Phosphatase and tensin homologue deficiency in glioblastoma confers resistance to radiation and temozolomide that is reversed by the protease inhibitor nelfinavir.

Glioblastomas are malignant brain tumors that are very difficult to cure, even with aggressive therapy consisting of surgery, chemotherapy, and radiation. Glioblastomas frequently have loss of the phosphatase and tensin homologue (PTEN), leading to the activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway. We examined whether PTEN deficiency leads to radioresistance and whether this can be reversed by nelfinavir, a protease inhibitor that decreases Akt signaling. Nelfinavir decreased Akt phosphorylation and enhanced radiosensitization in U251MG and U87MG glioblastoma cells, both of which are PTEN deficient. In the derivative line U251MG-PTEN, induction of wild-type PTEN with doxycycline decreased P-Akt expression and increased radiosensitivity to a similar extent as nelfinavir. Combining these two approaches had no greater effect on radiosensitivity than either alone. This epistasis-type analysis suggests that the nelfinavir acts along the Akt pathway to radiosensitize cells. However, nelfinavir neither decreased Akt phosphorylation in immortalized human astrocytes nor radiosensitized them. Radiosensitization was also assessed in vivo using a tumor regrowth delay assay in nude mice implanted with U87MG xenografts. The mean time to reach 1,000 mm(3) in the radiation + nelfinavir group was 71 days, as compared with 41, 34, or 45 days for control, nelfinavir alone, or radiation alone groups, respectively. A significant synergistic effect on tumor regrowth was detected between radiation and nelfinavir. (P = 0.01). Nelfinavir also increased the sensitivity of U251MG cells to temozolomide. These results support the clinical investigation of nelfinavir in combination with radiation and temozolomide in future clinical trials for patients with glioblastomas.

Clinical Outcomes of the HIV Protease Inhibitor Nelfinavir With Concurrent Chemoradiotherapy for Unresectable Stage IIIA/IIIB Non-Small Cell Lung Cancer: A Phase 1/2 Trial.

IMPORTANCE: Local failure after chemoradiotherapy (CT-RT) significantly contributes to mortality in patients with locally advanced non-small cell lung cancer (LA-NSCLC). One approach to improve local control is through targeted radiosensitization of the tumor. OBJECTIVE: To evaluate the dose-limiting toxic effects, maximally tolerated dose, and recommended phase 2 dose of the protease inhibitor nelfinavir mesylate, administered concurrently with CT-RT in patients with LA-NSCLC, and, in the phase 2 portion of the study, to estimate the objective response rate, local and distant failure rates, and overall survival. DESIGN, SETTING, AND PARTICIPANTS: This prospective, open-label, single-group, single-institution phase 1/2 trial tested the oral protease inhibitor nelfinavir in combination with concurrent CT-RT in 35 patients aged 18 to 89 years with biopsy-confirmed unresectable stage IIIA/IIIB LA-NSCLC and a minimum Karnofsky performance status from June 29, 2007, to February 22, 2012, with an analysis date of May 9, 2017. Median follow-up for all patients was 6.8 years, with a minimum 5 years of follow-up for all survivors. INTERVENTIONS: Oral nelfinavir mesylate, 625 mg, twice daily or 1250 mg, twice daily was administered for 7 to 14 days before and during concurrent CT-RT. MAIN OUTCOMES AND MEASURES: Graded toxic effects, overall survival, local failure, distant failure, objective response rate, and progression-free survival as measured by Response Evaluation Criteria in Solid Tumors, version 1.1. RESULTS: Thirty-five patients (16 women and 19 men; median age, 60 years [range, 39-79 years]) enrolled and met protocol-specified criteria for adherence, with 5 at a dose of 625 mg twice daily and 30 at a dose of 1250 mg twice daily. No dose-limiting toxic effects were observed. No grade 4 or higher nonhematologic toxic effects were observed. Thirty-three of the 35 patients had evaluable posttreatment computed tomographic scans, with an objective response rate of 94% (31 of 33; 95% CI, 86%-100%). The cumulative incidence of local failure was 39% (95% CI, 30.5%-47.5%). Median progression-free survival was 11.7 months (95% CI, 6.2-17.1 months). Median overall survival for all patients was 41.1 months (95% CI, 19.0-63.1 months); the 5-year mean (SE) overall survival rate was 37.1% (8.2%). CONCLUSIONS AND RELEVANCE: This study suggests that nelfinavir administered with concurrent CT-RT is associated with acceptable toxic effects and a promising objective response rate, local failure, progression-free survival, and overall survival in unresectable LA-NSCLC. These data suggest that nelfinavir may enhance the efficacy of standard CT-RT in this disease. Additional testing in the randomized phase 3 setting should be conducted to establish the improvement associated with nelfinavir with concurrent CT-RT. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00589056.

