Sorafenib and Mek inhibition is synergistic in medullary thyroid carcinoma in vitro

Clinical trials using kinase inhibitors have demonstrated transient partial responses and disease control in patients with progressive medullary thyroid cancer (MTC). The goal of this study was to identify potential combinatorial strategies to improve on these results using sorafenib, a multikinase inhibitor with activity in MTC, as a base compound to explore signaling that might predict synergystic interactions. Two human MTC cell lines, TT and MZ-CRC-1, which harbor endogenous C634W or M918T RET mutations, respectively, were exposed to sorafenib, everolimus, and AZD6244 alone and in combination. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and poly (ADP-ribose) polymerase (PARP) cleavage assays were performed to measure cell survival and apoptosis. Western blots were performed to confirm activity of the compounds and to determine possible mechanisms of resistance and predictors of synergy. As a solitary agent, sorafenib was the most active compound on MTT assay. Western blots confirmed that sorafenib, everolimus, and AZD6244 inhibited their anticipated targets. At concentrations below its IC50, sorafenib-treated TT and MZ-CRC-1 cells demonstrated transient inhibition and then re-activation of Erk over 6 h. In concordance, synergistic effects were only identified using sorafenib in combination with the Mek inhibitor AZD6244 (P<0.001 for each cell line). Cells treated with everolimus demonstrated activation of Akt and Ret via TORC2 complex-dependent and TORC2 complex-independent mechanisms respectively. Everolimus was neither additive nor syngergistic in combination with sorafenib or AZD6244. In conclusion, sorafenib combined with a Mek inhibitor demonstrated synergy in MTC cells in vitro. Mechanisms of resistance to everolimus in MTC cells likely involved TORC2-dependent and TORC2-independent pathways.

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

Neuro-Oncology ◽

10.1093/neuonc/noaa215.252 ◽

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.

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Clinical efficacy with dabrafenib and trametinib in a T599_V600insT poorly differentiated metastatic thyroid carcinoma

BMJ Case Reports ◽

10.1136/bcr-2021-243264 ◽

2021 ◽

Vol 14 (8) ◽

pp. e243264

Author(s):

Chung-Shien Lee ◽

Emily Miao ◽

Kasturi Das ◽

Nagashree Seetharamu

Keyword(s):

Thyroid Carcinoma ◽

Kinase Inhibitors ◽

Mek Inhibitor ◽

Mitogen Activated Protein Kinase ◽

Braf Mutations ◽

Effective Treatment Option ◽

Mitogen Activated Protein ◽

Treatment Data ◽

Poorly Differentiated

BRAF (v-raf murine sarcoma viral oncogene homolog B1) and MEK (mitogen-activated protein kinase kinase) inhibitors have been shown to improve clinical outcomes in tumours presenting with mutations in the BRAF gene. The most common form of BRAF mutation is V600E/K and has been shown to occur in thyroid cancers. Treatment data for patients harbouring less frequent BRAF mutations are limited. In vitro studies have shown that mutations in codons 599–601 increase kinase activity similar to that in V600E mutations, which suggests that BRAF and MEK inhibitors could be an effective treatment option. Here, we report a case of a patient with thyroid carcinoma harbouring a rare amino acid insertion in codon 599 of the BRAF gene (T599_V600insT) treated with a BRAF and MEK inhibitor.

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CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition

Journal of Oncology ◽

10.1155/2019/6486173 ◽

2019 ◽

Vol 2019 ◽

pp. 1-19 ◽

Cited By ~ 3

Author(s):

Cynthia M. Simbulan-Rosenthal ◽

Anirudh Gaur ◽

Hengbo Zhou ◽

Maryam AbdusSamad ◽

Qing Qin ◽

...

Keyword(s):

Drug Resistance ◽

Kinase Inhibitors ◽

Braf Inhibitor ◽

Mek Inhibitor ◽

Melanoma Cells ◽

Stem Cell Marker ◽

High Dose ◽

Recurrence Rates ◽

Mapk Inhibitors

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibitionin vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as “melanoma initiating cells,” resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

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Preclinical study of a PARP inhibitor in neuroblastoma.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.9570 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 9570-9570

Author(s):

Anissa Addioui ◽

Assila Belounis ◽

Sonia Cournoyer ◽

Carine Nyalendo ◽

Rose- Marie Brito ◽

...

