Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells.

OBJECTIVE: Lactobacillus rhamnosus GG (LGG), a probiotic, given by gavage is radioprotective of the mouse intestine. LGG-induced radioprotection is toll-like receptor 2 (TLR2) and cyclooxygenase-2 (COX-2)-dependent and is associated with the migration of COX-2+mesenchymal stem cells (MSCs) from the lamina propria of the villus to the lamina propria near the crypt epithelial stem cells. Our goals were to define the mechanism of LGG radioprotection including identification of the TLR2 agonist, and the mechanism of the MSC migration and to determine the safety and efficacy of this approach in models relevant to clinical radiation therapy. DESIGN: Intestinal radioprotection was modelled in vitro with cell lines and enteroids as well as in vivo by assaying clinical outcomes and crypt survival. Fractionated abdominal and single dose radiation were used along with syngeneic CT26 colon tumour grafts to assess tumour radioprotection. RESULTS: LGG with a mutation in the processing of lipoteichoic acid (LTA), a TLR2 agonist, was not radioprotective, while LTA agonist and native LGG were. An agonist of CXCR4 blocked LGG-induced MSC migration and LGG-induced radioprotection. LGG given by gavage induced expression of CXCL12, a CXCR4 agonist, in pericryptal macrophages and depletion of macrophages by clodronate liposomes blocked LGG-induced MSC migration and radioprotection. LTA effectively protected the normal intestinal crypt, but not tumours in fractionated radiation regimens. CONCLUSIONS: LGG acts as a 'time-release capsule' releasing radioprotective LTA. LTA then primes the epithelial stem cell niche to protect epithelial stem cells by triggering a multicellular, adaptive immune signalling cascade involving macrophages and PGE2 secreting MSCs. TRIAL REGISTRATION NUMBER: NCT01790035; Pre-results.

Impact of concurrent versus adjuvant chemotherapy on the severity and duration of lymphopenia in glioma patients treated with radiation therapy.

Prolonged severe lymphopenia has been shown to persist beyond a year in glioma patients after radiation therapy (RT) with concurrent and adjuvant chemotherapy. This study examines the differential impact of concurrent versus adjuvant chemotherapy on lymphopenia after RT. WHO grade II-III glioma patients who received RT with concurrent and/or adjuvant chemotherapy from 2007 to 2016 were retrospectively analyzed. Concurrent chemotherapy was temozolomide (TMZ), and adjuvant chemotherapy was either TMZ or procarbazine/lomustine/vincristine (PCV). Absolute lymphocyte count (ALC) was analyzed at baseline, 1.5, 3, 6, and 12 months after the start of RT. Univariable and multivariable logistic regression were used to identify the clinical variables in predicting acute or late lymphopenia. There were 151 patients with evaluable ALC: 91 received concurrent and adjuvant TMZ (CRT + ADJ), 32 received only concurrent TMZ (CRT), and 28 received only adjuvant TMZ or PCV (ADJ). There were 9 (10%) versus 6 (19%) versus 0 (0%) cases of grade 3 lymphopenia (ALC < 500/mm3) at 6 weeks and 4 (6%) versus 0 (0%) versus 3 (17%) cases at 12 months in CRT + ADJ, CRT and ADJ groups, respectively. On multivariable analyses, concurrent chemotherapy (odds ratio [OR] 72.3, p < 0.001), female sex (OR 10.8, p < 0.001), and older age (OR 1.06, p = 0.002) were the most significant predictors for any grade ≥ 1 lymphopenia (ALC < 1000/mm3) at 1.5 months. Older age (OR 1.08, p = 0.02) and duration of adjuvant chemotherapy (OR 1.19, p = 0.003) were significantly associated with grade ≥ 1 lymphopenia at 12 months. Thus, concurrent chemotherapy appears as the dominant contributor to the severity of acute lymphopenia after RT in WHO grade II-III glioma patients, and duration of adjuvant chemotherapy appears as the key factor to prolonged lymphopenia.

A Hypoxia-Targeted Boron Neutron Capture Therapy Agent for the Treatment of Glioma.

