Papers by Michael Chastkofsky
Journal of Investigative Dermatology, 2015
Protein Tyrosine Kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed i... more Protein Tyrosine Kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the epithelial linings of the gastrointestinal tract and skin, where it is expressed in nondividing differentiated cells. We found PTK6 expression increases in the epidermis following UVB treatment. To evaluate the roles of PTK6 in the skin following UVB-induced damage, we exposed back skin of Ptk6 +/+ and Ptk6−/− SENCAR mice to incremental doses of UVB for thirty weeks. Wild type mice were more sensitive to UVB and exhibited increased inflammation and greater activation of STAT3 than Ptk6−/− mice. Disruption of Ptk6 did not have an impact on proliferation, although PTK6 was expressed and activated in basal epithelial cells in wild type mice following UVB treatment. However, wild type mice exhibited shortened tumor latency and increased tumor load compared with Ptk6−/− mice, and STAT3 activation was increased in these tumors. PTK6 activation was detected in UVB-induced tumors, and this correlated with increased activating phosphorylation of FAK and BCAR1. Activation of PTK6 was also detected in human squamous cell carcinomas of the skin. Although PTK6 plays roles in normal differentiation, it also contributes to UVB induced injury and tumorigenesis in vivo.
Glioblastoma (GBM) is the most lethal primary brain tumor in adults. There is no treatment that p... more Glioblastoma (GBM) is the most lethal primary brain tumor in adults. There is no treatment that provides durable relief for the vast majority of GBM patients. In this study, we’ve tested a bispecific antibody comprised of single-chain variable regions (scFvs) against T cell CD3ε and GBM cell interleukin 13 receptor alpha 2 (IL13Rα2). We demonstrate that this BiTE (BiTELLON) engages peripheral and tumor-infiltrating lymphocytes harvested from patient’s tumors, and in so doing exerts anti-GBM activity ex vivo. The interaction of BiTELLON with T cells and engagement of IL13Rα2-expressing GBM cells stimulates T cell proliferation as well as production of pro-inflammatory cytokines INFγ and TNFα. We have modified neural stem cells (NSCs) to produce and secrete the BiTE (NSCsLLON). When injected intracranially in mice with brain tumor, NSCsLLON show tropism for tumor, secrete BiTELLON, and remain viable for several days. When injected directly into tumor, NSCLLON provide significant survi...
Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiati... more Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiation therapy is the standard of care treatment for DIPG, but offers only transient relief of symptoms for DIPG patients without providing significant survival benefit. Oncolytic virotherapy (OV) is an anticancer treatment that has been investigated for treating various types of brain tumors. Here, we have explored the use of mesenchymal stem cells (MSC) for OV delivery and evaluated treatment efficacy using preclinical models of DIPG. Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins that are important for OV entry, and that MSCs loaded with OV disseminate within and release OV throughout the tumor in mice bearing DIPG brainstem xenografts. When combining administration of OV-loaded MSCs with radiotherapy, mice bearing brainstem DIPG xenografts experience a significant survival benefit, relative to that conferred by either therapy alone (p<0....
Neuro-Oncology
Maternal embryonic leucine zipper kinase (MELK) is the second highest expressed kinase in diffuse... more Maternal embryonic leucine zipper kinase (MELK) is the second highest expressed kinase in diffuse intrinsic pontine glioma (DIPG). Inhibition of MELK by the small molecule OTSSP167 effectively reduces proliferation and induces cell death in primary DIPG cell lines at low nanomolar concentrations. RNA sequencing of DIPG cells treated with OTSSP167 reveals upregulation of genes associated with the PPAR transcription factors as well as upregulation of PPAR target genes. Western blot and immunofluorescence demonstrates reduced inhibitory phosphorylation and increased nuclear localization of PPARγ upon MELK inhibition. Combined treatment of DIPG cells with OTSSP167 and PPARγ agonists synergistically reduced cell survival. Given the importance of the blood-brain barrier in DIPG, brain penetration of OTSSP167 was investigated
Molecular cancer research : MCR, Jan 16, 2016
Disruption of the gene encoding Protein Tyrosine Kinase 6 (Ptk6) delayed differentiation and incr... more Disruption of the gene encoding Protein Tyrosine Kinase 6 (Ptk6) delayed differentiation and increased growth in the mouse intestine. However, Ptk6 null mice were also resistant to azoxymethane-induced colon tumorigenesis. To further explore functions of PTK6 in colon cancer, expression of epithelial and mesenchymal markers, as well as proliferation, migration, and xenograft tumor growth were examined in human colon tumor cell lines with knockdown or overexpression of PTK6. PTK6 protein, transcript, and activation were also examined in a human colon tumor tissue array, using immunohistochemistry (IHC) and qRT-PCR. Knockdown of PTK6 led to the epithelial-mesenchymal transition (EMT) in SW480 and HCT116 cells, while overexpression of PTK6 in SW620 cells restored an epithelial phenotype in a kinase-independent manner. PTK6 knockdown also increased xenograft tumor growth of SW480 cells, suggesting tumor suppressor functions. In clinical specimens, PTK6 expression was highest in normal d...
