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.

scholarly journals MODL-19. DIPG HARBOUR ALTERATIONS TARGETABLE BY MEK INHIBITORS, WITH ACQUIRED RESISTANCE MECHANISMS OVERCOME BY COMBINATORIAL UP- OR DOWN-STREAM INHIBITION

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii414-iii414
Author(s):  
Elisa Izquierdo ◽  
Diana Carvalho ◽  
Alan Mackay ◽  
Sara Temelso ◽  
Jessica K R Boult ◽  
...  
Keyword(s):  
Drug Exposure ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Synergistic Effects ◽  
Acquired Resistance ◽  
Mek Inhibitor ◽  
Genetic Alterations ◽  
Mesenchymal Transition

Abstract The survival of children with 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 (BIOMEDE), 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. Treatment of PDX models and the patient with trametinib at relapse, however, 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, with overexpression of key proteins involved in invasion/migration, such as collagen-family proteins, integrins, MMPs and AHNAK2, amongst others. Resistant clones were conversely sensitive to the upstream receptor tyrosine kinase inhibitor dasatinib (GI50 36-93nM), and combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed 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.

scholarly journals A screen for combination therapies inBRAF/NRASwild type melanoma identifies nilotinib plus MEK inhibitor as a synergistic combination

2017 ◽  
Author(s):  
Marco Ranzani ◽  
Kristel Kemper ◽  
Magali Michaut ◽  
Oscar Krijgsman ◽  
Nanne Aben ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Acquired Resistance ◽  
Mek Inhibitor ◽  
Resistance Mechanisms ◽  
Wild Type ◽  
Genome Wide ◽  
Melanoma Patients ◽  
Inhibitor Combination

AbstractDespite recent therapeutic advances in the management ofBRAFV600-mutant melanoma, there is still a compelling need for more effective treatments for patients who developedBRAF/NRASwild type disease. Since the activity of single targeted agents is limited by innate and acquired resistance, we performed a high-throughput drug screen using 180 drug combinations to generate over 18,000 viability curves, with the aim of identifying agents that synergise to killBRAF/NRASwild type melanoma cells. From this screen we observed strong synergy between the tyrosine kinase inhibitor nilotinib and MEK inhibitors and validated this combination in an independent cell line collection. We found that AXL expression was associated with synergy to the nilotinib/MEK inhibitor combination, and that both drugs work in concert to suppress pERK. This finding was supported by genome-wide CRISPR screening which revealed that resistance mechanisms converge on regulators of the MAPK pathway. Finally, we validated the synergy of nilotinib/trametinib combinationin vivousing patient-derived xenografts. Our results indicate that a nilotinib/MEK inhibitor combination may represent an effective therapy inBRAF/NRASwild type melanoma patients.

scholarly journals Triple blockade of EGFR, MEK and PD-L1 has antitumor activity in colorectal cancer models with constitutive activation of MAPK signaling and PD-L1 overexpression

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
S. Napolitano ◽  
N. Matrone ◽  
A. L. Muddassir ◽  
G. Martini ◽  
A. Sorokin ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Molecular Mechanisms ◽  
Mapk Pathway ◽  
Combined Treatment ◽  
Acquired Resistance ◽  
Mek Inhibitor ◽  
Gene Signature ◽  
Mapk Signaling

Abstract Background Molecular mechanisms driving acquired resistance to anti-EGFR therapies in metastatic colorectal cancer (mCRC) are complex but generally involve the activation of the downstream RAS-RAF-MEK-MAPK pathway. Nevertheless, even if inhibition of EGFR and MEK could be a strategy for overcoming anti-EGFR resistance, its use is limited by the development of MEK inhibitor (MEKi) resistance. Methods We have generated in vitro and in vivo different CRC models in order to underline the mechanisms of MEKi resistance. Results The three different in vitro MEKi resistant models, two generated by human CRC cells quadruple wild type for KRAS, NRAS, BRAF, PI3KCA genes (SW48-MR and LIM1215-MR) and one by human CRC cells harboring KRAS mutation (HCT116-MR) showed features related to the gene signature of colorectal cancer CMS4 with up-regulation of immune pathway as confirmed by microarray and western blot analysis. In particular, the MEKi phenotype was associated with the loss of epithelial features and acquisition of mesenchymal markers and morphology. The change in morphology was accompanied by up-regulation of PD-L1 expression and activation of EGFR and its downstream pathway, independently to RAS mutation status. To extend these in vitro findings, we have obtained mouse colon cancer MC38- and CT26-MEKi resistant syngeneic models (MC38-MR and CT26-MR). Combined treatment with MEKi, EGFR inhibitor (EGFRi) and PD-L1 inhibitor (PD-L1i) resulted in a marked inhibition of tumor growth in both models. Conclusions These results suggest a strategy to potentially improve the efficacy of MEK inhibition by co-treatment with EGFR and PD-L1 inhibitors via modulation of host immune responses.

