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.

Evofosfamide for the treatment of human papillomavirus-negative head and neck squamous cell carcinoma.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line-derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models. Biomarker analysis used RNA sequencing, whole-exome sequencing, and whole-genome CRISPR knockout screens. Five advanced/metastatic HNSCC patients received evofosfamide monotherapy (480 mg/m2 qw × 3 each month) in a phase 2 study. Evofosfamide was potent and highly selective for hypoxic HNSCC cells. Proliferative rate was a predominant evofosfamide sensitivity determinant and a proliferation metagene correlated with activity in CDX models. Evofosfamide showed efficacy as monotherapy and with radiotherapy in PDX models, augmented CTLA-4 blockade in syngeneic tumors, and reduced hypoxia in nodes disseminated from an orthotopic model. Of 5 advanced HNSCC patients treated with evofosfamide, 2 showed partial responses while 3 had stable disease. In conclusion, evofosfamide shows promising efficacy in aggressive HPV-negative HNSCC, with predictive biomarkers in development to support further clinical evaluation in this indication.

Metabolic and Physiologic Imaging Biomarkers of the Tumor Microenvironment Predict Treatment Outcome with Radiation or a Hypoxia-Activated Prodrug in Mice.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here, we define two imaging biomarkers that predict differences in tumor response to therapy: (i) partial oxygen pressure (pO2), measured by EPR imaging; and (ii) [1-13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa2, and Su.86.86 tumors was 9.1 ± 1.7, 11.1 ± 2.2, and 17.6 ± 2.6 mm Hg, and the rate of pyruvate-to-lactate conversion was 2.72 ± 0.48, 2.28 ± 0.26, and 1.98 ± 0.51 per minute, respectively (n = 6, each). These results are in agreement with steady-state data of matabolites quantified by mass spectroscopy and histologic analysis, indicating glycolytic and hypoxia profile in Hs766t, MiaPaca2, and Su.86.86 tumors. Fractionated radiotherapy (5 Gy × 5) resulted in a tumor growth delay of 16.7 ± 1.6 and 18.0 ± 2.7 days in MiaPaca2 and Su.86.86 tumors, respectively, compared with 6.3 ± 2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0 ± 3.5 and 25.0 ± 7.7 days increase in survival time, respectively) than MiaPaCa2 (2.7 ± 0.4 and 6.7 ± 0.7 days) and Su.86.86 (4.7 ± 0.6 and 0.7 ± 0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiotherapy and TH-302.Significance: pO2 imaging data and clinically available metabolic imaging data provide useful insight into predicting the treatment efficacy of chemotherapy, radiation, and a hypoxia-activated prodrug as monotherapies and combination therapies in PDAC tumor xenograft models. Cancer Res; 78(14); 3783-92. ©2018 AACR.

Hypoxia-activated evofosfamide for treatment of recurrent bevacizumab-refractory glioblastoma: a phase I surgical study.

Background: Anti-angiogenic therapy is known to induce a greater degree of hypoxia, including in glioblastoma (GBM). Evofosfamide (Evo) is a hypoxia-activated prodrug which is reduced, leading to the release of the alkylating agent bromo-isophosphoramide mustard. We assessed the safety, tolerability, preliminary efficacy, and biomarkers of Evo plus bevacizumab (Bev) in Bev-refractory GBM. Methods: Twenty-eight patients with Bev-refractory GBM were enrolled in a dose escalation study receiving from 240 mg/m2 (cohort 1) to 670 mg/m2 (cohort 4) of Evo every 2 weeks in combination with Bev. Patients deemed surgical candidates underwent a single dose of Evo or placebo with pimonidazole immediately prior to surgery for biomarker evaluation, followed by dose escalation upon recovery. Assessments included adverse events, response, and survival. Results: Evo plus Bev was well tolerated up to and including the maximum dose of 670 mg/m2, which was determined to be the recommended phase II dose. Overall response rate was 17.4%, with disease control (complete response, partial response, and stable disease) observed in 14 (60.9%) of the 23 patients. The ratio of enhancement to non-enhancement was significant on log-rank analysis with time to progression (P = 0.023), with patients having a ratio of less than 0.37 showing a median progression-free survival of 98 days versus 56 days for those with more enhancement. Conclusions: Evo plus Bev was well tolerated in patients with Bev-refractory GBM, with preliminary evidence of activity that merits further investigation.

