Tumor Growth Delay Studies Research Articles

Changing the Landscape of Solid Tumor Therapy from Apoptosis-Promoting to Apoptosis-Inhibiting Strategies.

The many limitations of implementing anticancer strategies under the term "precision oncology" have been extensively discussed. While some authors propose promising future directions, others are less optimistic and use phrases such as illusion, hype, and false hypotheses. The reality is revealed by practicing clinicians and cancer patients in various online publications, one of which has stated that "in the quest for the next cancer cure, few researchers bother to look back at the graveyard of failed medicines to figure out what went wrong". The message is clear: Novel therapeutic strategies with catchy names (e.g., synthetic "lethality") have not fulfilled their promises despite decades of extensive research and clinical trials. The main purpose of this review is to discuss key challenges in solid tumor therapy that surprisingly continue to be overlooked by the Nomenclature Committee on Cell Death (NCCD) and numerous other authors. These challenges include: The impact of chemotherapy-induced genome chaos (e.g., multinucleation) on resistance and relapse, oncogenic function of caspase 3, cancer cell anastasis (recovery from late stages of apoptosis), and pitfalls of ubiquitously used preclinical chemosensitivity assays (e.g., cell "viability" and tumor growth delay studies in live animals) that score such pro-survival responses as "lethal" events. The studies outlined herein underscore the need for new directions in the management of solid tumors.

MNP hyperthermia and hypofractionated radiation activate similar immunogenetic and cytotoxic pathways

Objective The goal of this study is to better understand the immunogenetic expression and related cytotoxic responses of moderate but clinically relevant doses of hypofractionated radiation (1x15 Gy and 3x8 Gy) and magnetic nanoparticle hyperthermia (mNPH, CEM43 30) Methods Genetic, protein, immunopathology and tumor growth delay assessments were used to determine the immune and cytotoxic responses following radiation and mNPH alone and in combination. Although the thermal dose used, 43 C°/30 min (CEM43 30), typically results in modest independent cytotoxicity, it has shown the ability to stimulate an immune response and enhance other cancer treatments. The radiation doses studied (15 Gy and 3x8 Gy) are commonly used in preclinical research and are effective in selected stereotactic and palliative treatment settings, however they are not commonly used as first-line primary tumor treatment regimens. Results Our RNA-based genetic results suggest that while many of the cytotoxic and immune gene and protein pathways for radiation and hyperthermia are similar, radiation, at the doses used, results in a more consistent and expansive anti-cancer immune/cytotoxic expression profile. These results were supported by immunohistochemistry based cytotoxic T-cell tumor infiltration and tumor growth delay studies. When used together radiation and hyperthermia led to greater immune and cytotoxic activity than either modality alone. Conclusion This study clearly shows that modest, but commonly used hypofractionated radiation and hyperthermia doses share many important immune and cytotoxic pathways and that combining the treatments, as compared to either treatment alone, results in genetic and biological anti-cancer benefits.

Abstract 1683: A comparative evaluation of immunotherapy responses in murine colon carcinoma

Abstract With the drive in oncology to develop molecules targeting the immune system, there is a need for reliable and well-characterized preclinical models. To this end, the impact of checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1, has been extensively investigated preclinically, and the landscape of available models varies greatly in response to these molecules. With several recent checkpoint inhibitor-based drug approvals, the focus of immuno-oncology drug development has shifted to immunometabolism targets and costimulatory molecules, particularly with combination strategies. Comparative preclinical tumor model validation with respect to these targets is generally lacking; however, the same level of granularity as was afforded anti-PD-1 and anti-PD-L1 would be needed with these molecules to allow confidence in designing preclinical proof of concept studies for future clinical development. To better understand the utility of these agents in preclinical development, we examined the response of two murine colon carcinoma models, CT26 and MC38, against a panel of costimulatory molecules and epacadostat, an IDO inhibitor, using in vivo tumor growth delay studies. Our data demonstrate that CT26 tumors are sensitive to the costimulatory molecules, anti-GITR, anti-CD137, and anti-OX40, resulting in 6/10, 7/10, and 2/10 complete responses (CRs), respectively. In contrast, the response of MC38 tumors to these agents was modest with no CRs and times to evaluation size (TES) extending only 1.4 to 6.9 days beyond control. Epacadostat treatment did not impact growth of CT26 or MC38, with TES's extended by 4.4 or 2.3 days, respectively, compared to control. Interestingly, addition of epacadostat did not improve the response of anti-PD-1 in either model. To determine whether the changes in response could be related to differences in immune infiltration of these tumors, immunophenotyping from untreated CT26 and MC38 tumors was evaluated. We found that immune cell populations also highlighted potential differences in these models. CT26 tumors were well infiltrated with a mean of 25.2% CD45+ cells, with 54.8% of CD3+ cells being CD8+ T cells. Comparatively, MC38 tumors were weakly infiltrated with CD45+ cells (5.3%) with 35.9% of CD3+ cells being CD8+ T cells. Macrophage and MSDC infiltrates, as a percent of CD11b+ cells, in CT26 tumors were generally double that found in MC38 tumors. Our results indicate that CT26 is more sensitive to the costimulatory molecules tested than MC38. Further, epacadostat as monotherapy or in combination with anti-PD-1 did not elicit clinically meaningful tumor growth delay in either model. Stark differences were also seen in overall lymphocyte infiltrate and composition between CT26 and MC38. The differences in baseline immune infiltrate and efficacy allow us to determine which models and immunotherapies are best suited for rational drug combination approaches. Citation Format: Sheri R. Barnes, Sumithra Urs, Mary Anne Meade, David Draper, Hillary Evens, Daniel Saims, Scott Wise, Maryland Rosenfeld Franklin. A comparative evaluation of immunotherapy responses in murine colon carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1683.

