David Dadey

The ATF6 pathway of the ER stress response contributes to enhanced viability in glioblastoma

Therapeutic resistance is a major barrier to improvement of outcomes for patients with glioblastoma. The endoplasmic reticulum stress response (ERSR) has been identified as a contributor to chemoresistance in glioblastoma; however the contributions of the ERSR to radioresistance have not been characterized. In this study we found that radiation can induce ER stress and downstream signaling associated with the ERSR. Induction of ER stress appears to be linked to changes in ROS balance secondary to irradiation. Furthermore, we observed global induction of genes downstream of the ERSR in irradiated glioblastoma. Knockdown of ATF6, a regulator of the ERSR, was sufficient to enhance radiation induced cell death. Also, we found that activation of ATF6 contributes to the radiation-induced upregulation of glucose regulated protein 78 (GRP78) and NOTCH1. Our results reveal ATF6 as a potential therapeutic target to enhance the efficacy of radiation therapy.

Autotaxin Inhibition with PF-8380 Enhances the Radiosensitivity of Human and Murine Glioblastoma Cell Lines.

Purpose: Glioblastoma multiforme (GBM) is an aggressive primary brain tumor that is radio-resistant and recurs despite aggressive surgery, chemo, and radiotherapy. Autotaxin (ATX) is over expressed in various cancers including GBM and is implicated in tumor progression, invasion, and angiogenesis. Using the ATX specific inhibitor, PF-8380, we studied ATX as a potential target to enhance radiosensitivity in GBM. Methods and Materials: Mouse GL261 and Human U87-MG cells were used as GBM cell models. Clonogenic survival assays and tumor transwell invasion assays were performed using PF-8380 to evaluate role of ATX in survival and invasion. Radiation dependent activation of Akt was analyzed by immunoblotting. Tumor induced angiogenesis was studied using the dorsal skin fold model in GL261. Heterotopic mouse GL261 tumors were used to evaluate the efficacy of PF-8380 as a radiosensitizer. Results: Pre-treatment of GL261 and U87-MG cells with 1 μM PF-8380 followed by 4 Gy irradiation resulted in decreased clonogenic survival, decreased migration (33% in GL261; P = 0.002 and 17.9% in U87-MG; P = 0.012), decreased invasion (35.6% in GL261; P = 0.0037 and 31.8% in U87-MG; P = 0.002), and attenuated radiation-induced Akt phosphorylation. In the tumor window model, inhibition of ATX abrogated radiation induced tumor neovascularization (65%; P = 0.011). In a heterotopic mouse GL261 tumors untreated mice took 11.2 days to reach a tumor volume of 7000 mm3, however combination of PF-8380 (10 mg/kg) with irradiation (five fractions of 2 Gy) took more than 32 days to reach a tumor volume of 7000 mm3. Conclusion: Inhibition of ATX by PF-8380 led to decreased invasion and enhanced radiosensitization of GBM cells. Radiation-induced activation of Akt was abrogated by inhibition of ATX. Furthermore, inhibition of ATX led to diminished tumor vascularity and delayed tumor growth. These results suggest that inhibition of ATX may ameliorate GBM response to radiotherapy.

An NAD+-dependent transcriptional program governs self-renewal and radiation resistance in glioblastoma

Significance Glioblastoma, the most common primary malignant brain tumor in adults, remains challenging despite multimodality therapy, necessitating the discovery of new therapies. Nicotinamide adenine dinucleotide (NAD+) plays a pivotal role in cancer cell metabolism, but how NAD+ impacts functional signaling events in glioblastoma is not well understood. We provide clinical evidence that high expression of NAMPT, the rate-limiting step in NAD+ biosynthesis, in glioblastoma tumors is associated with poor overall survival in patients, and demonstrate NAMPT and NAD+ are required for the maintenance of patient-derived glioblastoma stem-like cells (GSCs). Moreover, we delineate a NAD+-dependent transcriptional program that governs GSC self-renewal and dictates the radiation resistance of these cells. These findings identify potential new therapeutic avenues for the treatment of glioblastoma. Accumulating evidence suggests cancer cells exhibit a dependency on metabolic pathways regulated by nicotinamide adenine dinucleotide (NAD+). Nevertheless, how the regulation of this metabolic cofactor interfaces with signal transduction networks remains poorly understood in glioblastoma. Here, we report nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ synthesis, is highly expressed in glioblastoma tumors and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD+ levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD+-dependent network. Accordingly, we demonstrate E2F2 is required for GSC self-renewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix–loop–helix gene family. Finally, we find NAMPT mediates GSC radiation resistance. The identification of a NAMPT-E2F2-ID axis establishes a link between NAD+ metabolism and a self-renewal transcriptional program in glioblastoma, with therapeutic implications for this formidable cancer.