Radiotherapy Synergizes with the Hypoxia-Activated Prodrug Evofosfamide: In Vitro and In Vivo Studies.

AIMS: Evofosfamide (TH-302) is a hypoxia-activated prodrug (HAP) that releases the DNA-damaging bromo-isophosphoramide mustard (Br-IPM) moiety selectively under hypoxic conditions. Since solid tumors are known to have hypoxic regions, HAPs in combination with chemotherapy or radiotherapy (XRT) will be beneficial. We tested the oxygen dependence of release kinetics of Br-IPM using electron paramagnetic resonance (EPR) with spin trapping by monitoring redox cycling of the nitroimidazole moiety of TH-302, and oxygen dependence of TH-302 on in vitro cytotoxicity at different levels of hypoxia was also examined. Two tumor implants (SCCVII and HT29) in mice were studied. RESULTS: TH-302 fragmentation to release Br-IPM was noticed at oxygen levels <76 mmHg, which increased with higher levels of hypoxia. Enhanced cellular cytotoxicity was also observed at oxygen levels <76 mmHg. In vivo pO2 imaging in the two tumor implants showed that the SCCVII tumor implant had higher level of hypoxia compared with the HT29 xenograft. TH-302 as a monotherapy in vivo showed modest effects in SCCVII implants and minimal effects in HT29 xenografts, whereas TH-302 in combination with ionizing radiation showed significant benefit in both tumor models. INNOVATION: We examined the kinetics of redox cycling versus fragmentation of TH-302. The combination of oxygen-dependent XRT with TH-302 is effective even in tumors with significant hypoxia. CONCLUSIONS: Imaging studies identifying the magnitude of hypoxia in tumors indicated that the responsiveness to TH-302 and the antitumor effect of TH-302 were enhanced by combining with XRT in both the TH-302-sensitive SCCVII tumor and -resistant HT29 tumor. Antioxid. Redox Signal. 28, 131-140.

A Combination of Radiation and the Hypoxia-Activated Prodrug Evofosfamide (TH-302) is Efficacious against a Human Orthotopic Pancreatic Tumor Model.

This study was designed to investigate the effect of single-dose radiation therapy (RT) in combination with evofosfamide (TH-302), a hypoxia-activated prodrug, in a pre-clinical model of pancreatic cancer. AsPC1 tumors were implanted orthotopically in the pancreas of nude mice. Tumors were treated with 15 Gy of RT, using a 1 cm diameter field, and delivered as a continuous arc. Image-guidance to center the field on the tumor was based on CT imaging with intraperitoneal contrast. Evofosfamide (100 mg/kg, i.p.) was administered 3 hours before RT. Tumor volumes were measured using ultrasound, and regrowth curves were plotted. Tumor hypoxia and cell proliferation were measured using pimonidazole and the thymidine analog EdU, respectively. In vitro clonogenic assays were performed. Tumors were shown to contain substantial areas of hypoxia, as calculated by percent pimonidazole staining. Evofosfamide was active in these tumors, as demonstrated by a significant reduction in uptake of the thymidine analog EdU. This effect was visible in oxygenated tissue, consistent with the previously reported bystander effects of evofosfamide. RT produced significant regrowth delay, as did evofosfamide. The combination of both agents produced a growth delay that was at least equal to the sum of the two treatments given separately. The improvement in tumor response when evofosfamide is combined with RT supports the hypothesis that hypoxia is a cause of radioresistance in high dose RT for pancreatic cancer. Assessing the efficacy and safety of stereotactic radiation treatment and evofosfamide is warranted in patients with locally advanced pancreatic cancer.

Preclinical Benefit of Hypoxia-Activated Intra-arterial Therapy with Evofosfamide in Liver Cancer.

