Evaluation of antiproliferative and apoptotic effects of the rational combination of the MEK1/2 inhibitor selumetinib (AZD6244) and inhibitors of the hedgehog pathway in colorectal cancer (CRC) cell lines.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 422-422
Author(s):  
A. Spreafico ◽  
J. J. Tentler ◽  
A. Tan ◽  
T. M. Pitts ◽  
M. I. Kachaeva ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mapk Pathway ◽  
Mutant Cell ◽  
Gene Array ◽  
Combination Strategy ◽  
Cell Viability Assay ◽  
Inhibition Of Proliferation ◽  
Antiproliferative Effects ◽  
Rational Combination

422 Background: The MAPK pathway is a crucial regulator of cell proliferation, survival, and resistance to apoptosis. Hyperactivation of this pathway due to mutations in KRAS have been reported in up to 50% of CRC cases. Clinical trials have shown that KRAS patients do not benefit from therapies targeting EGFR, highlighting the need for new therapeutic options. Utilizing differential gene array analyses, we have identified the hedgehog (HH) signaling pathway as a potential mediator of resistance to AZD6244. Based on these results, we tested the rational combination of selumetinib and the HH inhibitor, cyclopamine against human CRC cell lines. Methods: CRC cell lines were exposed to varying concentrations of selumetinib and cyclopamine. For AZD6244, cell lines with IC50≤ 0.1 μM were considered extremely sensitive (ES) and those with IC50≥ 1μM were deemed extremely resistant (ER). Four KRAS mutant cell lines (2ES, 2ER) were selected for combination studies. The antiproliferative effects were assessed using the sulforhodamine B (SRB) cell viability assay, and potential synergy was evaluated using the Chou and Talalay method. Apoptosis was analyzed using bioluminescent caspase 3/7 detection. Results: In all four cell lines tested, synergistic antiproliferative effects of selumetinib and cyclopamine were observed, including resistant lines to selumetinib. We observed significant induction of apoptosis when cell lines were exposed to the combination treatment, independent of their responsiveness to selumetinib in the SRB assay. Conclusions: Treatment of KRAS mutant CRC cell lines with selumetinib and cyclopamine resulted in synergistic inhibition of proliferation, regardless of sensitivity to selumetinib. Interestingly, a significant increase in apoptosis was observed in response to the combination, which may explain the synergy observed by the combination index (CI). In vivo analyses of this combination in cell lines and human CRC explants are ongoing to further validate these results. These preclinical data may suggest a rational combination strategy for patients with KRAS mutant CRC. No significant financial relationships to disclose.

scholarly journals Brassinin Inhibits Proliferation in Human Liver Cancer Cells via Mitochondrial Dysfunction

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 332
Author(s):  
Taeyeon Hong ◽  
Jiyeon Ham ◽  
Jisoo Song ◽  
Gwonhwa Song ◽  
Whasun Lim
Keyword(s):  
Mitochondrial Dysfunction ◽  
Cancer Cells ◽  
Cell Lines ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Nuclear Antigen ◽  
Cruciferous Vegetable ◽  
Antiproliferative Effects ◽  
Hep3b Cells ◽  
Human Liver Cancer Cells

Brassinin is a phytochemical derived from Chinese cabbage, a cruciferous vegetable. Brassinin has shown anticancer effects on prostate and colon cancer cells, among others. However, its mechanisms and effects on hepatocellular carcinoma (HCC) have not been elucidated yet. Our results confirmed that brassinin exerted antiproliferative effects by reducing proliferating cell nuclear antigen (PCNA) activity, a proliferation indicator and inducing cell cycle arrest in human HCC (Huh7 and Hep3B) cells. Brassinin also increased mitochondrial Ca2+ levels and depolarized the mitochondrial membrane in both Huh7 and Hep3B cells. Moreover, brassinin generated high amounts of reactive oxygen species (ROS) in both cell lines. The ROS scavenger N-acetyl-L-cysteine (NAC) inhibited this brassinin-induced ROS production. Brassinin also regulated the AKT and mitogen-activated protein kinases (MAPK) signaling pathways in Huh7 and Hep3B cells. Furthermore, co-administering brassinin and pharmacological inhibitors for JNK, ERK1/2 and P38 decreased cell proliferation in both HCC cell lines more than the pharmacological inhibitors alone. Collectively, our results demonstrated that brassinin exerts antiproliferative effects via mitochondrial dysfunction and MAPK pathway regulation on HCC cells.