Nelfinavir down-regulates hypoxia-inducible factor 1alpha and VEGF expression and increases tumor oxygenation: implications for radiotherapy.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) expression. We examined the effect of nelfinavir, an HIV protease inhibitor that inhibits Akt signaling, on VEGF and HIF-1alpha expression and on angiogenesis, tumor oxygenation, and radiosensitization. Nelfinavir decreases VEGF expression under normoxia via the transcription factor Sp1, which regulates the proximal core VEGF promoter. Nelfinavir decreased Sp1 phosphorylation and decreased Sp1 binding to a probe corresponding to the proximal VEGF promoter in a gel shift assay. Nelfinavir also decreased the hypoxic induction of HIF-1alpha, which also regulates the VEGF promoter, most likely by decreasing its translation. The effect of nelfinavir on VEGF expression had the functional consequence of decreasing angiogenesis in an in vivo Matrigel plug assay. To determine the effect this might have on tumor radiosensitization, we did tumor regrowth assays with xenografts in nude mice. The combination of nelfinavir and radiation increased time to regrowth compared with radiation alone whereas nelfinavir alone had little effect on tumor regrowth. This radiosensitizing effect was greater than suggested by in vitro clonogenic survival assays. One possible explanation for the discordance is that nelfinavir has an effect on tumor oxygenation. Therefore, we examined this with the hypoxia marker EF5 and found that nelfinavir leads to increased oxygenation within tumor xenografts. Our results suggest that nelfinavir decreases HIF-1alpha/VEGF expression and tumor hypoxia, which could play a role in its in vivo radiosensitizing effect. These data support the use of nelfinavir in combination with radiation in future clinical trials.

Bortezomib sensitizes human head and neck carcinoma cells SQ20B to radiation.

The proteasome inhibitor bortezomib was tested in a cell screen as a single agent with good efficacy in multiple hematologic and solid cancer cell lines. Phase II/III studies have supported the use of bortezomib in hematologic malignancies. In solid tumors, however, the results have been poor. There is data that bortezomib can induce PTEN expression resulting in down-regulation of PI3K-Akt signaling. We and others have shown that down-regulation of Akt results in radiation sensitization. We therefore evaluated the use of bortezomib in the head and neck cancer cell line SQ20B as a radiation sensitizer. SQ20B have a constitutively active mutation in EGFR resulting in a robust Akt response. We found that 10 nM of bortezomib decreased Akt signaling to almost undetectable. This same concentration decreased the surviving fraction after 2 Gy (SF2) from 0.77 to 0.45. Given that radiation is usually given at 2 Gy increments daily for 30 or more treatments, the exponential difference in log kill could be as high as 7 logs. The dose of bortezomib is also 2 logs less as a sensitizer than that required for single agent efficacy. Further studies should be done to explore this model in vivo.

HIV protease inhibitors block Akt signaling and radiosensitize tumor cells both in vitro and in vivo.

In tumor cells with mutations in epidermal growth factor receptor (SQ20B), H-Ras (T24), or K-Ras (MIAPACA2 and A549), the inhibition of Akt phosphorylation increases radiation sensitivity in clonogenic assays, suggesting that Akt is a potential molecular target when combined with therapeutic radiation. Insulin resistance and diabetes are recognized side effects of HIV protease inhibitors (HPIs), suggesting that these agents may inhibit Akt signaling. Because activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is common in human cancers, we hypothesized that HPIs can inhibit Akt activity resulting in increased tumor cell sensitivity to ionizing radiation-induced cell death. Five first-generation HPIs were subsequently tested and three of the five (amprenavir, nelfinavir, and saquinavir but not ritonavir or indinavir) inhibited Akt phosphorylation at Ser473 at serum concentrations routinely achieved in HIV patients. In both tumor cell colony formation assays and tumor regrowth delay experiments, combinations of drug and radiation exerted synergistic effects compared with either modality alone. In addition, in vivo, doses of amprenavir or nelfinavir comparable with the therapeutic levels achieved in HIV patients were sufficient to down-regulate phosphorylation of Akt in SQ20B and T24 xenografts. Finally, overexpression of active PI3K in cells without activation of Akt resulted in radiation resistance that could be inhibited with HPIs. Because there is abundant safety data on HPIs accumulated in thousands of HIV patients over the last 5 years, these agents are excellent candidates to be tested as radiation sensitizers in clinical trials.

