John S. Jarboe

Expression of Interleukin-13 Receptor α2 in Glioblastoma Multiforme: Implications for Targeted Therapies

Glioblastoma multiforme is the most common primary malignant brain tumor and despite treatment with surgery, radiation, and chemotherapy, the median survival of patients with glioblastoma multiforme is approximately 1 year. Glioblastoma multiforme explants and cell lines have been reported to overexpress the interleukin-13 receptor alpha2 subunit (IL13Ralpha2) relative to nonneoplastic brain. Based on this finding, a recombinant cytotoxin composed of IL13 ligand and a truncated form of Pseudomonas aeruginosa exotoxin A (IL13-PE38QQR) was developed for the targeted treatment of glioblastoma multiforme tumors. In a recently completed phase III clinical trial, however, IL13-PE38QQR was found to be no more effective than an existing therapy in prolonging survival. To determine possible explanations for this result, we analyzed the relative expression levels of IL13Ralpha2 in glioblastoma multiforme and nonneoplastic brain specimens using publicly available oligonucleotide microarray databases, quantitative real-time reverse transcription PCR, and immunohistochemical staining. Increased expression of the IL13Ralpha2 gene relative to nonneoplastic brain was observed in 36 of 81 (44%) and 8 of 17 (47%) tumor specimens by microarray and quantitative real-time reverse transcription PCR analyses, respectively. Immunohistochemical staining of tumor specimens showed highly variable expression of IL13Ralpha2 protein both within and across specimens. These data indicate that prescreening of subjects may be of benefit in future trials of IL13Ralpha2 targeting therapies.

Mini-review: bmx kinase inhibitors for cancer therapy.

Kinase inhibitors are among the fastest growing class of anti-cancer therapies. One family of kinases that has recently gained attention as a target for treating malignant disorders is the Tec kinase family. Evidence has been published that one member of this family; the Bmx kinase, may play a role in the pathogenesis of glioblastoma, prostate, breast and lung cancer. Bmx has also shown potential as an anti-vascular therapy in combination with radiation or as a sensitizer to chemotherapeutic agents. Therefore, several companies such as Pharmacyclics, Avila Therapeutics, Merck and Co., Metaproteomics, IRM, and Moerae Matrix have developed compounds or peptides that function as Bmx kinase inhibitors. These companies have subsequently been issued patents for these inhibitors. Additionally, it has been shown that current clinical stage EGFR inhibitors can irreversibly inhibit Bmx, suggesting these compounds might be rapidly moved to clinical trials for other malignancies. This review will discuss current patents issued since 2009 that contain data specifically on inhibition of the Bmx kinase, and will also discuss the scientific literature that suggests their potential application as therapeutics in the treatment of the aforementioned malignancies.

Kinomic profiling approach identifies Trk as a novel radiation modulator.

BACKGROUND Ionizing radiation treatment is used in over half of all cancer patients, thus determining the mechanisms of response or resistance is critical for the development of novel treatment approaches. MATERIALS AND METHODS In this report, we utilize a high-content peptide array platform that performs multiplex kinase assays with real-time kinetic readout to investigate the mechanism of radiation response in vascular endothelial cells. We applied this technology to irradiated human umbilical vein endothelial cells (HUVEC). RESULTS We identified 49 specific tyrosine phosphopeptides that were differentially affected by irradiation over a time course of 1h. In one example, the Tropomyosin receptor kinase (Trk) family members, TrkA and TrkB, showed transient activation between 2 and 15 min following irradiation. When we targeted TrkA and TrkB using small molecule inhibitors, HUVEC were protected from radiation damage. Conversely, stimulation of TrkA using gambogic amide promoted radiation enhancement. CONCLUSIONS Thus, we show that our approach not only can identify rapid changes in kinase activity but also identify novel targets such as TrkA. TrkA inhibition resulted in radioprotection that correlated with enhanced repair of radiation-induced damage while TrkA stimulation by gambogic amide produced radiation sensitization.

