Hypoxia-activated Prodrug Research Articles

Evofosfamide sensitizes esophageal carcinomas to radiation without increasing normal tissue toxicity

Background and purposeEsophageal cancer incidence is increasing and is rarely curable. Hypoxic tumor areas cause resistance to conventional therapies, making them susceptible for treatment with hypoxia-activated prodrugs (HAPs). We investigated in vivo whether the HAP evofosfamide (TH-302) could increase the therapeutic ratio by sensitizing esophageal carcinomas to radiotherapy without increasing normal tissue toxicity. Materials and methodsTo assess therapeutic efficacy, growth of xenografted esophageal squamous cell (OE21) or adeno (OE19) carcinomas was monitored after treatment with TH-302 (50 mg/kg, QD5) and irradiation (sham or 10 Gy). Short- and long-term toxicity was assessed in a gut mucosa and lung fibrosis irradiation model, sensitive to acute and late radiation injury respectively. Mice were injected with TH-302 (50 mg/kg, QD5) and the abdominal area (sham, 8 or 10 Gy) or the upper part of the right lung (sham, 20 Gy) was irradiated. Damage to normal tissues was assessed 84 hours later by histology and blood plasma citrulline levels (gut) and for up to 1 year by non-invasive micro CT imaging (lung). ResultsThe combination treatment of TH-302 with radiotherapy resulted in significant tumor growth delay in OE19 (P = 0.02) and OE21 (P = 0.03) carcinomas, compared to radiotherapy only. Irradiation resulted in a dose-dependent decrease of crypt survival (P < 0.001), mucosal surface area (P < 0.01) and citrulline levels (P < 0.001) in both tumor and non-tumor bearing animals. On the long-term, irradiation increased CT density in the lung, indicating fibrosis, over time. TH-302 did not influence the radiation-induced short-term and long-term toxicity, confirmed by histological evaluation. ConclusionThe combination of TH-302 and radiotherapy might be a promising approach to improve the therapeutic index for esophageal cancer patients.

Mitomycin C modulates tumor microenvironment and enhances radiosensitivity in rectal cancer

Background: Neoadjuvant chemoradiation is the standard of care for locally advanced rectal cancer, however, pathological complete response (pCR) rate remains unsatisfactory. Tumor hypoxia in rectal cancer might be one factor of radioresistance. Mitomycin C (MMC), a hypoxia-activated prodrug (HAP), enhances radiosensitivity and is the standard adjunct to radiotherapy (RT) in anal cancer. Our study evaluated the effect of MMC and RT on rectal cancer and its tumor microenvironment (TME). Methods: In vitro hypoxia was induced by 1% O2 culture for CT26 rectal adenocarcinoma cells and HIF-1α expression was validated with Western blotting. Clonogenicity and MTT assays were used for radiation and drug survival. In syngeneic CT26 model, tumors were treated as follows: control, MMC (2 mg/kg/day), RT (2 Gy × 2 fractions), and combination with MMC 2 h prior to RT. Tumor volume, body weight, and white blood cell count were monitored for 1 month. Immune cells in TME were analyzed using flow cytometry. Results: MMC inhibited cell viability and enhanced radioresponse in CT 26 cells with delayed repair of DNA double strand break (P Ifnb1 was augmented by RT and upregulated with addition of MMC. In vivo treatment using MMC plus RT suppressed tumor growth with durable effect in comparison to MMC or RT alone (P Conclusions: The combination of MMC and RT may have synergistic therapeutic effect accompanied by modulation of TME towards favorable anti-tumor immunity.

