Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

Mapping Hypoxia in Renal Carcinoma with Oxygen-enhanced MRI: Comparison with Intrinsic Susceptibility MRI and Pathology.

Purpose To cross-validate T1-weighted oxygen-enhanced (OE) MRI measurements of tumor hypoxia with intrinsic susceptibility MRI measurements and to demonstrate the feasibility of translation of the technique for patients. Materials and Methods Preclinical studies in nine 786-0-R renal cell carcinoma (RCC) xenografts and prospective clinical studies in eight patients with RCC were performed. Longitudinal relaxation rate changes (∆R1) after 100% oxygen inhalation were quantified, reflecting the paramagnetic effect on tissue protons because of the presence of molecular oxygen. Native transverse relaxation rate (R2*) and oxygen-induced R2* change (∆R2*) were measured, reflecting presence of deoxygenated hemoglobin molecules. Median and voxel-wise values of ∆R1 were compared with values of R2* and ∆R2*. Tumor regions with dynamic contrast agent-enhanced MRI perfusion, refractory to signal change at OE MRI (referred to as perfused Oxy-R), were distinguished from perfused oxygen-enhancing (perfused Oxy-E) and nonperfused regions. R2* and ∆R2* values in each tumor subregion were compared by using one-way analysis of variance. Results Tumor-wise and voxel-wise ∆R1 and ∆R2* comparisons did not show correlative relationships. In xenografts, parcellation analysis revealed that perfused Oxy-R regions had faster native R2* (102.4 sec-1 vs 81.7 sec-1) and greater negative ∆R2* (-22.9 sec-1 vs -5.4 sec-1), compared with perfused Oxy-E and nonperfused subregions (all P < .001), respectively. Similar findings were present in human tumors (P < .001). Further, perfused Oxy-R helped identify tumor hypoxia, measured at pathologic analysis, in both xenografts (P = .002) and human tumors (P = .003). Conclusion Intrinsic susceptibility biomarkers provide cross validation of the OE MRI biomarker perfused Oxy-R. Consistent relationship to pathologic analyses was found in xenografts and human tumors, demonstrating biomarker translation. Published under a CC BY 4.0 license. Online supplemental material is available for this article.

Pre-clinical imaging of transgenic mouse models of neuroblastoma using a dedicated 3-element solenoid coil on a clinical 3T platform.

BACKGROUND: The use of clinical MRI scanners to conduct pre-clinical research facilitates comparisons with clinical studies. Here the utility and sensitivity of anatomical and functional MRI data/biomarkers acquired from transgenic mouse models of neuroblastoma using a dedicated radiofrequency (RF) coil on a clinical 3T scanner was evaluated. METHODS: Multiparametric MRI of transgenic mice bearing abdominal neuroblastomas was performed at 3T, and data cross-referenced to that acquired from the same mice on a pre-clinical 7T MRI system. T2-weighted imaging, quantitation of the native longitudinal relaxation time (T1) and the transverse relaxation rate (R2*), and dynamic contrast-enhanced (DCE)-MRI, was used to assess tumour volume, phenotype and response to cyclophosphamide or cabozantinib. RESULTS: Excellent T2-weighted image contrast enabled clear tumour delineation at 3T. Significant correlations of tumour volume (R=0.98, P<0.0001) and R2* (R=0.87, P<0.002) measured at 3 and 7T were established. Mice with neuroblastomas harbouring the anaplastic lymphoma kinase mutation exhibited a significantly slower R2* (P<0.001), consistent with impaired tumour perfusion. DCE-MRI was performed simultaneously on three transgenic mice, yielding estimates of Ktrans for each tumour (median Ktrans values of 0.202, 0.168 and 0.114 min-1). Cyclophosphamide elicited a significant reduction in both tumour burden (P<0.002) and native T1 (P<0.01), whereas cabozantinib induced significant (P<0.01) tumour growth delay. CONCLUSIONS: Simultaneous multiparametric MRI of multiple tumour-bearing animals using this coil arrangement at 3T can provide high efficiency/throughput for both phenotypic characterisation and evaluation of novel therapeutics, and facilitate the introduction of functional MRI biomarkers into aligned imaging-embedded clinical trials.

Diffusion-weighted MRI for early detection and characterization of prostate cancer in the transgenic adenocarcinoma of the mouse prostate model.