PURPOSE: Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation has been limited by the poor tumor selectivity of agents. To address this unmet need, a boronated 2-nitroimidazole derivative (B-381) was synthesized and evaluated for its capability of targeting hypoxic glioma cells. METHODS: B-381 has been synthesized from a 1-step reaction. Using D54 and U87 glioma cell lines, the in vitro cytotoxicity and cellular accumulation of B-381 has been evaluated under normoxic and hypoxic conditions compared to L-boronophenylalanine (BPA). Furthermore, tumor retention of B-381 was evaluated in vivo. RESULTS: B-381 had low cytotoxicity in normal and cancer cells. Unlike BPA, B-381 illustrated preferential retention in hypoxic glioma cells compared to normoxic glioma cells and normal tissues in vitro. In vivo, B-381 illustrated significantly higher long-term tumor retention compared to BPA, with 9.5-fold and 6.5-fold higher boron levels at 24 and 48 h, respectively. CONCLUSIONS: B-381 represents a new class of BNCT agents in which their selectivity to tumors is based on hypoxic tumor metabolism. Further studies are warranted to evaluate B-381 and similar compounds as preclinical candidates for future BNCT clinical trials for the treatment of glioma.

Radiation-induced circulating myeloid-derived suppressor cells induce systemic lymphopenia after chemoradiotherapy in patients with glioblastoma.

Severe and prolonged lymphopenia frequently occurs in patients with glioblastoma after standard chemoradiotherapy and has been associated with worse survival, but its underlying biological mechanism is not well understood. To address this, we performed a correlative study in which we collected and analyzed peripheral blood of patients with glioblastoma (n = 20) receiving chemoradiotherapy using genomic and immune monitoring technologies. RNA sequencing analysis of the peripheral blood mononuclear cells (PBMC) showed an elevated concentration of myeloid-derived suppressor cell (MDSC) regulatory genes in patients with lymphopenia when compared with patients without lymphopenia after chemoradiotherapy. Additional analysis including flow cytometry and single-cell RNA sequencing further confirmed increased numbers of circulating MDSC in patients with lymphopenia when compared with patients without lymphopenia after chemoradiotherapy. Preclinical murine models were also established and demonstrated a causal relationship between radiation-induced MDSC and systemic lymphopenia using transfusion and depletion experiments. Pharmacological inhibition of MDSC using an arginase-1 inhibitor (CB1158) or phosphodiesterase-5 inhibitor (tadalafil) during radiation therapy (RT) successfully abrogated radiation-induced lymphopenia and improved survival in the preclinical models. CB1158 and tadalafil are promising drugs in reducing radiation-induced lymphopenia in patients with glioblastoma. These results demonstrate the promise of using these classes of drugs to reduce treatment-related lymphopenia and immunosuppression.

A pilot phase Ib study to evaluate tadalafil to overcome immunosuppression during chemoradiotherapy for IDH-wild-type glioblastoma.

Background: Myeloid-derived suppressor cells (MDSCs) are critical regulators of immunosuppression and radioresistance in glioblastoma (GBM). The primary objective of this pilot phase Ib study was to validate the on-target effect of tadalafil on inhibiting MDSCs in peripheral blood and its safety when combined with chemoradiotherapy in GBM patients. Methods: Patients with newly diagnosed IDH-wild-type GBM received radiation therapy (RT) and temozolomide (TMZ) combined with oral tadalafil for 2 months. A historical cohort of 12 GBM patients treated with RT and TMZ was used as the comparison group. The ratio of MDSCs, T cells, and cytokines at week 6 of RT compared to baseline were analyzed using flow cytometry. Progression-free survival (PFS) and overall survival (OS) were estimated by the Kaplan-Meier method. Results: Tadalafil was well tolerated with no dose-limiting toxicity among 16 evaluable patients. The tadalafil cohort had a significantly lower ratio of circulating MDSCs than the control: granulocytic-MDSCs (mean 0.78 versus 3.21, respectively, P = 0.01) and monocytic-MDSCs (1.02 versus 1.96, respectively, P = 0.006). Tadalafil increased the CD8 ratio compared to the control (1.99 versus 0.70, respectively, P < 0.001), especially the PD-1-CD8 T cells expressing Ki-67, CD38, HLA-DR, CD28, and granzyme B. Proinflammatory cytokine IL-1ß was also significantly increased after tadalafil compared to the control. The tadalafil cohort did not have significantly different PFS and OS than the historical control. Conclusions: Concurrent tadalafil is well tolerated during chemoradiotherapy for GBM. Tadalafil is associated with a reduction of peripheral MDSCs after chemoradiotherapy and increased CD8 T-cell proliferation and activation.