Preclinical studies of SB 11285, a novel STING agonist for immuno-oncology.

e14616 Background: The activation of innate and adaptive immunity via Stimulator of Interferon Genes (STING) signaling is a potentially transformative immuno-therapeutic strategy in cancer. We report here in vivo efficacy and safety studies of SB 11285. Methods: Tumor Growth Inhibition (TGI) and Tumor Growth Delay (TGD) studies in syngeneic mouse models were initiated when mean tumor volume (MTV) reached 100mm3: A20 Lymphoma: (10 animals/Group); (A) Saline; (B) 100µg SB 11285, intratumoral (i.t.), days 3,4,6,8,10; (C) 100mg/kg Cyclophosphamide, intraperitoneal (i.p), days 1,2; and (D) combination of cyclophosphamide+SB 11285. CT26 Colon Carcinoma experimental design is shown in Table. Re-challenge study was initiated in tumor-free animals in A20 lymphoma model on day 73, and monitored for an additional 45 days. Presence of activated immune cells intumor tissues was evaluated by immuno-histochemistry. Cytokine response was evaluated in serum after a single i.p. injection of SB 11285 at 10mg/kg. Maximum Tolerated Dose (MTD) in mice was determined by daily i.p. injection of SB 11285 for 10 days. Results: A20 model: % TGI in the treatment groups were—A, 0; B, 86; C, 98, and D, 93; % TGD, day 70: A, 0; B, 64; C, 156; D, 288. In D, day 73, 90% of animals remained tumor-free. CT26 model: Table 1 shows MTV on day 19 and %TGD on day 43; Re-challenge study. All animals from the SB 11285-treated groups remained completely tumor-free on day 45 compared to control group (MTV,1666 mm3); Immuno-histochemistry of SB 11285-treated groups, revealed the infiltration of CD8+T and NK cells into tumor and surrounding tissues; Cytokine analysis did not show systemic inflammatory response; MTD of SB 11285 was 16 mg/kg/day. Conclusions: SB 11285, a novel STING agonist, showed very potent, and highly durable immune response-mediated anti-tumor activity. SB 11285 was well tolerated, safe, and is being advanced to IND-enabling studies. [Table: see text]

Dynamic treatment effect (DTE) curves reveal the mode of action for standard and experimental cancer therapies.

We present a method for estimating the empirical dynamic treatment effect (DTE) curves from tumor growth delay (TGD) studies. This improves on current common methods of TGD analysis, such as T/C ratio and doubling times, by providing a more detailed treatment effect and overcomes their lack of reproducibility. The methodology doesn't presuppose any prior form for the treatment effect dynamics and is shown to give consistent estimates with missing data. The method is illustrated by application to real data from TGD studies involving three types of therapy. Firstly, we demonstrate that radiotherapy induces a sharp peak in inhibition in a FaDu model. The height, duration and timing of the peak increase linearly with radiation dose. Second, we demonstrate that a combination of temozolomide and an experimental therapy in a glioma PDX model yields an effect, similar to an additive version of the DTE curves for the mono-therapies, except that there is a 30 day delay in peak inhibition. In the third study, we consider the DTE of anti-angiogenic therapy in glioma. We show that resulting DTE curves are flat. We discuss how features of the DTE curves should be interpreted and potentially used to improve therapy.

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins.

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell-specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies.