Tandem activated photodynamic and chemotherapy: Using pH-Sensitive nanosystems to realize different tumour distributions of photosensitizer/prodrug for amplified combination therapy

Photodynamic therapy (PDT) combined with hypoxia-activated prodrugs to overcome hypoxia environment has been recently explored as a promising clinical modality for cancer therapy. Nevertheless, delivering these two therapeutic agents together to different tumour areas that possess a number of biological barriers remains a considerable challenge. Herein, we used the semiconducting polyelectrolyte-based zwitterionic photosensitizer (PFNS) to modify the surface of upconversion nanoparticles (NPs) and prepare near-infrared (NIR) light-responsive PDT agents (UCNP@PFNS). A pH-sensitive Mn-Ca3(PO4)2 (MnCaP) layer was further coated onto UCNP@PFNS with the hypoxia-activated prodrug AQ4N incorporated inside. The final nanocomposites exhibited a diameter of 73 nm with high stability in the blood and a remarkably enhanced permeability and retention (EPR) effect in tumours. Importantly, when these nanoparticles reached the tumour site, the acidic tumour microenvironment (pH 6.5–6.8) decomposed the MnCaP layer, releasing both UCNP@PFNS (30 nm) and AQ4N. The relatively small size of UCNP@PFNS and AQ4N satisfied the different distribution requirements in tumour and achieved a high therapeutic effect, thereby reaching an inhibition rate of as high as 83%. In addition, Mn2+ ions can be released during the decomposition of CaP, leading to a significantly increased magnetic resonance (MR) signal in the tumour site. Overall, we report a nanoparticle guided by MRI and fluorescence imaging possesses of tandem active pattern of PDT and chemotherapy, which is promising for future clinical diagnosis and treatment.

Impact of Tumour Hypoxia on Evofosfamide Sensitivity in Head and Neck Squamous Cell Carcinoma Patient-Derived Xenograft Models.

Tumour hypoxia is a marker of poor prognosis and failure of chemoradiotherapy in head and neck squamous cell carcinoma (HNSCC), providing a strategy for therapeutic intervention in this setting. To evaluate the utility of the hypoxia-activated prodrug evofosfamide (TH-302) in HNSCC, we established ten early passage patient-derived xenograft (PDX) models of HNSCC that were characterised by their histopathology, hypoxia status, gene expression, and sensitivity to evofosfamide. All PDX models closely resembled the histology of the patient tumours they were derived from. Pimonidazole-positive tumour hypoxic fractions ranged from 1.7–7.9% in line with reported HNSCC clinical values, while mRNA expression of the Toustrup hypoxia gene signature showed close correlations between PDX and matched patient tumours, together suggesting the PDX models may accurately model clinical tumour hypoxia. Evofosfamide as a single agent (50 mg/kg IP, qd × 5 for three weeks) demonstrated antitumour efficacy that was variable across the PDX models, ranging from complete regressions in one p16-positive PDX model to lack of significant activity in the three most resistant models. Despite all PDX models showing evidence of tumour hypoxia, and hypoxia being essential for activation of evofosfamide, the antitumour activity of evofosfamide only weakly correlated with tumour hypoxia status determined by pimonidazole immunohistochemistry. Other candidate evofosfamide sensitivity genes—MKI67, POR, and SLFN11—did not strongly influence evofosfamide sensitivity in univariate analyses, although a weak significant relationship with MKI67 was observed, while SLFN11 expression was lost in PDX tumours. Overall, these data confirm that evofosfamide has antitumour activity in clinically-relevant PDX tumour models of HNSCC and support further clinical evaluation of this drug in HNSCC patients. Further research is required to identify those factors that, alongside hypoxia, can influence sensitivity to evofosfamide and could act as predictive biomarkers to support its use in precision medicine therapy of HNSCC.

