Sequencing of Sipuleucel-T and Androgen Deprivation Therapy in Men with Hormone-Sensitive Biochemically Recurrent Prostate Cancer: A Phase II Randomized Trial.

Purpose: STAND, a randomized, phase II, open-label trial (NCT01431391), assessed sequencing of sipuleucel-T (an autologous cellular immunotherapy) with androgen deprivation therapy (ADT) in biochemically recurrent prostate cancer (BRPC) patients at high risk for metastasis.Experimental Design: Men with BRPC following prostatectomy and/or radiotherapy, a PSA doubling time ≤12 months, and no metastasis were enrolled. Patients were randomized (34/arm) to sipuleucel-T followed by ADT (started 2 weeks after sipuleucel-T completion), or ADT followed by sipuleucel-T (started 12 weeks after ADT initiation); ADT continued for 12 months in both arms. The primary endpoint was PA2024-specific T-cell response [enzyme-linked immunospot (ELISPOT)] over time.Results: PA2024-specific ELISPOT responses over time were similar between groups, except at week 6, where responses were higher with sipuleucel-T→ADT versus ADT→sipuleucel-T (P = 0.013). PA2024-specific T-cell proliferation responses, averaged across time points, were approximately 2-fold higher with sipuleucel-T→ADT versus ADT→sipuleucel-T (P = 0.001). PA2024-specific cellular and humoral responses and prostatic acid phosphatase-specific humoral responses increased significantly versus baseline (P < 0.001) and were maintained for 24 months (both arms). Median time-to-PSA recurrence was similar between arms (21.8 vs. 22.6 months, P = 0.357). Development of a PA2024-specific humoral response correlated with prolonged time-to-PSA progression (HR, 0.22; 95% CI, 0.08-0.67; P = 0.007). Sipuleucel-T with ADT was generally well tolerated.Conclusions: Sipuleucel-T→ADT appears to induce greater antitumor immune responses than the reverse sequence. These results warrant further investigation to determine whether this sequence leads to improved clinical outcomes, as well as the independent contribution of ADT alone in terms of immune activation. Clin Cancer Res; 23(10); 2451-9. ©2016 AACR.

An analysis of leukapheresis and central venous catheter use in the randomized, placebo controlled, phase 3 IMPACT trial of Sipuleucel-T for metastatic castrate resistant prostate cancer.

PURPOSE: Sipuleucel-T is an autologous cellular immunotherapy. We review the safety of the leukapheresis procedure required for sipuleucel-T preparation and complications related to venous catheter use in the randomized, placebo controlled phase 3 IMPACT (IMmunotherapy for ProstAte Cancer Trial) study (NCT 00065442). MATERIALS AND METHODS: A total of 512 patients with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer were enrolled in the study. All patients were scheduled to undergo 3 standard 1.5 to 2.0 blood volume leukapheresis procedures at 2-week intervals. Leukapheresis related adverse events and those related to venous catheter use were reviewed. Immune cell counts were examined throughout the treatment course. RESULTS: Of 512 enrolled patients 506 underwent 1 or more leukapheresis procedures and were included in this analysis. Adverse events were comparable between the sipuleucel-T and control arms. Leukapheresis related adverse events were primarily associated with transient hypocalcemia (39.3%). Most leukapheresis related adverse events (97%) were of mild/moderate intensity. Median white blood cell count and absolute monocyte and lymphocyte counts were stable and within normal ranges throughout the treatment course. Of all patients 23.3% had a central venous catheter placed primarily for leukapheresis. Patients with vs without a central venous catheter had a higher risk of infection potentially related to catheter use (11.9% vs 1.3%, p <0.0001) and a trend toward a higher incidence of venous vascular events potentially related to catheter use, excluding the central nervous system (5.9% vs 2.1%, p = 0.06). CONCLUSIONS: Adverse events related to leukapheresis are manageable and quickly reversible. The majority of patients can undergo leukapheresis without a central venous catheter. Central venous catheters are associated with an increased risk of infections and venous vascular events. Peripheral intravenous access should be used when feasible.