Abstract 827: Camptothecins: Tissue penetration and implications for therapy

Abstract We have evaluated distribution and micro-regional activity of members of the camptothecin class of topoisomerase I targeting agents including topotecan (TPT), irinotecan (IRN), Irinophore C (IrC), a liposomal formulation of IRN and SN38, the active metabolite of IRN. Despite having a similar mechanism of action these agents are used against different cancers and possess significantly different pharmacological and physicochemical properties. The native fluorescence of each agent was used to visualize its distribution in HCT116 & HT29 tissue cryosections that were subsequently processed via multiplexed staining to relate drug exposure to its effect on tumor cell status (proliferation, apoptosis) and distance from vasculature. TPT exhibited the most uniform tumor distribution but it also experienced a significantly faster tumor wash-out while IRN was retained longer in tumors but exhibited a larger concentration gradient in relation to tumor blood vessels. IrC, the high molecular weight liposomal formulation, exhibited the poorest tissue distribution but it also displayed longer tumor retention due to its extended plasma half-life. At a depth of 100 µm away from vessels drug levels reached 94 ± 5 % (TPT), 41 ± 10 % (IRN) and 4.8 ± 2 % (IrC) of maximal levels seen near vessels. In terms of vessel permeability, IRN and IrC did not extravasate equally from all vessels, ∼50% of vessels showed little permeation by the two drugs and, because of this, some microregions of the tumors received much higher exposure than others. However, despite the significant differences in their tissue distribution, all 3 agents were able to exert a complete suppression of cycling tumor cells at 4 hours following treatment. At 24 hours following TPT or IRN tumor proliferation status returned to normal levels indicating that neither drug was retained long enough to exert activity on out of cycle cells. In contrast IrC benefited from an extended plasma exposure and was able to inhibit proliferation over a period of several days. The time to 50% recovery in proliferation was ∼3 days for TPT and IRN versus 14 days for IrC. Tumor growth delay studies showed that both TPT and IRN benefited somewhat from a dose splitting schedule designed to specifically target proliferation at peak levels two days in row. However, this strategy was still outperformed by IrC. Tumor growth delay following 50 mg/kg drug qwx2 was 9.6 ± 3 days (IRN) versus 27.8 ± 3.8 days (IrC). To determine the root cause of the discrepancy between the poor distribution but good microregional activity of IRN and IrC an in vitro 3D tissue-disc model was employed. It was found that IRN tissue penetration was limited by its high degree of cellular accumulation. SN38, the active metabolite of IRN, did not accumulate to the same extent and did penetrate tissue effectively. Hence it was concluded that the poor tissue distribution observed for both IRN and IrC was offset by systemic conversion to SN38, which was able to exert a more uniform activity within tumors. Citation Format: Alastair H. Kyle, Maria Jose Gandolfo, Jennifer H.E. Baker, Andrew I. Minchinton. Camptothecins: Tissue penetration and implications for therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 827. doi:10.1158/1538-7445.AM2014-827

Hsp90 inhibition enhances PI-3 kinase inhibition and radiosensitivity in glioblastoma

Combined targeting with a PI3-kinase inhibitor, BKM120, and an Hsp90 inhibitor, HSP990, was investigated as a multi-targeted approach to potentiate cell death in glioblastoma (GBM). Additionally, the effect of dual drug treatment combined with cytotoxic stress (radiation therapy) was examined. Four human GBM cell lines containing wild-type or mutated PTEN and/or p53 were studied. The effects of drug treatments on cell viability, apoptosis induction, pAKt activity, cell cycle arrest, clonogenicity, and tumor growth delay were studied. Combined concurrent treatment with both drugs produced more cell killing in cell viability and apoptosis assays than either drug alone. BKM120 plus HSP990 induced suppression of baseline Akt signaling as well as radiation (RT)-induced pAkt signaling in all cell lines. Cell cycle analysis revealed that HSP990 and BKM120, singly or combined, induced G2/M arrest leading to apoptosis/necrosis and polyploidy. Additionally, the drugs radiosensitized GBM cells in clonogenic assays. In vivo tumor growth delay studies demonstrated the effectiveness of combined drug treatment with HSP990 and BKM120 over single drug treatment, as well as the effectiveness of combined drug treatment in enhancing the effectiveness of radiation therapy. In conclusion, HSP990 and BKM120, with and without RT, are active agents against glioma tumors. The sensitivity to these agents does not appear to depend on PTEN/p53status in the cell lines tested. We suggest that the combined action of both drugs is a viable multi-targeted strategy with the potential to improve clinical outcome for patients with high-grade glioma.

