Dongqin Zhu

Novel Peripherally Derived Neural-Like Stem Cells as Therapeutic Carriers for Treating Glioblastomas

Glioblastoma (GBM), an aggressive grade IV astrocytoma, is the most common primary malignant adult brain tumor characterized by extensive invasiveness, heterogeneity, and angiogenesis. Standard treatment options such as radiation and chemotherapy have proven to be only marginally effective in treating GBM because of its invasive nature. Therefore, extensive efforts have been put forth to develop tumor‐tropic stem cells as viable therapeutic vehicles with potential to treat even the most invasive tumor cells that are harbored within areas of normal brain. To this end, we discovered a newly described NG2‐expressing cell that we isolated from a distinct pericyte subtype found abundantly in cultures derived from peripheral muscle. In this work, we show the translational significance of these peripherally derived neural‐like stem cells (NLSC) and their potential to migrate toward tumors and act as therapeutic carriers. We demonstrate that these NLSCs exhibit in vitro and in vivo GBM tropism. Furthermore, NLSCs did not promote angiogenesis or transform into tumor‐associated stromal cells, which are concerns raised when using other common stem cells, such as mesenchymal stem cells and induced neural stem cells, as therapeutic carriers. We also demonstrate the potential of NLSCs to express a prototype therapeutic, tumor necrosis factor α‐related apoptosis‐inducing ligand and kill GBM cells in vitro. These data demonstrate the therapeutic potential of our newly characterized NLSC against GBM. Stem Cells Translational Medicine 2017;6:471–481

Combined inhibition of AKT and HSF1 suppresses breast cancer stem cells and tumor growth

Breast cancer is the most common cancer in women and the second leading cause of cancer deaths in women. Over 90% of breast cancer deaths are attributable to metastasis. Our lab has recently reported that AKT activates heat shock factor 1 (HSF1), leading to epithelial-to-mesenchymal transition in HER2-positive breast cancer. However, it is unknown whether the AKT-HSF1 pathway plays an important role in other breast cancer subtypes, breast cancer stem cells, or breast cancer growth and metastasis. Herein, we showed AKT and HSF1 to be frequently co-activated in breast cancer cell lines and specimens across different subtypes. Activated AKT (S473) and HSF1 (S326) are strongly associated with shortened time to metastasis. Inhibition of the AKT-HSF1 signaling axis using small molecule inhibitors, HSF1 knockdown or the dominant-negative HSF1 mutant (S326A) reduced the growth of metastatic breast cancer cells and breast cancer stem cells. The combination of small molecule inhibitors targeting AKT (MK-2206) and HSF1 (KRIBB11) resulted in synergistic killing of breast cancer cells and breast cancer stem cells across different molecular subtypes. Using an orthotopic xenograft mouse model, we found that combined targeting of AKT and HSF1 to significantly reduce tumor growth, induce tumor apoptosis, delay time to metastasis, and prolong host survival. Taken together, our results indicate AKT-HSF1 signaling mediates breast cancer stem cells self-renewal, tumor growth and metastasis, and that dual targeting of AKT and HSF1 resulted in synergistic suppression of breast cancer progression thereby supporting future testing of AKT-HSF1 combination therapy for breast cancer patients.Breast cancer is the most common cancer in women and the second leading cause of cancer deaths in women. Over 90% of breast cancer deaths are attributable to metastasis. Our lab has recently reported that AKT activates heat shock factor 1 (HSF1), leading to epithelial-to-mesenchymal transition in HER2-positive breast cancer. However, it is unknown whether the AKT-HSF1 pathway plays an important role in other breast cancer subtypes, breast cancer stem cells, or breast cancer growth and metastasis. Herein, we showed AKT and HSF1 to be frequently co-activated in breast cancer cell lines and specimens across different subtypes. Activated AKT (S473) and HSF1 (S326) are strongly associated with shortened time to metastasis. Inhibition of the AKT-HSF1 signaling axis using small molecule inhibitors, HSF1 knockdown or the dominant-negative HSF1 mutant (S326A) reduced the growth of metastatic breast cancer cells and breast cancer stem cells. The combination of small molecule inhibitors targeting AKT (MK-2206) and HSF1 (KRIBB11) resulted in synergistic killing of breast cancer cells and breast cancer stem cells across different molecular subtypes. Using an orthotopic xenograft mouse model, we found that combined targeting of AKT and HSF1 to significantly reduce tumor growth, induce tumor apoptosis, delay time to metastasis, and prolong host survival. Taken together, our results indicate AKT-HSF1 signaling mediates breast cancer stem cells self-renewal, tumor growth and metastasis, and that dual targeting of AKT and HSF1 resulted in synergistic suppression of breast cancer progression thereby supporting future testing of AKT-HSF1 combination therapy for breast cancer patients.

