Vascular-targeted therapies have shown promise as adjuvant cancer treatment. As these agents undergo clinical evaluation, sensitive imaging biomarkers are need to assess drug target interaction and treatment response. In this study, dynamic contrast enhanced MRI (DCE-MRI) and diffusion-weighted MRI (DW-MRI) were evaluated for detecting response of intracerebral 9L gliosarcomas to the antivascular agent VEGF-Trap, a fusion protein designed to bind all forms of Vascular Endothelial Growth Factor-A (VEGF-A) and Placental Growth Factor (PGF). Rats with 9L tumors were treated twice weekly for two weeks with vehicle or VEGF-Trap. DCE- and DW-MRI were performed one day prior to treatment initiation and one day following each administered dose. Kinetic parameters (Ktrans: volume transfer constant, kep: efflux rate constant from extravascular/extracellular space to plasma, and vp: blood plasma volume fraction) and the apparent diffusion coefficient (ADC) over the tumor volumes were compared between groups. A significant decrease in kinetic parameters was observed 24 hours following the first dose of VEGF-Trapin treated versus control animals (p<0.05) and was accompanied by a decline in ADC values. In addition to the significant hemodynamic effect, VEGF-Trap treated animals exhibited significantly longer tumor doubling times (p<0.05) compared to the controls. Histological findings were found to support imaging response metrics. In conclusion, kinetic MRI parameters and change in ADC have been found to serve as sensitive and early biomarkers of VEGF-Trapanti-vascular targeted therapy.
anti-angiogenic therapy; VEGF-Trap; glioma; DCE-MRI; DW-MRI; hemodynamics; diffusion; preclinical
Diffusion MRI; Magnetic Resonance Spectroscopy (MRS); Perfusion MRI; Brain Tumor; Imaging Biomarkers; Translational Research
PURPOSE: In the current study we examined the ability of diffusion MRI (dMRI) to predict pathologic response in pancreatic cancer patients receiving neoadjuvant chemoradiation. METHODS: We performed a prospective pilot study of dMRI in patients with resectable pancreatic cancer. Patients underwent dMRI prior to neoadjuvant chemoradiation. Surgical specimens were graded according to the percent tumor cell destruction. Apparent diffusion coefficient (ADC) maps were used to generate whole-tumor derived ADC histogram distributions and mean ADC values. The primary objective of the study was to correlate ADC parameters with pathologic and CT response. RESULTS: Ten of the 12 patients enrolled on the study completed chemoradiation and had surgery. Three were found to be unresectable at the time of surgery and no specimen was obtained. Out of the 7 patients who underwent pancreaticoduodenectomy, 3 had a grade III histopathologic response (> 90% tumor cell destruction), 2 had a grade IIB response (51% to 90% tumor cell destruction), 1 had a grade IIA response (11% to 50% tumor cell destruction), and 1 had a grade I response (> 90% viable tumor). Median survival for patients with a grade III response, grade I-II response, and unresectable disease were 25.6, 18.7, and 6.1 months, respectively. There was a significant correlation between pre-treatment mean tumor ADC values and the amount of tumor cell destruction after chemoradiation with a Pearson correlation coefficient of 0.94 (P = .001). Mean pre-treatment ADC was 161 × 10− 5 mm2/s (n = 3) in responding patients (> 90% tumor cell destruction) compared to 125 × 10− 5 mm2/s (n = 4) in non-responding patients (> 10% viable tumor). CT imaging showed no significant change in tumor size in responders or non-responders. CONCLUSIONS: dMRI may be useful to predict response to chemoradiation in pancreatic cancer. In our study, tumors with a low ADC mean value at baseline responded poorly to standard chemoradiation and would be candidates for intensified therapy.
Bioluminescence imaging is utilized widely for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on high signal-to-background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, ATP-independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc, NL) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in TGF-β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development.
Bioluminescence; TGF-β; molecular imaging; luciferase; cell signaling
To determine the potential value of intravoxel water diffusion heterogeneity imaging for brain tumor characterization and evaluation of high-grade gliomas, by comparing an established heterogeneity indices (α value) measured in human high-grade gliomas to those of normal appearing white and grey matter landmarks.