18F-EF5 PET Imaging as an Early Response Biomarker for the Hypoxia-Activated Prodrug SN30000 Combined with Radiation Treatment in a Non–Small Cell Lung Cancer Xenograft Model

Hypoxia is a significant therapeutic problem for solid tumors because hypoxic cells are treatment-resistant and more aggressive. Hypoxia-activated prodrugs such as SN30000 use a mechanism of activation in hypoxic cells similar to that of 2-nitroimidazole hypoxia PET tracers. Therefore, we have evaluated the usefulness of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-(18)F-pentafluoropropyl)-acetamide ((18)F-EF5) PET to monitor and predict tumor response to SN30000 plus radiation treatment (RT). Human non-small cell lung cancer xenografts (H460) in athymic rats were imaged with (18)F-EF5 PET before and after treatment with SN30000 (90 mg/kg), with or without 15-Gy RT. The feasibility of imaging early changes in hypoxia in response to SN30000 was examined 24 h after treatment, followed by ex vivo γ-counting and immunohistochemical examination to study drug-induced apoptosis. Subsequently, the therapeutic effects of SN30000 with or without RT were evaluated in tumor growth delay studies and compared with early treatment-induced changes observed by (18)F-EF5 PET. Changes in tumor hemoglobin oxygen saturation as a function of time after treatment measured by optical spectroscopy were compared with PET data. The uptake of (18)F-EF5 was significantly lower in SN30000-treated tumors than in saline controls 24 h after treatment (mean standardized uptake value, 0.44 ± 0.08 vs. 0.56 ± 0.08 for control group; P < 0.05). Apoptosis was significantly higher in SN30000-treated tumors than in controls. Early treatment-induced changes in (18)F-EF5 uptake were indicative of tumor response in growth delay studies at the group level. SN30000 plus RT significantly decreased (18)F-EF5 uptake relative to baseline and resulted in complete tumor remission in 5 of 7 animals. SN30000 alone decreased (18)F-EF5 uptake, generally in tumors with high initial standardized uptake values, and showed a minor tumor growth delay effect. The changes induced by SN30000 with or without RT in (18)F-EF5 uptake correlated with baseline hypoxia levels. RT caused significant increases in tumor oxygen concentration and hemoglobin oxygen saturation. A hypoxia PET imaging agent can measure changes in tumor hypoxic fraction in response to SN30000. These results suggest the utility of (18)F-EF5 PET for monitoring early response to tumor treatment with SN30000 plus RT in the clinical development of this novel hypoxia-activated prodrug.

Abstract 5719: Improvement of radiotherapy for glioblastoma by co-targeting PI3K and HSP90

Abstract Introduction: Glioblastoma (GBM) tumors frequently contain mutations in the tumor suppressor gene, PTEN, leading to loss of PTEN activity. PTEN loss causes overactivation of the PI3K pathway, inducing inhibition of apoptosis and radioresistance. Heat-shock protein 90 (HSP90) is a molecular chaperone that is over-expressed in GBM and that has among its client proteins, PI3K and Akt. It was hypothesized that dual inhibition of Hsp90 and PI3K signaling would additively or synergistically radiosensitize GBM through inhibition of radiation-induced PI3K/Akt signaling, leading to enhanced apoptosis. This study investigated the radiosensitizing effect of the HSP 90 inhibitor, HSP990 in combination with the PI3K inhibitor, BKM-120 in U87 GBM xenografts. Methods: Human U87 (GBM) cells, null for PTEN, were studied. Radiosensitivity was determined by apoptotic assays (Annexin V kit) in vitro and by tumor growth delay studies in vivo. Immunoblots were performed to assess pAkt levels. HSP990 and BKM 120 were provided by Novartis Pharmaceuticals. Results: Apoptotic assays revealed the following percentages of cells undergoing apoptosis 48 hrs after treatments: radiation alone: 57%; HSP990 alone: 75%; HSP990 + radiation (RT): 86%; BKM120 alone: 59%; BKM120+ RT: 42%. The combination of BKM120 + HSP990 induced the highest percentage of apoptosis (89% of cell population). The addition of radiation to the combined drug treatment did not increase the percentage of cells undergoing apoptosis (66% for triple modality treatment vs. 89% for HSP990 + BKM120 treatment). In vivo, each individual treatment (RT, HSP990 or BKM120) slowed the rate of tumor growth compared with the control group and all pairs of treatments performed better than either of the component treatments alone (p&amp;lt;0.0001 for all comparisons). The combination of all three treatments performed significantly better than BKM+RTX and HSP+RTX (p=0.01 for both) but was not significantly better than BKM+HSP (p=0.06). Conclusions: The response of U87 glioblastoma to radiotherapy was enhanced in the presence of BKM120 and HSP990. Enhanced apoptosis contributed to the mechanism of cell death. This work was supported by a grant from Novartis Pharmaceuticals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5719. doi:1538-7445.AM2012-5719

111In-BnDTPA-F3: an Auger electron-emitting radiotherapeutic agent that targets nucleolin