Truncated Glioma-Associated Oncogene Homolog 1 (tGLI1) Mediates Mesenchymal Glioblastoma via Transcriptional Activation of CD44

The molecular pathways driving mesenchymal glioblastoma (GBM) are still not well understood. We report here that truncated glioma-associated oncogene homolog 1 (tGLI1) is a tumor-specific transcription factor that facilitates GBM growth, is enriched in the mesenchymal subtype of GBM and glioma stem cells (GSC), and promotes mesenchymal GSC by upregulating transcription of CD44. In an orthotopic GBM xenograft mouse model, tGLI1-overexpressing tumors grew more aggressively with increased proliferation and angiogenesis compared with control and GLI1-overexpressing xenografts. tGLI1 was highly expressed in GBM clinical specimens but undetectable in normal brains, whereas GLI1 was expressed in both tissues. A tGLI1 activation signature (tGAS) correlated with glioma grade, tumor angiogenesis, and poor overall survival, and GBMs with high tGAS were enriched with mesenchymal GBM/GSC gene signatures. Neurospheres contained increased levels of tGLI1, but not GLI1, compared with the monolayer culture; mesenchymal GSC expressed more tGLI1 than proneural GSC. Ectopic tGLI1 expression enhanced the ability of mesenchymal GSC to yield neurospheres in vitro and to form tumors in mouse brains. Selective tGLI1 knockdown reduced neurosphere formation of GBM cells. tGLI1 bound to and transactivated the promoter of the CD44 gene, a marker and mediator for mesenchymal GSC, leading to its expression. Collectively, these findings advance our understanding of GBM biology by establishing tGLI1 as a novel transcriptional activator of CD44 and a novel mediator of mesenchymal GBM and GSC.Significance: These findings highlight the role of a tumor-specific gain-of-function transcription factor tGLI1 in mesenchymal glioma stem cell maintenance and mesenchymal GBM growth. Cancer Res; 78(10); 2589-600. ©2018 AACR.

Abstract 2150: Therapeutic efficacy of an optimized quadruply mutated IL13-based bacterial cytotoxin in an orthotopic murine glioma model.