PURPOSE: To evaluate safety and characterize anticancer efficacy of hepatic hypoxia-activated intra-arterial therapy (HAIAT) with evofosfamide in a rabbit model. EXPERIMENTAL DESIGN: VX2-tumor-bearing rabbits were assigned to 4 intra-arterial therapy (IAT) groups (n = 7/group): (i) saline (control); (ii) evofosfamide (Evo); (iii) doxorubicin-lipiodol emulsion followed by embolization with 100-300 µm beads (conventional, cTACE); or (iv) cTACE and evofosfamide (cTACE + Evo). Blood samples were collected pre-IAT and 1, 2, 7, and 14 days post-IAT. A semiquantitative scoring system assessed hepatocellular damage. Tumor volumes were segmented on multidetector CT (baseline, 7/14 days post-IAT). Pathologic tumor necrosis was quantified using manual segmentation on whole-slide images. Hypoxic fraction (HF) and compartment (HC) were determined by pimonidazole staining. Tumor DNA damage, apoptosis, cell proliferation, endogenous hypoxia, and metabolism were quantified (γ-H2AX, Annexin V, caspase-3, Ki-67, HIF1α, VEGF, GAPDH, MCT4, and LDH). RESULTS: cTACE + Evo showed a similar profile of liver enzymes elevation and pathologic scores compared with cTACE. Neither hematologic nor renal toxicity were observed. Animals treated with cTACE + Evo demonstrated smaller tumor volumes, lower tumor growth rates, and higher necrotic fractions compared with cTACE. cTACE + Evo resulted in a marked reduction in the HF and HC. Correlation was observed between decreases in HF or HC and tumor necrosis. cTACE + Evo promoted antitumor effects as evidenced by increased expression of γ-H2AX, apoptotic biomarkers, and decreased cell proliferation. Increased HIF1α/VEGF expression and tumor glycolysis supported HAIAT. CONCLUSIONS: HAIAT achieved a promising step towards the locoregional targeting of tumor hypoxia. The favorable toxicity profile and enhanced anticancer effects of evofosfamide in combination with cTACE pave the way towards clinical trials in patients with liver cancer. Clin Cancer Res; 23(2); 536-48. ©2016 AACR.

Hypoxia Imaging With PET Correlates With Antitumor Activity of the Hypoxia-Activated Prodrug Evofosfamide (TH-302) in Rodent Glioma Models.

High-grade gliomas are often characterized by hypoxia, which is associated with both poor long-term prognosis and therapy resistance. The adverse role hypoxia plays in treatment resistance and disease progression has led to the development of hypoxia imaging methods and hypoxia-targeted treatments. Here, we determined the tumor hypoxia and vascular perfusion characteristics of 2 rat orthotopic glioma models using 18-fluoromisonidozole positron emission tomography. In addition, we determined tumor response to the hypoxia-activated prodrug evofosfamide (TH-302) in these rat glioma models. C6 tumors exhibited more hypoxia and were less perfused than 9L tumors. On the basis of these differences in their tumor hypoxic burden, treatment with evofosfamide resulted in 4- and 2-fold decreases in tumor growth rates of C6 and 9L tumors, respectively. This work shows that imaging methods sensitive to tumor hypoxia and perfusion are able to predict response to hypoxia-targeted agents. This has implications for improved patient selection, particularly in clinical trials, for treatment with hypoxia-activated cytotoxic prodrugs, such as evofosfamide.

Multimodal targeting of tumor vasculature and cancer stem-like cells in sarcomas with VEGF-A inhibition, HIF-1α inhibition, and hypoxia-activated chemotherapy.

Vascular endothelial growth factor A (VEGF-A) inhibition with pazopanib is an approved therapy for sarcomas, but likely results in compensatory pathways such as upregulation of hypoxia inducible factor 1α (HIF-1α). In addition, cancer stem-like cells can preferentially reside in hypoxic regions of tumors and be resistant to standard chemotherapies. In this study, we hypothesized that the combination of VEGF-A inhibition, HIF-1α inhibition, and hypoxia-activated chemotherapy with evofosfamide would be an effective multimodal strategy. Multimodal therapy was examined in one genetically engineered and two xenograft mouse models of sarcoma. In all three models, multimodal therapy showed greater efficacy than any single agent therapy or bimodality therapy in blocking tumor growth. Even after cessation of therapy, tumors treated with multimodal therapy remained relatively dormant for up to 2 months. Compared to the next best bimodality therapy, multimodal therapy caused 2.8-3.3 fold more DNA damage, 1.5-2.7 fold more overall apoptosis, and 2.3-3.6 fold more endothelial cell-specific apoptosis. Multimodal therapy also decreased microvessel density and HIF-1α activity by 85-90% and 79-89%, respectively, compared to controls. Sarcomas treated with multimodal therapy had 95-96% depletion of CD133(+) cancer stem-like ells compared to control tumors. Sarcoma cells grown as spheroids to enrich for CD133(+) cancer stem-like cells were more sensitive than monolayer cells to multimodal therapy in terms of DNA damage and apoptosis, especially under hypoxic conditions. Thus multimodal therapy of sarcomas with VEGF-A inhibition, HIF-1α inhibition, and hypoxia-activated chemotherapy effectively blocks sarcoma growth through inhibition of tumor vasculature and cancer stem-like cells.