IntroductionThe F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), a fragment of the human high mobility group protein 2, binds nucleolin. Nucleolin is expressed in the nuclei of normal cells but is also expressed on the membrane of some cancer cells. The goal was to investigate the use of 111In-labeled F3 peptide for Auger electron-targeted radiotherapy.MethodsF3 was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy and conjugated to p-SCN-benzyl-diethylenetriaminepentaacetic acid (BnDTPA) for labeling with 111In to form 111In-BnDTPA-F3. MDA-MB-231-H2N (231-H2N) human breast cancer cells were exposed to 111In-BnDTPA-F3 and used in cell fractionation, γH2AX immunostaining (a marker of DNA double-strand breaks), and clonogenic assays. In vivo, biodistribution studies of 111In-BnDTPA-F3 were performed in 231-H2N xenograft-bearing mice. In tumor growth delay studies, 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) was administered intravenously to 231-H2N xenograft-bearing mice once weekly for 3 weeks.ResultsMembrane-binding of FITC-F3 was observed in 231-H2N cells, and there was co-localization of FITC-F3 with nucleolin in the nuclei. After exposure of 231-H2N cells to 111In-BnDTPA-F3 for 2 h, 1.7% of 111In added to the medium was membrane-bound. Of the bound 111In, 15% was internalized, and of this, 37% was localized in the nucleus. Exposure of 231-H2N cells to 111In-BnDTPA-F3 (1 μM, 6 MBq/μg) resulted in a dose-dependent increase in γH2AX foci and in a significant reduction of clonogenic survival compared to untreated cells or cells exposed to unlabeled BnDTPA-F3 (46 ± 4.1%, 100 ± 1.8%, and 132 ± 7.7%, respectively). In vivo, tumor uptake of 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) at 3-h post-injection was 1% of the injected dose per gram (%ID/g), and muscle uptake was 0.5%ID/g. In tumor growth delay studies, tumor growth rate was reduced 19-fold compared to untreated or unlabeled BnDTPA-F3-treated mice (p = 0.023).Conclusion111In-BnDTPA-F3 is internalized into 231-H2N cells and translocates to the nucleus. 111In-BnDTPA-F3 has a potent cytotoxic effect in vitro and an anti-tumor effect in mice bearing 231-H2N xenografts despite modest total tumor accumulation.

Abstract B89: Molecular imaging using bacterial nitroreductase reporter genes by repurposing the clinical stage hypoxia PET probe EF5.

Abstract Oncolytic viruses and tumor-tropic bacteria offer promise as cancer therapeutics of the future. There is a need to develop technologies to monitor the spatio-temporal distribution of these live vectors in a manner that is predictive of normal tissue toxicity and antitumor efficacy. Positron emission tomography (PET) is the preferred non-invasive imaging modality (biomarker) but suitable advanced reporter gene/PET probe combinations are lacking. A range of 2-nitroimidazole (2-NI) probes are currently in clinical use for the detection of hypoxia (EF5, FMISO, HX4, FAZA) and thus have already attained a high level of clinical validation. We hypothesized that (2-NI) PET agents might be repurposed to monitor the biodistribution of replicating biological agents, thereby leveraging two decades of research efforts to optimise hypoxia PET probe pharmacology. Bacterial nitroreductases (NTRs; type I) are efficient O2-independent enzymes that provide the necessary catalytic flexibility to achieve this goal. Historically E. coli NfsB has been the focus of gene therapy applications. We cloned eleven candidate NTRs from E. coli namely; AzoR, KefF, MdaB, NemA, NfsA, NfsB, WrbA, YcaK, YcdI, YdjA and Yief. Using HCT116 cells engineered to express each NTR, we showed NfsA alone was able to metabolise a range of 2-NI probe molecules, including EF5 (pentafluoroetanidazole), leading to efficient cellular retention. We confirmed catalytic efficiency of EF5 reduction by recombinant NfsA (kcat/Km 97 s−1/mM). In contrast NfsB only weakly metabolised EF5 (kcat/Km 0.24 s−1/mM). We compared EF5 adduct retention in HCT116 cells, detected by mAb using flow cytometry, either under anoxia (&amp;lt; 10ppm O2) or engineered to express NfsA or NfsB. Following exposure to EF5 in monolayer, median fluorescence was &amp;gt;100-fold greater in NfsA expressing cells than anoxic cells, whereas NfsB cells were negative. Mixed Wt : NfsA multicellular layers containing various ratios of cells demonstrated the capacity to detect, with precision, single NfsA-positive cells in mixed cell populations. HCT116 xenografts composed of increasing proportions of NfsA cells (0% to 25%) were established in nude mice and analysed by immunohistochemistry and flow cytometry. Ex-vivo pimonidazole-binding confirmed all NfsA positive cells were detected in vivo following administration of EF5. NfsA, like NfsB, can bioactivate the clinical-stage prodrug PR-104. We determined the relationship between EF5 retention and PR-104 cytotoxicity in HCT116 xenografts harbouring variable proportions of NfsA cells (0%-25%). A correlation was observed between total EF5 retention and global clonogenic cell kill (r2 = 0.828; p &amp;lt;0.001) indicating EF5 binding by NfsA cells predicts for whole tumor sensitivity to PR-104. Tumor growth delay (TGD) studies confirmed the excellent efficacy of PR-104 (1.0 mmol/kg, ip) against tumors containing a minority (14.7%) of NfsA expressing cells (TGD = 386%; p&amp;lt;0.001). This marked tumor control achieved by single dose PR-104 confirmed the presence of a robust bystander cell killing effect able to control the majority (85%) of WT cells. In summary, use of E. coli NfsA enables the repurposing of hypoxia PET probes for reporter gene applications which has significant utility in the development of vector platforms and shows promise in combination with the prodrug PR-104. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B89.