Materials and Methods
Twenty patients with high-grade gliomas prospectively underwent diffusion-weighted magnetic resonance imaging using multiple b-values. The stretched-exponential model was used to generate α and distributed diffusion coefficient (DDC) maps. The α values and DDCs of the tumor and contralateral anatomic landmarks were measured in each patient. Differences between α values of tumors and landmark tissues were assessed using paired t tests. Correlation between tumor α and tumor DDC was assessed using Pearson’s correlation coefficient.
Mean α of tumors was significantly lower than that of contralateral frontal white matter (P = 0.0249), basal ganglia (P < 0.0001), cortical grey matter (P < 0.0001), and centrum semiovale (P = 0.0497). Correlation between tumor α and tumor DDC was strongly negative (Pearson correlation coefficient, −0.8493; P < 0.0001).
The heterogeneity index α of human high-grade gliomas is significantly different from those of normal brain structures, which potentially offers a new method for evaluating brain tumors. The observed negative correlation between tumor α and tumor DDC requires further investigation.
Diffusion-weighted magnetic resonance imaging; stretched-exponential; intravoxel water diffusion heterogeneity; brain tumor; glioma
Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and non-invasively measures ATM kinase activity in living cells and subjects.
Methods and Materials
Using the split luciferase technology we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence.
Treatment of ATMR expressing cells with ATM inhibitors resulted in a dose dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, while a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and siRNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models which correlated in a time-dependent fashion with changes in Chk2 activity.
We describe the development and validation of a novel, specific, non-invasive bioluminescent reporter that enables monitoring of ATM activity in real-time in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens, and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in pre-clinical models.
Quantitative quality control procedures were sought to evaluate technical variability in multi-center measurements of the diffusion coefficient of water as a prerequisite to use of the biomarker apparent diffusion coefficient (ADC) in multi-center clinical trials.
Materials and Methods
A uniform data acquisition protocol was developed and shared with 18 participating test sites along with a temperature-controlled diffusion phantom delivered to each site. Usable diffusion weighted imaging data of ice water at 5 b-values were collected on 35 clinical MRI systems from 3 vendors at 2 field strengths (1.5 and 3T) and analyzed at a central processing site.
Standard deviation of bore-center ADCs measured across 35 scanners was <2%; error range: −2% to +5% from literature value. Day-to-day repeatability of the measurements was within 4.5%. Intra-exam repeatability at the phantom center was within 1%. Excluding one outlier, inter-site reproducibility of ADC at magnet isocenter was within 3%, though variability increased for off-center measurements. Significant (>10%) vendor-specific and system-specific spatial non-uniformity ADC bias was detected for the off-center measurement that was consistent with gradient non-linearity.
Standardization of DWI protocol has improved reproducibility of ADC measurements and allowed identifying spatial ADC non-uniformity as a source of error in multi-site clinical studies.
diffusion; MRI; phantom; ice-water; quality control; gradient non-linearity
Recent clinical practice for the management for cancer patients has begun to change from a statistical “one-size fits all” approach to medicine to more individualized care. Pre-treatment biomarkers (i.e. genetically and histologically based) have a growing role in providing guidance related to the appropriate therapy and likelihood of response; they do not take into account heterogeneity within the tumor mass. Thus, a biomarker which could be utilized to measure actual tumor response early following treatment initiation would provide an important opportunity to evaluate treatment effects on an individual patient basis. Diffusion weighted magnetic resonance imaging (DW-MRI) offers the opportunity to monitor treatment-associated alterations in tumor microenvironment using quantification of changes in tumor water diffusion values as a surrogate imaging biomarker. Results obtained thus far using DW-MRI have shown that changes in tumor diffusion values can be detected early following treatment initiation which correlate with traditional outcome measures. Sensitive imaging biomarkers are providing for the first time a means of assessing 3 dimensional tumor response early in the treatment cycle which may also provide opportunities to visualize spatial heterogeneity of response within the tumor which could open up further opportunities for spatially modulating therapy. This review highlights the development of DW-MRI and its proposed usefulness in the clinical management of cancer patients. The utility of DW-MRI for assessing therapeutic-induced response is further evaluated on tumors residing in the brain, head and neck and bone.
