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PDF file - 116 KB, Combination index analysis of TH-302 combined with bortezomib at a non-constant ratio in MMS1 cells.

PDF file - 117 KB, Combination index analysis of TH-302 combined with bortezomib at a non-constant ratio in OPM2 cells.

TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of γH2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell–based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect. Mol Cancer Ther; 11(3); 740–51. ©2011 AACR.

Recently, we showed that hypoxia is a critical microenvironmental factor in multiple myeloma, and that the hypoxia-activated prodrug TH-302 selectively targets hypoxic multiple myeloma cells and improves multiple disease parameters in vivo. To explore approaches for sensitizing multiple myeloma cells to TH-302, we evaluated in this study the antitumor effect of TH-302 in combination with the clinically used proteasome inhibitor bortezomib. First, we show that TH-302 and bortezomib synergistically induce apoptosis in multiple myeloma cell lines in vitro. Second, we confirm that this synergism is related to the activation of caspase cascades and is mediated by changes of Bcl-2 family proteins. The combination treatment induces enhanced cleavage of caspase-3/8/9 and PARP, and therefore triggers apoptosis and enhances the cleavage of proapoptotic BH3-only protein BAD and BID as well as the antiapoptotic protein Mcl-1. In particular, TH-302 can abrogate the accumulation of antiapoptotic Mcl-1 induced by bortezomib, and decreases the expression of the prosurvival proteins Bcl-2 and Bcl-xL. Furthermore, we found that the induction of the proapoptotic BH3-only proteins PUMA (p53-upregulated modulator of apoptosis) and NOXA is associated with this synergism. In response to the genotoxic and endoplasmic reticulum stresses by TH-302 and bortezomib, the expression of PUMA and NOXA were upregulated in p53-dependent and -independent manners. Finally, in the murine 5T33MMvv model, we showed that the combination of TH-302 and bortezomib can improve multiple disease parameters and significantly prolong the survival of diseased mice. In conclusion, our studies provide a rationale for clinical evaluation of the combination of TH-302 and bortezomib in patients with multiple myeloma. Mol Cancer Ther; 12(9); 1763–73. ©2013 AACR.

PDF file - 120K, Supplemental Figure 5. Effects of hypoxia and drug treatment on ROS generation by AML cells.

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.

Plots of tumor growth delay vs HF10; pO2; lac/pyr

Supplementary Table 2. Time to reach 4 times start volume per treatment group for H460 bearing animals. Data represent mean {plus minus} SD, p-value and sensitization enhancement ratio.

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Supplementary Figure S1: Quantitative volumetric image analysis.

Median pO2 and HF10 of individual PDAC tumors in EPR oxygen imaging study.

Purpose: Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm. Recent evidence has shown the bone marrow microenvironment in patients with AML to be intrinsically hypoxic. Adaptive cellular responses by leukemia cells to survive under low oxygenation also confer chemoresistance. We therefore asked whether therapeutic exploitation of marrow hypoxia via the hypoxia-activated nitrogen mustard prodrug, TH-302, could effectively inhibit AML growth.Experimental Design: We assessed the effects of hypoxia and TH-302 on human AML cells, primary samples, and systemic xenograft models.Results: We observed that human AML cells and primary AML colonies cultured under chronic hypoxia (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic controls. TH-302 treatment resulted in dose- and hypoxia-dependent apoptosis and cell death in diverse AML cells. TH-302 preferentially decreased proliferation, reduced HIF-1α expression, induced cell-cycle arrest, and enhanced double-stranded DNA breaks in hypoxic AML cells. Hypoxia-induced reactive oxygen species by AML cells were also diminished. In systemic human AML xenografts (HEL, HL60), TH-302 [50 mg/kg intraperitoneally (i.p.) 5 times per week] inhibited disease progression and prolonged overall survival. TH-302 treatment reduced the number of hypoxic cells within leukemic bone marrows and was not associated with hematologic toxicities in nonleukemic or leukemic mice. Later initiation of TH-302 treatment in advanced AML disease was as effective as earlier TH-302 treatment in xenograft models.Conclusions: Our results establish the preclinical activity of TH-302 in AML and provide the rationale for further clinical studies of this and other hypoxia-activated agents for leukemia therapy. Clin Cancer Res; 19(23); 6506–19. ©2013 AACR.

Supplementary Figure S1. TH-302 has potent antitumor activity in a 3D in vitro co-culture system of leukemia cells and bone marrow-derived mesenchymal stromal cells that recapitulates the multidimensional BM niche Supplementary Figure S2. Hypoxia is present in spheroids as indicated by PIMO positive staining in spheroids incubated with PIMO for 3hr before harvesting Supplementary figure S3. In vitro synergistic activity of TH-302 in combination with chemotherapy and demethylating agents Supplementary Figure S4. TH-302 has anti-leukemia activity in an in vivo primary AML xenograft murine model. Supplementary figure S5. In vitro synergistic activity of TH-302 in combination with sorafenib Supplementary figure S6 A, B. Assessment of BM hypoxia in AML/ETO bearing mice treated with TH-302 or chemotherapy

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 [18F]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 [18F]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 [18F]HX4 hypoxia PET imaging for patient selection. Clin Cancer Res; 21(13); 2984–92. ©2015 AACR.