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP2) levels, consistent with prior evidence that MARCKS can regulate PIP2 levels. We also found increased staining for PIP2 in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP2 co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP2 levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.

Targeting the effector domain of the myristoylated alanine rich C-kinase substrate enhances lung cancer radiation sensitivity.

Lung cancer is the leading cause of cancer related deaths. Common molecular drivers of lung cancer are mutations in receptor tyrosine kinases (RTKs) leading to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pro-growth, pro-survival signaling pathways. Myristoylated alanine rich C-kinase substrate (MARCKS) is a protein that has the ability to mitigate this signaling cascade by sequestering the target of PI3K, phosphatidylinositol (4,5)-bisphosphate (PIP2). As such, MARCKS has been implicated as a tumor suppressor, though there is some evidence that MARCKS may be tumor promoting in certain cancer types. Since the MARCKS function depends on its phosphorylation status, which impacts its subcellular location, MARCKS role in cancer may depend highly on the signaling context. Currently, the importance of MARCKS in lung cancer biology is limited. Thus, we investigated MARCKS in both clinical specimens and cell culture models. Immunohistochemistry scoring of MARCKS protein expression in a diverse lung tumor tissue array revealed that the majority of squamous cell carcinomas stained positive for MARCKS while other histologies, such as adenocarcinomas, had lower levels. To study the importance of MARCKS in lung cancer biology, we used inducible overexpression of wild-type (WT) and non-phosphorylatable (NP)-MARCKS in A549 lung cancer cells that had a low level of endogenous MARCKS. We found that NP-MARCKS expression, but not WT-MARCKS, enhanced the radiosensitivity of A549 cells in part by inhibiting DNA repair as evidenced by prolonged radiation-induced DNA double strand breaks. We confirmed the importance of MARCKS phosphorylation status by treating several lung cancer cell lines with a peptide mimetic of the phosphorylation domain, the effector domain (ED), which effectively attenuated cell growth as measured by cell index. Thus, the MARCKS ED appears to be an important target for lung cancer therapeutic development.

Variants of Osteoprotegerin Lacking TRAIL Binding for Therapeutic Bone Remodeling in Osteolytic Malignancies

Osteolytic bone damage is a major cause of morbidity in several metastatic pathologies. Current therapies using bisphosphonates provide modest improvement, but cytotoxic side effects still occur prompting the need to develop more effective therapies to target aggressive osteoclastogenesis. Increased levels of receptor activator of NF–κB ligand (TNFSF11/RANKL), leading to RANKL-RANK signaling, remain the key axis for osteoclast activation and bone resorption. Osteoprotegerin (TNFRSF11B/OPG), a decoy receptor for RANKL, is significantly decreased in patients who present with bone lesions. Despite its potential in inhibiting osteoclast activation, OPG also binds to TNF-related apoptosis-inducing ligand (TNFSF10/TRAIL), making tumor cells resistant to apoptosis. Toward uncoupling the events of TRAIL binding of OPG and to improve its utility for bone remodeling without inducing tumor resistance to apoptosis, OPG mutants were developed by structural homology modeling based on interactive domain identification and by superimposing models of OPG, TRAIL, and its receptor DR5 (TNFRSF10B) to identify regions of OPG for rational design. The OPG mutants were purified and extensively characterized for their ability to decrease osteoclast damage without affecting tumor apoptosis pathway both in vitro and in vivo, confirming their potential in bone remodeling following cancer-induced osteolytic damage. Implications: OPG variants were developed that lack TRAIL binding, yet retain RANKL binding and suggest new possibilities for therapeutic targeting in osteolytic malignancies. Mol Cancer Res; 13(5); 819–27. ©2015 AACR.