Abstract 5011: Targeting hypoxia-induced immune suppression to overcome immunotherapy resistance in prostate cancer

Abstract Immune checkpoint blockade is effective in “hot” tumors like melanoma with pre-existing immune infiltrates; however, “cold” tumors like prostate cancer fail to respond. We found that prostate cancers harbor regions of hypoxia that resist T cell infiltration even in the context of anti-CTLA-4 (cytotoxic T lymphocyte associated protein-4) and anti-PD-1 (programmed cell death protein 1) blockade. These hypoxic zones serve as islands of immune privilege through the recruitment and suppressive polarization of immature myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM). We found that targeted hypoxia ablation using TH-302, a hypoxia-activated prodrug, sensitized both transplantable and spontaneous models of prostate cancer to checkpoint blockade, coincident with enhanced T cell infiltration and effector function and loss of MDSC recruitment and suppressive function. Tumors treated with the combination of TH-302 and checkpoint blockade showed a reduced capacity to suppressively polarize new myeloid immigrants, implying a durable reconditioning of the tumor microenvironment (TME) into an immune-infiltrated, pro-inflammatory milieu. T cells infiltrating combination-treated tumors exhibited increased mitochondrial respiration, consistent with creation of a metabolically favorable milieu for T cell function. Based on these findings, we hypothesized that other approaches capable of metabolically rewiring the TME should promote anti-tumor immunity and sensitize checkpoint blockade-resistant tumors to immunotherapy. With this in mind, we performed a longitudinal study comparing a panel of different mitochondrial respiration inhibitors and a glutaminase inhibitor for their efficacy in reducing hypoxia, improving T cell infiltration and decreasing myeloid cell recruitment and suppressive polarization using immunofluorescence staining and confocal microscopy. Our preliminary data suggests that inhibitors targeting mitochondrial respiration, rather than those targeting glutamine metabolism synergize with checkpoint blockade and exhibit the highest efficacy in increasing T cell recruitment. We continue to characterize the dynamics of hypoxia reduction, duration of normalization following drug withdrawal, and impact on the immune microenvironment of these diverse approaches to metabolic reconditioning. Citation Format: Priyamvada Jayaprakash, Midan Ai, Arthur Liu, Pratha Budhani, Todd Bartkowiak, Jie Sheng, Casey Ager, Courtney Nicholas, Ashvin Jaiswal, Yanqiu Sun, Krishna Shah, Sadhana Balasubramanyam, Nan Li, Guocan Wang, Jing Ning, Anna Zal, Tomasz Zal, Michael Curran. Targeting hypoxia-induced immune suppression to overcome immunotherapy resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5011.

Evaluation of 5-hydroxy-1,4-naphthoquinone-cobalt(III) complexes for hypoxia-activated drug delivery

Three novel Py2N2-cobalt(III) complexes with the 5-hydroxy-1,4-naphthoquinone nuclei (NQ) were evaluated as potential hypoxia-activated anticancer prodrugs. The complexes were synthesized and fully characterized by IR and UV–Visible spectroscopies, ESI mass spectrometry and CHN elemental analysis. Structural information was obtained from density functional theory (DFT) calculations. Cyclic voltammetry analysis in acetonitrile indicates that the ligand substituents (H, CH3 and p-tolylthio) do not have a relevant effect on the Co3+/Co2+ redox potential. Reactions with ascorbic acid in phosphate buffers were performed to simulate redox activation of the complexes in biological media. Fast and irreversible dissociation of the NQ ligands was observed for all complexes upon Co3+/Co2+ reduction. Cytotoxic activity of complexes 1 and 3 was evaluated in tumor cells (HT-29 and HCT-116) under hypoxic and normoxic conditions.

Schedule-dependent potentiation of chemotherapy drugs by the hypoxia-activated prodrug SN30000