MK-1775, a Novel Wee1 Kinase Inhibitor, Radiosensitizes p53-Defective Human Tumor Cells

Radiotherapy is commonly used to treat a variety of solid tumors. However, improvements in the therapeutic ratio for several disease sites are sorely needed, leading us to assess molecularly targeted therapeutics as radiosensitizers. The aim of this study was to assess the wee1 kinase inhibitor, MK-1775, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast, and prostate cancers were tested for radiosensitization by MK-1775 using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of MK-1775 to abrogate the radiation-induced G₂ block, thereby allowing cells harboring DNA lesions to prematurely progress into mitosis, was determined using flow cytometry and detection of γ-H2AX foci. The in vivo efficacy of the combination of MK-1775 and radiation was assessed by tumor growth delay experiments using a human lung cancer cell line growing as a xenograft tumor in nude mice. Clonogenic survival analyses indicated that nanomolar concentrations of MK-1775 radiosensitized p53-defective human lung, breast, and prostate cancer cells but not similar lines with wild-type p53. Consistent with its ability to radiosensitize, MK-1775 abrogated the radiation-induced G₂ block in p53-defective cells but not in p53 wild-type lines. MK-1775 also significantly enhanced the antitumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors. These results indicate that p53-defective human tumor cells are significantly radiosensitized by the potent and selective wee1 kinase inhibitor, MK-1775, in both the in vitro and in vivo settings. Taken together, our findings strongly support the clinical evaluation of MK-1775 in combination with radiation.

Evaluation of the Antitumor and Radiosynthetizing Activity of a Novel Quinoline Sulfonamide Derivative (PIQSA) as a Histone Deacetylase Inhibitor

Inhibition of histone deacetylases (HDACs) is emerging as a new strategy in cancer therapy. In the present work a novel pyrimido-quinoline benzene sulfonamide (PIQSA compound) was designed and synthesized postulating its ability to inhibit HDAC enzyme in cancer cells. This study was designed to examine the in vitro anti-tumor efficacy of PIQSA against Ehrlich Ascite carcinoma cells (EAC) and three of the human cancer cell lines (H460), brain (U251) and liver (HepG2). The results of Cytotoxic assays showed that PIQSA exhibited in vitro antitumor activity in a dose dependant manner. The tumor growth delay studies indicating that PIQSA resulted in significant regression in tumor growth, which was more pronounced when PIQSA treatment accompanied with radiation exposure. Also, the efficacy of PIQSA to influence radiation response in Ehrlich solid carcinoma (ESC) tumors was estimated. The results suggest that PIQSA exhibited antitumor activities and strong radioenhancing properties associated with inhibition of HDAC activity, DNA fragmentation followed by apoptotic cell death, preferential cell loss of cells particularly in G1/G0 phase through an apoptotic pathway.

Mutant p53-induced Up-regulation of Mitogen-activated Protein Kinase Kinase 3 Contributes to Gain of Function

Mitogen-activated protein kinase kinase 3 (MAP2K3) is a member of the dual specificity kinase group. Growing evidence links MAP2K3 to invasion and tumor progression. Here, we identify MAP2K3 as a transcriptional target of endogenous gain-of-function p53 mutants R273H, R175H, and R280K. We show that MAP2K3 modulation occurred at the mRNA and protein levels and that endogenous mutant p53 proteins are capable of binding to and activate the MAP2K3 promoter. In addition, we found that the studied p53 mutants regulate MAP2K3 gene expression through the involvement of the transcriptional cofactors NF-Y and NF-kappaB. Finally, functional studies showed that endogenous MAP2K3 knockdown inhibits proliferation and survival of human tumor cells, whereas the ectopic expression of MAP2K3 can rescue the proliferative defect induced by mutant p53 knockdown. Taken together, our findings define a novel player through which mutant p53 exerts its gain-of-function activity in cancer cells.