Laser interstitial thermal therapy for subependymal giant cell astrocytoma: technical case report

Subependymal giant cell astrocytoma (SEGA) is a rare tumor occurring almost exclusively in patients with tuberous sclerosis complex. Although open resection remains the standard therapy, complication rates remain high. To minimize morbidity, less invasive approaches, such as endoscope-assisted resection, radiosurgery, and chemotherapy with mTOR pathway inhibitors, are also used to treat these lesions. Laser interstitial thermal therapy (LITT) is a relatively new modality that is increasingly used to treat a variety of intracranial lesions. In this report, the authors describe two pediatric cases of SEGA that were treated with LITT. In both patients the lesion responded well to this treatment modality, with tumor shrinkage observed on follow-up MRI. These cases highlight the potential of LITT to serve as a viable minimally invasive therapeutic approach to the management of SEGAs in the pediatric population.

Tumor-Specific Binding of Radiolabeled PEGylated GIRLRG Peptide: A Novel Agent for Targeting Cancers

Cancer-specific targeting sparing normal tissues would significantly enhance cancer therapy outcomes and reduce cancer-related mortality. One approach is to target receptors or molecules that are specifically expressed on cancer cells. Peptides as cancer-specific targeting agents offer advantages such as ease of synthesis, low antigenicity, and enhanced diffusion into tissues. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum stress chaperone that regulates the unfolded protein response and is overexpressed in various cancers. In this study, we evaluated GIRLRG peptide that specifically targets GRP78 for cancer-specific binding (in vitro) and noninvasive tumor imaging (in vivo). Methods: GIRLRG peptide was modeled into the GRP78 ATPase domain using computational modeling. Surface plasmon resonance studies were performed to determine the affinity of GIRLRG peptide to GRP78 protein. GIRLRG was conjugated with PEG to prolong its circulation in mice. Tumor binding efficacy of PEG-GIRLRG peptide was evaluated in nude mice bearing heterotopic cervical (HT3), esophageal (OE33), pancreatic (BXPC3), lung (A549), and glioma (D54) tumors. Nano-SPECT/CT imaging of the mice was performed 48 and 72 h after injection with 111In-labeled PEG-GIRLRG or PEG-control peptide. Post-SPECT biodistribution studies were performed 96 h after injection of the radiolabeled peptides. Results: Using molecular modeling and surface plasmon resonance, we identified that GIRLRG was binding with an affinity constant of 2.16 × 10−3 M in the ATPase domain of GRP78. GIRLRG peptide specifically bound to cervical, lung, esophageal, and glioma cells. SPECT imaging revealed that 111In-PEG-GIRLRG specifically bound to cervical, esophageal, pancreatic, lung, and brain tumors. Post-SPECT biodistribution data also validated the SPECT imaging results. Conclusion: GIRLRG peptide specifically binds to the ATPase domain of GRP78. Radiolabeled PEG-GIRLRG could be used to target various cancers. Further studies would be required to translate PEG-GIRLRG peptide into the clinic.

Antibody targeting GRP78 enhances the efficacy of radiation therapy in human glioblastoma and non-small-cell lung cancer cell lines and tumor models.