scholarly journals Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 142 ◽  
Author(s):  
Mariusz L. Hartman ◽  
Malgorzata Sztiller-Sikorska ◽  
Anna Gajos-Michniewicz ◽  
Malgorzata Czyz
Keyword(s):  
Phenotypic Plasticity ◽  
Cell Lines ◽  
Mapk Pathway ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Genetic Alterations ◽  
Resistant Cell ◽  
Cross Resistance ◽  
Preclinical Model ◽  
Development Of Resistance

The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKThigh resistant cell lines. There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.

Synergistic growth inhibition of RCC and NSCLC cell lines by sorafenib plus vorinostat and induction of angiogenic genes by ER stress

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e16114-e16114
Author(s):  
H. A. Drabkin ◽  
G. Sharma ◽  
L. J. Costa ◽  
C. Korch ◽  
R. M. Gemmill
Keyword(s):  
Growth Inhibition ◽  
Er Stress ◽  
Cell Lines ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Angiogenic Factors ◽  
Nsclc Cell ◽  
Angiogenic Genes

e16114 Sorafenib, vorinostat and the combination were examined in 34 RCC and NSCLC cell lines. In growth assays, sorafenib at 8 μM resulted in synergy with multiple vorinostat doses, whereas no synergy was seen with lower doses. Changes in phospho-ERK and AKT were not predictive of growth inhibition, whereas frequent inhibition of cyclin D1 and upregulation of p21Waf1 was observed. To anticipate resistance mechanisms to the anti-angiogenic effects of sorafenib, we studied the expression of 13 angiogenic factors in 10 selected lines. At baseline, bFGF, VEGF and IL-8 were highly expressed in RCCs, whereas Gro-α, VEGF, and IL-8 predominated in NSCLCs. Multiple angiogenic factors were upregulated by sorafenib and vorinostat, especially VEGF, IL-6, CTGF, EDN1, PDGFβ, and IL-8. Importantly, sorafenib at 8 μM, but not lower doses, induced ER stress in these cell lines and thapsigargin or tunicamycin treatment recapitulated many, but not all, of the observed angiogenic gene responses to sorafenib. In fact, CHOP induction by sorafenib plus vorinostat was the only parameter, other than growth inhibition, that changed in a synergistic manner. In summary, sorafenib plus vorinostat potently inhibits the in vitro growth of RCC and NSCLC cell lines. Upregulation of multiple angiogenic genes, in part by an ER-stress mechanism, may contribute to acquired resistance in vivo. No significant financial relationships to disclose.

Effect of bypass kinase pathways on acquired CDK4/6 inhibitor resistance.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 379-379
Author(s):  
Renee De Leeuw ◽  
Christopher McNair ◽  
Matthew Joseph Schiewer ◽  
Neermala Poudel Neupane ◽  
Michael Augello ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Early Stage ◽  
Mapk Signaling ◽  
Cross Resistance ◽  
Mek Inhibitors ◽  
Mesenchymal Transition ◽  
Inhibitor Resistance ◽  
Tumor Types