N',N'-Dimethyl-N',N'-bis(phenylcarbonothioyl) Propanedihydrazide (Elesclomol) Selectively Kills Cisplatin Resistant Lung Cancer Cells through Reactive Oxygen Species (ROS).

Cisplatin is an important chemotherapeutic agent in lung cancer treatment. The mechanism of drug resistance to cisplatin is complex and historically has been difficult to overcome. We report here that cisplatin resistant lung cancer cell lines possess high basal levels of reactive oxygen species (ROS) when compared to normal cells and their parental cell counterparts. These resistant cells also have low thioredoxin (TRX) levels which may be one of the contributory factors to high ROS. N'(1),N'(3)-dimethyl-N'(1),N'(3)-bis(phenylcarbonothioyl) propanedihydrazide (elesclomol), an agent known to increase ROS is selectively toxic to cisplatin-resistant cells, while sparing normal cells and the parental counterpart. The cytotoxic effect of elesclomol in resistant cells is accompanied by further decreases in TRX and glutathione (GSH) antioxidant systems, while opposite results were found in parental cells. The ID(50) of elesclomol in cisplatin-resistant cells ranged from 5-10 nM, which is well within clinically achievable ranges. N-Acetylcysteine (NAC), which is known to neutralize ROS, can abolish the cytotoxic effect of elesclomol, suggesting that the cytotoxic effect results from increased ROS. Overall, our data suggest that elesclomol selectively kills cisplatin-resistant tumor cells through increased ROS. This agent may hold potential to overcome cisplatin resistance and should be further explored to treat patients who have failed cisplatin therapy.

Under normoxia, 2-deoxy-D-glucose elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N-linked glycosylation.

In tumor cells growing under hypoxia, inhibiting glycolysis with 2-deoxy-d-glucose (2-DG) leads to cell death, whereas under normoxic conditions cells similarly treated survive. Surprisingly, here we find that 2-DG is toxic in select tumor cell lines growing under normal oxygen tension. In contrast, a more potent glycolytic inhibitor, 2-fluorodeoxy-d-glucose, shows little or no toxicity in these cell types, indicating that a mechanism other than inhibition of glycolysis is responsible for their sensitivity to 2-DG under normoxia. A clue to this other mechanism comes from previous studies in which it was shown that 2-DG interferes with viral N-linked glycosylation and is reversible by exogenous addition of mannose. Similarly, we find that 2-DG interferes with N-linked glycosylation more potently in the tumor cell types that are sensitive to 2-DG under normoxia, which can be reversed by exogenous mannose. Additionally, 2-DG induces an unfolded protein response, including up-regulation of GADD153 (C/EBP-homologous protein), an unfolded protein response-specific mediator of apoptosis, more effectively in 2-DG-sensitive cells. We conclude that 2-DG seems to be toxic in select tumor cell types growing under normoxia by inhibition of N-linked glycosylation and not by glycolysis. Because in a phase I study 2-DG is used in combination with an anticancer agent to target hypoxic cells, our results raise the possibility that in certain cases, 2-DG could be used as a single agent to selectively kill both the aerobic (via interference with glycosylation) and hypoxic (via inhibition of glycolysis) cells of a solid tumor.

Differential toxic mechanisms of 2-deoxy-D-glucose versus 2-fluorodeoxy-D-glucose in hypoxic and normoxic tumor cells.

The dependence of hypoxic tumor cells on glycolysis as their main means of producing ATP provides a selective target for agents that block this pathway, such as 2-deoxy-D-glucose (2-DG) and 2-fluoro-deoxy-D-glucose (2-FDG). Moreover, it was demonstrated that 2-FDG is a more potent glycolytic inhibitor with greater cytotoxic activity than 2-DG. This activity correlates with the closer structural similarity of 2-FDG to glucose than 2-DG, which makes it a better inhibitor of hexokinase, the first enzyme in the glycolytic pathway. In contrast, because of its structural similarity to mannose, 2-DG is known to be more effective than 2-FDG in interfering with N-linked glycosylation. Recently, it was reported that 2-DG, at a relatively low dose, is toxic to certain tumor cells, even under aerobic conditions, whereas 2-FDG is not. These results indicate that the toxic effects of 2-DG in selected tumor cells under aerobic conditions is through inhibition of glycosylation rather than glycolysis. The intention of this minireview is to discuss the effects and potential clinical impact of 2-DG and 2-FDG as antitumor agents and to clarify the differential mechanisms by which these two glucose analogues produce toxicity in tumor cells growing under anaerobic or aerobic conditions.