Farnesyltransferase inhibitors: an overview of the results of preclinical and clinical investigations.

This article presents an overview of preclinical studies and clinical trials of a number of independently derived farnesyltransferase inhibitors (FTIs). Potential targets and biological modes of action of FTIs are discussed, and the results of clinical trials are summarized. The significant efficacy of FTIs as single or combined agents in preclinical studies stands in contrast with only moderate effects in clinical Phase II-III trials. These results reveal a substantial gap in the understanding of the complex activity of FTIs and their interactions with cytotoxic agents. We conclude that the rational combination of FTIs with other therapies, taking into account the biological activities of the individual agents, may improve the clinical results obtained with FTIs.

The RAS signal transduction pathway and its role in radiation sensitivity.

RAS has been shown to increase radiation resistance. Upstream and downstream pathways from RAS could thus be targets for manipulation of radiosensitivity. EGFR expression and AKT phosphorylation are also associated with the response to radiation. A retrospective study evaluating EGFR and AKT in patients treated with multimodality therapy found a significant association between P-AKT and treatment failure. Moreover, these data are strengthened by in vitro studies showing that inhibition of EGFR, RAS, PI3K, and AKT radiosensitized cancer cell lines. We have previously shown that PI3K is a mediator of RAS-induced radiation resistance. We now suggest that EGFR, which is upstream of PI3K, may also mediate resistance through a common pathway. In addition to EGFR and RAS, PTEN can also regulate the PI3K pathway. Identifying a common signal for EGFR, RAS, or PTEN that results in radiation resistance may uncover targets for developing molecular-based radiosensitization protocols for tumors resistant to radiation and thus improve local control.

Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction.

PURPOSE: Locoregional recurrence is the dominant form of treatment failure in head and neck (H&N) cancer. The epidermal growth factor receptor (EGFR) is frequently amplified in this disease (

A phase I trial of the dual farnesyltransferase and geranylgeranyltransferase inhibitor L-778,123 and radiotherapy for locally advanced pancreatic cancer.

PURPOSE: Preclinical and clinical studies have demonstrated that inhibition of prenylation can radiosensitize cell lines with activation of Ras and produce clinical response in patients with cancer. The aim of this study was to determine the maximally tolerated dose of the dual farnesyltransferase and geranylgeranyltransferase I inhibitor L-778,123 in combination with radiotherapy for patients with locally advanced pancreatic cancer. EXPERIMENTAL DESIGN: L-778,123 was given by continuous intravenous infusion with concomitant radiotherapy to 59.4 Gy in standard fractions. Two L-778,123 dose levels were tested: 280 mg/m2/day over weeks 1, 2, 4, and 5 for dose level 1; and 560 mg/m2/day over weeks 1, 2, 4, 5, and 7 for dose level 2. RESULTS: There were no dose-limiting toxicities observed in the eight patients treated on dose level 1. Two of the four patients on dose level 2 experienced dose-limiting toxicities consisting of grade 3 diarrhea in one case and grade 3 gastrointestinal hemorrhage associated with grade 3 thrombocytopenia and neutropenia in the other case. Other common toxicities were mild neutropenia, dehydration, hyperglycemia, and nausea/vomiting. One patient on dose level 1 showed a partial response of 6 months in duration. Both reversible inhibition of HDJ2 farnesylation and radiosensitization of a study patient-derived cell line were demonstrated in the presence of L-778,123. K-RAS mutations were found in three of the four patients evaluated. CONCLUSIONS: The combination of L-778,123 and radiotherapy at dose level 1 showed acceptable toxicity in patients with locally advanced pancreatic cancer. Radiosensitization of a patient-derived pancreatic cancer cell line was observed.

C-Raf-1 protein kinase is not essential for Ras transformation of mouse embryo fibroblasts.

