scholarly journals Ciona Brachyury proximal and distal enhancers have different FGF dose-response relationships

PLoS Genetics ◽  
2021 ◽  
Vol 17 (1) ◽  
pp. e1009305
Author(s):  
Matthew J. Harder ◽  
Julie Hix ◽  
Wendy M. Reeves ◽  
Michael T. Veeman
Keyword(s):  
Dose Response ◽  
Mapk Pathway ◽  
Expression Patterns ◽  
Mek Inhibitor ◽  
Distal Enhancer ◽  
Endogenous Gene ◽  
Similar Expression ◽  
Upstream Regulator ◽  
Ets Family ◽  
Expression Loss

Many genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not equivalent genetic elements.

scholarly journals Ciona Brachyury proximal and distal enhancers have different FGF dose-response relationships

2020 ◽  
Author(s):  
Matthew J. Harder ◽  
Julie Hix ◽  
Wendy M. Reeves ◽  
Michael T. Veeman
Keyword(s):  
Dose Response ◽  
Mapk Pathway ◽  
Expression Patterns ◽  
Mek Inhibitor ◽  
Distal Enhancer ◽  
Endogenous Gene ◽  
Similar Expression ◽  
Upstream Regulator ◽  
Ets Family ◽  
Expression Loss

AbstractMany genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not redundant genetic elements.

Treatment of a BRAF V600E Positive Ameloblastoma in a Pediatric Patient with MEK Inhibitor Monotherapy

FACE ◽  
2021 ◽  
pp. 273250162110051
Author(s):  
Steven Daws ◽  
Kongkrit Chaiyasate ◽  
Arshi Lehal
Keyword(s):  
Mapk Pathway ◽  
Case Reports ◽  
Radical Resection ◽  
Mek Inhibitor ◽  
Braf V600e Mutation ◽  
Braf V600e ◽  
Targeted Chemotherapy ◽  
Odontogenic Epithelium ◽  
Genetic Landscape ◽  
Frequent Presence

Ameloblastomas are uncommon tumors of the odontogenic epithelium standardly treated with radical resection. Recent studies of the genetic landscape of ameloblastoma have revealed the frequent presence of the BRAF V600E mutation, suggesting a possible role for targeted chemotherapy. We present the case of a primary mandibular ameloblastoma found in a 13-year-old female with confirmed BRAF V600E mutation. Prior to invasive surgical intervention she was treated for 8 weeks with the MEK inhibitor trametinib, but her tumor demonstrated little radiographic, clinical, or histologic response. Previous case reports have shown ameloblastoma in adult patients to be responsive to other agents targeting the MAPK pathway. Our observations in the presented case demonstrate the need for further research into the utility of targeted chemotherapy in ameloblastoma treatment.

scholarly journals Analysis of maxillopedia Expression Pattern and Larval Cuticular Phenotype in Wild-Type and Mutant Tribolium

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 721-731 ◽  
Author(s):  
Teresa D Shippy ◽  
Jianhua Guo ◽  
Susan J Brown ◽  
Richard W Beeman ◽  
Robin E Denell
Keyword(s):  
Rna Interference ◽  
Expression Pattern ◽  
Tribolium Castaneum ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
Homeotic Gene ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Similar Expression ◽  
Mutant Phenotypes

Abstract The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

scholarly journals Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell Lines

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2012
Author(s):  
Kathryn M. Appleton ◽  
Charuta C. Palsuledesai ◽  
Sean A. Misek ◽  
Maja Blake ◽  
Joseph Zagorski ◽  
...  
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Therapeutic Potential ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Intrinsic Resistance ◽  
Primary Resistance ◽  
Induced Apoptosis ◽  
Melanoma Cell Lines

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (≈50% BRAFV600 mutations and ≈30% NRAS mutations). While drugs targeting the MAPK pathway have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in patients with NRAS mutant melanomas in part due to their cytostatic effects and primary resistance. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. A combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells, which are highly resistant to trametinib. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlight the therapeutic potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

scholarly journals DDRE-07. DIPG HARBOUR ALTERATIONS TARGETABLE BY MEK INHIBITORS, WITH ACQUIRED RESISTANCE MECHANISMS OVERCOME BY COMBINATORIAL INHIBITION

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii62-ii62
Author(s):  
Elisa Izquierdo ◽  
Diana Carvalho ◽  
Alan Mackay ◽  
Sara Temelso ◽  
Jessica K R Boult ◽  
...  
Keyword(s):  
Drug Exposure ◽  
Mapk Pathway ◽  
Synergistic Effects ◽  
Acquired Resistance ◽  
Mek Inhibitor ◽  
Resistance Mechanisms ◽  
Genetic Alterations ◽  
Mesenchymal Transition

Abstract The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 16-50nM) in samples, which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and of a patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones (62-188-fold, GI50 2.4–5.2µM) in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted an observed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