Hypoxia-inducible factor-1 confers resistance to the glycolytic inhibitor 2-deoxy-D-glucose.

Hypoxic regions within solid tumors harbor cells that are resistant to standard chemotherapy and radiotherapy. Because oxygen is required to produce ATP by oxidative phosphorylation, under hypoxia, cells rely more on glycolysis to generate ATP and are thereby sensitive to 2-deoxy-d-glucose (2-DG), an inhibitor of this pathway. Universally, cells respond to lowered oxygen tension by increasing the amount of glycolytic enzymes and glucose transporters via the well-characterized hypoxia-inducible factor-1 (HIF). To evaluate the effects of HIF on 2-DG sensitivity, the following three models were used: (a) cells treated with oligomycin to block mitochondrial function in the presence (HIF(+)) or absence (HIF(-)) of hypoxia, (b) cells treated with small interfering RNA specific for HIF-1alpha and control cells cultured under hypoxia, and (c) a mutant cell line unable to initiate the HIF response and its parental HIF(+) counterpart under hypoxic conditions. In all three models, HIF increased resistance to 2-DG and other glycolytic inhibitors but not to other chemotherapeutic agents. Additionally, HIF reduced the effects of 2-DG on glycolysis (as measured by ATP and lactate assays). Because HIF increases glycolytic enzymes, it follows that greater amounts of 2-DG would be required to inhibit glycolysis, thereby leading to increased resistance to it under hypoxia. Indeed, hexokinase, aldolase, and lactate dehydrogenase were found to be increased as a function of HIF under the hypoxic conditions and cell types we used; however, phosphoglucose isomerase was not. Although both hexokinase and phosphoglucose isomerase are known to interact with 2-DG, our findings of increased levels of hexokinase more likely implicate this enzyme in the mechanism of HIF-mediated resistance to 2-DG. Moreover, because 2-DG is now in phase I clinical trials, our results suggest that glycolytic inhibitors may be more effective clinically when combined with agents that inhibit HIF.

Efficacy of 2-halogen substituted D-glucose analogs in blocking glycolysis and killing "hypoxic tumor cells".

PURPOSE: Since 2-deoxy-D-glucose (2-DG) is currently in phase I clinical trials to selectively target slow-growing hypoxic tumor cells, 2-halogenated D-glucose analogs were synthesized for improved activity. Given the fact that 2-DG competes with D-glucose for binding to hexokinase, in silico modeling of molecular interactions between hexokinase I and these new analogs was used to determine whether binding energies correlate with biological effects, i.e. inhibition of glycolysis and subsequent toxicity in hypoxic tumor cells. METHODS AND RESULTS: Using a QSAR-like approach along with a flexible docking strategy, it was determined that the binding affinities of the analogs to hexokinase I decrease as a function of increasing halogen size as follows: 2-fluoro-2-deoxy-D-glucose (2-FG) > 2-chloro-2-deoxy-D-glucose (2-CG) > 2-bromo-2-deoxy-D-glucose (2-BG). Furthermore, D-glucose was found to have the highest affinity followed by 2-FG and 2-DG, respectively. Similarly, flow cytometry and trypan blue exclusion assays showed that the efficacy of the halogenated analogs in preferentially inhibiting growth and killing hypoxic vs. aerobic cells increases as a function of their relative binding affinities. These results correlate with the inhibition of glycolysis as measured by lactate inhibition, i.e. ID50 1 mM for 2-FG, 6 mM for 2-CG and > 6 mM for 2-BG. Moreover, 2-FG was found to be more potent than 2-DG for both glycolytic inhibition and cytotoxicity. CONCLUSIONS: Overall, our in vitro results suggest that 2-FG is more potent than 2-DG in killing hypoxic tumor cells, and therefore may be more clinically effective when combined with standard chemotherapeutic protocols.