Molecular Cancer Research, 2016
Disruption of the gene encoding Protein Tyrosine Kinase 6 (Ptk6) delayed differentiation and incr... more Disruption of the gene encoding Protein Tyrosine Kinase 6 (Ptk6) delayed differentiation and increased growth in the mouse intestine. However, Ptk6-null mice were also resistant to azoxymethane-induced colon tumorigenesis. To further explore functions of PTK6 in colon cancer, expression of epithelial and mesenchymal markers, as well as proliferation, migration, and xenograft tumor growth, was examined in human colon tumor cell lines with knockdown or overexpression of PTK6. PTK6 protein, transcript, and activation were also examined in a human colon tumor tissue array, using immunohistochemistry and qRT-PCR. Knockdown of PTK6 led to the epithelial-mesenchymal transition (EMT) in SW480 and HCT116 cells, whereas overexpression of PTK6 in SW620 cells restored an epithelial phenotype in a kinase-independent manner. PTK6 knockdown also increased xenograft tumor growth of SW480 cells, suggesting tumor suppressor functions. In clinical specimens, PTK6 expression was highest in normal differentiated epithelial cells and reduced in tumors. In contrast, overexpression of constitutively active PTK6 promoted STAT3 and ERK5 activation in colon cancer cells, and endogenous PTK6 promoted cell survival and oncogenic signaling in response to DNA-damaging treatments. These data indicate that PTK6 has complex, context-specific functions in colon cancer; PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner, whereas activation of PTK6 promotes oncogenic signaling. Implications: Understanding context-specific functions of PTK6 is important, because although it promotes cell survival and oncogenic signaling after DNA damage, expression of PTK6 in established tumors may maintain the epithelial phenotype, preventing tumor progression. Mol Cancer Res; 14(6); 563-73. Ó2016 AACR.
Genes & Development, 2005
In eukaryotes and archaea, uridines in various RNAs are converted to pseudouridines by RNA-guided... more In eukaryotes and archaea, uridines in various RNAs are converted to pseudouridines by RNA-guided RNA modification complexes termed H/ACA RNPs. Guide RNAs within the complexes base-pair with target RNAs to direct modification of specific ribonucleotides. Cbf5, a protein component of the complex, likely catalyzes the modification. However, little is known about the organization of H/ACA RNPs and the roles of the multiple proteins thought to comprise the complexes. We have reconstituted functional archaeal H/ACA RNPs from recombinant components, defined the components necessary and sufficient for function, and determined the direct RNA-protein and protein-protein interactions that occur between the components. The results provide substantial insight into the functional organization of this RNP. The functional complex requires a guide RNA and each of four proteins: Cbf5, Gar1, L7Ae, and Nop10. Two proteins interact directly with the guide RNA: L7Ae and Cbf5. L7Ae does not interact with other H/ACA RNP proteins in the absence of the RNA. We have defined two novel functions for Cbf5. Cbf5 is the protein that specifically recognizes and binds H/ACA guide RNAs. In addition, Cbf5 recruits the two other essential proteins, Gar1 and Nop10, to the pseudouridylation guide complex.
Journal of Investigative Dermatology, 2015
Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed i... more Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the epithelial linings of the gastrointestinal tract and the skin, where it is expressed in nondividing differentiated cells. We found that PTK6 expression increases in the epidermis following UVB treatment. To evaluate the roles of PTK6 in the skin following UVB-induced damage, we exposed back skin of Ptk6 +/+ and Ptk6 -/- SENCAR mice to incremental doses of UVB for 30 weeks. Wild-type mice were more sensitive to UVB and exhibited increased inflammation and greater activation of signal transducer and activator of transcription-3 (STAT3) than Ptk6-/- mice. Disruption of Ptk6 did not have an impact on proliferation, although PTK6 was expressed and activated in basal epithelial cells in wild-type mice following UVB treatment. However, wild-type mice exhibited shortened tumor latency and increased tumor load compared with Ptk6-/- mice, and STAT3 activation was increased in these tumors. PTK6 activation was detected in UVB-induced tumors, and this correlated with increased activating phosphorylation of focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1). Activation of PTK6 was also detected in human squamous cell carcinomas of the skin. Although PTK6 has roles in normal differentiation, it also contributes to UVB-induced injury and tumorigenesis in vivo.
Uploads
Papers by Michael Chastkofsky