Blocking the functional domain of TIP1 by antibodies sensitizes cancer to radiation therapy.

Non-small-cell lung cancer (NSCLC) and glioblastoma (GB) have poor prognoses. Discovery of new molecular targets is needed to improve therapy. Tax interacting protein 1 (TIP1), which plays a role in cancer progression, is overexpressed and radiation-inducible in NSCLC and GB. We evaluated the effect of an anti-TIP1 antibody alone and in combination with ionizing radiation (XRT) on NSCLC and GB in vitro and in vivo. NSCLC and GB cells were treated with anti-TIP1 antibodies and evaluated for proliferation, colony formation, endocytosis, and cell death. The efficacy of anti-TIP1 antibodies in combination with XRT on tumor growth was measured in mouse models of NSCLC and GB. mRNA sequencing was performed to understand the molecular mechanisms involved in the action of anti-TIP1 antibodies. We found that targeting the functional domain of TIP1 leads to endocytosis of the anti-TIP1 antibody followed by reduced proliferation and increased apoptosis-mediated cell death. Anti-TIP1 antibodies bound specifically (with high affinity) to cancer cells and synergized with XRT to significantly increase cytotoxicity in vitro and reduce tumor growth in mouse models of NSCLC and GB. Importantly, downregulation of cancer survival signaling pathways was found in vitro and in vivo following treatment with anti-TIP1 antibodies. TIP1 is a new therapeutic target for cancer treatment. Antibodies targeting the functional domain of TIP1 exhibited antitumor activity and enhanced the efficacy of radiation both in vitro and in vivo. Anti-TIP1 antibodies interrupt TIP1 function and are effective cancer therapy alone or in combination with XRT in mouse models of human cancer.

Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli.

Translation initiation from the ribosomal P-site is the specialty of the initiator tRNAs (tRNA(fMet)). Presence of the three consecutive G-C base pairs (G29-C41, G30-C40 and G31-C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P-site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNA(fMet) containing mutations in one, two or all the three G-C base pairs. The strain shows a severe deficiency of methionine and S-adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.

Long-Acting Recombinant Human Interleukin-7, NT-I7, Increases Cytotoxic CD8 T Cells and Enhances Survival in Mouse Glioma Models.

PURPOSE: Patients with glioblastoma (GBM) are treated with radiotherapy (RT) and temozolomide (TMZ). These treatments may cause prolonged systemic lymphopenia, which itself is associated with poor outcomes. NT-I7 is a long-acting IL7 that expands CD4 and CD8 T-cell numbers in humans and mice. We tested whether NT-I7 prevents systemic lymphopenia and improves survival in mouse models of GBM. EXPERIMENTAL DESIGN: C57BL/6 mice bearing intracranial tumors (GL261 or CT2A) were treated with RT (1.8 Gy/day × 5 days), TMZ (33 mg/kg/day × 5 days), and/or NT-I7 (10 mg/kg on the final day of RT). We followed the mice for survival while serially analyzing levels of circulating T lymphocytes. We assessed regulatory T cells (Treg) and cytotoxic T lymphocytes in the tumor microenvironment, cervical lymph nodes, spleen, and thymus, and hematopoietic stem and progenitor cells in the bone marrow. RESULTS: GBM tumor-bearing mice treated with RT+NT-I7 increased T lymphocytes in the lymph nodes, thymus, and spleen, enhanced IFNγ production, and decreased Tregs in the tumor which was associated with a significant increase in survival. NT-I7 also enhanced central memory and effector memory CD8 T cells in lymphoid organs and tumor. Depleting CD8 T cells abrogated the effects of NT-I7. Furthermore, NT-I7 treatment decreased progenitor cells in the bone marrow. CONCLUSIONS: In orthotopic glioma-bearing mice, NT-I7 mitigates RT-related lymphopenia, increases cytotoxic CD8 T lymphocytes systemically and in the tumor, and improves survival. A phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957).

GSK-3ß inhibition promotes cell death, apoptosis, and in vivo tumor growth delay in neuroblastoma Neuro-2A cell line.