ABSTRACTHypoxia-activated prodrugs (HAPs) are hypothesized to improve the therapeutic index of chemotherapy drugs that are ineffective against tumor cells in hypoxic microenvironments. SN30000 (CEN-209) is a benzotriazine di-N-oxide HAP that potentiates radiotherapy in preclinical models, but its combination with chemotherapy has not been explored. Here we apply multiple models (monolayers, multicellular spheroids and tumor xenografts) to identify promising SN30000/chemotherapy combinations (with chemotherapy drugs before, during or after SN30000 exposure). SN30000, unlike doxorubicin, cisplatin, gemcitabine or paclitaxel, was more active against cells in spheroids than monolayers by clonogenic assay. Combinations of SN30000 and chemotherapy drugs in HCT116/GFP and SiHa spheroids demonstrated hypoxia-and schedule-dependent potentiation of gemcitabine or doxorubicin in growth inhibition and clonogenic assays. Co-administration with SN30000 suppressed clearance of gemcitabine in NIH-III mice, likely due to SN30000-induced hypothermia which also modulated extravascular transport of gemcitabine in tumor tissue as assessed from its diffusion through HCT116 multicellular layer cultures. Despite these systemic effects, the same schedules that gave therapeutic synergy in spheroids (SN30000 3 h before or during gemcitabine, but not gemcitabine 3 h before SN30000) enhanced growth delay of HCT116 xenografts without increasing host toxicity. Identification of hypoxic and S-phase cells by immunohistochemistry and flow cytometry established that hypoxic cells initially spared by gemcitabine subsequently reoxygenate and re-enter the cell cycle, and that this repopulation is prevented by SN30000 only when administered with or before gemcitabine. This illustrates the value of spheroids in modeling tumor microenvironment-dependent drug interactions, and the potential of HAPs for overcoming hypoxia-mediated drug resistance.

Hypoxia-active nanoparticles used in tumor theranostic.

Hypoxia is a hallmark of malignant tumors and often correlates with increasing tumor aggressiveness and poor treatment outcomes. Therefore, early diagnosis and effective killing of hypoxic tumor cells are crucial for successful tumor control. There has been a surge of interdisciplinary research aimed at developing functional molecules and nanomaterials that can be used to noninvasively image and efficiently treat hypoxic tumors. These mainly include hypoxia-active nanoparticles, anti-hypoxia agents, and agents that target biomarkers of tumor hypoxia. Hypoxia-active nanoparticles have been intensively investigated and have demonstrated advanced effects on targeting tumor hypoxia. In this review, we present an overview of the reports published to date on hypoxia-activated prodrugs and their nanoparticle forms used in tumor-targeted therapy. Hypoxia-responsive nanoparticles are inactive during blood circulation and normal physiological conditions but are activated by hypoxia once they extravasate into the hypoxic tumor microenvironment. Their use can enhance the efficiency of tumor chemotherapy, radiotherapy, fluorescence and photoacoustic intensity, and other imaging and therapeutic strategies. By targeting the broad habitats of tumors, rather than tumor-specific receptors, this strategy has the potential to overcome the problem of tumor heterogeneity and could be used to design diagnostic and therapeutic nanoparticles for a broad range of solid tumors.

Studies Towards Hypoxia-Activated Prodrugs of PARP Inhibitors.

Poly(ADP-ribose)polymerase (PARP) inhibitors (PARPi) have recently been approved for the treatment of breast and ovarian tumors with defects in homologous recombination repair (HRR). Although it has been demonstrated that PARPi also sensitize HRR competent tumors to cytotoxic chemotherapies or radiotherapy, normal cell toxicity has remained an obstacle to their use in this context. Hypoxia-activated prodrugs (HAPs) provide a means to limit exposure of normal cells to active drug, thus adding a layer of tumor selectivity. We have investigated potential HAPs of model PARPi in which we attach a bioreducible “trigger” to the amide nitrogen, thereby blocking key binding interactions. A representative example showed promise in abrogating PARPi enzymatic activity in a biochemical assay, with a ca. 160-fold higher potency of benzyl phthalazinone 4 than the corresponding model HAP 5, but these N-alkylated compounds did not release the PARPi upon one-electron reduction by radiolysis. Therefore, we extended our investigation to include NU1025, a PARPi that contains a phenol distal to the core binding motif. The resulting 2-nitroimidazolyl ether provided modest abrogation of PARPi activity with a ca. seven-fold decrease in potency, but released the PARPi efficiently upon reduction. This investigation of potential prodrug approaches for PARPi has identified a useful prodrug strategy for future exploration.