Abstract B76: Cellular metabolism, murine pharmacokinetics and preclinical antitumor activity of SN29966, a novel hypoxia-activated irreversible pan-HER inhibitor

Abstract Hypoxia occurs in most human tumors and is associated with disease progression, resistance to conventional therapies and poor patient outcome. Hypoxia can up-regulate HER1 by several known mechanisms, including increased mRNA translation (Franovic et al., PNAS,2007;104:13092) and delayed receptor endocytosis (Wang et al., Nat Med.,2009;15:319). We have developed SN29966, a hypoxia-activated prodrug of the irreversible pan-HER inhibitor SN29926, to target and exploit hypoxic cells and thereby broaden the therapeutic index of this class of agent. Quaternization of the aminebearing Michael acceptor masks activity SN29926, and one-electron reduction provides selective release under hypoxia. This prodrug design confers 62-fold deactivation of inhibitor activity with respect to HER1 autophosphorylation in A431 cells (Smaill et al., this meeting). In a panel of HER1/2 expressing cell lines SN29966 showed hypoxia-dependent inhibition of proliferation (hypoxic/oxic IC50 ratios of 63, 38, 31 and 19 in BT474, A431, SKOV3 and SKBR3 cells, respectively), a property SN29926 lacked (IC50 ratios 0.7–1.1). SN29926 was generated from SN29966 under hypoxic conditions at a rate of 200–500 pmol/hr/106 cells. Oxic production was ∼1 pmol/hr/106 cells. Plasma and A431 tumor pharmacokinetics (PK) of prodrug SN29966 and inhibitor were measured in nude mice by LC/MS/MS detection (with D6 internal standards) following administration at their respective MTDs (133 and 75 umol/kg; ip). Prodrug SN29966 gave a plasma AUC0-72h of 2016 umol-h/L, some ∼110-fold greater than achieved for administration of inhibitor SN29926 (18 umol-h/L). The latter gave a tumor AUC0-inf of 100 umol-h/kg with a half-life (t½) of 9 h. In contrast the prodrug SN29966 gave a tumor AUC0-72h of 2245 umol-h/kg with a stable tumor tissue concentration of ∼ 30 umol/kg out to 72 h, such that a t½ could not be determined. Consistent with this long prodrug residency, SN29926 released from prodrug had a t½ in tumor tissue of &amp;gt;72h, providing an AUC0-72h of 464 umol-h/kg. Thus the AUC of SN29926 in A431 tumors was at least 4.6-fold higher after administration of prodrug SN29966 than following administration of inhibitor SN29926 itself at equivalent toxicity. In A431 tumor growth delay studies, SN29966 (113 umol/kg, q4dx6, ip) induced tumor regressions (12/12) with no recovery of growth by day 36 (tumor volume 44 ±19 mm3), whereas controls grew rapidly (day 8 tumor volume 1043 ±109 mm3). Comparative administration of inhibitor SN29926 (63 umol/kg, q4dx6, ip) provided tumor stasis, but with rapid growth following cessation of treatment (day 36 tumor volume 642 ±191 mm3), a difference that was significant from prodrug (p&amp;lt;0.01). On this dose and schedule toxicity parameters were comparable, with body weight loss at nadir (day 23) of 5.4% ±1.3% (0/12 deaths) for SN29966 versus 9.8% ±1.3% (2/12 deaths) for SN29926. Superior SN29966 activity was also seen in the SKOV3 xenograft model and was associated with suppression of HER2 phosphorylation. Collectively the data indicate a marked therapeutic advantage for prodrug SN29966 over inhibitor SN29926 in both A431 and SKOV3 tumor models. The PK parameters of SN29966 and the durable release of inhibitor in tumors may explain the robust antitumor activity of this new class of agent. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B76.

Radiation-guided Drug Delivery of Nanoparticle Albumin-bound Paclitaxel to Lung Cancer

Radiation-guided Drug Delivery of Nanoparticle Albumin-bound Paclitaxel to Lung Cancer

Enhancement of xenograft tumor radiosensitivity by the histone deacetylase inhibitor MS-275 and correlation with histone hyperacetylation.

Histone deacetylase (HDAC) inhibitors are undergoing clinical evaluation in cancer therapy. Because HDAC modulation has been shown to enhance the radiosensitivity of tumor cells in vitro, we investigated the effects of the HDAC inhibitor MS-275 on the radioresponse of DU145 prostate carcinoma xenografts. As an indicator of HDAC inhibition in vivo, the histone acetylation status in tumor lysates was determined after two, four, and six injections of MS-275 delivered at 12-hour intervals, as well as 24 and 48 hours after the last injection. Tumor growth delay studies were then performed using this DU-145 xenograft model with radiation administered to leg tumors after the fourth dose of MS-275, which corresponded to the time of maximum histone hyperacetylation. An increase in histone hyperacetylation was detected in each tumor after two injections of MS-275 with a maximum hyperacetylation occurring after four to six injections. In tumor growth delay studies, the combination of MS-275 and radiation resulted in a greater than additive inhibition of tumor growth as compared with the individual modalities. As alternative sources for an indicator of drug radiosensitizing activity, histone hyperacetylation was determined in a series of normal tissues, including lymphocytes. Each of the normal tissues also had a maximal histone hyperacetylation after four to six injections of MS-275. These studies show that MS-275 enhances the radiosensitivity of DU145 xenografts and suggest that histone hyperacetylation status can serve as a useful marker for drug radiosensitizing activity.