Keyword(s):

Cell Survival ◽

Synergistic Effects ◽

Parp Inhibitor ◽

Chemotherapeutic Agents ◽

Tumor Initiating Cells ◽

Apoptotic Pathway ◽

Western Blots ◽

Optimal Drug ◽

Parp 1

9570 Background: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In spite of many therapeutic improvements, only 60% survive long term despite aggressive combinations of multi-agent chemotherapy. In previous studies, we have demonstrated that tumor initiating cells (TIC) expressing CD133 (CD133high) in NB are more resistant to chemotherapy. Moreover, these cells express higher levels of PARP-1, a central protein involved in DNA repair. PARP-1 expression is significantly lower in NB usually showing spontaneous regression than in standard NB, suggesting an implication of PARP-1 in NB progression. The objective of this study is to determine the efficacy in vitro of AG-014699 (AG), a PARP- inhibitor, used in monotherapy or in combination to cisplatine (CP) and doxorubicine (DR), classical chemotherapeutic agents used in NB treatment, on NB cell survival. Methods: Six NB cell lines (parental or CD133high purified by flow cytometry (FACS)) were treated with AG alone or in association to CP or DR. PARP-1 ELISA protein assay was used to determine the optimal drug concentration needed to inhibit the protein. Cell survival was measured by MTT test. Western Blots were done to evaluate any apoptotic or autophagic pathway modulations. Quantification of DNA damage in treated cell was done by immunofluorescence of H2A-X protein. Results: We showed that a 4µM concentration of AG is sufficient for PARP-1 inhibition. One third of celllines presented a sensitivity to AG when used in monotherapy with an IC50 lower than 5µM. However, AG demonstrated synergistic effects when associated to DR, decreasing the IC50 by half, although none is observed when combined to CP. Sentitivity of the TIC did not appear to be more important than the bulk cells. With increasing concentration of AG, our WB showed no increase in cleaved Caspase-3 suggesting no modulation of the apoptotic pathway. However, autophagy seemed to be upregulated confirmed by an increase in cleaved LC3 II protein. Double strand breaks increased 2.5 folds when 4µM AG is added to the IC50 of DR. Conclusions: AG used in combination at potentially therapeutic doses shows promising results in NB. These results will allow for the improvement of NB treatments by introducing a new therapeutic strategy.

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The Relevance of Aurora Kinase Inhibition in Hematological Malignancies

Cancer Diagnosis & Prognosis ◽

10.21873/cdp.10016 ◽

2021 ◽

Vol 1 (3) ◽

pp. 111-126

Author(s):

CAIO BEZERRA MACHADO ◽

EMERSON LUCENA DA SILVA ◽

BEATRIZ MARIA DIAS NOGUEIRA ◽

JEAN BRENO SILVEIRA DA SILVA ◽

MANOEL ODORICO DE MORAES FILHO ◽

...

Keyword(s):

Clinical Trials ◽

Hematological Malignancies ◽

Kinase Inhibitors ◽

Synergistic Effects ◽

Aurora Kinase ◽

Aurora Kinases ◽

The Past ◽

Oncologic Patients

Aurora kinases are a family of serine/threonine protein kinases that play a central role in eukaryotic cell division. Overexpression of aurora kinases in cancer and their role as major regulators of the cell cycle quickly inspired the idea that their inhibition might be a potential pathway when treating oncologic patients. Over the past couple of decades, the search for designing and testing of molecules capable of inhibiting aurora activities fueled many pre-clinical and clinical studies. In this study, data from the past 10 years of in vitro and in vivo investigations, as well as clinical trials, utilizing aurora kinase inhibitors as therapeutics for hematological malignancies were compiled and discussed, aiming to highlight potential uses of these inhibitors as a novel monotherapy model or alongside conventional chemotherapies. While there is still much to be elucidated, it is clear that these kinases play a key role in oncogenesis, and their manageable toxicity and potentially synergistic effects still render them a focus of interest for future investigations in combinatorial clinical trials

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Targeting of Janus Kinases Limits Pro-Inflammatory but Also Immunosuppressive Circuits in the Crosstalk between Synovial Fibroblasts and Lymphocytes

Biomedicines ◽

10.3390/biomedicines9101413 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1413

Author(s):

Nina Yao ◽

Theresa Tretter ◽

Peter Kvacskay ◽

Wolfgang Merkt ◽

Norbert Blank ◽

...

Keyword(s):

Immune Cells ◽

Kinase Inhibitors ◽

Synergistic Effects ◽

Synovial Fibroblasts ◽

Janus Kinase ◽

Response To Treatment ◽

Th Cells ◽

Proven Efficacy ◽

Clinical Responses

Crosstalk between synovial fibroblasts (SF) and immune cells plays a central role in the development of rheumatoid arthritis (RA). Janus kinase inhibitors (JAKi) have proven efficacy in the treatment of RA, although clinical responses are heterogeneous. Currently, little is known regarding how JAKi affect pro- and anti-inflammatory circuits in the bidirectional interplay between SF and immune cells. Here, we examined the effects of tofacitinib, baricitinib and upadacitinib on crosstalk between SF and T or B lymphocytes in vitro and compared them with those of biologic disease modifying anti-rheumatic drugs (bDMARDs). JAKi dose-dependently suppressed cytokine secretion of T helper (Th) cells and decreased interleukin (IL)-6 and matrix metalloproteinase (MMP)3 secretion of SF stimulated by Th cells. Importantly, JAK inhibition attenuated the enhanced memory response of chronically stimulated SF. Vice versa, JAKi reduced the indoleamine-2,3-dioxygenase (IDO)1-mediated suppression of T cell-proliferation by SF. Remarkably, certain bDMARDs were as efficient as JAKi in suppressing the IL-6 and MMP3 secretion of SF stimulated by Th (adalimumab, secukinumab) or B cells (canakinumab) and combining bDMARDs with JAKi had synergistic effects. In conclusion, JAKi limit pro-inflammatory circuits in the crosstalk between SF and lymphocytes; however, they also weaken the immunosuppressive functions of SF. Both effects were dose-dependent and may contribute to heterogeneity in clinical response to treatment.