Purpose: Non–small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM) have poor median survival. NSCLC and GBM overexpress glucose regulated protein 78 (GRP78), which has a role in radioresistance and recurrence. In this study, we determined the effect of anti-GRP78 antibody and the combined effect of the anti-GRP78 antibody with ionizing radiation (XRT) on NSCLC and GBM cell lines both in vitro and in vivo. Experimental Design: NSCLC and GBM cancer cell lines were treated with anti-GRP78 antibodies and evaluated for proliferation, colony formation, cell death, and PI3K/Akt/mTOR signaling. The efficacy of anti-GRP78 antibodies on tumor growth in combination with XRT was determined in vivo in mouse xenograft models. Results: GBM and NSCLC cells treated with anti-GRP78 antibodies showed attenuated cell proliferation, colony formation, and enhanced apoptosis. GBM and NSCLC cells treated with anti-GRP78 antibodies also showed global suppression of PI3K/Akt/mTOR signaling. Combining antibody with XRT resulted in significant tumor growth delay in both NSCLC and GBM heterotopic tumor models. Conclusions: Antibodies targeting GRP78 exhibited antitumor activity and enhanced the efficacy of radiation in NSCLC and GBM both in vitro and in vivo. GRP78 is a promising novel target, and anti-GRP78 antibodies could be used as an effective cancer therapy alone or in combination with XRT. Clin Cancer Res; 23(10); 2556–64. ©2016 AACR.

Abstract 4599: Antibody targeting PDZ domain of TIP-1 induces proliferation arrest through AKT/mTOR signaling inhibition in lung cancer and glioblastoma

Antigens that are over-expressed in cancer in response to radiation are being used as novel targets. We showed tax interacting protein 1 (TIP-1) to be radiation-inducible that translocated to the surface of the cancer cell following irradiation. TIP-1, which consists of a single PDZ domain plays an important role in cell signaling, cancer development, and progression. TIP-1’s involvement in various survival pathways makes it an attractive target for anticancer therapeutics. We used antibodies specific to this PDZ domain to determine its role in cancer cell survival. We monitored proliferation in lung cancer (A549 and H460) and glioblastoma (D54 and U251) cells after 24, 48, 72 and 96h treatment with the anti-PDZ antibody. We observed a time-dependent proliferation arrest with anti-PDZ antibody treatment which was associated with increased apoptosis. The anti-PDZ antibody when combined with radiation (3Gy) led to reduced proliferation and colony formation. Anti-PDZ antibody had no effect on the proliferation of normal lung (MRC-5) and endothelial (HUVEC) cells. Cells treated with anti-PDZ antibody showed decreased levels of the phosphorylated forms of AKT, mTOR, and a downstream substrate of mTOR, 4EBP1. Anti-PDZ antibody treatment also led to an overall reduction in basal levels of AKT, mTOR, and 4EBP1. Further, we evaluated the effect of the anti-PDZ antibody on tumor growth in heterotopic mouse models of lung cancer (A549) and glioma (U251). We observed significant growth delay in mice treated with anti-PDZ antibody treatment when compared to mice treated with the isotype control. The combination of the anti-PDZ antibody with radiation showed an additive effect. Immunoblot analysis of tumor tissues also showed downregulation of phosphorylated and total levels of AKT, mTOR and 4EBP1 in the tumors treated with anti-PDZ antibody. Overall, our results suggest that TIP-1 is a promising therapeutic target for treatment of lung cancer and glioblastoma. Antibodies specific to the PDZ domain of TIP-1 enhance the efficacy of radiotherapy. The anti-PDZ antibodies need to be optimized further before translating it into the clinic. Citation Format: Vaishali Kapoor, David Dadey, Kelly Hoye, Andrea Collins, Dinesh Thotala, Dennis Hallahan. Antibody targeting PDZ domain of TIP-1 induces proliferation arrest through AKT/mTOR signaling inhibition in lung cancer and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4599. doi:10.1158/1538-7445.AM2017-4599

Abstract 4202: Peptide probes binding to radiation-inducible cell surface GRP78 as a novel targeting strategy for various tumors

