Abstract
Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a ...panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF–MEK–ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF–MEK–ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.
Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of
...mutant RMS cells and correlates
efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to
dependency in RMS cells; however, there is poor
response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF-MEK-ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis.
assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of
RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF-MEK-ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress
mutant RMS tumor growth.
K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the ...cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P. falciparum and P. vivax in vitro, is efficacious against P. falciparum in in vivo rodent models, produces parasite reduction ratios equivalent to dihydroartemisinin and has pharmacokinetic and pharmacodynamic characteristics compatible with a single-dose cure. In vitro studies with transgenic parasites expressing variant forms of K13 show no cross-resistance with the C580Y mutation, the primary variant observed in Southeast Asia. E209 is a superior next generation endoperoxide with combined pharmacokinetic and pharmacodynamic features that overcome the liabilities of artemisinin derivatives.
Current antipsychotics are effective in treating the positive symptoms associated with schizophrenia, but they remain suboptimal in targeting cognitive dysfunction. Recent studies have suggested that ...positive allosteric modulation of the M
muscarinic acetylcholine receptor (mAChR) may provide a novel means of improving cognition. However, very little is known about the potential of combination therapies in extending coverage across schizophrenic symptom domains. This study investigated the effect of the M
mAChR positive allosteric modulator BQCA 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, alone or in combination with haloperidol (a first-generation antipsychotic), clozapine (a second-generation atypical antipsychotic), or aripiprazole (a third-generation atypical antipsychotic), in reversing deficits in sensorimotor gating and spatial memory induced by the N-methyl-d-aspartate receptor antagonist, MK-801 (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzoa,dcyclohepten-5,10-imine. Sensorimotor gating and spatial memory induction are two models that represent aspects of schizophrenia modeled in rodents. In prepulse inhibition (an operational measure of sensorimotor gating), BQCA alone had minimal effects but exhibited different levels of efficacy in reversing MK-801-induced prepulse inhibition disruptions when combined with a subeffective dose of each of the three (currently prescribed) antipsychotics. Furthermore, the combined effect of BQCA and clozapine was absent in M
mice. Interestingly, although BQCA alone had no effect in reversing MK-801-induced memory impairments in a Y-maze spatial test, we observed a reversal upon the combination of BQCA with atypical antipsychotics, but not with haloperidol. These findings provide proof of concept that a judicious combination of existing antipsychotics with a selective M
mAChR positive allosteric modulator can extend antipsychotic efficacy in glutamatergic deficit models of behavior.
Abstract
Rhabdomyosarcoma (RMS) is the most commonly diagnosed soft tissue sarcoma in children. While the 5-year event-free survival for localized rhabdomyosarcoma is over 70%, it is less than 30% ...for patients presenting with metastatic disease. Given that the current treatment modalities for RMS patients remain cytotoxic therapies with profound life-long lasting complications, identification of less toxic and more effective therapies is highly desired. Whole-genome and RNA sequencing of human rhabdomyosarcoma have identified frequent RAS genes oncogenic mutations in fusion-negative rhabdomyosarcoma (FN-RMS), which comprises 65% of RMS. This high incidence of RAS mutations present opportunity for design of targeted therapeutic strategies for FN-RMS. By siRNA-mediated knock down of individual RAS isoforms we showed that H/NRAS Q61X-mutant FN-RMS cell lines exhibited oncogenic RAS dependency and that knockdown of the Q61X-mutant H/NRAS inhibited ERK MAPK, but not PI3K-AKT pathway activity. Knockdown of the Q61X-mutant H/NRAS phenocopied ERK inhibitor treatment by inducing myogenic differentiation. Further, as compared to RAS-wild type RMS cells, H/NRAS-Q61X FN-RMS cell lines were preferentially more sensitive to MEK or ERK, but not PI3K or AKT inhibitors. RNA sequencing analysis revealed significant overlap of genes signatures associated with NRAS-Q61H knockdown and ERK inhibitor treatment in the FN-RMS cell line RD. These results indicated a central role of the ERK MAPK pathway in mediating H/NRAS Q61X-dependency in FN-RMS. However, in vivo evaluation of clinically relevant exposers with the MEK inhibitor trametinib demonstrated poor response, which correlated with inefficient ERK inhibition in pharmacodynamic assays. A trametinib-sensitizing CRISPR screen pointed to vertical inhibition of the RAF-MEK-ERK cascade by co-suppression of MEK and either CRAF or ERK. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically blocked ERK activity and induced cell cycle arrest, myogenic differentiation and cell death. In vivo evaluation of low dose pan-RAFi + ERKi or MEKi + ERKi potently suppressed H/NRAS Q61X RMS tumor growth, with pan-RAFi + ERKi being more effective and better tolerated, suggesting wider therapeutic window. These results support the clinical translation of pan-RAFi + MEKi/ERKi for H/NRAS Q61X-mutant FN-RMS.