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1

Blood ◽  
2021 ◽  
Author(s):  
Roger Belizaire ◽  
Sebastian Hassan John Koochaki ◽  
Namrata D. Udeshi ◽  
Alexis Vedder ◽  
Lei Sun ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Therapeutic Potential ◽  
Mutant Cell ◽  
E3 Ubiquitin Ligase ◽  
Akt Signaling ◽  
Allelic Series ◽  
Genetic Ablation

CBL encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and non-receptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10-20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, PIK3R1 recruitment, and downstream PI3K/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine-kinase binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

Depsipeptide in the Treatment of Relapsed and Refractory Multiple Myeloma (MM): A Prospective Evaluation of the Cell Cycle.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1497-1497
Author(s):  
Zoe Goldberg* ◽  
Scott Ely ◽  
Selina Chen-Kiang ◽  
Martha Chesi ◽  
Peter L. Bergsagel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Phase Ii ◽  
Caspase 3 ◽  
Gene Array ◽  
Cyclin D3 ◽  
Proliferation Index ◽  
G1 Arrest

Abstract Background: Dysregulation of the cell cycle and apoptosis are two critical events in the pathophysiology of MM. This notion is supported by: 1)A high tumor burden is often present despite a low rate of tumor cell proliferation. 2)G1 arrest is common in MM cells while normal plasma cells are permanently withdrawn from cell cycle. 3) Cyclin D1 is often overexpressed without a defined genetic substrate. Herein, we show that cell cycle evaluation in vivo is feasible and that the histone-deacetylase inhibitor depsipeptide might be effective in selected patients with MM. Patients and Methods: In vitro studies were performed in 12 human MM cell lines with defined cytogenetic abnormalities. The IC50 for depsipeptide was determined by evaluation of apoptosis by standard methods. In vivo studies where done as correlates in a phase II protocol. These include: Immunohistochemistry (IHC) for co-expression of CD138/Ki-67 as a proliferation index (PCPI), cyclin D1, D3, caspase 3 cleavege, CD31 and bcl-2 before treatment and at 24 hrs and 30 days after treatment. Gene array studies are being performed on selected patients at those timepoints. To date, four stage III patients (PTS) with relapsed MM with four or fewer prior lines of therapy have been treated with one to three cycles of depsipeptide at a dose of 13mg/m2,as a 4-hour infusion on days 1, 8, and 15, repeated every 28 days. Mean age was 63 years (range, 56 to 72). KPS of >80%. Mean albumin was 3.5, (range, 3.2 to 4), mean LDH was 243 (range, 179 to 315). Results: 1)Depsipeptide induces apoptosis in several MM cell lines. All lines were susceptible to depsipeptide, however, differential sensitivities were noted. Three cell lines (ie U266) that contained 11q13 translocation (cyclin D1 overexpression) were the most sensitive with IC50s at least 2 fold lower than other lines. 2) Cell cycle changes are induced by depsipeptide: In 2/4 PTS, a significant increase of the PCPI was seen, whereas a marked reduction in the PCPI in a patient with cyclin D3 overexpression (27% to 16%) was also noted. One patient had an increase of cyclin D1 post treatment. No changes where seen in bcl-2, CD-31, or cleaved caspase-3 expression. 3) Depsipeptide is safe in a limited cohort of MM PTS: Grade 2 fatigue and anorexia were the most common toxicities. Mild thrombocytopenia (mean of 67) did not require transfusions. One patient had stable disease after 3 cycles of treatment, one patient had progression of disease after 3 cycles, one patient progressed after the 1st cycle, and one patient is too early for evaluation. Conclusions: 1)Patients with 11q13 translocation should be a target for treatment with depsipeptide. 2)Depsipeptide given on this schedule is safe and can stabilize tumor-mass in PTS with otherwise progressive relapsed and refractory disease.3) Evidence of cell cycle modulation can be seen during treatment with depsipeptide. No profound changes in apoptosis is evident.4)Further studies may help to understand the mechanism of transcriptional regulation by depsipeptide and will help design rational therapy and combinations. This study continues to accrue patients as part of New York Phase II Consortium. Supported by NCI grant (SAIC1N01-CO-12400-02) and a SCOR for Myeloma grant from the Leukemia and Lymphoma Society of America.

Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis and Inhibition of Proliferation of Ba/F3 FLT-3 ITD Mutant Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1589-1589
Author(s):  
Jenny E. Hernandez ◽  
Junling Li ◽  
Ru-Qi Wei ◽  
Paul Tapang ◽  
Steven K. Davidsen ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Mutant Cell ◽  
Scid Mice ◽  
Myelogenous Leukemia ◽  
Parp Cleavage ◽  
Mutant Cells

Abstract FLT3 is an receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of the differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with Acute myelogenous leukemia (AML) and confer poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations on the FLT3 kinase activation loop (e.g. D835V). Our laboratory is studying the signaling pathways associated with a newly identified multi-targeted tyrosine kinase receptor small molecule inhibitor (RTKI), ABT-869. Recently published work in our laboratory showed that using ABT-869 to treat MV4-11, a human AML FLT-3 ITD mutant cell line, resulted in the inhibition of phosphorylation of FLT-3 with a downstream inhibitory effect on the activation of STAT5, ERK, and Pim-1. Cell viability assays determined that MV-411 cells responded to ABT-869 in a concentration dependent manner (IC50 = 10nM). Apoptosis studies also showed an induction of apoptosis in ABT-869 treated cells. In vivo studies involving xenograft injections of MV-411 cells into SCID mice and subsequent treatment with ABT-869 demonstrated regression of tumor formation. In this study, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation is used as an isolated Flt-3 mutant model system. In vitro, ABT-869 is effective in inhibiting the proliferation of Ba/F3 Flt-3 ITD mutant cells when compared to Ba/F3 Flt-3 D835V mutant and Ba/F3 Flt-3 WT cells. Trypan Blue Exclusion and Alamar Blue assays were used to demonstrate that there is 50% inhibition of growth and proliferation (IC50) of Ba/F3 FLT3 ITD mutant cells at a concentration of 1nM after 48 hours of treatment. Ba/F3 FLT3 D835V mutant cells show an IC50 between 1μM and 10μM after 48 hours of treatment. In contrast, Ba/F3 FLT3 WT cells demonstrate an IC50 of 10μM only after 72 hours of treatment. Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis (41.2%) occurred in Ba/F3 Flt-3 ITD mutant cells treated with 10nM ABT-869 after 24 hours when compared to untreated (6.5%) or vehicle control (6.1%) cells. Staining of Ba/F3 Flt-3 WT treated cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cell only and DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells following treatment with ABT-869 whereas no cleavage was observed with Ba/F3 WT cells treated with ABT-869. In vivo, the activity of ABT-869 treatment of SCID mice injected with Baf3 Flt-3 ITD, Baf3 Flt-3 D835V, or Baf3 Flt-3 WT cells is also being evaluated. Using bioluminescence imaging, it was determined that Ba/F3 FLT-3 ITD mutant and Ba/F3 Flt-3 D835Vmutant cell lines result in metastases and subsequent death in SCID mice after 2 weeks for ITD and 5 weeks for D835V, whereas mice injected with Ba/F3 WT survive longer than 5 weeks. Preliminary data demonstrated that ABT-869 prolonged survival in mice injected with the Ba/F3 FLT3-ITD cells compared to controls. Our preclinical data demonstrate that ABT-869 is effective specifically with FLT-3 ITD mutant cell lines in an isolated system. These studies provide rationale for the treatment of AML patients and the prevention of relapse.