Traditional radiation therapy is often associated with significant toxicity to normal cells that could limit the success of cancer therapy. The greater understanding in the molecular differences between cancer cells and normal cells has lead to targeted therapies in cancer treatment. We have discovered novel radiation-inducible antigens in cancer using phage-display peptide libraries. One such inducible-antigen is Glucose regulated protein 78kDa (GRP78) that was shown to bind the hexapeptide GIRLRG. GRP78 is known to regulate cellular stresses, including hypoglycemia, hypoxia and the ER stress response. It is over-expressed in different cancer subtypes and has been correlated to their poor prognosis. In this study, we evaluated the cell surface induction of GRP78 post irradiation (IR) on lung cancer (A549 and LLC), glioma (D54 and GL261) and endothelial (HUVEC and 3B11) cell lines using flow cytometry. Significantly higher surface expression of GRP78 was observed in all cell lines. We determined the specificity of GIRLRG to bind tumors in vivo using nano SPECT technology. GIRLRG was conjugated to a 40KDa PEG (for longer circulation time) and radiolabelled with 111Indium (111In) using diethylene triamine pentaacetic acid (DTPA) as the chelator. The A549 tumor bearing nude mice were irradiated (3Gy x 3 times) or sham irradiated prior to tail vein injections of 111In labeled GIRLRG. The mice were imaged 48h and 96h post injection using nano CT-SPECT imager. The SPECT images revealed that GIRLRG specifically bound to the irradiated A549 tumors while little or no binding was seen in the sham irradiated tumors. The post-SPECT imaging bio-distrubution also revealed maximum uptake in irradiated tumors. We further evaluated tumor binding of GIRLRG in Glioma (D54), esophageal cancer (OE33), cervical cancer (HT3), and pancreatic cancer (BxPC3). The nano-SPECT imaging showed that GIRLRG specifically bound to all the irradiated tumors tested. Phosphor images of the tumor sections showed that GIRLRG specifically bound to the tumors and not to normal tissues. In conclusion, GIRLRG peptide has high affinity to GRP78 in vitro and in vivo. Radiolabeled GIRLRG is a novel tool for imaging tumors and may be developed further as a therapeutic agent to deliver cancer specific drugs or therapeutic radioisotopes. Citation Format: Vaishali Kapoor, David Dadey, Kim Nguyen, Hua Li, Buck Rogers, Dinesh Thotala, Dennis Hallahan. Peptide probes binding to radiation-inducible cell surface GRP78 as a novel targeting strategy for various tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4202.

Abstract 3326: Targeting lysophosphatidic acid receptor 1 (LPAR1) radiosensitizes poor prognosis cancers

Therapies for poor prognosis cancers, such as lung cancer and glioblastoma, are limited due to radio-resistance and tumor recurrence. Development of molecular targeted therapy can serve as a potential method to improve the efficacy of radiation therapy in both glioblastoma and lung cancer. Ionizing radiation (IR) can activate a series of pro-survival pathways which contributes to the pathogenesis of cancer cells. Among these pathways, cytosolic phospholipase A2 (cPLA2) is an integral component which is activated by IR. Following activation, cPLA2 cleaves arachidonic acid to form phosphatidylcholine (PC) and yields lysophosphatidylcholine (LPC). Autotaxin (ATX) then converts LPC to lysophosphatidic acid (LPA). LPA acts through G-protein-coupled receptors (GPCR) such as LPA1, LPA2, and LPA3, influencing various fundamental cellular functions. Specifically, these receptors can modulate the ability of cancer cells to proliferate, differentiate, and survive. Earlier we have shown that inhibition of ATX decreases invasion and enhances radiation sensitivity of cancer cells. In the present study we investigated the role of LPA receptors and its effect on radioresistance in human A549, murine Lewis Lung Carcinoma (LLC), and rat C6 glioma cells. We analyzed the expression levels of the three LPA receptors in various cancer cell lines including lung, brain, breast and pancreatic cancer cells. We found high expression of LPA1 in LLC, C6 and A549, which we thus chose for further study. Using inhibitors of LPA1/LPA3, Ki16425 and VPC 12249, we determined the effect of LPA1 inhibition on the AKT and ERK activation. Inhibition of LPA1 attenuated phosphorylation of both AKT and ERK in irradiated lung cancer cells. This implicated LPA1 in the regulation of pro-survival signaling, and we hypothesized that LPA1 regulation could influence radioresistance in lung cancer and glioma cells. We targeted LPA1 using siRNA in LLC, C6 and A549 cells which led to an average of 50% reduction in clonogenic survival and cell proliferation after irradiation. Similar results were obtained when LLC and A549 cells were treated with 10μM Ki16425 or 10μM VPC 12249. In addition, LLC and A549 cells which were treated with combinations of radiation and LPA1 inhibitors and analyzed using cell invasion assays showed a 70% reduction in cancer cell invasion. These results indicate that LPA1 could serve as a novel target for cancer treatment due to its ability to enhance the efficacy of radiotherapy in multiple cancer cell lines. Citation Format: Arpine Khudanyan, David Dadey, Rowan Karvas, Rama Kotipatruni, Dennis Hallahan, Dinesh Thotala. Targeting lysophosphatidic acid receptor 1 (LPAR1) radiosensitizes poor prognosis cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3326. doi:10.1158/1538-7445.AM2015-3326