Optimised synthesis of a nitroCBI hypoxia-activated prodrug with substantial anticancer activity

An optimised synthesis of a hypoxia-activated anticancer prodrug related to the duocarmycin family of natural products is described. The improved 10-step synthesis increases the overall yield from 4.4% to over 40% while requiring just 2 chromatography-based purifications. The most significant improvements optimise the isomer distribution in a key chlorosulfonylation reaction, and facilitate the removal of tin residues from a tributyltin hydride-mediated radical reaction. Improved preparations of two side chains are also reported. The new method provides practical access to sufficient material to support advanced efficacy and toxicology assessments.

Combined Cancer Chemo-Photodynamic and Photothermal Therapy Based on ICG/PDA/TPZ-Loaded Nanoparticles.

Although photodynamic therapy (PDT) has been an attractive strategy for several cancer treatments in the clinical setting, PDT efficacy is attenuated by consumption of oxygen. To address this photodynamic issue, we adopted a phototherapy-chemotherapy combination strategy based on targeted delivery of the near-infrared photosensitizer indocyanine green (ICG), photothermal conversion agent polydopamine (PDA), and tirapazamine (TPZ), a hypoxia-activated prodrug. Under laser irradiation, ICG consumption of oxygen and aggravated hypoxia in tumor sites can activate TPZ to damage DNA. In parallel, ICG produces reactive oxygen species which work in synergy with PDA to enhance phototherapeutic efficiency. Herein, hybrid CaCO3/TPGS nanoparticles delivering ICG, PDA, and TPZ (ICG-PDA-TPZ NPs) were designed for effective and safe cancer therapy. ICG-PDA-TPZ NPs showed significantly improved cellular uptake and accumulation in tumors. Furthermore, we demonstrated that ICG-PDA-TPZ NPs showed intensive photodynamic and photothermal effects in vitro and in vivo, which synergized with TPZ in subcutaneous U87 malignant glioma growth and orthotopic B16F10 tumor inhibition, with negligible side effects. Thus, ICG-PDA-TPZ NPs could be an effective strategy for improvement of PDT.

Functional CRISPR and shRNA Screens Identify Involvement of Mitochondrial Electron Transport in the Activation of Evofosfamide.

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ 0 (ρ zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ 0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.

Programmable Therapeutic Nanodevices with Circular Amplification of H2 O2 in the Tumor Microenvironment for Synergistic Cancer Therapy.

Tumor microenvironment activated nanodevices have remarkable superiority to enhance therapeutic efficacy and minimize side effects, but their practical applications are dramatically reduced by the low abundance and heterogeneous distribution of specific stimuli at the tumor site. Herein, programmable vesicular nanodevices based on the triblock copolymer containing poly(ethylene glycol) (PEG) and poly(caprolactone) (PCL) with peroxalate esters (PO) as hydrogen peroxide-responsive linkage (PEG-PO-PCL-PO-PEG), are developed for co-delivery of hypoxia-activated prodrug (AQ4N) and glucose oxidase (GOD). The obtained nanodevices (PAG) can be activated by the high level of H2 O2 in tumor microenvironment to improve the permeability of membranes for glucose entrance. Afterward, the oxidation of glucose catalyzed by GOD produces amplified H2 O2 amounts which in turn induce complete destruction of PAG for fast release of AQ4N and GOD. Ultimately, the PAG can exert programmable therapeutic effects from the following aspects: 1) starvation therapy by cutting off the energy supply from glucose through GOD catalysis; 2) oxidative cytotoxicity after H2 O2 amplification; 3) chemotherapy of AQ4N activated by the intensified tumor hypoxia microenvironment after oxygen consumption. The stimuli amplification, controlled drug release, synergistic therapy, and corresponding mechanisms of PAG are demonstrated. Therefore, the presented work could provide significant new insights for cancer treatment.

Investigational Hypoxia-Activated Prodrugs: Making Sense of Future Development.

