Pathological activation of the thyroid-stimulating hormone receptor (TSHR) is caused by thyroid-stimulating antibodies in patients with Graves' disease (GD) or by somatic and rare genomic mutations ...that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study, we describe the identification and
characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH-inhibition bioassay. TSHR-expressing CHO cells, which also expressed a luciferase-tagged CRE response element, were optimized using bovine TSH as the activator, in a 384 well plate format, which had a
score of 0.3-0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7 μM. The selection criteria for a positive hit were based on a mean signal threshold of ≥50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin - a post receptor activator of adenylyl cyclase - confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC
of 12.3 μM and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations.
docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by GD patient sera and monoclonal-stimulating TSHR antibodies. In conclusion, we report the identification of a novel small molecule TSHR inhibitor, which has the potential to be developed as a therapeutic antagonist for abrogation of TSHR signaling by TSHR autoantibodies in GD.
Our ability to discover effective drug combinations is limited, in part by insufficient understanding of how the transcriptional response of two monotherapies results in that of their combination. We ...analyzed matched time course RNAseq profiling of cells treated with single drugs and their combinations and found that the transcriptional signature of the synergistic combination was unique relative to that of either constituent monotherapy. The sequential activation of transcription factors in time in the gene regulatory network was implicated. The nature of this transcriptional cascade suggests that drug synergy may ensue when the transcriptional responses elicited by two unrelated individual drugs are correlated. We used these results as the basis of a simple prediction algorithm attaining an AUROC of 0.77 in the prediction of synergistic drug combinations in an independent dataset.
Phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger ...RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells. TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc–dependent transcription. The synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kδ inhibitors, inhibited casein kinase-1 ε (CK1ε). Targeting CK1ε using a selective chemical inhibitor or short hairpin RNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. These results suggest that TGR-1202 is a dual PI3Kδ/CK1ε inhibitor, which may in part explain the clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1ε should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc.
•A novel PI3Kδ inhibitor TGR-1202 synergizes with proteasome inhibitor carfilzomib by silencing c-Myc in preclinical models of lymphoma.•The unique activity of TGR-1202 as a single agent and in combination with carfilzomib is driven by an unexpected activity targeting CK1ε.
Bromodomain and extraterminal protein inhibitors (BETi) are epigenetic therapies aimed to target dysregulated gene expression in cancer cells. Despite early successes of BETi in a range of ...malignancies, the development of drug resistance may limit their clinical application. Here, we evaluated the mechanisms of BETi resistance in uveal melanoma, a disease with little treatment options, using two approaches: a high-throughput combinatorial drug screen with the clinical BET inhibitor PLX51107 and RNA sequencing of BETi-resistant cells. NF-κB inhibitors synergistically sensitized uveal melanoma cells to PLX51107 treatment. Furthermore, genes involved in NF-κB signaling were upregulated in BETi-resistant cells, and the transcription factor CEBPD contributed to the mechanism of resistance. These findings suggest that inhibitors of NF-κB signaling may improve the efficacy of BET inhibition in patients with advanced uveal melanoma. SIGNIFICANCE: These findings provide evidence that inhibitors of NF-κB signaling synergize with BET inhibition in
and
models, suggesting a clinical utility of these targeted therapies in patients with uveal melanoma.
Abstract
SARS-CoV-2 hijacks the host cell transcriptional machinery to induce a phenotypic state amenable to its replication. Here we show that analysis of Master Regulator proteins representing ...mechanistic determinants of the gene expression signature induced by SARS-CoV-2 in infected cells revealed coordinated inactivation of Master Regulators enriched in physical interactions with SARS-CoV-2 proteins, suggesting their mechanistic role in maintaining a host cell state refractory to virus replication. To test their functional relevance, we measured SARS-CoV-2 replication in epithelial cells treated with drugs predicted to activate the entire repertoire of repressed Master Regulators, based on their experimentally elucidated, context-specific mechanism of action. Overall, 15 of the 18 drugs predicted to be effective by this methodology induced significant reduction of SARS-CoV-2 replication, without affecting cell viability. This model for host-directed pharmacological therapy is fully generalizable and can be deployed to identify drugs targeting host cell-based Master Regulator signatures induced by virtually any pathogen.