Abstract 1791: Targeting radiation-inducible cell surface GRP78 using GIRLRG peptide as a novel imaging and therapeutic strategy for tumors

The greatest challenge in radiotherapy is to maximize radiation doses to tumors while sparing the normal tissues. Ionizing radiation has been shown to induce expression of proteins on surface of tumor cells. Using this strategy, drug delivery can be targeted specifically to cancer and guided by use of a beam of radiation. We used phage-displayed peptide libraries to discover radiation-inducible antigens in different cancer models. One such inducible-antigen is Tax interacting protein-1 (Tip-1) which was shown to bind the hexapeptide HVGGSSV. TIP-1 is an atypical PDZ protein consisting of only a single PDZ domain that is predominantly cytosolic protein and expressed in all types of cells. Overall TIP-1 expression levels in tumor cells and non-tumor cells are similar; however it is over-expressed on the tumor cell surface following irradiation. In this study, we evaluated the affinity of binding of HVGGSSV and a scrambled peptide to TIP-1 using surface plasmon resonance (SPR) technology. A KD (dissociation constant) of 3.3e-8M for HVGGSSV and 1.37e-3M for the scrambled peptide was obtained. We next determined the specificity of HVGGSSV to bind tumors in vivo using nano SPECT technology. HVGGSSV was conjugated to a 40KDa PEG (for longer circulation time) and radiolabelled with 111Indium (111In) using diethylene triamine pentaacetic acid (DTPA) as the chelator. The A549 tumor bearing nude mice were irradiated (3Gy x 3 times) or sham irradiated prior to tail vein injections of 111In labeled HVGGSSV. The mice were imaged 48h and 96h post injection using nano CT-SPECT imager. The SPECT images revealed that HVGGSSV specifically bound to the irradiated A549 tumors while little or no binding was seen in the sham irradiated tumors. The post-SPECT imaging bio-distrubution also revealed maximum uptake in irradiated tumors. We further evaluated tumor binding of HVGGSSV in Glioma (D54), esophageal cancer (OE33), cervical cancer (HT3), and pancreatic cancer (BxPC3). The nano-SPECT imaging showed that HVGGSSV specifically bound to all the irradiated tumors tested. Phosphor images of the tumor sections showed that HVGGSSV specifically bound to the tumors and not to normal tissues. In conclusion, HVGGSSV peptide has high affinity to TIP-1 in vitro and in vivo. Radiolabeled HVGGSSV is a novel tool for imaging tumors and may be developed further as a therapeutic agent to deliver cancer specific drugs or therapeutic radioisotopes. Citation Format: Vaishali Kapoor, David Dadey, Kim Nguyen, Hua Li, Buck Rogers, Dinesh Thotala, Dennis Hallahan. Targeting radiation-inducible cell surface TIP-1 using HVGGSSV peptide as a novel imaging and therapeutic strategy for tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1491. doi:10.1158/1538-7445.AM2015-1491