more
Introduction. Acute erythroid leukemia (AEL) is a high-risk leukemia subtype of poorly understood genetic basis. In integrated comparative genomic analysis of 159 AEL and 1509 non AEL myeloid tumors, we identified 5 age-related AEL subtypes with distinct genomic features and outcome: adult AEL with bi-allelic alterations in TP53 (31%), frequently co-occurring with alterations in DNMT3A,BCOR, EZH2, RB1, or NFIX; NPM1-mutated (12%); KMT2A-mutated/rearranged (11%), mostly co-mutated with STAG2; pediatric, NUP98-rearranged (5%) and adult, DDX41-mutated (3%). Thirty-eight percent of cases lacked an identifiable exclusive recurrent founding alteration but were enriched in mutations in ASXL1, TET2 and splicing factors. Methods. To explore the roles and cooperativity of the identified alterations in leukemogenesis we used CRISPR-Cas9 genome editing to induce combinations of loss-of-function mutations in 9 recurrently mutated genes in AEL (Tp53, Tet2, Dnmt3a, Asxl1, Ezh2, Stag2, Bcor, Ppm1d,...
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Infants with MLL rearranged (MLLr) acute lymphoblastic leukemia (ALL) have a poor prognosis, with an event free survival of only 23-44%. Whole genome sequencing (WGS) of this subtype has revealed a paucity of cooperating mutations, with... more
Infants with MLL rearranged (MLLr) acute lymphoblastic leukemia (ALL) have a poor prognosis, with an event free survival of only 23-44%. Whole genome sequencing (WGS) of this subtype has revealed a paucity of cooperating mutations, with an average of 2.2 somatic single nucleotide variations and/or insertions/deletions per case. Despite recent progress in defining the epigenetic alterations that result from the expression of the MLL fusion protein, these insights have only recently begun to be extrapolated into the development of new therapeutic approaches whose benefits have yet to be defined. Thus, there remains an urgent need for the development of alternative approaches to improve outcomes in these patients. To identify compounds that are active in MLLr disease, we established in vitro and in vivo assays to evaluate drug sensitivity of primary infant ALL patient samples. 15 infant MLLr leukemia samples that have previously undergone WGS were xenografted into NOD/SCID/IL2Rγnull (N...
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10535 Background: Rhabdomyosarcoma (RMS) is an aggressive malignancy of childhood with a poor prognosis in patients with metastatic or recurrent disease. Inhibitors of Wee1 kinase and heat shock protein 90 (HSP90) have in vitro activity... more
10535 Background: Rhabdomyosarcoma (RMS) is an aggressive malignancy of childhood with a poor prognosis in patients with metastatic or recurrent disease. Inhibitors of Wee1 kinase and heat shock protein 90 (HSP90) have in vitro activity in RMS and have emerged as potential novel treatment strategies. We performed a comprehensive preclinical phase III study to compare the Wee1 inhibitor AZD1775 and HSP90 inhibitor ganetespib (GANET) in combination with irinotecan (IRN) and vincristine (VCR). Methods: Orthotopic xenografts (O-PDXs) were created by injecting luciferase labeled RMS cells into the hind-leg muscle of CD-1 nude mice. Pharmacokinetic studies on RMS O-PDXs were performed to determine matched human AUC-guided dosing. A total of 540 O-PDXs derived from 4 high risk RMS patients, 2 alveolar and 2 embryonal, were randomly enrolled into 14 treatment groups. Six courses of blinded placebo-controlled therapy were given on a clinically relevant schedule. Mice were classified as havin...
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The first challenge in the 2014 competition launched by the Teach-Discover-Treat (TDT) initiative asked for the development of a tutorial for ligand-based virtual screening, based on data from a primary phenotypic high-throughput screen... more
The first challenge in the 2014 competition launched by the Teach-Discover-Treat (TDT) initiative asked for the development of a tutorial for ligand-based virtual screening, based on data from a primary phenotypic high-throughput screen (HTS) against malaria. The resulting Workflows were applied to select compounds from a commercial database, and a subset of those were purchased and tested experimentally for anti-malaria activity. Here, we present the two most successful Workflows, both using machine-learning approaches, and report the results for the 114 compounds tested in the follow-up screen. Excluding the two known anti-malarials quinidine and amodiaquine and 31 compounds already present in the primary HTS, a high hit rate of 57% was found.