We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on ...the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.
Abstract
Quantitative metrics to objectively assess the fidelity of cancer models, such as cell lines, organoids, or patient-derived xenografts (PDXs), remain elusive, with histological criteria or ...the presence of specific mutations often used as driving principles. However, focusing on individual mutations inevitably ignores the effect of a large complement of additional, model-specific genetic and epigenetic events. As a result, effective model fidelity assessment is best performed a posteriori, for instance, by determining whether dependencies identified in a specific tumor model (e.g., a cell line or organoid) are recapitulated in vivo in PDXs or in patients, via pre-clinical or clinical trials. Unfortunately, such an approach is inefficient and time-consuming, creating an urgent need for methodologies capable of effectively and quantitatively assessing model fidelity a priori.
This unmet need motivated us to develop and test a quantitative, molecular-level framework (OncoMatch), to assess the fidelity of a given tumor model in the context of a specific biological question, and in particular for addressing issues of drug sensitivity. We addressed this challenge by integrating two independent computational metrics to assess: (a) conservation of regulatory networks inferred from patient-derived samples in a model of interest, and; (b) overlap of master regulator (MR) proteins–i.e., proteins representing the mechanistic determinants of the transcriptional state associated with the phenotype of interest–as inferred from patient-derived and model-derived samples.
We show that these molecular-level criteria can effectively identify cell lines that recapitulate patient-specific drug mechanism of action and drug sensitivity, independent of histological consideration. By leveraging gene expression profiles of drug-perturbations in primary cells and explants derived from gastroenteropancreatic-neuroendocrine tumor (GEP-NET) patients, we show that H-STS, an EBV-immortalized lymphoblastoid cell line, represents a high-fidelity model for the assessment of drug mechanism of action and drug sensitivity in these tumors. In particular, our analysis shows highly significant conservation of drug mechanism of action for 60 of 95 profiled drugs (63%, p < 10-10, Bonferroni's corrected), and higher conservation among drugs exhibiting greater bioactivity in this context. This rate is comparable and, in fact higher than what is achieved using tumor-type-matched cell line pairs representative of glioma, pancreatic and prostate carcinoma, and dramatically higher than the conservation observed between unrelated models, which we used as negative controls.
Based on this systematic analysis, OncoMatch represents a valuable addition to our repertoire of tools for prioritizing cell lines, organoids and patient-derived xenograft models as high-fidelity human cancer models. We provide comprehensive prioritization of 921 cell lines as potential high-fidelity models for 10,024 human tumor samples in TCGA. This represents an actionable resource to guide selection of cell line models for specific drug mechanism of action and drug sensitivity studies.
Citation Format: Mariano J. Alvarez, Yan Pengrong, Mary L. Alpaugh, Michaela Bowden, Ewa T. Sicinska, Chensheng W. Zhou, Charles Karan, Ronald B. Realubit, Prabhjot S. Mundi, Adina Grunn, Jager Dirk, John A. Chabot, Antonio T. Fojo, Paul E. Oberstein, Hanina Hibshoosh, Jeffrey W. Milsom, Matthew H. Kulke, Massimo Loda, Gabriela Chiosis, Diane L. Reidy-Lagunes, Andrea Califano. OncoMatch: Unbiased, quantitative assessment of cancer model fidelity for drug sensitivity and mechanism of action elucidation abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-003.