Hypoxia, which occurs in most cancer cases, disrupts the efficacy of anticarcinogens. Fortunately, hypoxia itself is a potential target for cancer treatment. Hypoxia-activated prodrugs (HAPs) can be selectively activated by reductase under hypoxia. Some promising HAPs have been already achieved, and many clinical trials of HAPs in different types of cancer are ongoing. However, none of them has been approved in clinic to date. From the studies on HAPs began, some achievements are obtained but more challenges are put forward. In this paper, we reviewed the research progress of HAPs to discuss the strategies for HAPs development. According to the research status and results of these studies, administration pattern, reductase activity, and patient selection need to be taken into consideration to further improve the efficacy of existing HAPs. As the requirement of new drug research and development, design of optimal preclinical models and clinical trials are quite important in HAPs development, while different drug delivery systems and anticancer drugs with different mechanisms can be sources of novel HAPs.

Prototyping kinase inhibitor-cytotoxin anticancer mutual prodrugs activated by tumour hypoxia: A chemical proof of concept study

Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was shown to initiate a spontaneous cyclisation/fragmentation reaction that simultaneously released the kinase inhibitor semaxanib (SU5416) and the amine- or alcohol-linked cytotoxin from the prodrugs. Preliminary cell testing and reduction potential measurements support optimisation of the compounds towards tumour-selective mutual prodrugs.

Overcoming Radioresistance: Small Molecule Radiosensitisers and Hypoxia-activated Prodrugs

The role of hypoxia in radiation resistance is well established and many approaches to overcome hypoxia in tumours have been explored, with variable success. Two small molecule strategies for targeting hypoxia have dominated preclinical and clinical efforts. One approach has been the use of electron-affinic nitroheterocycles as oxygen-mimetic sensitisers. These agents are best exemplified by the 5-nitroimidazole nimorazole, which has limited use in conjunction with radiotherapy in head and neck squamous cell carcinoma. The second approach seeks to leverage tumour hypoxia as a tumour-specific address for hypoxia-activated prodrugs. These prodrugs are selectively activated by reductases under hypoxia to release cytotoxins, which in some instances may diffuse to kill surrounding oxic tumour tissue. A number of these hypoxia-activated prodrugs have been examined in clinical trial and the merits and shortcomings of recent examples are discussed. There has been an evolution from delivering DNA-interactive cytotoxins to molecularly targeted agents. Efforts to implement these strategies clinically continue today, but success has been elusive. Several issues have been identified that compromised these clinical campaigns. A failure to consider the extravascular transport and the micropharmacokinetic properties of the prodrugs has reduced efficacy. One key element for these ‘targeted’ approaches is the need to co-develop biomarkers to identify appropriate patients. Hypoxia-activated prodrugs require biomarkers for hypoxia, but also for appropriate activating reductases in tumours, as well as markers of intrinsic sensitivity to the released drug. The field is still evolving and changes in radiation delivery and the impact of immune-oncology will provide fertile ground for future innovation.

Hypoxia-Activated Prodrugs of PERK Inhibitors.

Tumour hypoxia plays an important role in tumour progression and resistance to therapy. Under hypoxia unfolded proteins accumulate in the endoplasmic reticulum (ER) and this stress is relieved through the protein kinase R-like ER kinase (PERK) signalling arm of the unfolded protein response (UPR). Targeting the UPR through PERK kinase inhibitors provides tumour growth inhibition, but also elicits on-mechanism normal tissue toxicity. Hypoxia presents a target for tumour-selective drug delivery using hypoxia-activated prodrugs. We designed and prepared hypoxia-activated prodrugs of modified PERK inhibitors using a 2-nitroimidazole bioreductive trigger. The new inhibitors retained PERK kinase inhibitory activity and the corresponding prodrugs were strongly deactivated. The prodrugs were able to undergo fragmentation following radiolytic reduction, or bioreduction in HCT116 cells, to release their effectors, albeit inefficiently. We examined the effects of the prodrugs on PERK signalling in hypoxic HCT116 cells. This study has identified a 2-substituted nitroimidazole carbamate prodrug with potential to deliver PERK inhibitors in a hypoxia-selective manner.