Interleukin 13 receptor alpha 2 (IL13Rα2) is a tumor-restricted plasma membrane receptor that is overexpressed on greater than 70% of Glioblastoma Multiformes (GBMs) as well as in other malignancies, including melanoma, adenocarcinoma, ovarian cancer and renal cell carcinoma. We and others have therapeutically exploited this attractive target using IL13-based targeting strategies to deliver bacterial toxins, chemotherapeutics, nanoparticles, viruses and immunotherapy directly to the IL13Rα2 tumor-restricted biomarker. In past work, we found multiple mutational hotspots on IL13 that (a) abrogated its binding to the physiologically abundant IL13Rα1/IL4Rα receptor heterodimer, and (b) increased its binding to the tumor-restricted IL13Rα2. Based on these findings, we created a delivery platform established as an optimized IL13Rα2-Targeted Quadruple Mutant of IL13 (TQM13; IL13.E13K/R66D/S69D/K105R) that we demonstrated binds with high affinity towards IL13Rα2, but not the IL13Rα1/IL4Rα receptor heterodimer. Of importance, we demonstrated the ability of radiolabeled TQM13 to target IL13Rα2-expressing tumors in vivo using positron emission tomography (PET). The objective of this current work was to demonstrate for the first time the in vivo IL13Rα2-targeted killing effect of our optimized TQM13 ligand genetically fused to a potent derivative of Pseudomonas exotoxin (PE). We successfully expressed TQM13 and TQM13-PE proteins in an E. coli expression system, denatured, refolded and purified them via Nickel-based affinity and ion exchange chromatography. We demonstrated IL13Rα2-specific affinity of TQM13 using a novel IL13Rα2-inducible system in which B16 melanoma cells expressed varying amounts of IL13Rα2 in proportion to the concentration of doxycycline. Of importance, the number of induced TQM13 binding sites (in vitro and in vivo) was comparable to the range seen in GBM and other malignancies (50,000-4,500,000 receptors/cell). In addition, we demonstrated that TQM13-PE killed cells in direct proportion to IL13Rα2 expression. Therefore, we tested the efficacy of TQM13-PE in an orthotopic murine GBM model by injecting mice with 1 μg of TQM13-PE intracranially on day 7 and 14 post stereotactic tumor cell implantation. We found a significant increase in survival only in mice bearing IL13Rα2-expressing orthotopic tumors, in contrast to mice bearing IL13Rα2-negative tumors. Thus, we have successfully generated an optimized, biomarker-targeted anti-GBM agent that demonstrated specific therapeutic potential against the tumor-associated IL13Rα2 biomarker in vivo. Citation Format: Van Nguyen, Dongqin Zhu, Jesse M. Conyers, Waldemar Debinski, Akiva Mintz. Therapeutic efficacy of an optimized quadruply mutated IL13-based bacterial cytotoxin in an orthotopic murine glioma model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2150. doi:10.1158/1538-7445.AM2013-2150

Abstract B112: Personalizing anti-IL13R 2 therapy with real-time immunoPET visualization of biomarker expression.

Interleukin 13 Receptor 2 (IL13R 2) is a tumor-restricted plasma membrane receptor that is overexpressed on greater than 70% of Glioblastoma Multiformes (GBM) as well as in other malignancies, including melanoma, adenocarcinoma, ovarian cancer and renal cell carcinoma. We and others have therapeutically exploited this attractive tumor-restricted biomarker in a number of ways, including with bacterial toxins, chemotherapeutics, nanoparticles, viruses and immunotherapies. Our objective here is to for the first time develop a positron emission tomography (PET)-based molecular imaging probe that can non-invasively visualize IL13R 2 expression in order to determine if it is expressed on a particular patient9s tumor, which can then potentially be therapeutically targeted. To accomplish our goal, we created a novel IL13R 2-Targeted Quadruple Mutant of IL13 (TQM13; IL13.E13K/R66D/S69D/K105R) based on our prior work in which we identified functional “hotspot” amino acid mutations of IL13 that separately increased its affinity towards the tumor-restricted IL13R 2 but abrogated its binding to the physiologically abundant IL13R 1/IL4R heterodimer. In addition, we created a matching negative control, IL13R-Binding Deficient mutant (BDef13; IL13.E13K/R66D/S69D/K105A) with disrupted binding to both IL13R 1 and IL13R 2. In these experiments, our goals were to confirm the binding profile of this optimized ligand in vitro and its potential as a non-invasive PET probe to visualize the cancer-restricted IL13R 2 biomarker in vivo. We demonstrated that 125I-TQM13, but not 125I-BDef13, specifically bound to IL13R 2 via autoradiography on frozen GBM sections and electrophoretic mobility shift assay. An in vitro cell binding assay confirmed 125I-TQM13 had strong affinity to IL13R 2-expressing cells (Kd ∼ 5nM). This binding assay also established that G48a GBM cell line, which we utilized in our in vivo studies, highly expressed IL13R 2 with approximately three million binding sites per cell. Importantly, in an in vivo biodistribution study, 125I-TQM13 bound to IL13R 2-expressing G48a tumors at a 7-to-1 ratio compared to background muscles 24 hours after intravenous injection, in contrast to 125I-BDef13, which remained at background levels. We therefore radiolabeled TQM13 with the positron emitter 124I and intravenously delivered it to mice bearing subcutaneous G48a tumors. Subsequent microPET images at 24 and 48 hours post-injection demonstrated specific tumor localization with tumor-to-background ratio of 7:1 at 48 hours. In contrast, 124I-BDef13 only demonstrated background signal levels. In conclusion, we have successfully generated an optimized, biomarker-targeted IL13 derivative and demonstrated for the first time the potential to non-invasively image the attractive cancer-restricted biomarker, IL13R 2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B112.