Preclinical activity of cobimetinib alone or in combination with chemotherapy and targeted therapies in renal cell carcinoma

2021 ◽  
Author(s):  
Lichen Zhang ◽  
Deqiong Xie ◽  
Yonghua Lei ◽  
Aoli Na ◽  
Lei Zhu
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Mapk Pathway ◽  
Standard Of Care ◽  
Underlying Mechanism ◽  
Mek Inhibitor ◽  
Tumor Formation ◽  
Pathway Inhibition ◽  
New Treatments

Background: The poor outcome of advanced renal cell carcinoma (RCC) necessitates new treatments. Cobimetinib is a MEK inhibitor and approved for the treatment of melanoma. This work investigated the efficacy of cobimetinib alone and in combination with anti-RCC drugs. Methods: Proliferation and apoptosis assays were performed, and combination index was analyzed on RCC cell lines (CaKi-2, 786-O, A-704, ACHN and A489) and xenograft models. Immunoblotting analysis was conducted to investigate the MAPK pathway. Results: Cobimetinib was active against RCC cells, with IC50 at 0.006–0.8μM, and acted synergistically with standard-of-care therapy. Cobimetinib at nontoxic doses prevented tumor formation, inhibited tumor growth and enhanced efficacy of 5-fluorouracil, sorafenib and sunitinib via suppressing Raf/MEK/ERK, leading to MAPK pathway inhibition. Conclusion: Our findings demonstrate the potent anti-RCC activity of cobimetinib and its synergism with RCC standard-of-care drugs, and confirm the underlying mechanism of the action of cobimetinib.

Phosphatidylinositol 3-kinase mediates integrin-dependent NF-(κ)B and MAPK activation through separate signaling pathways

2001 ◽  
Vol 114 (8) ◽  
pp. 1579-1589 ◽  
Author(s):  
M. Reyes-Reyes ◽  
N. Mora ◽  
A. Zentella ◽  
C. Rosales
Keyword(s):  
Signaling Pathways ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Monocytic Leukemia ◽  
Monocytic Cells ◽  
Mapk Activation

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, (β)1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via (β)1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor (κ)B (NF-(κ)B) activation was then analyzed. We found that integrins activated both NF-(κ)B and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-(κ)B activation. In contrast, a dominant negative mutant of Rac completely prevented NF-(κ)B activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-(κ)B is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Ρ augmented NF-(κ)B activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-(κ)B, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.

Conserved and distinct roles of kreisler in regulation of the paralogous Hoxa3 and Hoxb3 genes

Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 759-769 ◽  
Author(s):  
M. Manzanares ◽  
S. Cordes ◽  
L. Ariza-McNaughton ◽  
V. Sadl ◽  
K. Maruthainar ◽  
...  
Keyword(s):  
Binding Sites ◽  
Hox Genes ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Enhancer Activity ◽  
Anteroposterior Patterning ◽  
Endogenous Gene ◽  
Transgenic Embryos ◽  
The Individual ◽  
Segmental Expression

During anteroposterior patterning of the developing hindbrain, the anterior expression of 3′ Hox genes maps to distinct rhombomeric boundaries and, in many cases, is upregulated in specific segments. Paralogous genes frequently have similar anterior boundaries of expression but it is not known if these are controlled by common mechanisms. The expression of the paralogous Hoxa3 and Hoxb3 genes extends from the posterior spinal cord up to the rhombomere (r) 4/5 boundary and both genes are upregulated specifically in r5. However, in this study, we have found that Hoxa3 expression is also upregulated in r6, showing that there are differences in segmental expression between paralogues. We have used transgenic analysis to investigate the mechanisms underlying the pattern of segmental expression of Hoxa3. We found that the intergenic region between Hoxa3 and Hoxa4 contains several enhancers, which summed together mediate a pattern of expression closely resembling that of the endogenous Hoxa3 gene. One enhancer specifically directs expression in r5 and r6, in a manner that reflects the upregulation of the endogenous gene in these segments. Deletion analysis localized this activity to a 600 bp fragment that was found to contain a single high-affinity binding site for the Maf bZIP protein Krml1, encoded by the kreisler gene. This site is necessary for enhancer activity and when multimerized it is sufficient to direct a kreisler-like pattern in transgenic embryos. Furthermore the r5/r6 enhancer activity is dependent upon endogenous kreisler and is activated by ectopic kreisler expression. This demonstrates that Hoxa3, along with its paralog Hoxb3, is a direct target of kreisler in the mouse hindbrain. Comparisons between the Krml1-binding sites in the Hoxa3 and Hoxb3 enhancers reveal that there are differences in both the number of binding sites and way that kreisler activity is integrated and restricted by these two control regions. Analysis of the individual sites revealed that they have different requirements for mediating r5/r6 and dorsal roof plate expression. Therefore, the restriction of Hoxb3 to r5 and Hoxa3 to r5 and r6, together with expression patterns of Hoxb3 in other vertebrate species suggests that these regulatory elements have a common origin but have later diverged during vertebrate evolution.