Anti-CD138-IFNα Fusion Proteins Are Effective in Treating Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 939-939
Author(s):  
Esther Yoo ◽  
Alex Vasuthasawat ◽  
Danh Tran ◽  
Alan Lichtenstein ◽  
Sherie Morrison
Keyword(s):  
Multiple Myeloma ◽  
Fusion Protein ◽  
Cell Lines ◽  
Dna Content ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Inhibition Of Proliferation ◽  
Discontinue Therapy

Abstract Abstract 939 Although IFNα has shown some efficacy in the treatment of multiple myeloma (MM), this efficacy has been limited in large part because systemic toxicity makes it difficult if not impossible to reach therapeutically effective doses at the site of the tumor. The short half-life of IFN also makes it difficult to sustain high levels during treatment, and because of the side effects, the patients often discontinue therapy. To address these issues, we have genetically fused IFNα2 to a chimeric IgG1 antibody specific for the antigen CD138 expressed on the surface of MM cells, yielding anti-CD138-IFNα. We have also produced a fusion protein (anti-CD138-mutIFNα) using a mutant IFNα that binds the IFN receptor (IFNAR) more tightly. The fusion proteins continued to bind CD138 and retained IFN activity and showed anti-proliferative activity against a broad panel of myeloma cell lines (HMCL) representing MM with different characteristic. To investigate the events responsible for the inhibition of proliferation, 8226/S, ANBL-6, MM1-144, H929, OCI-My5 and U266 cells were incubated with 500 pM anti-CD138-IFNα for 72 h and their DNA content analyzed by FLOW cytometry following permeabilization and staining with PI. The different cell lines exhibited different responses. All of the cell lines except OCI-My5 underwent apoptosis. For 8226/S, OCI-My5 and U266 there was little change in DNA content following treatment. ANBL-6 showed a slight increase in the number of cells in S. However, MM1-144 and H929 showed a marked accumulation in G2 with H929 also showing accumulation of cells with sub-G0content of DNA. Therefore, there is heterogeneity in the response of different HMCL to treatment with targeted IFNα2. For many but not all of the cell lines, anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inhibiting proliferation and causing DNA fragmentation. Anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inducing senescence-associated β-galactosidase and STAT1 activation in OCI-My5 cells. Treatment with anti-CD138-IFNα or anti-CD138-mutIFNα resulted in a decrease in the amount of IRF4 present in U266, suggesting that this may be responsible for the efficacy of the fusion proteins in this cell line. Treatment of the other cell lines did not alter the level of IRF4 present, but anti-CD138-IFNα and anti-CD138-mutIFNα treatment caused a decrease in the amount of ppRB present in 8226/S, OCI-My5 and MM1-144, and to a lesser extent in H929. To determine the in vivo efficacy of fusion protein treatment, SCID mice were injected subcutaneously with OCI-My5 cells and treated intravenously on days 14, 16 and 18 with 100 μg of the indicated proteins and monitored for tumor growth (Figure 1). Mice were sacrificed when tumors exceeded 1.5 cm in diameter. Treatment with anti-CD138-IFNα provided some protection (p ≤ 0.0001 compared to PBS). However, treatment with anti-CD138-mutIFNα was even more effective (p = 0.0004 compared to anti-CD138-IFNα). Anti-CD138-mutIFNα was also found to be more effective than anti-CD138-IFNα against primary MM cells. Patients with active myeloma were biopsied while off therapy and the marrow cells isolated by a negative antibody selection to >95% purity. After 72 h incubation with 25 nM of protein, anti-CD138 was found to have little effect. In contrast treatment with anti-CD138-IFNα caused a decrease in viability with anti-CD138-mutIFNα treatment leading to an even greater decrease in cell viability. Following 72 h of treatment, 25 nM of anti-CD138-mutIFNα was found to have more potent cytoreductive effects than 100 nM of anti-CD138-IFNα. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Anti-Tumor Activity of ASTX029, a Dual Mechanism Inhibitor of ERK1/2, in Preclinical AML Models

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-8
Author(s):  
Christopher J. Hindley ◽  
Lynsey Fazal ◽  
Joanne M. Munck ◽  
Vanessa Martins ◽  
Alpesh D. Shah ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Activating Mutations ◽  
Ic50 Value ◽  
Ras Family ◽  
Dual Mechanism ◽  
Anti Tumor Activity