Neuroblastoma is the most common extracranial solid tumor of childhood. While survival rates are high for localized disease, treatment response remains poor for a subset of patients with large tumors or disseminated disease. Thus, there remains much room for improvement in treatment strategies for this disease. Using in vitro and in vivo systems, we present glycogen synthase kinase-3ß (GSK-3ß) inhibition as a potential mechanism to treat neuroblastoma. Using the specific GSK-3ß inhibitor SB415286, we demonstrate that GSK-3ß inhibition decreases the viability of Neuro-2A cells, as determined by cell proliferation assay and clonogenic survival. Moreover, we show that GSK-3ß inhibition induces apoptosis in neuroblastoma cells, as determined by Annexin V staining and confirmed with DAPI staining. Using flow cytometry, we are able to demonstrate that SB415286 induces the accumulation of cells in the G2/M phase of the cell cycle. Finally, we show that these in vitro results translate into delayed tumor growth in vivo using a heterotopic tumor model in nude mice treated with SB415286. These findings suggest that GSK-3ß is a potential molecular target for the treatment of neuroblastoma.

Targeting a Radiosensitizing Antibody-Drug Conjugate to a Radiation-Inducible Antigen.

PURPOSE: We recently discovered that anti-TIP1 antibody activates endocytosis in cancer cells, which facilitates retention of antibody and dissociation of a conjugated drug. To improve the pharmacokinetics and cancer specificity of radiosensitizing drugs, we utilized antibody-drug conjugates (ADCs) that bind specifically to radiation-inducible antigen, TIP1, on non-small cell lung cancer (NSCLC). This approach exploits the long circulation time of antibodies to deliver a radiosensitizing drug to cancer each day during radiotherapy. EXPERIMENTAL DESIGN: Antibodies to TIP1 were prioritized based on affinity, cancer-specific binding, and internalization. The lead antibody, 7H5, was conjugated with a cytotoxic drug MMAE because of its ability to radiosensitize cancer. Cytotoxicity, colony formation, and tumor growth studies were performed with 7H5-VcMMAE in combination with radiation. RESULTS: 7H5 showed a high affinity to recombinant TIP1 protein and radiation-inducible TIP1 on the cancer cell surface. 7H5 undergoes endocytosis in NSCLC cells in vitro. We obtained an average drug-to-antibody ratio (DAR) of 4.25 for 7H5-VcMMAE. A 70% reduction in viable cells was observed following 7H5-VcMMAE treatment compared with 7H5 alone in both A549 and H1299 cells. 7H5-VcMMAE sensitized NSCLC cells to radiation, thereby significantly decreasing the surviving fraction. The ADC combined with radiation showed a prolonged delay in tumor growth and improved survival in A549 and H1299 tumor models. CONCLUSIONS: Targeting radiation-inducible TIP1 with a radiosensitizing ADC is a promising strategy to enhance the therapeutic efficacy of NSCLC. This novel approach of targeting with ADCs to radiation-inducible antigens will lead to clinical trials in lung cancer patients treated with radiotherapy.

TAF15 contributes to the radiation-inducible stress response in cancer.

Resistance to radiation therapy is a significant problem in the treatment of non-small cell lung cancer (NSCLC). There is an unmet need to discover new molecular targets for drug development in combination with standard of care cancer therapy. We found that TAF15 was radiation-inducible using phage-displayed peptide libraries. In this study, we report that overexpression of TAF15 is correlated with worsened survival in NSCLC patients. Radiation treatment led to surface induction of TAF15 in vitro and in vivo. We genetically silenced TAF15 which led to a significant reduction in proliferation of NSCLC cells. Cells depleted of TAF15 exhibited cell cycle arrest and enhanced apoptosis through activation and accumulation of p53. In combination with radiation, TAF15 knockdown led to a significant reduction in the surviving fraction of NSCLC cell lines. To determine the importance of TAF15 surface expression, we targeted TAF15 with an antibody. In combination with radiation, the anti-TAF15 antibody led to a reduction in the surviving fraction of cancer cells. These studies show that TAF15 is a radiation-inducible molecular target that is accessible to anti-cancer antibodies and enhances cell viability in response to radiation.

Interferon-Induced IDO1 Mediates Radiation Resistance and Is a Therapeutic Target in Colorectal Cancer.

