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.

scholarly journals Selumetinib normalizes Ras/MAPK signaling in clinically relevant Neurofibromatosis type 1 minipig tissues in vivo

2021 ◽  
Author(s):  
Sara H Osum ◽  
Alexander W Coutts ◽  
Dylan J Duerre ◽  
Barbara R Tschida ◽  
Mark N Kirstein ◽  
...  
Keyword(s):  
Neurofibromatosis Type 1 ◽  
Neurofibromatosis Type ◽  
Mapk Signaling

Abstract A version of this article was published in error on 9 February 2021 and has been temporarily removed. The Publisher apologizes for any inconvenience.

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.

Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis and Suppresses Proliferation of Ba/F3 FLT-3 ITD Mutant Cells in Vitro and in Vivo through Inhibition of FLT3 and AKT.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1938-1938
Author(s):  
Jenny E Hernandez ◽  
Joan Zape ◽  
Keith Glaser ◽  
Elliot Landaw ◽  
Cecilia Fu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Mutant Cell ◽  
Vehicle Control ◽  
Ic50 Value ◽  
Mutant Cells

Abstract FLT3 is a receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with acute myelogenous leukemia (AML) and confer a poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations in the FLT3 kinase activation loop, e.g. D835V. We are studying the signaling pathways associated with a small molecule multi-targeted receptor tyrosine kinase inhibitor (RTKI), ABT-869. To determine the effects of ABT-869 in vitro and in vivo, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation was 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 (IC50 value of 1 nM) when compared to Ba/F3 Flt-3 D835V mutant (IC50 value between 1 and 10 μM) and Ba/F3 Flt-3 wildtype (WT) cells (IC50 value of 10 μM). Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis occurred in Ba/F3 Flt-3 ITD mutant cells treated with 1μM ABT-869 for 24 hours (42.8%) when compared to untreated (4.7%) or vehicle control (4.0%) cells. Ba/F3 Flt-3 D835V mutant cell lines demonstrated a 12.5% rate of apoptosis at 1μM, compared to untreated (1.99%) and vehicle control (2.1%) cell lines. Propidium iodide staining of treated Ba/F3 Flt-3 WT cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cells or DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells, following 6 hours of treatment with 1 to 100 nM ABT-869, whereas no cleavage was observed in Ba/F3 WT cells treated with ABT-869. To study the effects of ABT-869 in vivo, we treated SCID mice injected with Ba/F3 Flt-3 ITD, Ba/F3 Flt-3 D835V, or Ba/F3 Flt-3 WT cells and monitored disease progression using bioluminescence imaging. The mice injected with the Ba/F3 FLT-3 ITD mutant cells and treated with vehicle control developed metastases and had a median survival time of 2 weeks. In contrast, the ABT-869 treated group had slower disease progression with median survival of 6.2 weeks (P<0.008). Both control and treated mice injected with Ba/F3 FLT-3 D835V mutant cell lines developed metastases and had similar survival (median 1.7 and 1.9 weeks, respectively). Survival times of control and treated mice injected with Ba/F3 FLT-3 WT cells were also similar (median 8.4 and 8.1 weeks, respectively). Previous work identified that ABT-869 induced apoptosis of acute myeloid leukemia cells through inhibition of FLT-3 reception phosphorylation, which is observed as early as 3 hours after treatment. In Ba/F3 cells expressing FLT-3 ITD, ABT-869 also inhibited phosphorylation of AKT, which is upstream of the pro-apoptotic protein Bad. Therefore, our preclinical data suggest that ABT-869 induces apoptosis of FLT-3 ITD mutant cells both in vitro and in vivo. These studies provide rationale for the treatment of acute leukemia patients harboring the FLT3-ITD mutation with ABT-869 and the potential benefit of combining small molecule inhibitors that target both RTKs and AKT.