DW-MRI; apparent diffusion coefficient; imaging biomarker; cancer; treatment response
We have recently synthesized a peptide called Disruptin, which comprised the SVDNPHVC segment of the epidermal growth factor receptor (EGFR) that inhibits binding of heat shock protein 90 (Hsp90) to the EGFR and EGF-dependent EGFR dimerization to cause EGFR degradation. The effect is specific for EGFR versus other Hsp90 client proteins [Ahsan et al. (2013). Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization. J Biol Chem
288, 26879–26886]. Here, we show that Disruptin decreases the clonogenicity of a variety of EGFR-dependent cancer cells in culture but not of EGFR-independent cancer or noncancerous cells. The selectivity of Disruptin toward EGFR-driven cancer cells is due to the high level of EGF stimulation of EGFR in EGFR-dependent tumor cells relative to normal cells. When administered by intraperitoneal injection into nude mice bearing EGFR-driven human tumor xenografts, Disruptin causes extensive degradation of EGFR in the tumor but not in adjacent host tissue. Disruptin markedly inhibits the growth of EGFR-driven tumors without producing the major toxicities caused by the Hsp90 inhibitor geldanamycin or by cisplatin. These findings provide proof of concept for development of a new Disruptin-like class of antitumor drugs that are directed specifically against EGFR-driven tumors.
Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, β-transducing repeat containing protein 1 (β-TrCP1). Conversely, β-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, β-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutant of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.
Imaging biomarkers capable of early quantification of tumor response to therapy would provide an opportunity to individualize patient care. Image registration of longitudinal scans provides a method of detecting treatment associated changes within heterogeneous tumors by monitoring alterations in the quantitative value of individual voxels over time, which is unattainable by traditional volumetric-based histogram methods. The concepts involved in the use of image registration for tracking and quantifying breast cancer treatment response using parametric response mapping (PRM), a voxel-based analysis of diffusion-weighted magnetic resonance imaging (DW-MRI) scans, are presented. Application of PRM to breast tumor response detection is described, wherein robust registration solutions for tracking small changes in water diffusivity in breast tumors during therapy are required. Methodologies that employ simulations are presented for measuring expected statistical accuracy of PRM for response assessment. Test-retest clinical scans are used to yield estimates of system noise to indicate significant changes in voxel-based changes in water diffusivity. Overall, registration-based PRM image analysis provides significant opportunities for voxel-based image analysis to provide the required accuracy for early assessment of response to treatment in breast cancer patients receiving neoadjuvant chemotherapy.
Ovarian cancer is the fifth leading cause of cancer deaths among American women. Platinum-based chemotherapy, such as cisplatin, represents the standard of care for ovarian cancer. However, toxicity and acquired resistance to cisplatin have proven challenging in the treatment of ovarian cancer patients.
Using a genetically engineered mouse (GEM) model of ovarian endometrioid adenocarcinoma (OEA) in combination with molecular imaging technologies, we studied the activation of the AKT serine/threonine kinase in response to long-term cisplatin therapy.
Treatment of cells in culture and tumor-bearing animals with cisplatin resulted in activation of AKT, a key mediator of cell survival. Based on these results we investigated the therapeutic utility of AKT inhibition in combination with cisplatin, which resulted in enhanced and prolonged induction of apoptosis and in significantly improved tumor control compared to either agent alone.
These results provide an impetus for clinical trials using combination therapy. To facilitate these trials, we also demonstrate the utility of diffusion-weighted MRI as an imaging biomarker for evaluation of therapeutic efficacy in OEA.
bioluminescence imaging; diffusion-weighted MR imaging; ovarian carcinoma; AKT; cisplatin
The future of personalized oncological therapy will likely rely on evidence-based medicine to integrate all of the available evidence to delineate the most efficacious treatment option for the patient. To undertake evidence-based medicine through use of targeted therapy regimens, identification of the specific underlying causative mutation(s) driving growth and progression of a patient's tumor is imperative. Although molecular subtyping is important for planning and treatment, intraclonal genetic diversity has been recently highlighted as having significant implications for biopsy-based prognosis. Overall, delineation of the clonal architecture of a patient's cancer and how this will impact on the selection of the most efficacious therapy remain a topic of intense interest.