Differential sensitivity to 2-deoxy-D-glucose between two pancreatic cell lines correlates with GLUT-1 expression.

OBJECTIVES: To determine whether the differential growth inhibition of pancreatic tumor cells to the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) correlates with uptake, expression of GLUT-1 transporter, and levels of hypoxic-inducible factor-1alpha. METHODS: Growth inhibition assays with 2-DG, lactic acid analysis, Western blots of GLUT-1, and hypoxic-inducible factor-1alpha were correlated with each other and with uptake and accumulation of radio-labeled 2-DG in 2 pancreatic cell lines. RESULTS: Under normal oxygen tension, we find that the pancreatic cell line MIA PaCa2 (1420) is 7 times more sensitive to 2-DG and equally sensitive to oxamate, as compared with Panc-1 (1469). Lactate levels in both cell types are similarly low, indicating that mitochondria are functioning normally in these cells and that they are not solely dependent on glycolysis for survival under an aerobic microenvironment. Since oxamate does not use glucose transporters for entry into the cell, the equal sensitivity to this drug suggests that the selective growth inhibition of 2-DG in these 2 cell types might be reflective of differential expression of glucose transporters. Indeed, we find that GLUT-1 is more highly expressed in 1420 and that this cell line accumulates 2-DG twice as much as 1469. Additionally, hypoxic-inducible factor, which is known to upregulate the expression of GLUT-1, is found at equally low levels in both cell types. Thus, the increased expression of GLUT-1 in 1420 appears to be independent of hypoxia-inducible factor. CONCLUSION: Overall, our results suggest that certain pancreatic tumors may be inherently sensitive to 2-DG, even under normal oxygen tension, due to greater intracellular accumulation of this inhibitor. Moreover, if 2-DG shows clinical efficacy, it may be possible to predict which pancreatic tumors would be sensitive to this agent based on their GLUT-1 expression profile and their increased uptake of 2-fluoro-deoxy-D-glucose currently used to image tumors via PET scanning.

Greater cell cycle inhibition and cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under hypoxic vs aerobic conditions.

PURPOSE: In order to investigate the hypothesis that cells found in hypoxic areas of solid tumors are more sensitive to glycolytic inhibitors than cells growing aerobically, we have previously characterized three distinct in vitro models (A, B and C) that simulate this condition. In all of the models it was shown that cells growing under hypoxic conditions are hypersensitive to the glycolytic inhibitor 2-deoxy- d-glucose (2-DG). However, in those studies cytostatic and cytotoxic effects were not distinguished from one another. Since successful treatment of cancer includes not only slowing down but also actually killing tumor cells, studies were undertaken to assess the effects of 2-DG on cell cycle progression and cell death. METHODS AND RESULTS: Using flow cytometry and cell viability assays, it was found that 2-DG caused significantly greater cell cycle inhibition and cell death in all three hypoxic models as compared to aerobically growing control cells. In model A (a chemically induced model of hypoxia in which rhodamine-123 is used to block oxidative phosphorylation), 1200 microg/ml of 2-DG was shown to induce more cell cycle arrest in late S/G(2) and more cell death than in the aerobic cell counterpart treated with 3600 microg/ml 2-DG. In rho(0) cells which are genetically constructed to be unable to perform oxidative phosphorylation (model B), an even greater window of selectivity (more than tenfold) between hypoxic and aerobic cells was found when considering 2-DG's effects on cell cycle arrest and cell death. In the environmental model (model C), where cells were grown under reduced amounts of external oxygen (0.1%), hypersensitivity to the effects of 2-DG with respect to cell cycle arrest and cell death were also observed. CONCLUSIONS: Overall, these results indicate that cells growing under anaerobic conditions respond with greater sensitivity to the effects of 2-DG on cell cycle inhibition and cell death than those growing under aerobic conditions. This supports our contention that glycolytic inhibitors added to standard chemotherapeutic protocols should increase treatment efficacy by selectively killing the slow-growing cells, which are found in the hypoxic portions of solid tumors, while sparing most of the normal cells that are also slow-growing but are living under aerobic conditions.