Colorectal cancer is a major cause of mortality worldwide. Chemotherapy and radiation remain standard treatment for locally advanced disease, with current immune-targeting therapies applying to only a small subset of patients. Expression of the immuno-oncology target indoleamine 2,3 dioxygenase 1 (IDO1) is associated with poor colorectal cancer clinical outcomes but is understudied as a potential treatment target. In this study, we examined the interaction between the IDO1 pathway and radiotherapy in colorectal cancer. We used human and mouse colorectal cancer cell lines, organoids, mouse syngeneic colorectal cancer tumor graft models, and colorectal cancer tissues from patients who received radiotherapy. IDO1 activity was blocked using the clinical IDO1 inhibitor epacadostat and by genetic disruption. We found that radiation induced IDO1 overexpression in colorectal cancer through type I and II IFN signaling. IDO1 enzymatic activity directly influenced colorectal cancer radiation sensitivity. IDO1 inhibition sensitized colorectal cancer to radiation-induced cell death, whereas the IDO1 metabolite kynurenine promoted radioprotection. IDO1 inhibition also potentiated Th1 cytokines and myeloid cell-modulating factors in the tumor microenvironment and promoted an abscopal effect on tumors outside the radiation field. Conversely, IDO1 blockade protected the normal small intestinal epithelium from radiation toxicity and accelerated recovery from radiation-induced weight loss, indicating a role in limiting side effects. These data demonstrated that IDO1 inhibition potentiates radiotherapy effectiveness in colorectal cancer. The findings also provide rationale and mechanistic insight for the study of IDO1 inhibitors as adjuvant therapy to radiation in patients with locally advanced sporadic and colitis-associated colorectal cancer.

Radiation induces iatrogenic immunosuppression by indirectly affecting hematopoiesis in bone marrow.

The immune system plays a vital role in cancer therapy, especially with the advent of immunotherapy. Radiation therapy induces iatrogenic immunosuppression referred to as radiation-induced lymphopenia (RIL). RIL correlates with significant decreases in the overall survival of cancer patients. Although the etiology and severity of lymphopenia are known, the mechanism(s) of RIL are largely unknown. We found that irradiation not only had direct effects on circulating lymphocytes but also had indirect effects on the spleen, thymus, and bone marrow. We found that irradiated cells traffic to the bone marrow and bring about the reduction of hematopoietic stem cells (HSC) and progenitor cells. Using mass cytometry analysis (CyTOF) of the bone marrow, we found reduced expression of CD11a, which is required for T cell proliferation and maturation. RNA Sequencing and gene set enrichment analysis of the bone marrow cells following irradiation showed down-regulation of genes involved in hematopoiesis. Identification of CD11a and hematopoietic genes involved in iatrogenic immune suppression can help identify mechanisms of RIL.

Therapy-Induced Senescence Drives Bone Loss.

Chemotherapy is important for cancer treatment, however, toxicities limit its use. While great strides have been made to ameliorate the acute toxicities induced by chemotherapy, long-term comorbidities including bone loss remain a significant problem. Chemotherapy-driven estrogen loss is postulated to drive bone loss, but significant data suggests the existence of an estrogen-independent mechanism of bone loss. Using clinically relevant mouse models, we showed that senescence and its senescence-associated secretory phenotype (SASP) contribute to chemotherapy-induced bone loss that can be rescued by depleting senescent cells. Chemotherapy-induced SASP could be limited by targeting the p38MAPK-MK2 pathway, which resulted in preservation of bone integrity in chemotherapy-treated mice. These results transform our understanding of chemotherapy-induced bone loss by identifying senescent cells as major drivers of bone loss and the p38MAPK-MK2 axis as a putative therapeutic target that can preserve bone and improve a cancer survivor's quality of life. SIGNIFICANCE: Senescence drives chemotherapy-induced bone loss that is rescued by p38MAPK or MK2 inhibitors. These findings may lead to treatments for therapy-induced bone loss, significantly increasing quality of life for cancer survivors.

Tumor microenvironment-targeted nanoparticles loaded with bortezomib and ROCK inhibitor improve efficacy in multiple myeloma.