Transfection of primary cells with mutated oncogenic ras plus a cooperating oncogene such as myc results in the acquisition of the transformed cell phenotype. The pathways downstream of Ras that are required for transformation are an active topic of research. The Raf-MEKK-MAP kinase pathway is triggered by activation of Ras and thought to be important in Ras transformation of rodent fibroblasts. To further explore the involvement of this pathway, fibroblasts from homozygous knock out c-Raf-1 mouse embryos (20 KO) and wild-type c-Raf-1 mouse embryos (16 WT) were transfected with H-ras and myc(v). The resulting cell line derived from the knock out cells grew slower both in tissue culture and had a longer latency period as tumors than the transformed cell line from the wild-type cells. Both cell lines were however able to form tumors in nude mice. These results suggest that c-Raf-1 is not required for Ras transformation in this system.

Farnesyltransferase inhibitors as radiation sensitizers.

Activation of Ras, by mutation, overexpression, or by signaling through tyrosine kinase receptors, is associated with radioresistance. Thus, therapies that inhibit Ras function could be an effective means to radiosensitize selected types of solid tumors. Inhibition of Ras prenylation using a variety of farnesyltransferase inhibitors resulted in radiosensitization of tumor cells that expressed activated Ras, both in vitro and in xenograft models. Farnesyltransferase inhibitor treatment could also inhibit tumor regrowth following irradiation of mice bearing T24 tumor xenografts that express activated Ras. In a phase I trial of the farnesyltransferase inhibitor L-778-123 and radiotherapy in patients with locally advanced head and neck cancer and non-small cell lung cancer, a high response rate was observed coupled with a mild toxicity profile. Additional clinical trials should shed light on the potential of this and other farnesyltransferase inhibitors to serve as radiosensitizers and may identify molecular markers that could predict a response to these agents.

Radiation sensitization of human cancer cells in vivo by inhibiting the activity of PI3K using LY294002.

Multiple genetic alterations such as in Ras or EGFR can result in sustained signaling through PI3K. Our previous experiments have shown that resistance to radiation results from PI3K activity in cells in culture. Here we examined whether inhibition of PI3K in vivo would sensitize tumors to radiation. The human bladder cancer cell line T24 has amplified and mutated H-Ras resulting in sustained PI3K activity and phosphorylation of the downstream target of PI3K, Akt. Nude mice bearing T24 tumor cell xenografts were randomly assigned to one of four groups: control, radiation alone, the PI3K inhibitor LY294002 alone, or combined LY294002 and radiation. The LY294002 was delivered intraperitoneally to the mice. Downregulation of Akt was documented by Western blot analysis of tumor lysates. In vivo sensitization was measured using clonogenic assays or regrowth assays.A dose of 100 mg/kg of LY294002, but not 50 mg/kg, consistently eliminated the phosphorylation of Akt. This inhibition was transient, and Akt activity returned after 30 min. This dose resulted in severe respiratory depression and lethargy resolving without lethality. It is not possible to tell whether these side effects of LY294002 were mechanism-based or idiosyncratic. The PI3K inhibitor LY294002 by itself had minimal antitumor effect. The combination of LY294002 and radiation resulted in significant and synergistic reduction in clonogenicity and growth delay. Inhibition of PI3K by LY294002 can synergistically enhance radiation efficacy. This acts as a proof of principle that inhibition of the Ras to PI3K pathway could be useful clinically.

Outcomes after the use of gastrostomy tubes in patients whose head and neck cancer was managed with radiation therapy.

BACKGROUND: The purpose of this study was to examine the effect of continued oral intake and duration of gastrostomy tube placement on posttreatment nutritional outcomes in patients being irradiated for head and neck cancer. METHODS: Factors associated with continued oral intake and its association with posttreatment outcomes were analyzed. RESULTS: Patients with no oral intake (39.6% of 91) were more likely to have laryngeal tumors, advanced disease, and pretreatment gastrostomy tube placement. Of the 55 patients whose gastrostomy tubes had been removed, those with continued oral intake and shorter gastrostomy tube placement were more likely to maintain their weight and report eating scores in the higher-functioning category, but have more restricted diets. Observed survival was significantly better for the continued-oral-intake group (p = .001). CONCLUSION: The beneficial effects of continued oral intake and shorter gastrostomy tube placement on posttreatment outcomes shown in this study suggest that clinicians involved in these patients' care should emphasize oral intake during treatment.