Selectively Potentiating Hypoxia Levels by Combretastatin A4 Nanomedicine: Toward Highly Enhanced Hypoxia-Activated Prodrug Tirapazamine Therapy for Metastatic Tumors.

Hypoxia-activated prodrugs (HAPs) have the potential to selectively kill hypoxic cells and convert tumor hypoxia from a problem to a selective treatment advantage. However, HAPs are unsuccessful in most clinical trials owing to inadequate hypoxia within the treated tumors, as implied by a further substudy of a phase II clinical trial. Here, a novel strategy for the combination of HAPs plus vascular disrupting agent (VDA) nanomedicine for efficacious solid tumor therapy is developed. An effective VDA nanomedicine of poly(l-glutamic acid)-graft-methoxy poly(ethylene glycol)/combretastatin A4 (CA4-NPs) is prepared and can selectively enhance tumor hypoxia and boost a typical HAP tirapazamine (TPZ) therapy against metastatic 4T1 breast tumors. After treatment with the combination of TPZ plus CA4-NPs, complete tumor reduction is observed in 4T1 xenograft mice (initial tumor volume is 180 mm3 ), and significant tumor shrinkage and antimetastatic effects are observed in challenging large tumors with initial volume of 500 mm3 . The report here highlights the potential of using a combination of HAPs plus VDA nanomedicine in solid tumor therapy.

Hypoxia-activated prodrugs and (lack of) clinical progress: The need for hypoxia-based biomarker patient selection in phase III clinical trials

Hypoxia-activated prodrugs (HAPs) are designed to specifically target the hypoxic cells of tumors, which are an important cause of treatment resistance to conventional therapies. Despite promising preclinical and clinical phase I and II results, the most important of which are described in this review, the implementation of hypoxia-activated prodrugs in the clinic has, so far, not been successful. The lack of stratification of patients based on tumor hypoxia status, which can vary widely, is sufficient to account for the failure of phase III trials. To fully exploit the potential of hypoxia-activated prodrugs, hypoxia stratification of patients is needed. Here, we propose a biomarker-stratified enriched Phase III study design in which only biomarker-positive (i.e. hypoxia-positive) patients are randomized between standard treatment and the combination of standard treatment with a hypoxia-activated prodrug. This implies the necessity of a Phase II study in which the biomarker or a combination of biomarkers will be evaluated. The total number of patients needed for both clinical studies will be far lower than in currently used randomize-all designs. In addition, we elaborate on the improvements in HAP design that are feasible to increase the treatment success rates.

ONECUT2 is a driver of neuroendocrine prostate cancer

Neuroendocrine prostate cancer (NEPC), a lethal form of the disease, is characterized by loss of androgen receptor (AR) signaling during neuroendocrine transdifferentiation, which results in resistance to AR-targeted therapy. Clinically, genomically and epigenetically, NEPC resembles other types of poorly differentiated neuroendocrine tumors (NETs). Through pan-NET analyses, we identified ONECUT2 as a candidate master transcriptional regulator of poorly differentiated NETs. ONECUT2 ectopic expression in prostate adenocarcinoma synergizes with hypoxia to suppress androgen signaling and induce neuroendocrine plasticity. ONEUCT2 drives tumor aggressiveness in NEPC, partially through regulating hypoxia signaling and tumor hypoxia. Specifically, ONECUT2 activates SMAD3, which regulates hypoxia signaling through modulating HIF1α chromatin-binding, leading NEPC to exhibit higher degrees of hypoxia compared to prostate adenocarcinomas. Treatment with hypoxia-activated prodrug TH-302 potently reduces NEPC tumor growth. Collectively, these results highlight the synergy between ONECUT2 and hypoxia in driving NEPC, and emphasize the potential of hypoxia-directed therapy for NEPC patients.