Drug resistance and dose-limiting toxicities are significant barriers for treatment of multiple myeloma (MM). Bone marrow microenvironment (BMME) plays a major role in drug resistance in MM. Drug delivery with targeted nanoparticles have been shown to improve specificity and efficacy and reduce toxicity. We aim to improve treatments for MM by (1) using nanoparticle delivery to enhance efficacy and reduce toxicity; (2) targeting the tumor-associated endothelium for specific delivery of the cargo to the tumor area, and (3) synchronizing the delivery of chemotherapy (bortezomib; BTZ) and BMME-disrupting agents (ROCK inhibitor) to overcome BMME-induced drug resistance. We find that targeting the BMME with P-selectin glycoprotein ligand-1 (PSGL-1)-targeted BTZ and ROCK inhibitor-loaded liposomes is more effective than free drugs, non-targeted liposomes, and single-agent controls and reduces severe BTZ-associated side effects. These results support the use of PSGL-1-targeted multi-drug and even non-targeted liposomal BTZ formulations for the enhancement of patient outcome in MM.

PEGylated peptide to TIP1 is a novel targeting agent that binds specifically to various cancers in vivo.

Targeted molecular imaging allows specific visualization and monitoring of tumors. Cancer-specific peptides have been developed for imaging and therapy. Peptides that specifically target cancer have several advantages including, ease of synthesis, low antigenicity, and enhanced diffusion into tissues. We developed the HVGGSSV peptide as a molecular targeting/imaging agent. HVGGSSV targets Tax interacting protein 1 (TIP1) which is a 14 kDa PDZ domain-containing protein that is overexpressed in cancer. We docked HVGGSSV in silico using the three-dimensional structure of TIP1 and found the binding energy was -6.0 kCal/mol. The binding affinity of HVGGSSV to TIP1 protein was found to have a KD of 3.3 × 10-6 M using surface plasmon resonance. We conjugated a 40 kDa PEG to HVGGSSV to enhance the circulation and evaluated the tumor binding in nude mice bearing heterotopic cervical (HT3), esophageal (OE33), pancreatic (BXPC3), lung (A549) and glioma (D54) tumors. NanoSPECT/CT imaging of the mice was performed 48 h and 72 h after injecting with 111Indium (111In) labeled PEG-HVGGSSV or PEG-control peptide. SPECT imaging revealed that 111In-PEG-HVGGSSV specifically bound to cervical, esophageal, pancreatic, lung and brain tumors. Post SPECT biodistribution data further validated tumor-specific binding. Overall, HVGGSSV peptide specifically binds to the major groove of the TIP1 protein surface. PEGylated-HVGGSSV could be used to target cancers that overexpress TIP1.

Differential efficacy of combined therapy with radiation and AEE788 in high and low EGFR-expressing androgen-independent prostate tumor models.

PURPOSE: To determine the efficacy of combining radiation (XRT) with a dual epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor inhibitor, AEE788, in prostate cancer models with different levels of EGFR expression. METHODS AND MATERIALS: Immunoblotting was performed for EGFR, phosphorylated-EGFR, and phosphorylated-AKT in prostate cancer cells. Clonogenic assays were performed on DU145, PC-3, and human umbilical vein endothelial cells treated with XRT +/- AEE788. Tumor xenografts were established for DU145 and PC-3 on hind limbs of athymic nude mice assigned to four treatment groups: (1) control, (2) AEE788, (3) XRT, and (4) AEE788 + XRT. Tumor blood flow and growth measurements were performed using immunohistochemistry and imaging. RESULTS: AEE788 effectively decreased phosphorylated-EGFR and phosphorylated-AKT levels in DU145 and PC-3 cells. Clonogenic assays showed no radiosensitization for DU145 and PC-3 colonies treated with AEE788 + XRT. However, AEE788 caused decreased proliferation in DU145 cells. AEE788 showed a radiosensitization effect in human umbilical vein endothelial cells and increased apoptosis susceptibility. Concurrent AEE788 + XRT compared with either alone led to significant tumor growth delay in DU145 tumors. Conversely, PC-3 tumors derived no added benefit from combined-modality therapy. In DU145 tumors, a significant decrease in tumor blood flow with combination therapy was shown by using power Doppler sonography and tumor blood vessel destruction on immunohistochemistry. Maldi-spectrometry (MS) imaging showed that AEE788 is bioavailable and heterogeneously distributed in DU145 tumors undergoing therapy. CONCLUSIONS: AEE788 + XRT showed efficacy in vitro/in vivo with DU145-based cell models, whereas PC-3-based models were adequately treated with XRT alone without added benefit from combination therapy. These findings correlated with differences in EGFR expression and showed effects on both tumor cell proliferation and vascular destruction.