Oncogenic mutations in genes such as the RAS family (KRAS, NRAS or HRAS) or receptor tyrosine kinases (RTKs) drive tumor growth through aberrant activation of the mitogen activated protein kinase (MAPK) signaling pathway. Acute myeloid leukemia (AML) patients frequently exhibit activating mutations in MAPK pathway members, such as NRAS and KRAS, suggesting that these malignancies may be driven by aberrant activation of the MAPK pathway. Targeting of the MAPK pathway has been clinically validated in solid tumors, with agents targeting BRAF and MEK approved for the treatment of BRAF-mutant melanoma. However, there is currently no approved therapy directly targeting activated RAS family members and resistance to MAPK pathway inhibitors is frequently associated with reactivation of MAPK signaling. ERK1/2 (ERK) is a downstream node in the MAPK pathway and therefore represents an attractive therapeutic target for inhibition of MAPK signaling in these settings. We have recently described in vivo anti-tumor activity in MAPK-activated solid tumor models following treatment with ASTX029, a highly potent ERK inhibitor developed using fragment-based drug design. ASTX029 has a distinctive ERK binding mode which confers dual mechanism inhibition of ERK, inhibiting both the catalytic activity of ERK and its phosphorylation by MEK. Here, we demonstrate that ASTX029 is also active in AML models and potently inhibits in vitro and in vivo MAPK signaling and growth in these models. Using a panel of 15 AML cell lines, we investigated sensitivity to ASTX029 in vitro. We observed that 8 cell lines bearing mutations leading to increased MAPK pathway signaling were sensitive to treatment with ASTX029 with an average IC50 value of 47 nM, in contrast to an average IC50 value of 1800 nM for cell lines without activating mutations. The phosphorylation of RSK, a direct substrate of ERK, was suppressed for up to 24 h following treatment with ASTX029 in vitro. We have previously demonstrated good oral bioavailability for ASTX029 and once daily dosing resulted in significant tumor growth inhibition in AML cell line xenograft models. To confirm target engagement in vivo, we examined MAPK signaling in xenograft tissue and observed inhibition of the phosphorylation of RSK and of ERK itself, consistent with the dual mechanism of action proposed for ASTX029. In summary, the ERK inhibitor, ASTX029, has potent activity against MAPK-activated tumor models, including AML models, and is now being tested in a Phase 1/2 clinical trial in advanced solid tumors (NCT03520075). These data highlight its therapeutic potential for the treatment of AML in patients with mutations leading to MAPK pathway activation and support further investigation in these patient populations. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Fazal:Astex Pharmaceuticals: Current Employment. Munck:Astex Pharmaceuticals: Current Employment. Martins:Astex Pharmaceuticals: Current Employment. Shah:Astex Pharmaceuticals: Current Employment. Wilsher:Astex Pharmaceuticals: Current Employment. Wallis:Astex Pharmaceuticals: Current Employment. Keer:Astex Pharmaceuticals, Inc.: Current Employment. Lyons:Astex Pharmaceuticals: Current Employment.

scholarly journals Aspirin-Based Organoiron Dendrimers as Promising Anti-Inflammatory, Anticancer, and Antimicrobial Drugs

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1568
Author(s):  
Alaa S. Abd-El-Aziz ◽  
Maysun R. Benaaisha ◽  
Amani A. Abdelghani ◽  
Rabin Bissessur ◽  
Laila H. Abdel-Rahman ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Anticancer Activity ◽  
Cell Lines ◽  
Cancer Cell ◽  
Inflammatory Activity ◽  
Breast Cancer Cell Lines ◽  
Cell Viability Assay ◽  
Reference Drug ◽  
Anti Inflammatory