MR Imaging Biomarkers to Monitor Early Response to Hypoxia-Activated Prodrug TH-302 in Pancreatic Cancer Xenografts.

TH-302 is a hypoxia-activated prodrug known to activate selectively under the hypoxic conditions commonly found in solid tumors. It is currently being evaluated in clinical trials, including two trials in Pancreatic Ductal Adenocarcinomas (PDAC). The current study was undertaken to evaluate imaging biomarkers for prediction and response monitoring of TH-302 efficacy in xenograft models of PDAC. Dynamic contrast-enhanced (DCE) and diffusion weighted (DW) magnetic resonance imaging (MRI) were used to monitor acute effects on tumor vasculature and cellularity, respectively. Three human PDAC xenografts with known differential responses to TH-302 were imaged prior to, and at 24 h and 48 hours following a single dose of TH-302 or vehicle to determine if imaging changes presaged changes in tumor volumes. DW-MRI was performed at five b-values to generate apparent diffusion coefficient of water (ADC) maps. For DCE-MRI, a standard clinically available contrast reagent, Gd-DTPA, was used to determine blood flow into the tumor region of interest. TH-302 induced a dramatic decrease in the DCE transfer constant (Ktrans) within 48 hours after treatment in the sensitive tumors, Hs766t and Mia PaCa-2, whereas TH-302 had no effect on the perfusion behavior of resistant SU.86.86 tumors. Tumor cellularity, estimated from ADC, was significantly increased 24 and 48 hours after treatment in Hs766t, but was not observed in the Mia PaCa-2 and SU.86.86 groups. Notably, growth inhibition of Hs766t was observed immediately (day 3) following initiation of treatment, but was not observed in MiaPaCa-2 tumors until 8 days after initiation of treatment. Based on these preclinical findings, DCE-MRI measures of vascular perfusion dynamics and ADC measures of cell density are suggested as potential TH-302 response biomarkers in clinical trials.

Comparison of hypoxia-activated prodrug evofosfamide (TH-302) and ifosfamide in preclinical non-small cell lung cancer models.

Evofosfamide (TH-302) is a hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide. In hypoxic conditions Br-IPM is released and alkylates DNA. Ifosfamide is a chloro-isophosphoramide prodrug activated by hepatic Cytochrome P450 enzymes. Both compounds are used for the treatment of cancer. Ifosfamide has been approved by the FDA while evofosfamide is currently in the late stage of clinical development. The purpose of this study is to compare efficacy and safety profile of evofosfamide and ifosfamide in preclinical non-small cell lung cancer H460 xenograft models. Immunocompetent CD-1 mice and H460 tumor-bearing immunocompromised nude mice were used to investigate the safety profile. The efficacy of evofosfamide or ifosfamide, alone, and in combination with docetaxel or sunitinib was compared in ectopic and intrapleural othortopic H460 xenograft models in animals exposed to ambient air or different oxygen concentration breathing conditions. At an equal body weight loss level, evofosfamide showed greater or comparable efficacy in both ectopic and orthotopic H460 xenograft models. Evofosfamide, but not ifosfamide, exhibited controlled oxygen concentration breathing condition-dependent antitumor activity. However, at an equal body weight loss level, ifosfamide yielded severe hematologic toxicity when compared to evofosfamide, both in monotherapy and in combination with docetaxel. At an equal hematoxicity level, evofosfamide showed superior antitumor activity. These results indicate that evofosfamide shows superior or comparable efficacy and a favorable safety profile when compared to ifosfamide in preclinical human lung carcinoma models. This finding is consistent with multiple clinical trials of evofosfamide as a single agent, or in combination therapy, which demonstrated both anti-tumor activity and safety profile without severe myelosuppression.

Hypoxia-Activated Prodrug TH-302 Targets Hypoxic Bone Marrow Niches in Preclinical Leukemia Models.

PURPOSE: To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models. EXPERIMENTAL DESIGN: Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models. RESULTS: Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a "hypoxia index" compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells. CONCLUSIONS: These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins.