Investigation of the pharmacokinetics of 3'-deoxy-3'-[18F]fluorothymidine uptake in the bone marrow before and early after initiation of chemoradiation therapy in head and neck cancer.

INTRODUCTION: The kinetics of the bone marrow uptake of 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) before and early after initiation of chemoradiation therapy was investigated in patients with head and neck cancer. METHODS: Fourteen subjects with head and neck cancer underwent FLT positron emission tomography (PET) at baseline and after 10 Gy of radiation therapy. Thirteen subjects also received one cycle of platinum-based chemotherapy before the second FLT PET. Kinetic parameters, including the flux constant based on compartmental analysis (K(FLT)) and the Patlak constant (K(Patlak)) for cervical marrow, were calculated. Standardized uptake values (SUVs) for the cervical marrow (inside the radiation field) and lumbar spine marrow (outside the radiation field) were also determined. RESULTS: There was a significant drop in FLT uptake in the bone marrow inside the radiation field. Mean pretreatment uptake values for the cervical spine were SUV=3.08+/-0.66, K(FLT)=0.045+/-0.016 min(-1) and K(Patlak)=0.039+/-0.013 min(-1). After treatment, these values were SUV=0.74+/-0.19, K(FLT)=0.011+/-0.005 min(-1) and K(Patlak)=0.005+/-0.002 min(-1). Compartmental analysis revealed a significant drop in k(3) in irradiated cervical marrow. FLT uptake in the bone marrow outside the radiation field exhibited a significantly smaller decrease. CONCLUSIONS: There is a marked decrease in FLT uptake in irradiated bone marrow after 10 Gy of radiation therapy to the head and neck. The drop in FLT uptake in irradiated marrow is due to a significant decrease in the net phosphorylation rate of FLT.

Radiation response in two HPV-infected head-and-neck cancer cell lines in comparison to a non-HPV-infected cell line and relationship to signaling through AKT.

PURPOSE: Human papilloma virus (HPV)-associated cancers of the head and neck (H&N) are increasing in frequency and are often treated with radiation. There are conflicting data in the literature regarding the radiation response in the presence of HPV infection, with some data suggesting they may be more sensitive to radiation. There are few studies looking at in vitro effects of HPV and further sensitization by inhibitors of specific signaling pathways. We are in the process of starting a clinical trial in H&N cancer patients using nelfinavir (NFV) (which inhibits Akt) and it would be important to know the effect of HPV on radiation response +/- NFV. METHODS AND MATERIALS: Two naturally infected HPV-16 cell lines (UPCI-SCC90 and UMSCC47) and the HPV-negative SQ20B H&N squamous carcinoma cells were used. Western blots with or without 10 uM NFV were done to evaluate signaling from the PI3K-Akt pathway. Clonogenic assays were done in the three cell lines with or without NFV. RESULTS: Both UPCI-SCC90 and UMSCC47 cells were sensitive to radiation as compared with SQ20B and the degree corresponded to Akt activation. The SQ20B cell line has an activating mutation in EGFR resulting in phosphorylation (P) of Akt; UMSCC47 has decreased P-phosphatase and TENsin (PTEN), resulting in increased P-Akt; UPCI-SCC90 had overexpression of P-PTEN and decreased P-Akt. NFV resulted in downregulation of Akt in all three cell lines, resulting in sensitization to radiation. CONCLUSIONS: HPV-infected H&N cancers are sensitive to radiation. The degree of sensitivity correlates to Akt activation and they can be further sensitized by NFV.

Signaling pathways in adenoid cystic cancers: implications for treatment.

Adenoid cystic cancers (ACC) in the head and neck are rare yet present a clinical dilemma. Although 5-y survivals are excellent, they have a propensity for late recurrences. Most of these cancers are initially treated with surgery followed by radiation. When recurrences happen, treatment options are limited both by the morbidity and low efficacy of re-irradiation and repeated surgical resection. Reported response rates to chemotherapy are low and targeted therapies may be one option. We, therefore, investigated signaling pathways that may be active in adenoid cystic cancers. Tissues from the last nine ACC patients resected at the University of Iowa were immunohistochemically stained with antibodies for EGFR, phosphorylated (P) Akt, and P-MAPK in order to molecularly characterize these tumors. An ACC cell line (ACC3) was also characterized by western blot. We found that seven of the nine tumor samples had strong expression of P-Akt and 5/9 had P-MAPK. None of them had EGFR expression. In the ACC3 cell line, similar data was found in that there was P-Akt and P-MAPK but no EGFR expression. We tested the HIV protease inhibitor nelfinavir (NFV) which has been shown to inhibit Akt signaling to see its effect on ACC3 cells. Both P-Akt and P-MAPK were inhibited with NFV in ACC3 cells and this resulted in growth inhibition and clonogenic death. In patients where re-irradiation or further surgery is not an option, a trial of NFV may be warranted.