Studies investigating dynamic susceptibility contrast magnetic resonance imaging-determined relative cerebral blood volume (rCBV) maps as a metric of treatment response assessment have generated conflicting results. We evaluated the potential of various analytical techniques to predict survival of patients with glioma treated with chemoradiation. rCBV maps were acquired in patients with high-grade gliomas at 0, 1, and 3 weeks into chemoradiation therapy. Various analytical techniques were applied to the same cohort of serial rCBV data for early assessment of survival. Three different methodologies were investigated: 1) percentage change of whole tumor statistics (i.e., mean, median, and percentiles), 2) physiological segmentation (low rCBV, medium rCBV, or high rCBV), and 3) a voxel-based approach, parametric response mapping (PRM). All analyses were performed using the same tumor contours, which were determined using contrast-enhanced T1-weighted and fluid attenuated inversion recovery images. The predictive potential of each response metric was assessed at 1-year and overall survival. PRM was the only analytical approach found to generate a response metric significantly predictive of patient 1-year survival. Time of acquisition and contour volume were not found to alter the sensitivity of the PRM approach for predicting overall survival. We have demonstrated the importance of the analytical approach in early response assessment using serial rCBV maps. The PRM analysis shows promise as a unified early and robust imaging biomarker of treatment response in patients diagnosed with high-grade gliomas.
RATIONALE: Treatment of glioblastoma (GBM) remains challenging due in part to its histologic intratumoral heterogeneity that contributes to its overall poor treatment response. Our goal was to evaluate a voxel-based biomarker, the functional diffusion map (fDM), as an imaging biomarker to detect heterogeneity of tumor response in a radiation dose escalation protocol using a genetically engineered murine GBM model. EXPERIMENTAL DESIGN: Twenty-four genetically engineered murine GBM models [Ink4a-Arf-/-/Ptenloxp/loxp/Ntv-a RCAS/PDGF(+)/Cre(+)] were randomized in four treatment groups (n = 6 per group) consisting of daily doses of 0, 1, 2, and 4 Gy delivered for 5 days. Contrast-enhanced T1-weighted and diffusion-weighted magnetic resonance imaging (MRI) scans were acquired for tumor delineation and quantification of apparent diffusion coefficient (ADC) maps, respectively. MRI experiments were performed daily for a week and every 2 days thereafter. For each animal, the area under the curve (AUC) of the percentage change of the ADC (AUCADC) and that of the increase in fDM values (AUCfDM+) were determined within the first 5 days following therapy initiation. RESULTS: Animal survival increased with increasing radiation dose. Treatment induced a dose-dependent increase in tumor ADC values. The strongest correlation between survival and ADC measurements was observed using the AUCfDM+ metric (R2 = 0.88). CONCLUSION: This study showed that the efficacy of a voxel-based imaging biomarker (fDM) was able to detect spatially varying changes in tumors, which were determined to be a more sensitive predictor of overall response versus whole-volume tumor measurements (AUCADC). Finally, fDM provided for visualization of treatment-associated spatial heterogeneity within the tumor.
Induction of apoptosis plays a crucial role in the response of tumors to treatment. Thus, we investigated the pharmacodynamics and tumor saturation kinetics of a death receptor 5 antibody (anti-DR5) when combined with chemotherapeutics. For our investigations, we applied an imaging method that allows monitoring of apoptosis noninvasively in living mice. A stably transfected apoptosis reporter based on split luciferase technology facilitates to screen various chemotherapeutics and anti-DR5 on their ability to induce apoptosis in glioblastoma cells in vitro as well as in vivo. We found that doxorubicin (DOX) treatment in vitro led to significant apoptosis induction within 48 hours and to a 2.3-fold increased anti-DR5 binding to the cell surface. In contrast, cisplatin and 5-fluorouracil (5-FU) treatment altered anti-DR5 binding only marginally. Induction of apoptosis by treatment with anti-DR5 was dose- and time-dependent (both in vitro and in vivo). Simultaneous visualization of fluorescence-labeled anti-DR5 in tumor tissue and apoptosis revealed maximal apoptosis induction immediately after the compound had reached tumor site. Regarding combination therapy of anti-DR5 and DOX, we found that the sequential application of DOX before anti-DR5 resulted in synergistically enhanced apoptosis reporter activity. In striking contrast, anti-DR5 given before DOX did not lead to increased apoptosis induction. We suggest that DOX-induced recruitment of DR5 to the cell surface impacts the enhanced apoptotic effect that can be longitudinally monitored by apoptosis imaging. This study demonstrates that the combination of apoptosis and fluorescence imaging is an excellent method for optimizing dosing and treatment schedules in preclinical cancer models.