Citation Format: Natalia Garcia, Vanessa Del-Pozo, Marielle Yohe, Craig Goodwin, Terry Shackleford, Long Wang, Kunal Baxi, Yidong Chen, Myron Ignatius, Kris Wood, Peter Houghton, Angelina Vaseva. Vertical inhibition of the RAF MEK ERK cascade induces myogenic differentiation, apoptosis and tumor regression in H/NRAS Q61X mutant rhabdomyosarcoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB251.
Abstract
Background: Pediatric bone and soft tissue sarcomas are heavily dependent on the Type 1 insulin-like growth factor receptor (IGF-1R) signaling axis, which promotes tumorigenic properties ...such as proliferation, survival, angiogenesis and metastasis. While a substantial proportion of patients demonstrate intrinsic resistance to monoclonal antibodies targeting IGF-1R, a strong and sustained response is seen in a limited number of patients, and disease stabilization in about 30% of patients. Understanding these resistance mechanisms may identify combinations of targeted therapies that optimize therapeutic efficiency of IGF-1R-targeted antibodies. Methods: For intrinsic resistance, we used a panel of Ewing sarcoma (ES; n=10) and rhabdomyosarcoma (RMS; n=10) cell lines that, with the exception of Rh41, are all intrinsically resistant to TZ-1, a monoclonal antibody that targets IGF-1R. For acquired resistance, we developed a TZ-1 resistant RMS cell line, Rh41/TZ-1. We utilized Receptor Tyrosine Kinase (RTK) Arrays to analyze the phosphorylation status of 49 RTKs. The cell lines were treated with TZ-1 and changes in RTK expression were assessed by quantitative PCR. Results: RMS cells intrinsically resistant to TZ-1 treatment expressed multiple activated RTKs including EGFR family of receptors, IGF-1R, IR, PDGFR, FGFR4, HGFR4, RYK, ALK, DDR1, AXL, DTK. Interestingly, ES cells (n=5) had either a complex pattern of activated RTK expression similar to RMS cells or a relatively less complex set (n=5) of activated RTKs (IGF-1R, IR, HGFR, ERBB4, AXL, DTK). TZ-1 treatment of these cell lines with intrinsic resistance demonstrated decreased phosphorylation of IGF-1R, but had little effect on other RTKs. In contrast TZ-1 treatment of Rh41 cells rapidly induced activation of several RTKs including: AXL, PDGFRB, FGFR4 and ALK. Increased expression of these receptors was confirmed by qPCR. In contrast, Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed activated RTKs (PDGFRB, FGFR4, AXL, DTK, VEGFR1). In Rh41 cells treated with TZ-1, the BRD4 inhibitor, JQ1 (500 nM, 24 Hr), blocked the induction of IGF-1R, IRB, AXL, PDGFR-A/-B, VEGFR1 and ALK. However, it had less effect on the induction of FGFR 2/3/4 receptors. Further, combined treatment of TZ-1 and JQ1 reduced cellular proliferation of Rh41 cells more than TZ-1 treatment alone. Conclusions: We demonstrate that intrinsic resistance to the IGF-1R targeted antibody TZ-1 is characterized by constitutive activation of multiple RTKs. In response to TZ-1 treatment in a sensitive cell line there is rapid induction of multiple RTKs indicating a dynamic response to inhibition of IGF-1R. Our data suggests that BRD4 is involved in induction of these signaling molecules and that combined inhibition of BRD4 along with the IGF-1R targeted therapy can overcome this resistance and warrants further investigation. Support: US Public Health Service Grant CA165995.
Citation Format: Terry J. Shackleford, Seethalakshmi Hariharan, Doris Phelps, Hemant Bid, Hiahong Zhong, Peter Houghton. Mechanisms of intrinsic and acquired resistance to antibodies targeting IGF-1R in pediatric sarcoma cell lines abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5825. doi:10.1158/1538-7445.AM2017-5825
The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite's life cycle, thus impacting prevention, treatment, and transmission of the ...disease. The 4(1H)-quinolone-3-diarylethers are selective potent inhibitors of the parasite's mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.
Abstract
Background: Insulin-like growth factor-I receptor (IGFR) signaling promotes tumorigenic properties which makes targeting IGFR an attractive therapeutic target. IGF signaling has been linked ...to proliferation, survival, angiogenesis, metastasis and resistance to anticancer therapies, and contributes to pediatric malignancies including Ewing sarcoma and rhabdomyosarcoma. Recently, monoclonal antibodies targeting IGFR have shown promise in a limited number of sarcoma patients. A substantial proportion of patients demonstrate intrinsic resistance, and those that do respond will often lose clinical benefit (acquired resistance). Intrinsic and acquired resistance ultimately results in failure to respond to treatment and continued growth of the tumor. Understanding these resistance mechanisms will identify combinations of targeted therapy to optimize therapeutic efficiency of this targeted agent.