Maximizing therapeutic gain with gemcitabine and fractionated radiation

Purpose/Objective: The nucleoside analogue gemcitabine inhibits cellular repair and repopulation, induces apoptosis, causes tumor growth delay, and enhances radiation-induced growth delay. After single doses of drug and radiation, maximum enhancement of tumor response was obtained when gemcitabine preceded radiation by at least 24 h. Conversely, the cellular radioresponse of the normal gastrointestinal epithelium was slightly protected when gemcitabine and radiation were separated by 24 h. This differential response created a time frame within which therapeutic gain could be maximized. In our present investigation, we sought to define the most therapeutically beneficial scheme of gemcitabine administration when combined with fractionated radiotherapy. Methods and Materials: C3Hf/Kam mice were given identical drug and radiation schedules of administration, and both normal tissue (jejunal mucosa) and tumor (Sa-NH) responses were measured. Irradiation was given once per day for 5 days in normal tissue and tumor growth delay studies and twice per day for the tumor cure endpoint. A total dose of 25 mg/kg gemcitabine was given i.p. in 1 of 3 schedules: a single dose of 25 mg/kg 24 h before the start of fractionated irradiation, 12.5 mg/kg 24 h before the first and third radiation doses, or 24 h before each of 5 radiation doses. Groups of mice bearing 7- or 8-mm diameter tumors were treated with gemcitabine alone or in combination with fractionated irradiation under ambient or hypoxic conditions. The survival response of the jejunal mucosa was quantified by the microcolony assay and histologically by quantifying apoptosis, mitosis, S-phase fraction, and crypt cellularity. Results: For tumor growth delay, dose-modifying factors (DMFs) were similar (1.34–1.46) for all 3 schedules of drug administration. In contrast, the response of the jejunum was strongly dependent on the schedule of gemcitabine administration. A single dose of gemcitabine before the start of fractionated radiotherapy resulted in slight radioprotection (DMF 0.96). Two doses and 5 daily doses of gemcitabine enhanced radiation response by factors of 1.09 and 1.23, respectively. Major factors affecting the response of the jejunal mucosa were apoptotic death of S-phase cells exposed to gemcitabine and cell cycle synchrony of surviving cells. Tumor reoxygenation was found to be a major mechanism for tumor radioenhancement, in addition to those reported earlier. Conclusion: All 3 schedules of drug administration produced therapeutic gain; however, when gemcitabine was given more than once in a 5-fraction radiation treatment schedule, normal tissue toxicity increased. The highest therapeutic gain (1.4) was achieved by giving a single dose of gemcitabine (25 mg/kg) 24 h before the start of fractionated radiotherapy.

Evaluation of Chemotherapy and Radiation Enhancement and P NMR Spectral Changes Induced by Biochemical Modulation

The combination of N-(phosphonacetyl)-L-aspartate (PALA), 6-methylmercap-topurine riboside (MMPR), and 6-aminonicotinamide (6AN) has been shown to be an effective antineoplastic regimen and also to enhance the effects of other antineoplastic agents (1-1). To further enhance the effect of this combination, we investigated the effects of adding adriamycin, at its maximally tolerated dose, to this regimen. The response rate (complete regression + partial regression) for the four-drug regimen was higher than for the three-drug regimen, and the tumor growth delay was also significantly higher than for treatment with PALA, MMPR, 6AN, or after treatment with maximally tolerated doses of adriamycin alone (11 mg/kg). The addition of adriamycin to PALA, MMPR, 6AN did not result in enhancement of the effect of radiation, as measured by tumor growth delay studies and tumor control (complete and partial regression rate). The mechanism of action of the combination of PALA, MMPR, and 6AN is not known definitively, but a possible mechanism previously suggested is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Treatment with PALA, MMPR, 6AN, and adriamycin (at 2.5 hr post MMPR, 6AN) resulted in a nadir NTP/Pi value, as determined by 31P NMR spectroscopy, at approximately 10 hrpost MMPR + 6AN (7.5 hrpost adriamycin), which was not significantly different from the NTP/Pi value determined after treatment with the three-drug combination.