Adverse events associated with concurrent chemoradiation therapy in patients with head and neck cancer.

OBJECTIVE: To assess toxicities, functional outcomes, and health-related quality of life associated with concurrent chemoradiation therapy (CRT) in patients with head and neck cancer. DESIGN: Prospective and retrospective outcomes study. SETTING: Tertiary care institution. PATIENTS: Participants in the longitudinal Outcomes Assessment Project whose head and neck cancer was treated with CRT between February 1, 2000, and March 1, 2007 (n = 104). INTERVENTIONS: Patients prospectively provided functional and health-related quality of life information, including data from the 1-year and most current follow-up visits. Medical records were reviewed to determine toxicity and survival rates. MAIN OUTCOME MEASURES: Well-defined acute and late toxicities; functional outcomes (diet, dentition, tracheostomies); head and neck cancer-specific, general health, and depression outcomes; and survival rates. RESULTS: Most patients had oropharyngeal or laryngeal tumors (87.5%) and advanced-stage disease (75.0%). Approximately one-half had hematologic toxicities and toxicity-related treatment delays. Approximately one-quarter had neurotoxicities and/or ototoxicites, moist desquamation, pneumonia, nausea and vomiting requiring hospitalization or intravenous fluids, dehydration or malnutrition requiring hospitalization, and mild or moderate fever. Although patients receiving the current intensity-modulated radiation therapy (IMRT) protocol using the Pinnacle(3) planning system had more toxicity-related treatment delays, they had fewer toxicities and better functional and health-related quality of life outcomes compared with those receiving conventional lateral opposing-field radiation or the initial IMRT protocol using the Best nomos PEACOCK planning system. CONCLUSIONS: Patients receiving CRT experience a substantial number of treatment-related adverse events, primarily affecting oropharyngeal and laryngeal function, with improvement noted for the current IMRT protocol. Improving dental prosthetic rehabilitation and including evaluations with speech and swallowing pathologists before and during treatment may enhance patient outcomes.

Validation and toxicity of PI3K/Akt pathway inhibition by HIV protease inhibitors in humans.

PURPOSE: Activation of the phosphatidylinositol 3-kinase/Akt pathway in tumors leads to radiation resistance, and inhibition of this pathway radiosensitizes tumors in laboratory models. Several first-generation human immunodeficiency virus (HIV) protease inhibitors (HPIs) inhibit Akt activation and are radiosensitizers. In order to validate a biomarker of Akt activity in anticipation of clinical trials using HPIs combined with radiotherapy, we sought to determine whether Akt activation was inhibited in leukocytes of HIV+ patients that were already taking these agents. RESULTS: Patients taking these "active" radiosensitizing protease inhibitors had low levels of phospho-Akt compared to HIV+ patients taking either no medications or other anti-retroviral regimens. We found no significant differences in acute toxicities or in the ability to finish radiation treatment between 14 patients taking radiosensitizing HPIs and the 28 controls. METHODS AND MATERIALS: Peripheral blood mononuclear cells from HIV+ patients either taking radiosensitizing HPIs (nelfinavir, saquinavir, amprenavir) or not were analyzed by Western blotting for phospho-Akt. In order to determine whether these radiosensitizing HPIs increase the toxicity of radiotherapy, we performed a retrospective cohort study of HIV+ cancer patients treated with radiation and compared patients on radiosensitizing HPIs to controls not taking these agents. CONCLUSIONS: These results demonstrate the proof of principle that HPIs can inhibit Akt activation in patients taking normally prescribed anti-retroviral doses and are not associated with excessive toxicity. Radiosensitizing HPIs are excellent candidates for Phase I clinical trials as radiation sensitizers, and peripheral blood mononuclear cells can be used as a drug activity biomarker for Akt pathway inhibition.