Lithium treatment prevents neurocognitive deficit resulting from cranial irradiation.

Curative cancer treatment regimens often require cranial irradiation, resulting in lifelong neurocognitive deficiency in cancer survivors. This deficiency is in part related to radiation-induced apoptosis and decreased neurogenesis in the subgranular zone of the hippocampus. We show that lithium treatment protects irradiated hippocampal neurons from apoptosis and improves cognitive performance of irradiated mice. The molecular mechanism of this effect is mediated through multiple pathways, including Akt/glycogen synthase kinase-3beta (GSK-3beta) and Bcl-2/Bax. Lithium treatment of the cultured mouse hippocampal neurons HT-22 induced activation of Akt (1.5-fold), inhibition of GSK-3beta (2.2-fold), and an increase in Bcl-2 protein expression (2-fold). These effects were sustained when cells were treated with lithium in combination with ionizing radiation. In addition, this combined treatment led to decreased expression (40%) of the apoptotic protein Bax. The additional genes regulated by lithium were identified by microarray, such as decorin and Birc1f. In summary, we propose lithium treatment as a novel therapy for prevention of deleterious neurocognitive consequences of cranial irradiation.

PERK Regulates Glioblastoma Sensitivity to ER Stress Although Promoting Radiation Resistance.

The aggressive nature and inherent therapeutic resistance of glioblastoma multiforme (GBM) has rendered the median survival of afflicted patients to 14 months. Therefore, it is imperative to understand the molecular biology of GBM to provide new treatment options to overcome this disease. It has been demonstrated that the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is an important regulator of the endoplasmic reticulum (ER) stress response. PERK signaling has been observed in other model systems after radiation; however, less is known in the context of GBM, which is frequently treated with radiation-based therapies. To investigate the significance of PERK, we studied activation of the PERK-eIF2α-ATF4 pathway in GBM after ionizing radiation (IR). By inhibiting PERK, it was determined that ionizing radiation (IR)-induced PERK activity led to eIF2α phosphorylation. IR enhanced the prodeath component of PERK signaling in cells treated with Sal003, an inhibitor of phospho-eIF2α phosphatase. Mechanistically, ATF4 mediated the prosurvival activity during the radiation response. The data support the notion that induction of ER stress signaling by radiation contributes to adaptive survival mechanisms during radiotherapy. The data also support a potential role for the PERK/eIF2α/ATF4 axis in modulating cell viability in irradiated GBM.Implications: The dual function of PERK as a mediator of survival and death may be exploited to enhance the efficacy of radiation therapy.Visual Overview: http://mcr.aacrjournals.org/content/16/10/1447/F1.large.jpg Mol Cancer Res; 16(10); 1447-53. ©2018 AACR.

Post-operative radiation effects on lymphopenia, neutrophil to lymphocyte ratio, and clinical outcomes in palatine tonsil cancers.

OBJECTIVE: To evaluate radiation-induced lymphopenia associated with unilateral vs. bilateral neck radiation and to test post-treatment neutrophil to lymphocyte ratio (NLR) as a prognostic clinical biomarker. METHODS: This was a single academic center retrospective review of palatine tonsil squamous cell cancer patients treated with post-operative intensity modulated radiation therapy (IMRT) from 1997 to 2013. Absolute lymphocyte count (ALC) and NLR were evaluated during and after radiation for up to a year. Correlations of lab values with loco-regional control (LRC), freedom from distant metastases (FFDM), and overall survival (OS) were assessed. RESULTS: Ninety-nine patients with median follow up 5.8 years had ALC recorded at least at baseline and within one year of starting RT. Acute grade 3-4 lymphopenia (<10 weeks from RT start) occurred in 79% of bilateral neck RT patients (n = 70) and 58% of unilateral neck RT patients (n = 29), p = 0.03. There was no significant difference in late grade 3-4 (p = 0.12) lymphopenia. In a multivariable Cox regression model, acute NLR > 11.875 correlated with worse OS (HR = 4.4, 95% CI 1.2-16). Late NLR > 6.875 independently correlated with significantly worse FFDM (HR = 16, 95% CI 1.9-137) and OS (HR = 12, 95% CI 3.0-48). CONCLUSIONS: Unilateral neck radiation may prevent acute iatrogenic immunosuppression. In exploratory analyses, elevated post-treatment NLR was associated with risk for distant metastases and death.