Designing nanocarriers with actions directed at a specific organ or tissue is a very promising strategy since it can significantly reduce the toxicity of a bioactive drug. In this study, an organometallic dendrimer was used to synthesize a biocompatible drug delivery system by attaching aspirin to the periphery of the dendrimer. Our goal is to enhance the bioavailability and anticancer activity of aspirin and reduce its toxicity through successive generations of organoiron dendrimers. The biological activity of aspirin-based dendrimer complexes was evaluated. The result of antimicrobial activity of the synthesized dendrimers also demonstrated an increase in their antimicrobial activity with increased generation of the dendrimers for most types of microorganisms. This study reveals for the first time that organoiron dendrimers linked with aspirin exhibit an excellent Gram-negative activity comparable to the reference drug Gentamicin. All synthesized dendrimers were tested for their anticancer activity against breast cancer cell lines (MCF-7), hepatocellular cell lines (Hep-G2), and a non-cancer cell line, Human Embryonic Kidney (HEK293), using the MTT cell viability assay and compared against a standard anticancer drug, Doxorubicin. Compounds G3-D9-Asp and G4-D12-Asp exhibited noticeable activity against both cell lines, both of which were more effective than aspirin itself. In addition, the in vivo anti-inflammatory activity and histopathology of swollen paws showed that the designed aspirin-based dendrimers displayed significant anti-inflammatory activity; however, G2-D6-Asp showed the best anti-inflammatory activity, which was more potent than the reference drug aspirin during the same period. Moreover, the coupling of aspirin to the periphery of organoiron dendrimers showed a significant reduction in the toxicity of aspirin on the stomach.

scholarly journals Synthetic lethal screening identifies existing drugs with selective viability effects on Neurofibromatosis type-1 model systems

2021 ◽  
Author(s):  
Yuanli Wang ◽  
Megan Stevens ◽  
Torrey R Mandigo ◽  
Stephanie J Bouley ◽  
Aditi Sharma ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Neurofibromatosis Type 1 ◽  
Mutant Cell ◽  
Neurofibromatosis Type ◽  
Model Systems ◽  
Synthetic Lethal ◽  
Mutant Cells

Neurofibromatosis type 1 (NF1) is a genetic multi-system disorder. Symptoms include near universal benign neurofibromas, as well as malignant tumours, including generally fatal malignant peripheral nerve sheath tumours. There are limited therapies for any NF1-associated tumours; therefore, there is a clear clinical need to discover new drugs that specifically target NF1-deficient tumour cells. Using a Drosophila NF1-KO cell model, we used synthetic lethal screening to identify candidate drug targets for NF1-deficient tumours and performed statistical enrichment analysis to identify further targets. We then assessed the top 72 candidate synthetic lethal partner genes to NF1 using Variable Dose Analysis, resulting in 15 candidate genes that decreased NF1-KO viability by >10% and were novel druggable targets for NF1. Autophagy inhibitors Chloroquine (CQ) and Bafilomycin A1 resulted in a significant reduction in NF1-KO cell viability, which was conserved in a panel of human NF1 mutant cell lines. AZT and Enzalutamide also selectively reduced NF1 mutant cell viability in human cell lines. Furthermore, the effect of CQ was conserved in a Drosophila NF1-mutant in vivo model. This study highlights two key points: 1) The use of Drosophila cells as a model to screen for drugs specifically targeting NF1 mutant cells was highly successful as candidate interactions were conserved across a panel of human NF1 mutant cells and an in vivo fly NF1 mutant model, and 2) NF1-deficient cells have vulnerability to disruption of the autophagy pathway, telomerase activity, and AR activity. These pathways/drugs represent promising targets for the potential treatment of NF1-associated tumours.

Genetic alterations in HRAS gene in relation to outcome and response to cetuximab in head and neck squamous cell carcinoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 5574-5574
Author(s):  
Theodoros Rampias ◽  
Athina Giagini ◽  
Hiroumi Matsuzaki ◽  
Valentina Bartzi ◽  
Spyros Siolos ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mapk Pathway ◽  
Small Sample Size ◽  
Mutant Cell ◽  
Disease Free Survival ◽  
Genetic Alterations ◽  
Resistant Cell ◽  
Small Sample ◽  
The Impact ◽  
Hras Gene