Loss of bone mass due to disease, such as osteoporosis and metastatic cancer to the bone, is a leading cause of orthopedic complications and hospitalization. Onset of bone loss resulting from disease increases the risk of incurring fractures and subsequent pain, increasing medical expenses while reducing quality of life. Although current standard CT-based protocols provide adequate prognostic information for assessing bone loss, many of the techniques for evaluating CT scans rely on measures based on whole-bone summary statistics. This reduces the sensitivity at identifying local regions of bone resorption, as well as formation. In this study, we evaluate the effectiveness of a voxel-based image post-processing technique, called the Parametric Response Map (PRM), for identifying local changes in bone mass in weight-bearing bones on CT scans using an established animal model of osteoporosis. Serial CT scans were evaluated weekly using PRM subsequent to ovariectomy or sham surgeries over the period of one month. For comparison, bone volume fraction and mineral density measurements were acquired and found to significantly differ between groups starting 3 weeks post-surgery. High resolution ex vivo measurements acquired four weeks post-surgery validated the extent of bone loss in the surgical groups. In contrast to standard methodologies for assessing bone loss, PRM results were capable of identifying local decreases in bone mineral by week 2, which were found to be significant between groups. This study concludes that PRM is able to detect changes in bone mineral with higher sensitivity and spatial differentiation than conventional techniques for evaluating CT scans, which may aid in clinical decision making for patients suffering from bone loss.
ovariectomy; osteoporosis; imaging; CT; response; biomarker
Receptor tyrosine kinases (RTK) are therapeutic targets for the treatment of malignancy. However, tumor cells develop resistance to targeted therapies through the activation of parallel signaling cascades. Recent evidence has shown that redundant or compensatory survival signals responsible for resistance are initiated by nontargeted glycoprotein RTKs coexpressed by the cell. We hypothesized that disrupting specific functions of the posttranslational machinery of the secretory pathway would be an effective strategy to target both primary and redundant RTK signaling. Using the N-linked glycosylation inhibitor, tunicamycin, we show that expression levels of several RTKS (EGFR, ErbB2, ErbB3, and IGF-IR) are exquisitely sensitive to inhibition of N-linked glycosylation. Disrupting this synthetic process reduces both cellular protein levels and receptor activity in tumor cells through retention of the receptors in the endoplasmic reticulum/Golgi compartments. Using U251 glioma and BXPC3 pancreatic adenocarcinoma cell lines, two cell lines resistant to epidermal growth factor receptor–targeted therapies, we show that inhibiting N-linked glycosylation markedly reduces RTK signaling through Akt and radiosensitizes tumor cells. In comparison, experiments in nontransformed cells showed neither a reduction in RTK-dependent signaling nor an enhancement in radiosensitivity, suggesting the potential for a therapeutic ratio between tumors and normal tissues. This study provides evidence that enzymatic steps regulating N-linked glycosylation are novel targets for developing approaches to sensitize tumor cells to cytotoxic therapies.
In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50–100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.