scholarly journals TATA-Binding Protein (TBP)-Like Factor (TLF) Is a Functional Regulator of Transcription: Reciprocal Regulation of the Neurofibromatosis Type 1 and c-fos Genes by TLF/TRF2 and TBP

2005 ◽  
Vol 25 (7) ◽  
pp. 2632-2643 ◽  
Author(s):  
Jayhong A. Chong ◽  
Magdalene M. Moran ◽  
Martin Teichmann ◽  
J. Stefan Kaczmarek ◽  
Robert Roeder ◽  
...  
Keyword(s):  
Neurofibromatosis Type 1 ◽  
Binding Protein ◽  
Neurofibromatosis Type ◽  
Tata Binding Protein ◽  
Related Factor ◽  
Reciprocal Regulation ◽  
In Cells

ABSTRACT The lack of direct targets for TATA-binding protein (TBP)-like factors (TLFs) confounds the understanding of their role in gene expression. Here we report that human TLF (also called TBP-related factor 2 [TRF2]) activates a number of different genes, including the neurofibromatosis type 1 (NF1) gene. The overexpression of TLF increases the amount of NF1 mRNA in cells. In vivo, TLF binds to and upregulates transcription from a fragment of the NF1 promoter. In vitro, purified TLF-TFIIA binds directly to the same NF1 promoter fragment that is required for TLF responsiveness in cells. Furthermore, targeted deletion of TLF in mice reduces NF1 levels. In contrast, TLF inhibits transcription driven by a fragment from the TATA-containing c-fos promoter by sequestering TFIIA. TBP affects the NF1 and c-fos promoters in a manner reciprocal to that of TLF, stimulating the c-fos promoter and inhibiting NF1 transcription. We conclude that TLF is a functional regulator of transcription with targets distinct from those of TBP.

scholarly journals Genetic disruption of the small GTPase RAC1 prevents plexiform neurofibroma formation in mice with neurofibromatosis type 1

2020 ◽  
Vol 295 (29) ◽  
pp. 9948-9958
Author(s):  
Julie A. Mund ◽  
SuJung Park ◽  
Abbi E. Smith ◽  
Yongzheng He ◽  
Li Jiang ◽  
...  
Keyword(s):  
Schwann Cells ◽  
Neurofibromatosis Type 1 ◽  
Plexiform Neurofibroma ◽  
Neurofibromatosis Type ◽  
Tumor Formation ◽  
Genetically Engineered ◽  
Small Gtpase ◽  
Cancer Predisposition Syndrome

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome caused by mutations in the NF1 tumor suppressor gene. NF1 encodes neurofibromin, a GTPase-activating protein for RAS proto-oncogene GTPase (RAS). Plexiform neurofibromas are a hallmark of NF1 and result from loss of heterozygosity of NF1 in Schwann cells, leading to constitutively activated p21RAS. Given the inability to target p21RAS directly, here we performed an shRNA library screen of all human kinases and Rho-GTPases in a patient-derived NF1−/− Schwann cell line to identify novel therapeutic targets to disrupt PN formation and progression. Rho family members, including Rac family small GTPase 1 (RAC1), were identified as candidates. Corroborating these findings, we observed that shRNA-mediated knockdown of RAC1 reduces cell proliferation and phosphorylation of extracellular signal–regulated kinase (ERK) in NF1−/− Schwann cells. Genetically engineered Nf1flox/flox;PostnCre+ mice, which develop multiple PNs, also exhibited increased RAC1-GTP and phospho-ERK levels compared with Nf1flox/flox;PostnCre– littermates. Notably, mice in which both Nf1 and Rac1 loci were disrupted (Nf1flox/floxRac1flox/flox;PostnCre+) were completely free of tumors and had normal phospho-ERK activity compared with Nf1flox/flox;PostnCre+ mice. We conclude that the RAC1-GTPase is a key downstream node of RAS and that genetic disruption of the Rac1 allele completely prevents PN tumor formation in vivo in mice.

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