5574 Background: Aberrant signaling through RAS/MAPK pathway is implicated in resistance to EGFR-targeted agents in cancer. Genetic alterations in Hras gene such as mutations and specific polymorphisms are associated with aggressive phenotype in several smoking-related malignancies. We sought to determine the impact of Hras genetic alterations on response to cetuximab and prognosis in HNSCC. Methods: Clinical outcome according to Hras status was investigated in a retrospective cohort of 140 HNSCC specimens. Primary endpoints were overall survival (OS) and disease-free survival (DFS) and secondary endpoint was treatment response. For statistical analysis, T-test was used for continuous data and x2 –test for categorical data. Cetuximab-resistant cell lines harboring mutant Hras (BB49, T24) were infected with lentivirus expressing shRNA targeting the Hras or a scrambled- shRNA. MTT assay was used to determine the effect of cetuximab on growth of lentivirus infected cells. Biochemical analysis involved immunoblotting for pERK1/2. Results: Mutationanalysis of tumor samples showed that 5.7% participants harbored Hras mutations and 16.42% harbored Hras polymorphisms (rs12628, rs41258054) that are associated with tumorigenesis. Patients bearing tumors with mutated Hras had inferior mean OS ( 22.13vs 35.20, p=0.02) and a non-significant trend for inferior mean DFS. Patients with tumors containing Hras genetic alterations (mutation or polymorphism) had significantly inferior mean OS (p=0.02) compared to those harboring wt Hras and trended towards inferior DFS (p=0.07). Patients had received various treatments such as surgery plus/minus RT and various chemotherapy regimens. A subgroup analysis of 38 patients treated with cetuximab-based regimens showed that wt Hras was associated with higher likelihood of attaining CR or PR to treatment of borderline significance (p=0.06) due to small sample size. Silencing of Hras in Hras-mutant cell lines restored sensitivity to cetuximab and caused a direct downregulation of pERK1/2 levels. Conclusions: Hras genetic alterations are associated with aggressive clinical course and may affect response to cetuximab in HNSCC.

LMTK3 as a novel regulator of oncogenic KIT in KIT-mutant cancers.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 11044-11044
Author(s):  
Lillian Rose Klug ◽  
Jeffrey Tyner ◽  
Michael C. Heinrich
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Mutant Cell ◽  
Xenograft Model ◽  
Melanoma Cells ◽  
Resistance Mutations ◽  
Human Kinase ◽  
Candidate Target

11044 Background: Multiple cancers, such as gastrointestinal stromal tumors (GIST) and melanoma, have been shown to be caused by somatic activating mutations in the receptor tyrosine kinase KIT. The major cause of death in patients with advanced KIT -mutant cancers is due to the development of KIT tyrosine kinase inhibitor-resistant (TKI-resistant) metastatic disease. Drug resistance arises almost exclusively from secondary mutations within KIT, highlighting the importance of KIT in the proliferation and survival of these tumors. Methods: We performed a human kinase siRNA screen in multiple KIT -mutant cancer cell lines using viability as a read out. We defined candidate targets as those whose knockdown decreased viability in all cell lines. Validation and mechanistic studies were done using a library of KIT-mutant GIST and melanoma cells. Results: We identified lemur tyrosine kinase 3 (LMTK3) as candidate target in three KIT -mutant cell lines. LMTK3 silencing reduced the viability of all KIT -mutant GIST and melanoma cells tested to date, including cell lines with KIT TKI-resistance mutations. Importantly, LMTK3 silencing decreased the viability of KIT -mutant cells specifically, but not that of KIT-independent GIST and melanoma cells. Further, we found that decreased cell viability was due to induction of apoptosis, as assessed by measuring caspase 3 and 7 activity within 96 hours of LMTK3 silencing. LMTK3 knockdown also reduced tumor growth in vivo in a GIST xenograft model. Because these cells depend so heavily on KIT and the loss of KIT signaling results in cell death, we hypothesized that LMTK3 silencing may affect this pathway. Indeed, LMTK3 silencing decreased levels of autophosphorylated KIT. We also observed a significant decrease in total KIT protein expression. This phenotype and corresponding viability was rescued with exogenous expression of full length LMTK3. Conclusions: LMTK3 is an important regulator of oncogenic KIT expression and activity in KIT-mutant GIST and melanoma and represents a novel, tractable target.

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