Project description:Induced pluripotent stem cells were gene edited to introduce heterozygous (promoter deletion, start site deletion, and exon 27 frameshift) and homozygous (exon 27 frameshift) mutations. iPSCs were differentiated into cardiomyocytes for transcriptome analysis.
Project description:This study employs a 2-stage design that aims to evaluate the efficacy and safety of ENV- 101, a potent Hedgehog (Hh) pathway inhibitor, in patients with refractory advanced solid tumors characterized by loss of function (LOF) mutations in the Patched-1 (PTCH1) gene. Stage 1 of this study will enroll approximately 44 patients randomized between two dose levels. As appropriate, Stage 2 of the study will expand enrollment based on the results of Stage 1.
Project description:The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. Whilst the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Using knock-in (KI) mice harbouring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. The dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN-loss driven cancers.
Project description:The goal of the study was to evaluate the influence of mutations in MLK4 on the protein function and the process of tumorigenesis in colorectal cancers. Biochemical data imply that a majority of MLK4 mutations in colon cancer are loss-of-function, including, E314K and Y330H mutations. Two colorectal cell lines, HCT15 and HT115, harboring MLK4 loss-of-function mutations, Y330H and E314K respectively, were used in the study. To evaluate the consequences of loss-of-function MLK4 mutations in those colon cancer cell lines, HCT15 and HT115 cell lines were generated where expression of MLK4-WT (in HCT15 and HT115 cell lines) or MLK4-Y330H (in HCT15 cells) or MLK4-E314K (in HT115 cells) can be induced by tetracycline. The reconstitution of MLK4-WT in colon cancer cells reduced viability, proliferation and ability to form colonies as well as significantly slowed down the tumor growth in mouse xenografts. To investigate the downstream effectors of MLK4 that mediate the above effects, we transcriptionally profiled colon cancer cell lines after MLK4-WT reconstitution. The data indicates that re-expression of functional MLK4 leads to reduction in cell proliferation, likely due to increased expression of cJUN and cdk inhibitors (p15 or p21). HCT15 and HT115 were treated with tetracycline for 24 hours to induce expression of MLK4. Cells were harvested and mRNA was isolated using Qiagen RNeasy Plus Mini Kit. Samples were hybridized to Affymetrix Human U133 Plus 2.0 array. Experiments were performed in triplicates for every sample. Induction of Y330H in HCT15 cells and MLK4-E314K in HT115 cells was used as a control.
Project description:Purpose: Recently discovered activating Interleukin-6 receptor subunit beta (IL6ST, encoding glycoprotein 130 (gp130)) mutations, as well as germline Signal transducer and activator of transcription 3 (STAT3) gain-of-function mutations are associated with multi-organ autoimmunity, severe morbidity, and adverse prognosis, resulting in an unmet medical need. Methods: To decipher the crucial cellular subsets and disease biology associated with STAT3 gain-of-function mutations, we examined the gene expression profile of STAT3 gain-of-function and loss-of-function mutations in unstimulated, IL10- or IL21-stimulated CD4 T cells and compared them to healthy donor cells. Results: Activating gp130 signaling in vivo resulted in fatal early-onset multi-organ autoimmunity, resembling numerous clinical features of human STAT3 gain-of-function disease. We observed strong T-cell activation and effector differentiation, accompanied by TH17 expansion and interferon-gamma production. Transcriptome profiling of murine CD4+ and CD8+ T-cells revealed commonly dysregulated genes and a STAT3 gain-of-function signature that was used to discriminate between STAT3 gain-of-function and healthy control patients. Conclusions: Hyperactive gp130/STAT3 signaling leads to strong TH17-mediated autoimmunity phenotypically resembling human STAT3 gain-of-function disease and identify TH17-cells as a key cellular subset for initiation and maintenance of autoimmunity
Project description:The goal of the study was to evaluate the influence of mutations in MLK4 on the protein function and the process of tumorigenesis in colorectal cancers. Biochemical data imply that a majority of MLK4 mutations in colon cancer are loss-of-function, including, E314K and Y330H mutations. Two colorectal cell lines, HCT15 and HT115, harboring MLK4 loss-of-function mutations, Y330H and E314K respectively, were used in the study. To evaluate the consequences of loss-of-function MLK4 mutations in those colon cancer cell lines, HCT15 and HT115 cell lines were generated where expression of MLK4-WT (in HCT15 and HT115 cell lines) or MLK4-Y330H (in HCT15 cells) or MLK4-E314K (in HT115 cells) can be induced by tetracycline. The reconstitution of MLK4-WT in colon cancer cells reduced viability, proliferation and ability to form colonies as well as significantly slowed down the tumor growth in mouse xenografts. To investigate the downstream effectors of MLK4 that mediate the above effects, we transcriptionally profiled colon cancer cell lines after MLK4-WT reconstitution. The data indicates that re-expression of functional MLK4 leads to reduction in cell proliferation, likely due to increased expression of cJUN and cdk inhibitors (p15 or p21).
Project description:Choroideremia (CHM) is a progressive X-linked retinopathy caused by mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1), an escort protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs, as a result of loss of function mutations in REP-1, could affect vesicular trafficking, exocytosis and secretion. To evaluate this hypothesis, intracellular vesicle transport, lysosomal acidification and rates of proteolytic degradation were studied in monocytes (CD14+ fraction) and primary skin fibroblasts from the nine age-matched controls and thirteen CHM patients carrying 10 different loss-of-function mutations.
Project description:Analysis of genes regulated by canonical Wnt signaling in the murine primary Schwann cells. Total RNA from b-catenin fl/fl Schwann cells, after introducing loss-of-function mutations with HTN-cre, or mimicking gain-of-function mutations with Chir98014 or Wnt3a-treatments, was compared to the respective controls.