Supplementary Figure S1-S3 legends

Supplemental Methods and Tables 1) mRNA Hybridization and Gene-expression Profiling. 2) Gene expression analysis by Nanostring technology. 3) Hyperpolarized Magnetic Resonance Spectroscopy. 4) In Vitro 1-13C Pyruvic Acid Feeding Studies and High Resolution Nuclear Magnetic Resonance (NMR) Experiments 5) Three-dimensional spheroids 6) Murine tumor models 7) Immunohistochemistry Supplementary Table S1. Cell line characteristics Supplementary Table S2. Patient characteristics Supplemental Table S3. List of upregulated DEPs in hypoxia. Supplemental Table S4. 1-13C pyruvate uptake and 1-13C lactate production in cell culture. Supplemental Table S5. TH-302 cytotoxic activity against leukemia cell lines

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.

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. Clin Cancer Res; 22(7); 1687–98. ©2015 AACR.

Supplementary figure 4. Body weight was monitored during and after treatment for both rhabdomyosarcoma (A) and H460 (B) tumor-bearing animals. The change in body weight is represented in percentage. Data represent mean {plus minus} SEM.

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Supplementary figure 3. A) Tumor growth of rhabdomyosarcoma bearing animals (n=8) treated with TH-302 (25mg/kg) in combination with 4, 8 or 12Gy of RT. Data represent mean {plus minus} SEM. B) T4xSV for rhabdomyosarcoma bearing animals treated with TH-302 in combination with RT 0, 4, 8 or 12Gy. C) T4xSV for rhabdomyosarcoma bearing animals exposed to different oxygen concentrations in combination with TH-302 and RT. D) T4xSV for H460 bearing animals exposed to different oxygen concentrations in combination with TH-302 and RT.

Supplementary figure 1. A) Treatment schedule for the rhabdomyosarcoma and H460 models. All animals were randomized over either the treatment or the control group. These groups were both treated with either NaCl or TH-302 (rhabdomysarcoma 25mg/kg, H460 50mg/kg). Rhabdomyosarcoma bearing animals started with oxygen modification at day 0 and a [18F]HX4 hypoxia PET scan. For histological controls only, this scan was repeated on day 4. Animals from the treatment group were exposed to radiotherapy (RT) at the end of treatment day 3 and their tumor volume was monitored until 4 times start volume was reached. H460 tumor bearing animals from the histological control group were injected with pimonidazole and Hoechst on the last treatment day and sacrificed. Animals from the treatment group were exposed to radiotherapy at day 4 and monitored until a tumor volume of 4 times start treatment volume was reached.

PDF file - 66K, Supplemental Table 1. Cell cycle analysis of AML cells treated with TH-302. AML cells were treated with vehicle (PBS) or TH- 302 at the indicated concentrations for 24 hours under hypoxic (1% O2) or normoxic (21% O2) conditions in triplicate wells. Cells were stained with propidium iodide staining and analyzed by flow cytometry. Mean plus-minus SEM percentages of cells in each cell cycle phase are shown. Similar results were obtained in three independent experiments. Supplemental Table 2. TH-302 induces dose and hypoxia-dependent apoptosis in representative human AML cell lines. Shown are the mean plus-minus SEM percentages of total apoptotic and dead cells as determined by flow cytometry for 7-AAD/annexin-V expression in a panel of seven human AML cell lines. Known molecular and cytogenetic characteristics of each cell line is provided. Cells were incubated with vehicle or TH-302 at doses ranging from 0.1-10 μM under normoxia (21% O2) or hypoxia (1% O2) for 72 hours. Cells were plated in multiple wells per condition with summarized results of at least two independent experiments provided. Results of student t test analysis comparing results under normoxia vs. hypoxia are provided.

PDF file - 372K, Supplemental Figure 1. AML marrow progression is associated with increased hypoxia.

PDF file - 635K, Supplemental Figure 4. Hypoxia attenuates cytarabine-induced apoptosis of human AML cells. Hypoxia attenuates chemotherapy-induced apoptosis of human AML cells.

Supplementary Table 1. Time to reach 4 times start volume per treatment group for rhabdomyosarcoma bearing animals. Data represent mean {plus minus} SD, p-value and sensitization enhancement ratio.

PDF file - 179K, Supplemental Figure 2. The number of hypoxic marrow cells correlate with AML burden.

Supplementary figure 5. Quantification of H460 histological staining for A) necrotic fraction (NF), B) relative vessel area (RVA), C) perfusion fraction (PF) and D) relative perfusion area (RPA). Data represent mean {plus minus} SEM.

PDF file - 98K, Supplemental Figure 3. Cytarabine induces apoptosis of human AML (HL60/PAR) cells in a dose and time-dependent manner.

Powassan virus (POWV) is a tick-borne neuroinvasive flavivirus endemic to North America. It is generally transmitted by the tick

On the Ground • Once mesquite encroachment is initiated it is difficult to reverse and continually degrades grasslands, hindering grass production that benefits both livestock and wildlife. • We evaluated the effectiveness of Sendero herbicide in the treatment of western honey mesquite. • We compared two treatment types (Sendero and Sendero plus Remedy Ultra) and two application methods (individual plant treatment and aerial broadcast). • Percent cover of grasses and some forbs increased throughout our study sites post treatment. • All treatment types were successful at decreasing the percent canopy cover of western honey mesquite, and we found no difference between treatment types.

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 ρ0 (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 ρ0 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.