Methods: To test intrinsic and acquired mechanisms of resistance, we utilized Receptor Tyrosine Kinase (RTK) Arrays to analyze the phosphorylation status of 49 RTKs. For intrinsic resistance, we used a panel of Ewing Sarcoma (EWS) cell lines that are unresponsive to TZ1, a monoclonal antibody that targets IGFR. For acquired resistance, we developed a TZ1 resistant rhabdomyosarcoma (RMS) cell line from TZ1 sensitive RH41 cells. The cell lines were treated with TZ1, an anti-IGFR monoclonal antibody. Signaling changes were assessed by comparing phosphorylation status of the RTKs in the untreated versus treated cells in the inherent resistance model and in the parental versus resistant cells in the acquired resistance model. Results: TZ-1 treatment of the EWS cell lines with intrinsic resistance decreased pIGFR, but these cells were characterized by expression of multiple phosphorylated RTKs. In the Rh41 acquired resistance model, increased activation of signaling pathways was detected in the resistance cells including: AXL, PDGFR, FGFR4 and ALK. Treatment of parental Rh41 cells with TZ1 also rapidly induced several activated RTKs. In TZ1-resistant Rh41 cells combined treatment of TZ1 with the PDGFR inhibitor, crenolanib, and the pan-FGFR inhibitor LY2874455 resulted in significant decrease in cellular proliferation.
Conclusions: Intrinsic resistance to TZ1 in EWS cell lines appears to be due to redundancy through multiple alternative activated RTKs. Systematic knockdown to determine which activated RTKs confer resistance is being undertaken. In Rh41 RMS cells acquired resistance to TZ1 is characterized by induction of several alternative activated RTKs, some of which are induced rapidly after TZ1 exposure. Combined treatment with TZ1 and drugs targeting PDGFR and FGFR was effective at overcoming resistance. These results suggest that identification of signaling pathways that compensate for IGFR inhibition can lead to effective potent multi-target combination therapy. Supported by USPHS Grant CA165995.
Citation Format: Terry J. Shackleford, Seethalakshmi Hariharan, Hemant K. Bid, Peter J. Houghton. Mechanisms of resistance to IGFR-targeted therapy in pediatric sarcomas. 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 2472.
Abstract
Ewing’s sarcoma (ES) is a rare but aggressive cancer typically occurring in the bone or soft tissue of children and adolescents. Current treatment regimens composed of surgery, radiation, ...and high-dose chemotherapy have improved outcomes for localized disease cases; however, ES continues to present a clinical challenge in the recurrent or metastatic setting. ES is often characterized by a chromosomal translocation that results in a chimeric EWS/ETS transcription factor. This fusion protein may contribute to tumorigenesis through modulation of downstream targets leading to cell cycle dysregulation, due in part to the overexpression of Cyclin D1. In addition, ES also has a high degree of DNA methylation when compared to other tumor types, which can lead to epigenetic silencing of key tumor suppressor genes. Abemaciclib is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 and 6) currently under clinical evaluation for metastatic breast cancer, non-small cell lung cancer, and other adult solid tumors. Abemaciclib shows antitumor activity in preclinical models of human adult and pediatric cancer with functional Rb and high cyclin D expression, and has been reported to elicit an immune response in addition to its cell cycle effects in breast cancer. This study was designed to assess abemaciclib-induced antitumor effects, in addition to epigenetic and immunogenic changes, in preclinical ES models.
Six ES cell lines were treated with abemaciclib and the resulting changes in protein expression and methylation were evaluated by Western blot and DNA promoter methylation analysis, respectively. In addition, tumor-secreted factors from conditioned media were measured by ELISA and activation of the interferon (IFN) pathway was assessed through Western blot, qRT-PCR, and RNAseq analysis. In vivo studies of abemaciclib as a single agent or in combination with ES standard-of-care (SOC) in multiple mouse xenograft models were also performed to measure abemaciclib-mediated antitumor effects and epigenetic and immunogenic changes. Single-agent abemaciclib elicited tumor growth delay or tumor stasis, while the combination with SOC was generally superior to either monotherapy arm, often resulting in objective responses. Abemaciclib induced global DNA demethylation through the Rb-E2F-DNMT1 axis, and a ligand-independent type III IFN response. In addition, abemaciclib increased expression of the tumor cell antigen presentation protein MHC-1, and this effect was more durable than abemaciclib-dependent cell cycle effects. These data demonstrate that abemaciclib activity in ES goes beyond cell cycle arrest to include a measurable and durable immunogenic response. Therefore, the multipronged antitumor effects of abemaciclib, namely CDK4/6 inhibition, global DNA demethylation, and resulting immunogenic changes, may be a viable therapeutic option for ES patients when used in combination with appropriate chemotherapy, and additional studies to better understand this unique biology are ongoing.
Citation Format: Michele Dowless, Caitlin Lowery, Matthew Renschler, Wayne Blosser, Jennifer Stephens, Robert Flack, Kelly Credille, Mia Chen, Frank Dorsey, Lillian Sams, Philip Ebert, Jonathan Olsen, Terry Shackleford, Peter Houghton, Louis Stancato. The CDK4 and 6 inhibitor abemaciclib induces both epigenetic and immunogenic responses in preclinical models of Ewing’s sarcoma 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 B43.
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