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MEK1/2 in Rhabdomyosarcoma

10.1101/629766 ◽

2019 ◽

Cited By ~ 1

Author(s):

Kenneth A. Crawford ◽

Megan M. Cleary ◽

Cora A. Ricker ◽

Matthew N. Svalina ◽

John F. Shern ◽

...

Keyword(s):

Cell Growth ◽

Myogenic Differentiation ◽

Synergistic Effects ◽

Mek Inhibitor ◽

Differentiation Therapy ◽

Muscle Stem Cells ◽

Response To Stress ◽

High Content Analysis ◽

Growth Evaluation

AbstractAlveolar and embryonal rhabdomyosarcoma (RMS) are soft-tissue cancers that affect children, adolescents, and young adults. Sometimes referred to as muscle cancer, RMS is a cancer of muscle and non-muscle origin that phenocopies incompletely differentiated myoblasts or activated satellite (muscle stem) cells. Interestingly, embryonal RMS (ERMS) has been observed to undergo terminal myogenic differentiation in response to stress induced by chemotherapy and radiation therapy4, 9, 24. Given the propensity of rhabdomyosarcoma to differentiation, in this report we explore the use of differentiation therapy combining MEK inhibitor (MEKi) cobimetinib and chemotherapy as a strategy to halt RMS growth. We evaluated a representative panel of RMS cell lines with cobimetinib and chemotherapy in two dosing schedules that mimic clinical use followed by cell growth evaluation and high content analysis (differentiation) assays. We uncovered that cobimetinib does not have significant additive or synergistic effects on cell differentiation or cell growth with chemotherapy in RMS and can have unanticipated antagonistic effects; specifically, pre-exposure of cobimetinib to cells can decrease the effectiveness of chemotherapy-mediated cell growth inhibition in vitro. Although differentiation-therapy is still a potential viable strategy in RMS, our data do not support MEKi/chemotherapy co-treatment in this context.

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Mechanisms of Resistance to Mitogen-Activated Protein Kinase Pathway Inhibition in BRAF-Mutant Melanoma

American Society of Clinical Oncology Educational Book ◽

10.14694/edbook_am.2012.32.189 ◽

2012 ◽

pp. 680-684 ◽

Cited By ~ 4

Author(s):

Eva M. Goetz ◽

Levi A. Garraway

Keyword(s):

Protein Kinase ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Mapk Pathway ◽

Drug Binding ◽

Mitogen Activated Protein Kinase ◽

Braf V600e ◽

Mechanisms Of Resistance ◽

Mitogen Activated Protein

Overview: Anticancer drug resistance remains a crucial impediment to the care of many patients with cancer. Although the exact mechanisms of resistance may differ for each therapy, common mechanisms of resistance predominate, including drug inactivation or modification, mutation of the target protein, reduced drug accumulation, or bypass of target inhibition. With the discovery and use of targeted therapies (such as small-molecule kinase inhibitors), resistance has received renewed attention—especially in light of the dramatic responses that may emerge from such therapeutics in particular genetic or molecular contexts. Recently, the mitogen-activated protein kinase (MAPK) pathway has become exemplary in this regard, since it is activated in many different cancers. Drugs targeting RAF and MAPK kinase (MEK) are currently in clinical trials for the treatment of several types of cancer. Vemurafenib, a selective RAF kinase inhibitor recently approved for the treatment of BRAF(V600E) melanoma, shows strong efficacy initially; however, the development of resistance is nearly ubiquitous. In vitro testing and analysis of patient samples have uncovered several mechanisms of resistance to RAF inhibition. Surprisingly, mutations in the drug-binding pocket have not thus far been observed; however, other alterations at the level of RAF, as well as downstream activation of MEK and bypass of MEK/extracellular signal-regulated kinase (ERK) signaling altogether, confer resistance to vemurafenib. Looking forward, combined RAF and MEK inhibitor treatments may improve efficacy—yet we must anticipate mechanisms of resistance to this combination as well. Therefore, understanding and/or determining the mechanism of resistance are paramount to effective cancer treatment.

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

Neuro-Oncology ◽

10.1093/neuonc/noaa222.592 ◽

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.

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