The endoplasmic reticulum (ER) provides a specialized environment for the folding and modification of trans-membrane proteins, including receptor tyrosine kinases (RTKs), which are vital for the growth and survival of malignancies. To identify compounds which disrupt the function of the ER and thus could potentially impair cancer cell survival signaling, we adapted a set of glycosylation-sensitive luciferase reporters for the development and optimization of a cell-based high-throughput screen (HTS). Secondary screens for false-positive luciferase activation and tertiary lectin-based and biochemical analyses were also devised for compound triage. Through a pilot screen of 2802 compounds from the National Cancer Institute (NCI) chemical libraries, we identified aclacinomycin (Acm) as a compound that preferentially affects ER function. We report that Acm reduces plasma membrane expression of glycoproteins including epidermal growth factor receptor (EGFR) and Met but does not inhibit N-linked glycosylation or generalized protein translation. Fluorescence microscopy co-localization experiments were also performed and demonstrated Acm accumulation in the ER in further support of the overall HTS design. The consequences of Acm treatment on cell survival were analyzed through clonogenic survival analysis. Consistent with the reduction of EGFR levels, pretreatment with Acm sensitizes the EGFR-mutant non-small cell lung cancer (NSCLC) cell lines HCC827 and HCC2935 to ionizing radiation and did not affect the sensitivity of the RTK-independent and KRAS-mutant A549 NSCLC cell line. Thus, Acm and similar compounds targeting the ER may represent a novel approach for radiosensitizing tumor cells dependent on RTK function.
There is an urgent need for the development of novel therapies to treat pancreatic cancer, which is among the most lethal of all cancers. KRAS activating mutations, which are found in >90% of pancreatic adenocarcinomas, drive tumor dependency on the Ras/MAPK and Akt signaling pathways. Radiation is currently being explored as a component of the standard treatment regimen for pancreatic cancer. This study’s purpose was to test the hypothesis that MEK inhibitors will offer clear therapeutic benefit when integrated into radiotherapy treatment regimens for treatment of this disease. We explored the activation of the MAPK and Akt pathways in response to radiation in multiple pancreatic tumor cell lines. Small molecule inhibitors of MEK (PD0325901) and Akt (API-2) were subsequently evaluated for their radiosensitizing potential alone and in combination. In vivo efficacy was tested in subcutaneous MIA-PaCa2 xenografts. Phosphorylated levels of ERK-1/2 and Akt were found to increase in response to radiation treatment in our pancreatic tumor cell line panel. MEK inhibitor-induced radiosensitization was observed in vitro and in vivo. The further addition of an Akt inhibitor to the MEK inhibitor/radiation regimen resulted in enhanced therapeutic gain as determined by increased radiosensitization and tumor cell death. In conclusion, MEK inhibition results in growth arrest, apoptosis, and radiosensitization of multiple preclinical pancreatic tumor models, and the effects can be enhanced by combination with an Akt inhibitor. These results provide rationale for further testing of a treatment regimen in pancreatic cancer that combines MEK inhibition with radiation, optimally in conjunction with Akt inhibition.
MEK-1/2; Akt; PI3-kinase; Radiation; Pancreatic Cancer
Chronic obstructive pulmonary disease (COPD) is increasingly being recognized as a highly heterogeneous disorder, composed of varying pathobiology. Accurate detection of COPD subtypes by image biomarkers are urgently needed to enable individualized treatment thus improving patient outcome. We adapted the Parametric Response Map (PRM), a voxel-wise image analysis technique, for assessing COPD phenotype. We analyzed whole lung CT scans of 194 COPD individuals acquired at inspiration and expiration from the COPDGene Study. PRM identified the extent of functional small airways disease (fSAD) and emphysema as well as provided CT-based evidence that supports the concept that fSAD precedes emphysema with increasing COPD severity. PRM is a versatile imaging biomarker capable of diagnosing disease extent and phenotype, while providing detailed spatial information of disease distribution and location. PRMs ability to differentiate between specific COPD phenotypes will allow for more accurate diagnosis of individual patients complementing standard clinical techniques.