379 Background: Cyclin Dependent Kinase-4/6 (CDK4/6) kinase inhibitors have shown clinical benefit in treatment of solid tumor types, including breast cancer. However, resistance is common, and the underpinning mechanisms of action are not well understood. Given the dependence of CDK4/6 inhibitors on retinoblastoma tumor suppressor (RB) function for activity, this class of agents may be particularly effective in tumor types for which RB loss is infrequent or occurs late in tumor progression. Methods: Here, models of acquired palbociclib resistance were generated in early stage, RB positive cancers, wherein it was shown acquired palbociclib resistance resulted in cross-resistance to other CDK4/6 inhibitors under clinical testing. Results: Cells showing acquired resistance exhibited aggressive in vitro and in vivo phenotypes without genetic loss of RB or RB pathway members, including enhanced proliferative capacity, migratory potential, and characteristics of epithelial to mesenchymal transition. Further analyses through integration of RNA sequencing and phospho-proteomics identified activation of the MAPK signaling pathway as a mediator of CDK4/6 inhibitor resistance, capable of bypassing CDK4/6 activity. However, this altered kinase dependence resulted in sensitization to MEK inhibitors, suggestive of new clinical opportunities in CDK4/6 resistant tumors. Conclusions: In sum, the studies herein not only identify activation of the MAPK pathway as capable of bypassing the CDK4/6 requirement and promoting aggressive tumor characteristics, but nominate MEK inhibitors as potential mechanisms to treat or prevent CDK4/6 inhibitor resistance.

scholarly journals SPDR-1 HSP90 inhibition overcomes resistant to molecular targeted therapy in BRAFV600E mutant glioblastoma

2021 ◽  
Vol 3 (Supplement_6) ◽  
pp. vi3-vi3
Author(s):  
Jo Sasame ◽  
Naoki Ikegaya ◽  
Yohei Miyake ◽  
Takahiro Hayashi ◽  
Akito Oshima ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Molecular Targeted Therapy ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Mtor Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Hsp90 Inhibition ◽  
Pdx Models

Abstract The BRAFV600E mutation results in the constitutive activation of downstream mitogen activated protein kinase (MAPK) pathway that promotes tumor growth. Recently, molecular targeted therapy using BRAF/MEK inhibitor has been reported for BRAFV600E mutant high-grade glioma, but the therapeutic effect is limited by the emergence of drug resistance. Herein, we established paired BRAFV600E mutant glioblastoma (GBM) patient-derived xenograft (PDX) models, which were derived from tumors at prior to and recurrence after molecular targeted therapy. These PDX models were found to extensively recapitulate the histology, genetic abnormalities, and even the clinical course of the patients. Furthermore, BRAF/MEK inhibitor gradually caused resistance in cell lines derived from specimens that initially responded to molecular targeted therapy. In this study, genomic and epigenomic changes had little effect on the resistance mechanism. On the other hand, we found that hyperactivation of the MAPK pathway through c-Raf and the AKT/mTOR pathway primarily caused resistance to molecular targeted therapy in BRAFV600E mutant GBM. Through a high throughput drug screening, we find that HSP90 inhibitor with BRAF/MEK inhibitor coordinately deactivates MAPK pathway and AKT/mTOR pathway, and mediates potent toxicity in vitro and in vivo in refractory and acquired resistant models. These findings support that this therapeutic approach can overcome the limitation of current molecular targeted therapy in BRAFV600E mutant GBM.

scholarly journals MEK-inhibitor-mediated rescue of skeletal myopathy caused by activating Hras mutation in a Costello syndrome mouse model

2021 ◽  
Author(s):  
William E. Tidyman ◽  
Alice F. Goodwin ◽  
Yoshiko Maeda ◽  
Ophir D. Klein ◽  
Katherine A. Rauen
Keyword(s):  
Mouse Model ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Costello Syndrome ◽  
Skeletal Myopathy ◽  
Mitogen Activated Protein

Costello syndrome (CS) is a congenital disorder caused by heterozygous activating germline HRAS mutations in the canonical Ras/mitogen-activated protein kinase (Ras/MAPK) pathway. CS is one of the RASopathies, a large group of syndromes due to mutations within various components of the Ras/MAPK pathway. An important part of the phenotype that greatly impacts quality of life is hypotonia. To gain a better understanding of the mechanisms underlying hypotonia in CS, a mouse model with an activating HrasG12V allele was utilized. We identified a skeletal myopathy that was due in part to an inhibition of embryonic myogenesis and myofiber formation, resulting in a reduction of myofiber size and number that led to reduced muscle mass and strength. In addition to hyperactivation of the Ras/MAPK and PI3K/AKT pathways, there was a significant reduction of p38 signaling, as well as global transcriptional alterations consistent with the myopathic phenotype. Inhibition of Ras/MAPK pathway signaling using a MEK inhibitor rescued the HrasG12V myopathy phenotype both in vitro and in vivo, demonstrating that increased MAPK signaling is the main cause of the muscle phenotype in CS.