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Investigate antileukemic activity of artemisinins, artesunate (ART), and dihydroartemisinin (DHA), in combination with cytarabine, a key component of acute myeloid leukemia (AML) chemotherapy using in vitro and in vivo models. Using ten... more
Investigate antileukemic activity of artemisinins, artesunate (ART), and dihydroartemisinin (DHA), in combination with cytarabine, a key component of acute myeloid leukemia (AML) chemotherapy using in vitro and in vivo models. Using ten human AML cell lines, we conducted a high-throughput screen to identify antimalarial agents with antileukemic activity. We evaluated effects of ART and DHA on cell viability, cytotoxicity, apoptosis, lysosomal integrity, and combination effects with cytarabine in cell lines and primary patient blasts. In vivo pharmacokinetic studies and efficacy of single-agent ART or combination with cytarabine were evaluated in three xenograft models. ART and DHA had the most potent activity in a panel of AML cell lines, with selectivity toward samples harboring MLL rearrangements and FLT3-ITD mutations. Combination of ART or DHA was synergistic with cytarabine. Single-dose ART (120 mg/kg) produced human equivalent exposures, but multiple dose daily administration required for in vivo efficacy was not tolerated. Combination treatment produced initial regression, but did not prolong survival in vivo. The pharmacology of artemisinins is problematic and should be considered in designing AML treatment strategies with currently available agents. Artemisinins with improved pharmacokinetic properties may offer therapeutic benefit in combination with conventional therapeutic strategies in AML.
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With combination therapies becoming increasingly vital to understanding and combatting disease, a reliable method for analyzing combined dose response is essential. The importance of combination studies both in basic and translational... more
With combination therapies becoming increasingly vital to understanding and combatting disease, a reliable method for analyzing combined dose response is essential. The importance of combination studies both in basic and translational research necessitates a method that can be applied to a wide range of experimental and analytical conditions. However, despite increasing demand, no such unified method has materialized. Here we introduce the Bivariate Response to Additive Interacting Doses (BRAID) model, a response surface model that combines the simplicity and intuitiveness needed for basic interaction classifications with the versatility and depth needed to analyze a combined response in the context of pharmacological and toxicological constraints. We evaluate the model in a series of simulated combination experiments, a public combination dataset, and several experiments on Ewing’s Sarcoma. The resulting interaction classifications are more consistent than those produced by traditi...
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Research Interests: Biochemistry, Bioinformatics, Environmental Science, Computer Science, Developmental Biology, and 15 moreClimate Change, Computational Biology, Biotechnology, Cancer, Biology, Cell Cycle, Ecology, Drug Discovery, Astrophysics, Astronomy, DNA, Drug Design, Cell Signalling, Earth Science, and Biochemistry and cell biology
Research Interests: Chemistry, Natural Products, Mass Spectrometry, Drug Discovery, Pharmacognosy, and 12 moreMedicine, Biological Sciences, Automation, Herbs, High throughput screening, Natural Product, CHEMICAL SCIENCES, Very high throughput, Natural, Fractionation, Medical and Health Sciences, and Biological products
Research Interests: Chemistry, Organic Chemistry, Medicinal Chemistry, Modeling, Drug Discovery, and 15 moreMedicine, Cell line, In Vitro, Humans, Animals, Protease Inhibitors, Medicinal, Molecular Model, Cysteine endopeptidases, Protein Conformation, Biological activity, Chemical Synthesis, purines, Pharmacology and pharmaceutical sciences, and cathepsin L
Human African trypanosomiasis (HAT), a major health concern in sub-Saharan Africa, is caused by the protozoan parasite Trypanosoma brucei. Recent studies have shown that a cathepsin B like protease, TbcatB, is essential to the survival of... more
Human African trypanosomiasis (HAT), a major health concern in sub-Saharan Africa, is caused by the protozoan parasite Trypanosoma brucei. Recent studies have shown that a cathepsin B like protease, TbcatB, is essential to the survival of T. brucei in vitro (Mackey, Z. B.; O&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;Brien, T. C.; Greenbaum, D. C.; Blank, R. B.; McKerrow, J. H. J. Biol. Chem. 2004, 279, 48426-48433). Herein, we describe the first inhibitors of TbcatB, a series of purine nitriles. The compounds are potent trypanocides, killing the parasite with a high degree of selectivity over a panel of three human cell lines. In addition, a predictive model of trypanocidal activity was developed on the basis of potency against TbcatB and various calculated physical property descriptors.