Combination treatment with hypoxia-activated prodrug evofosfamide (TH-302) and mTOR inhibitors results in enhanced antitumor efficacy in preclinical renal cell carcinoma models.

Tumors often consist of hypoxic regions which are resistant to chemo- and radiotherapy. Evofosfamide (also known as TH-302), a 2-nitroimidazole triggered hypoxia-activated prodrug, preferentially releases the DNA cross-linker bromo-isophosphoramide mustard in hypoxic cells. The intracellular kinase mTOR plays a key role in multiple pathways which are important in cancer progression. Here we investigated the enhanced efficacy profile and possible mechanisms of evofosfamide in combination with mTOR inhibitor (mTORi) everolimus or temsirolimus in renal cell carcinoma (RCC) xenograft models. The antitumor activities of the mTORi everolimus or temsirolimus alone, evofosfamide alone, or the combination were investigated in the 786-O and Caki-1 RCC cells in vitro and in vivo xenograft models. Two schedules were tested in which evofosfamide was started on the same day as the mTORi or 1 week after. Combination mechanisms were investigated by measuring a panel of pharmacodynamic biomarkers by immunohistochemistry. Antitumor efficacy in both RCC xenograft models was enhanced by the combination of evofosfamide and mTORi. Evofosfamide reduced the increased hypoxia induced by mTORi. Combination treatment induced increased DNA damage, decreased cell proliferation, and decreased survivin. Addition of mTORi did not change evofosfamide-mediated cytotoxicity in 786-O or Caki-1 cells in vitro which might suggest cell non-autonomous effects, specifically increased tumor hypoxia, are important for the in vivo combination activity. Taken together, evofosfamide potentiates the antitumor efficacy of mTOR inhibitors and inhibits the increased tumor hypoxia caused by mTOR inhibition. These studies provide a translational rationale for combining evofosfamide with mTOR inhibitors in clinical studies.

Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition.

BACKGROUND: The hypoxia-activated prodrug TH-302 is reduced at its nitroimidazole group and selectively under hypoxic conditions releases the DNA cross-linker bromo-isophosphoramide mustard (Br-IPM). Here, we have explored the effect of Chk1 inhibition on TH-302-mediated pharmacological activities. METHODS: We employed in vitro cell viability, DNA damage, cellular signaling assays and the in vivo HT29 human tumor xenograft model to study the effect of Chk1inhibition on TH-302 antitumor activities. RESULTS: TH-302 cytotoxicity is greatly enhanced by Chk1 inhibition in p53-deficient but not in p53-proficient human cancer cell lines. Chk1 inhibitors reduced TH-302-induced cell cycle arrest via blocking TH-302-induced decrease of phosphorylation of histone H3 and increasing Cdc2-Y15 phosphorylation. Employing the single-cell gel electrophoresis (comet) assay, we observed a potentiation of the TH-302 dependent tail moment. TH-302 induced γH2AX and apoptosis were also increased upon the addition of Chk1 inhibitor. Potentiation of TH-302 cytotoxicity by Chk1 inhibitor was only observed in cell lines proficient in, but not deficient in homology-directed DNA repair. We also show that combination treatment led to lowering of Rad51 expression levels as compared to either agent alone. In vivo data demonstrate that Chk1 inhibitor enhances TH-302 anti-tumor activity in p53 mutant HT-29 human tumor xenografts, supporting the hypothesis that these in vitro results can translate to enhanced in vivo efficacy of the combination. CONCLUSIONS: TH-302-mediated in vitro and in vivo anti-tumor activities were greatly enhanced by the addition of Chk1 inhibitors. The preclinical data presented in this study support a new approach for the treatment of p53-deficient hypoxic cancers by combining Chk1 inhibitors with the hypoxia-activated prodrug TH-302.

TH-302 in Combination with Radiotherapy Enhances the Therapeutic Outcome and Is Associated with Pretreatment [18F]HX4 Hypoxia PET Imaging.