An oral androgen receptor PROTAC degrader for prostate cancer.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 381-381 ◽  
Author(s):  
Taavi Neklesa ◽  
Lawrence B Snyder ◽  
Ryan R Willard ◽  
Nicholas Vitale ◽  
Kanak Raina ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Drug Exposure ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Significant Inhibition ◽  
Castration Resistant Prostate Cancer ◽  
Orally Bioavailable

381 Background: The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most patients initially respond to inhibitors of the AR pathway, but the response is often short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Methods: Here we report an orally bioavailable small molecule AR PROTAC that leads to ubiquitination and degradation of AR. This molecule has been characterized in in vitro degradation and functional assays, DMPK, toxicology and preclinical efficacy studies. Results: This AR PROTAC completely degrades AR in all cell lines tested, with an observed 50% degradation concentration (DC50) < 1 nM. PROTAC-mediated AR degradation suppresses the expression of the AR-target gene PSA, inhibits AR-dependent cell proliferation, and induces potent apoptosis in VCaP cells. The AR PROTAC degrades all clinically relevant mutant AR proteins and retains activity in a high androgen environment. In mouse xenograft studies, greater than 90% AR degradation is observed at a 1 mg/kg PO QD dose. Significant inhibition of tumor growth and AR signaling can be achieved in both an intact and castrate setting. Further, the AR PROTAC demonstrates in vivo efficacy and reduction of oncogenic Erg protein in a long term, castrate, enzalutamide-resistant VCaP tumor model. DMPK and exploratory toxicology studies show robust oral, dose proportional drug exposure in rodent and non-rodent species. Conclusions: In summary, we report preclinical data on an orally bioavailable AR PROTAC degrader that demonstrates efficacy in enzalutamide-resistant prostate cancer.

scholarly journals Fermitin family member 2 promotes melanoma progression by enhancing the binding of p-α-Pix to Rac1 to activate the MAPK pathway

Oncogene ◽  
2021 ◽  
Author(s):  
Shaobin Huang ◽  
Wuguo Deng ◽  
Peng Wang ◽  
Yue Yan ◽  
Chuanbo Xie ◽  
...  
Keyword(s):  
Family Member ◽  
Mapk Pathway ◽  
Specific Binding ◽  
Acquired Resistance ◽  
Clinical Samples ◽  
Nucleotide Exchange ◽  
Melanoma Progression ◽  
Melanoma Growth

AbstractWe identified fermitin family member 2 (FERMT2, also known as kindlin-2) as a potential target in A375 cell line by siRNA library screening. Drugs that target mutant BRAF kinase lack durable efficacy in the treatment of melanoma because of acquired resistance, thus the identification of novel therapeutic targets is needed. Immunohistochemistry was used to identify kindlin-2 expression in melanoma samples. The interaction between kindlin-2 and Rac1 or p-Rac/Cdc42 guanine nucleotide exchange factor 6 (α-Pix) was investigated. Finally, the tumor suppressive role of kindlin-2 was validated in vitro and in vivo. Analysis of clinical samples and Oncomine data showed that higher levels of kindlin-2 predicted a more advanced T stage and M stage and facilitated metastasis and recurrence. Kindlin-2 knockdown significantly inhibited melanoma growth and migration, whereas kindlin-2 overexpression had the inverse effects. Further study showed that kindlin-2 could specifically bind to p-α-Pix(S13) and Rac1 to induce a switch from the inactive Rac1-GDP conformation to the active Rac1-GTP conformation and then stimulate the downstream MAPK pathway. Moreover, we revealed that a Rac1 inhibitor suppressed melanoma growth and metastasis and the combination of the Rac1 inhibitor and vemurafenib resulted in a better therapeutic outcome than monotherapy in melanoma with high kindlin-2 expression and BRAF mutation. Our results demonstrated that kindlin-2 promoted melanoma progression, which was attributed to specific binding to p-α-Pix(S13) and Rac1 to stimulate the downstream MAPK pathway. Thus, kindlin-2 could be a potential therapeutic target for treating melanoma.

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