PURPOSE: The inherent treatment resistance of glioblastoma (GBM) can involve multiple mechanisms including checkpoint kinase (Chk1/2)-mediated increased DNA repair capability, which can attenuate the effects of genotoxic chemotherapies and radiation. The goal of this study was to evaluate diffusion-weighted magnetic resonance imaging (DW-MRI) as a biomarker for Chk1/2 inhibitors in combination with radiation for enhancement of treatment efficacy in GBM. EXPERIMENTAL DESIGN: We evaluated a specific small molecule inhibitor of Chk1/2, AZD7762, in combination with radiation using in vitro human cell lines and in vivo using a genetically engineered GBM mouse model. DW-MRI and T1-contrast MRI were used to follow treatment effects on intracranial tumor cellularity and growth rates, respectively. RESULTS: AZD7762 inhibited clonal proliferation in a panel of GBM cell lines and increased radiosensitivity in p53-mutated GBM cell lines to a greater extent compared to p53 wild-type cells. In vivo efficacy of AZD7762 demonstrated a dose-dependent inhibitory effect on GBM tumor growth rate and a reduction in tumor cellularity based on DW-MRI scans along with enhancement of radiation efficacy. CONCLUSION: DW-MRI was found to be a useful imaging biomarker for the detection of radiosensitization through inhibition of checkpoint kinases. Chk1/2 inhibition resulted in antiproliferative activity, prevention of DNA damage-induced repair, and radiosensitization in preclinical GBM tumor models, both in vitro and in vivo. The effects were found to be maximal in p53-mutated GBM cells. These results provide the rationale for integration of DW-MRI in clinical translation of Chk1/2 inhibition with radiation for the treatment of GBM.
The prognosis for patients with high grade gliomas is poor, with a median survival of 1 year. Treatment efficacy assessment is typically unavailable until 5-6 months post diagnosis. Investigators hypothesize that quantitative magnetic resonance imaging can assess treatment efficacy 3 weeks after therapy starts, thereby allowing salvage treatments to begin earlier. The purpose of this work is to build a predictive model of treatment efficacy by using quantitative magnetic resonance imaging data and to assess its performance. The outcome is 1-year survival status. We propose a joint, two-stage Bayesian model. In stage I, we smooth the image data with a multivariate spatiotemporal pairwise difference prior. We propose four summary statistics that are functionals of posterior parameters from the first-stage model. In stage II, these statistics enter a generalized non-linear model as predictors of survival status. We use the probit link and a multivariate adaptive regression spline basis. Gibbs sampling and reversible jump Markov chain Monte Carlo methods are applied iteratively between the two stages to estimate the posterior distribution. Through both simulation studies and model performance comparisons we find that we can achieve higher overall correct classification rates by accounting for the spatiotemporal correlation in the images and by allowing for a more complex and flexible decision boundary provided by the generalized non-linear model.
Bayesian analysis; Image analysis; Multivariate adaptive regression splines; Multivariate pairwise difference prior; Quantitative magnetic resonance imaging; Spatiotemporal model
Genetically engineered mouse (GEM) models of ovarian cancer that closely recapitulate their human tumor counterparts may be invaluable tools for preclinical testing of novel therapeutics. We studied murine ovarian endometrioid adenocarcinomas (OEAs) arising from conditional dysregulation of canonical WNT and PI3K/AKT/mTOR pathway signaling to investigate their response to conventional chemotherapeutic drugs and mTOR or AKT inhibitors.
OEAs were induced by injection of adenovirus expressing Cre recombinase (AdCre) into the ovarian bursae of Apcflox/flox;Ptenflox/flox mice. Tumor-bearing mice or murine OEA-derived cell lines were treated with cisplatin and paclitaxel, mTOR inhibitor rapamycin, or AKT inhibitors API-2 or perifosine. Treatment effects were monitored in vivo by tumor volume and bioluminescence imaging, in vitro by WST-1 proliferation assays, and in OEA tissues and cells by immunoblotting and immunostaining for levels and phosphorylation status of PI3K/AKT/mTOR signaling pathway components.
Murine OEAs developed within 3 weeks of AdCre injection and were not preceded by endometriosis. OEAs responded to cisplatin + paclitaxel, rapamycin, and AKT inhibitors in vivo. In vitro studies showed that response to mTOR and AKT inhibitors, but not conventional cytotoxic drugs, was dependent on the status of PI3K/AKT/mTOR signaling. AKT inhibition in APC−/PTEN− tumor cells resulted in compensatory up-regulation of ERK signaling.
The studies demonstrate the utility of this GEM model of ovarian cancer for pre-clinical testing of novel PI3K/AKT/mTOR signaling inhibitors and provide evidence for compensatory signaling, suggesting that multiple rather than single agent targeted therapy will be more efficacious for treating ovarian cancers with activated PI3K/AKT/mTOR signaling.
ovarian carcinoma; endometriosis; PI3K/AKT/mTOR signaling; mouse model; bioluminescence imaging