Background: c-Myc is one of the most frequently altered genes across a vast array of human cancers. Overexpression of c-Myc is observed in up to 30% of cases of diffuse large B-cell lymphoma (DLBCL), ...the most common type of aggressive lymphoma. Although DLBCL can be cured in 60-70% patients, a substantial minority of patients with DLBCL still die from their lymphoma. There is emerging evidence that c-Myc expression is an adverse risk factor independent of the cell-of-origin classification. To date no drugs that directly target c-Myc have been approved for the treatment of any cancer. In fact, since c-Myc is involved in many normal cellular functions, direct c-Myc inhibitors may be associated with significant toxicity. The goal of our study is to develop novel strategies targeting c-Myc that will have an improved therapeutic index. The c-Myc protein has a very short half-life of less than 30 minutes, and its translation is highly dependent on the eukaryotic initiation factor 4F (eIF4F). eIF4F is activated by the mammalian target of rapamycin (mTOR), which is regulated not only by the PI3K-AKT pathway but also the proteasome pathway. These observations led us to hypothesize that if the proteasome and PI3K pathways cooperate in the activation of mTOR and its downstream target eIF4F, then combinations of proteasome and PI3K inhibitors should potently suppress eIF4F dependent translation of c-Myc and the growth of c-Myc dependent lymphoma.
Methods: Cytotoxicity was studied in lymphoma cell lines and primary lymphoma cells using Cell TiterGlo. The Bliss additivism model was used to determine the expected inhibition of cell growth. The difference of the expected and observed levels of inhibition was used to calculate the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Mechanisms of synergy were determined through interrogation of PI3K/AKT/mTOR pathway and its downstream targets. Expression of c-Myc was investigated at the translation and transcription levels, using a combination of Western blot, qPCR, and a bi-cistronic luciferase reporter we developed to study cap dependent translation. Gene expression profiling (GEP) studies were conducted using RNAseq, and analyzed by the Fisher t-test and running enrichment score (RES) between different treatment groups.
Results: We used a high-throughput platform to screen the cytotoxicity of two PI3Kdelta inhibitors (TGR-1202 & idelalisib/Cal-101), two proteasome inhibitors (bortezomib & carfilzomib), and four combination pairs using these drugs. We found that TGR-1202 and Cal-101 caused only minimal to mild inhibition of lymphoma cells, while bortezomib and carfilzomib caused potent inhibition as single agents. The combination of TGR-1202 and carfilzomib was consistently the most synergistic doublet, while the combination of Cal-101 and bortezomib the least synergistic in numerous lymphoma cell lines studied to date (Figure 1). TGR-1202 and carfilzomib were also highly synergistic in primary lymphoma cells, while Cal-101 and bortezomib were not. Importantly, normal lymphocytes were resistant to the combination of TGR-1202 and carfilzomib. At the molecular level, only the combination of TGR-1202 and carfilzomib potently inhibited mTORC1 dependent phosphorylation of 4E-BP1, leading to marked reduction of c-Myc protein (Figures 2 & 3). In contrast, the combination of TGR-1202 and carfilzomib produced no reduction of the mRNA level of c-Myc (Figures 3). A luciferase reporter demonstrated that the synergistic combination TGR-1202 and carfilzomib specifically inhibited the translation downstream of the 5'UTR of c-Myc (Figure 3). GEP studies confirmed that the canonical c-Myc target genes were potently downregulated at the level of transcription by the combination of TGR-1202 and carfilzomib (Figure 3). These results demonstrate that TGR-1202 and carfilzomib in combination potently inhibited the translation of c-Myc and the c-Myc transcriptional program, which appears to be primarily through disruption of the PI3Kdelta and proteasome pathways that converge on mTOR. Ongoing experiments are focused on confirmation of these observations in vivo. Further, a phase I/II clinical trial evaluating this combination regimen in aggressive c-Myc driven lymphomas is being planned.
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Deng:TG Therapeutics: Research Funding; Gilead: Research Funding; Amgen/Onyx: Research Funding. Lentzsch:Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Axiom: Honoraria. O'Connor:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Research Funding; Acetylon: Consultancy, Other: Consultancy fee; Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Other: Consultancy fee; Takeda Millenium: Consultancy, Honoraria, Other: Consultancy fee, Research Funding; Novartis: Consultancy, Honoraria, Other: Consultancy fee; Seattle Genetics: Research Funding; Bayer: Consultancy, Honoraria.
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