MARCKS phosphorylation is modulated by a peptide mimetic of MARCKS effector domain leading to increased radiation sensitivity in lung cancer cell lines

Lung cancer is the leading cause of cancer-associated mortality in the United States. Kinase hyperactivation is a known mechanism of tumorigenesis. The phosphorylation status of the plasma membrane-associated protein myristoylated alanine rich C-kinase substrate (MARCKS) effector domain (ED) was previously established as being important in the sensitivity of lung cancer to radiation. Specifically, when MARCKS ED was in a non-phosphorylated state, lung cancer cells were more susceptible to ionizing radiation and experienced prolonged double-strand DNA breaks. Additional studies demonstrated that the phosphorylation status of MARCKS ED is important for gene expression and in vivo tumor growth. The present study used a peptide mimetic of MARCKS ED as a therapeutic intervention to modulate MARCKS phosphorylation. Culturing A549, H1792 and H1975 lung cancer cell lines with the MARCKS ED peptide led to reduced levels of phosphorylated MARCKS and phosphorylated Akt serine/threonine kinase 1. Further investigation demonstrated that the peptide therapy was able to reduce lung cancer cell proliferation and increase radiation sensitivity. In addition, the MARCKS peptide therapy was able to prolong double-strand DNA breaks following ionizing radiation exposure. The results of the present study demonstrate that a peptide mimetic of MARCKS ED is able to modulate MARCKS phosphorylation, leading to an increase in sensitivity to radiation.

Abstract 3391: Regenerative stem cell therapy for multiple myeloma osteolytic bone damage

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Multiple Myeloma (MM) remains the second most common hematologic malignancy occurring in adults, which affects primarily the skeletal system. Current therapies, which include chemotherapy, radiotherapy, autologouos stem cell transplantation and in some cases surgery, have the extended median survival between 3 and 10 years. However, MM is still incurable and therefore improving current therapies or developing novel ones to extend survival rate will be highly beneficial for patient management. The long term goal of this study is to develop a novel therapeutic approach to the treatment of MM using genetically-engineered mesenchymal stem cells (MSC) for inhibiting osteoclast activity by stable expression of osteoprotegin (OPG). Studies have shown that osteoclast activity is increased in myeloma patients through increased expression of Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) leading to RANKL/RANK signaling, resulting in osteoclast activation and ultimately bone resorption. Osteoprotegerin is a soluble decoy receptor for RANKL, and is decreased in expression in myeloma patients possibly because of a marked decrease in osteoblast and or MSC, which produce OPG. Thus, we hypothesize that therapy with MSC, overexpressing OPG, will greatly decrease osteolytic damage and reduce morbidity. Despite the potential of OPG in inhibiting osteoclast activation, OPG also binds to tumor related apoptosis-inducing ligand (TRAIL) making MM cells resistant to apoptosis. In order to eliminate TRAIL binding while still possessing RANKL binding, we have created mutant OPG constructs by site-directed mutagenesis based on interactive domain identification, and superimposing structural models of TRAIL, OPG and DR5, the death receptor that binds to TRAIL. The mutant OPGs were produced in HEK 293 cells for characterization of the five potential mutants and their TRAIL binding ability. One of the mutants abolished TRAIL binding while possessing RANKL binding as determined by TRAIL assay and osteoclast assay respectively. Finally, to achieve biphasic effects of MSC-OPG therapy with anti-tumor activity, the regenerative MSC therapy will be tested in combination with chemotherapy and anti-angiogenic therapy. These studies are performed in preclinical mouse models of MM. Currently, other OPG mutants are being characterized for their RANKL and TRAIL binding properties which will be confirmed via the assays mentioned and immunoprecipitation. Thus far, we conclude that using site-directed mutagenesis targeted at the N-terminal of OPG effectively retains RANKL binding and abolishes TRAIL binding. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3391. doi:10.1158/1538-7445.AM2011-3391

Abstract A87: Myristoylated alanine rich C-kinase substrate (MARCKS) expression in lung cancer cells influences immune cell populations in tumor microenvironment in murine models