Abstract 3639: In vivo targeting of a Glioblastoma Multiforme restricted biomarker

Our objective is to develop an optimized ligand-based delivery system that targets IL13Rα2, a tumor-restricted plasma membrane receptor overexpressed on greater than 70% of Glioblastoma Multiforme (GBM) patients but not present in normal brain tissues. In addition, IL13Rα2 is overexpressed in a variety of other cancers, including melanoma, adenocarcinoma, ovarian cancer and renal cell carcinoma. Based on our prior experience identifying functionally relevant “hotspot” amino acid mutations of IL13, we designed a novel IL13Rα2-Targeted Quadruple Mutant of IL-13 (TQM13) that we hypothesized will bind with a heightened affinity towards the tumor-restricted IL13Rα2 but not to the physiologically abundant IL13Rα1/IL4Rα heterodimer. In addition, we created a matching negative control, IL13R-Binding Null mutant (BN13, IL13.E13K/R66D/S69D/K105A) with disrupted binding to both IL13Rα1 and IL13Rα2. We successfully expressed these proteins in an E. coli expression system, denatured, refolded and purified them via Nickel-based affinity chromatography. Our experimental goals were to confirm the ligand secondary structure and assess its functional activity in vitro and in vivo. The α-helical-enriched secondary structure of TQM13 was confirmed to be highly similar to that of wild-type IL13 via circular dichroism spectroscopy measurement. TQM13 demonstrated high affinity towards the tumor-restricted IL13Rα2 in a surface plasmon resonance (Biacore) study (Kd ≈ 3 nM versus Kd ≈ 800 nM for BN13). To further confirm binding specificity, we radioiodinated TQM13 using the IODO-GEN method and demonstrated that 125I-TQM13, but not 125I-BN13, bound to the tumor-restricted IL13Rα2 via electrophoretic mobility shift assay. However, as demonstrated by surface plasmon resonance, TQM13 did not exhibit binding activity to the physiologically abundant IL13Rα1/IL4Rα heterodimer, in contrast to wild-type IL13. Importantly, in an in vivo biodistribution study, 125I-TQM13 bound to IL13Rα2-expressing tumors at a 2-to-1 ratio in comparison to background muscles only one hour after intravenous delivery of radiolabeled ligands. The tumor-to-muscle binding ratio improved to 5-to-1 at four hours after injection and 7-to-1 at the 24-hour timepoint. In contrast, 125I-BN13 did not bind to IL13Rα2-expressing tumors as its activity remained similar to background muscles at 24 hours after injection In conclusion, we have successfully generated an optimized, biomarker-targeted IL13 derivative and demonstrated for the first time via independent biomolecular studies its secondary structure and specific binding towards the tumor-associated IL13Rα2 in vitro and in vivo, but not towards the physiologically abundant IL13Rα1/IL4Rα heterodimer. This novel ligand is therefore suitable to deliver high doses of diagnostic and therapeutic radioactivity specifically to GBMs. 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 3639. doi:10.1158/1538-7445.AM2011-3639