PURPOSE: Conventional anticancer treatments are often impaired by the presence of hypoxia. TH-302 selectively targets hypoxic tumor regions, where it is converted into a cytotoxic agent. This study assessed the efficacy of the combination treatment of TH-302 and radiotherapy in two preclinical tumor models. The effect of oxygen modification on the combination treatment was evaluated and the effect of TH-302 on the hypoxic fraction (HF) was monitored using [(18)F]HX4-PET imaging and pimonidazole IHC stainings. EXPERIMENTAL DESIGN: Rhabdomyosarcoma R1 and H460 NSCLC tumor-bearing animals were treated with TH-302 and radiotherapy (8 Gy, single dose). The tumor oxygenation status was altered by exposing animals to carbogen (95% oxygen) and nicotinamide, 21% or 7% oxygen breathing during the course of the treatment. Tumor growth and treatment toxicity were monitored until the tumor reached four times its start volume (T4×SV). RESULTS: Both tumor models showed a growth delay after TH-302 treatment, which further increased when combined with radiotherapy (enhancement ratio rhabdomyosarcoma 1.23; H460 1.49). TH-302 decreases the HF in both models, consistent with its hypoxia-targeting mechanism of action. Treatment efficacy was dependent on tumor oxygenation; increasing the tumor oxygen status abolished the effect of TH-302, whereas enhancing the HF enlarged TH-302's therapeutic effect. An association was observed in rhabdomyosarcoma tumors between the pretreatment HF as measured by [(18)F]HX4-PET imaging and the T4×SV. CONCLUSIONS: The combination of TH-302 and radiotherapy is promising and warrants clinical testing, preferably guided by the companion biomarker [(18)F]HX4 hypoxia PET imaging for patient selection.

Efficacy and safety of the hypoxia-activated prodrug TH-302 in combination with gemcitabine and nab-paclitaxel in human tumor xenograft models of pancreatic cancer.

Tumors often contain hypoxic regions resistant to chemo- and radiotherapy. TH-302 (T) is an investigational hypoxia-activated prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. This study evaluated the efficacy and safety profile of combining T with gemcitabine (G) and nab-paclitaxel (nP) in human pancreatic ductal adenocarcinoma (PDAC) xenograft models in mice. Antitumor activity of the G + nP + T triplet was assessed and compared with T-alone or the G + nP doublet in the Hs766t, MIA PaCa-2, PANC-1, and BxPC-3 PDAC xenograft models. Efficacy was assessed by tumor growth kinetic analysis. Body weight, blood cell counts, blood chemistry, and the von Frey neuropathy assay were analyzed to evaluate safety profiles. Pharmacodynamic changes after the treatment were determined by immunohistochemistry of cell proliferation, DNA damage, apoptosis, hypoxia, and tumor stroma density. The G + nP + T triplet exhibited enhanced efficacy compared with T-alone or the G + nP doublet. Compared with vehicle (V), G + nP induced body weight loss, reduced neutrophil and lymphocyte counts, increased the levels of liver function parameters, and induced neurotoxicity. However, when T was added to G + nP, there was no statistically increased impairment compared to G + nP. The triplet significantly increased DNA damage, apoptosis, and tumor necrosis. Furthermore, the triplet further inhibited cell proliferation and reduced stroma density and intratumoral hypoxia. The triplet combination of G + nP + T exhibited superior efficacy but additive toxicity was not evident compared to the G + nP doublet in this study. This study provides a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety. A Phase I clinical trial of the triplet combination is currently underway (NCT02047500).

Evaluation of the "steal" phenomenon on the efficacy of hypoxia activated prodrug TH-302 in pancreatic cancer.

Pancreatic ductal adenocarcinomas are desmoplastic and hypoxic, both of which are associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are specifically activated in hypoxic environments to release cytotoxic or cytostatic effectors. TH-302 is a HAP that is currently being evaluated in a Phase III clinical trial in pancreatic cancer. Using animal models, we show that tumor hypoxia can be exacerbated using a vasodilator, hydralazine, improving TH-302 efficacy. Hydralazine reduces tumor blood flow through the "steal" phenomenon, in which atonal immature tumor vasculature fails to dilate in coordination with normal vasculature. We show that MIA PaCa-2 tumors exhibit a "steal" effect in response to hydralazine, resulting in decreased tumor blood flow and subsequent tumor pH reduction. The effect is not observed in SU.86.86 tumors with mature tumor vasculature, as measured by CD31 and smooth muscle actin (SMA) immunohistochemistry staining. Combination therapy of hydralazine and TH-302 resulted in a reduction in MIA PaCa-2 tumor volume growth after 18 days of treatment. These studies support a combination mechanism of action for TH-302 with a vasodilator that transiently increases tumor hypoxia.