Abstract 837: Beta1 And Beta2 Adrenergic Receptor Transgenic Mice Display Distinct MicroRNA Expression Patterns That Converge On The Hypertrophic Signature

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Danish Sayed ◽  
Shweta Rane ◽  
Leng-Yi Chen ◽  
Minzhen He ◽  
Jacqueline Lypowy ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Adrenergic Receptor ◽  
Mapk Pathway ◽  
Expression Patterns ◽  
Pressure Overload ◽  
Mapk Signaling ◽  
Compensatory Hypertrophy ◽  
Over Expression ◽  
Mrna Targets ◽  
Dose Dependent

MicroRNA (miRNA) are ~22 ribonucleotides-long, with a potential to recognize multiple mRNA targets guided by sequence complimentarity. This class of molecules is functionally versatile, with the capacity to specifically inhibit translation, as well as, induce mRNA degradation, through targeting the 3′-untranslated regions. The levels of individual miRNA vary under different developmental, biological, or pathological conditions, thus, implicating them in normal and pathological cellular attributes. We have previously reported a miRNA signature that distinguishes pressure-overload compensatory hypertrophy by recapitulating the neonatal pattern. We hypothesized that this ’signature’ might aid in discriminating the underlying molecular differences in genetic models of cardiac hypertrophy, as seen in the beta1 and 2 adrenergic receptor (B1AR and B2AR) transgenic (Tg) mice. To address this, we used microarray analysis of RNA isolated from the hearts of 3 months old B1AR and B2AR mice. In general, while both mice exhibited an overlap with the hypertrophy signature including, upregulation of miR-21 and downregulation of miR-133a, miR-133b, and miR-185, the B2-AR Tg exhibited a more extensive overlap with the hypertrophy pattern, which further included upregulation of miR-199a*, miR-214, and miR-15b. To understand the functional significance of these miRNA in myocyte hypertrophy, we cloned them and their anti-sense sequences into adenoviral vectors. Significantly, over-expression miR-21 resulted in a, dose-dependent, branching (sprouting) of the cells. Computational predictions by ’TargetScanS’ identified sprouty as potential target. Subsequently, we confirmed down-regulation of sprouty by over-expression of miR-21 and vice versa. Sprouty is a known inhibitor of the Ras-MAPK signaling pathway and is, concordantly, downregulated in many forms of cancer. In the heart, sprouty has been suggested to control myocyte size and vascularization during cardiac hypertrophy. Thus, we propose that B1AR and B2AR Tg models exhibit distinct miRNA profiles that converge on that of pressure-overload cardiac hypertrophy. Moreover, the commonly over-expressed miR-21 plays a role in downregulating sprouty, an antagonist of the Ras-MAPK pathway.

scholarly journals LGG-26. DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR (DLGNT) IN CHILDREN: DIFFERENT CLINICAL PRESENTATIONS AND OUTCOMES

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii371-iii371
Author(s):  
Andge Valiakhmetova ◽  
Ludmila Papusha ◽  
Ludmila Yasko ◽  
Alexander Druy ◽  
Alexander Karachunsky ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Braf Inhibitor ◽  
Complete Response ◽  
Mek Inhibitor ◽  
Braf V600e ◽  
Glioneuronal Tumor ◽  
Low Grade ◽  
Conventional Chemotherapy ◽  
Diffuse Leptomeningeal Glioneuronal Tumor ◽  
Braf Fusion

Abstract Diffuse leptomeningeal glioneuronal tumor (DLGNT) is an extremely rare disease, newly recognized in the 2016 WHO classification of tumors of the CNS. Most DLGNTs are low-grade neuroepithelial tumors with variable elements of neuronal/neurocytic and glial differentiation, have diffuse leptomeningeal enhancement on MRI, and typically harbor KIAA1549-BRAF fusions. Other alterations, such as the BRAF V600E substitution, are less common. Here, we present three cases of DLGNT with different presentations and outcomes. The first patient is a 2yr-old male with KIAA1549-BRAF fusion, and was treated with Carbo/VCR chemotherapy after a biopsy, with resultant ongoing stable disease for 3.5 years. The second patient, an 8yr-old male had the BRAF V600E point mutation and was treated with conventional chemotherapy (VCR/carboplatin). On progression, he received the BRAF inhibitor vemurafenib, achieving a complete response which last 14 month. The third patient, a 27 month old male, harbored a KIAA1549-BRAF fusion and was treated at diagnosis with the MEK inhibitor trametinib. The tumor has been radiographically stable in the context of clinical improvement for 21 months since the treatment initiation, ongoing 24 month. In summary, we present further evidence of MAPK pathway alterations in children with DLGNT. We describe a range of molecular presentations and clinical outcomes, including one patient treated with conventional chemotherapy with further stabilization of disease during 3.5 years and two patients who were successfully treated with targeted therapy.

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