Purpose: To determine if MARCKS expression in lung cancer cells alters the recruitment of immune cells to the tumor microenvironment while influencing outcome in in vivo models. Background: Lung cancer is the leading cause of cancer related deaths in the United States. There is increasing support that aberrant immune cell recruitment and activation in the tumor microenvironment leads to pro-tumor behavior. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) is an intracellular protein that has been described to alter cellular proliferation as well as be involved in lung epithelial cytokine secretion and immune cell migration. Depending on the type of cancer, MARCKS has either pro-tumor or anti-tumor properties. Currently, it is unknown whether MARCKS expression is beneficial or detrimental in lung cancer. We wanted to investigate if MARCKS expression was able to delay tumor burden as well as influence the immune cell populations being recruited to the tumor microenvironment. Methods: The C57BL/6 murine lung cancer cell line Lewis Lung Carcinoma (LLC) was engineered with lentiviral particles to over-express MARCKS (LLC-MARCKS). Western blot confirmed over-expression of MARCKS. Tumors were implanted into the flank of C57BL/6 mice and survival data was collected comparing LLC-MARCKS over-expression tumors versus empty lentiviral plasmid containing LLC tumors (LLC-Ctrl). Survival data was graphed by Kaplan-Meier and statistics calculated with log-rank survival test. To analyze immune cells in the tumor microenvironment, orthotopic lung nodules were established by tail-vein injections. Three weeks after tail-vein injections, lungs were resected from LLC-MARCKS and LLC-Ctrl tumors bearing mice for flow cytometry analysis. Differences in immune cells between groups were calculated by student9s t-test. Results: Mice with LLC-MARCKS tumors had an increase in median survival time compared to LLC-Ctrl tumors (31 days vs. 26 days) and a trend toward increase survival (p=0.058). While collecting lungs, it was grossly apparent that mice with LLC-Ctrl tumors had greater tumor burden and signs of disease, such as bloody exudates, compared to mice with LLC-MARCKS tumors. H & E staining confirmed that LLC-Ctrl tumors had higher tumor burden then LLC-MARCKS tumors. Flow cytometry identified a decrease in F4/80+ macrophages (p Conclusion: MARCKS expression in the murine lung cancer LLC cell line prolonged survival while also decreasing F4/80+ macrophages and neutrophils in the tumor microenvironment. Citation Format: Timothy D. Rohrbach, Travis D. Hull, John S. Jarboe, Nicholas Eustace, Yong Wang, Jessy S. Deshane, Christopher D. Willey. Myristoylated alanine rich C-kinase substrate (MARCKS) expression in lung cancer cells influences immune cell populations in tumor microenvironment in murine models. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A87.

Abstract 4943: Genetically engineered osteoprotegerin for multiple myeloma osteolytic bone damage

Current treatments for osteolytic cancers include a combination of radiation, chemotherapy and cytotoxic products, but toxic side effects are still of major concern. Studies have shown that osteoclast activity is increased in patients with osteolytic cancers such as Multiple Myeloma (MM), through increased expression of Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) leading to RANKL/RANK signaling, resulting in osteoclast activation and ultimately bone resorption. Moreover, Osteoprotegerin (OPG) is drastically decreased in these patients who presents with bone lesion. Thus, the use of OPG as a therapeutic molecule would greatly decrease osteolytic damage and reduce morbidity. However, despite the of OPG potential in inhibiting the activation of bone resorbing osteoclast, OPG also binds to tumor related apoptosis-inducing ligand (TRAIL) making tumor cells resistant to apoptosis. TRAIL binds to the Death Receptor 4 and 5 (DR4, DR5) and initiate cell death of Transformed cells such as MM. The present study was designed to develop a novel therapeutic approach to the treatment of osteolytic bone damage by use of genetically altered OPG retaining RANKL binding but abolished of TRAIL binding. In order to eliminate TRAIL binding while maintaining RANKL binding, we created mutant OPG-Fc constructs by site directed mutagenesis based on interactive domain identification and by superimposing structural models of TRAIL, OPG and DR5. The mutant OPGs were produced in HEK 293 cells for characterization of potential mutants and their TRAIL binding ability. We conclude that using site-directed mutagenesis at the N-terminal of OPG effectively retains RANKL binding property but abolishes TRAIL binding property as determined by osteoclast and TRAIL assays respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4943. doi:1538-7445.AM2012-4943