PURPOSE: To improve early diagnosis of prostate cancer to aid clinical decision-making. Diffusion-weighted magnetic resonance imaging (DW-MRI) is sensitive to water diffusion throughout tissues, which correlates with Gleason score, a histological measure of prostate cancer aggressiveness. In this study the ability of DW-MRI to detect prostate cancer onset and development was evaluated in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. MATERIALS AND METHODS: T2 -weighted and DW-MRI were acquired using a 7T MR scanner, 200 mm bore diameter; 10 TRAMP and 6 C57BL/6 control mice were scanned every 4 weeks from 8 weeks of age until sacrifice at 28-30 weeks. After sacrifice, the genitourinary tract was excised and sectioned for histological analysis. Histology slides registered with DW-MR images allowed for validation of DW-MR images and the apparent diffusion coefficient (ADC) as tools for cancer detection and disease stratification. An automated early assessment tool based on ADC threshold values was developed to aid cancer detection and progression monitoring. RESULTS: The ADC differentiated between control prostate ((1.86 ± 0.20) × 10(-3) mm(2) /s) and normal TRAMP prostate ((1.38 ± 0.10) × 10(-3) mm(2) /s) (P = 0.0001), between TRAMP prostate and well-differentiated cancer ((0.93 ± 0.18) × 10(-3) mm(2) /s) (P = 0.0006), and between well-differentiated cancer and poorly differentiated cancer ((0.63 ± 0.06) × 10(-3) mm(2) /s) (P = 0.02). CONCLUSION: DW-MRI is a tool for early detection of cancer, and discrimination between cancer stages in the TRAMP model. The incorporation of DW-MRI-based prostate cancer stratification and monitoring could increase the accuracy of preclinical trials using TRAMP mice.

Dependence of Wilms tumor cells on signaling through insulin-like growth factor 1 in an orthotopic xenograft model targetable by specific receptor inhibition.

We have previously demonstrated an increased DNA copy number and expression of IGF1R to be associated with poor outcome in Wilms tumors. We have now tested whether inhibiting this receptor may be a useful therapeutic strategy by using a panel of Wilms tumor cell lines. Both genetic and pharmacological targeting resulted in inhibition of downstream signaling through PI3 and MAP kinases, G(1) cell cycle arrest, and cell death, with drug efficacy dependent on the levels of phosphorylated IGF1R. These effects were further associated with specific gene expression signatures reflecting pathway inhibition, and conferred synergistic chemosensitisation to doxorubicin and topotecan. In the in vivo setting, s.c. xenografts of WiT49 cells resembled malignant rhabdoid tumors rather than Wilms tumors. Treatment with an IGF1R inhibitor (NVP-AEW541) showed no discernable antitumor activity and no downstream pathway inactivation. By contrast, Wilms tumor cells established orthotopically within the kidney were histologically accurate and exhibited significantly elevated insulin-like growth factor-mediated signaling, and growth was significantly reduced on treatment with NVP-AEW541 in parallel with signaling pathway ablation. As a result of the paracrine effects of enhanced IGF2 expression in Wilms tumor, this disease may be acutely dependent on signaling through the IGF1 receptor, and thus treatment strategies aimed at its inhibition may be useful in the clinic. Such efficacy may be missed if only standard ectopic models are considered as a result of an imperfect recapitulation of the specific tumor microenvironment.

Evaluation of clinically translatable MR imaging biomarkers of therapeutic response in the TH-MYCN transgenic mouse model of neuroblastoma.

PURPOSE: To evaluate noninvasive and clinically translatable magnetic resonance (MR) imaging biomarkers of therapeutic response in the TH-MYCN transgenic mouse model of aggressive, MYCN-amplified neuroblastoma. MATERIALS AND METHODS: All experiments were performed in accordance with the local ethical review panel and the UK Home Office Animals Scientific Procedures Act 1986 and with the UK National Cancer Research Institute guidelines for the welfare of animals in cancer research. Multiparametric MR imaging was performed of abdominal tumors found in the TH-MYCN model. T2-weighted MR imaging, quantitation of native relaxation times T1 and T2, the relaxation rate R2*, and dynamic contrast-enhanced MR imaging were used to monitor tumor response to cyclophosphamide (25 mg/kg), the vascular disrupting agent ZD6126 (200 mg/kg), or the antiangiogenic agent cediranib (6 mg/kg, daily). Any significant changes in the measured parameters, and in the magnitude of the changes after treatment between treated and control cohorts, were identified by using Student two-tailed paired and unpaired t test, respectively, with a 5% level of significance. RESULTS: Treatment with cyclophosphamide or cediranib induced a 54% or 20% reduction in tumor volume at 48 hours, respectively (P < .005 and P < .005, respectively; P < .005 and P < .005 versus control, respectively). Treatment with ZD6126 induced a 45% reduction in mean tumor volume 24 hours after treatment (P < .005; P < .005 versus control). The antitumor activity of cyclophosphamide, cediranib, and ZD6126 was consistently associated with a decrease in tumor T1 (P < .005, P < .005, and P < .005, respectively; P < .005, P < .005, and P < .005 versus control, respectively) and with a correlation between therapy-induced changes in native T1 and changes in tumor volume (r = 0.56; P < .005). Tumor response to cediranib was also associated with a decrease in the dynamic contrast-enhanced MR imaging-derived volume transfer constant (P = .07; P < .05 versus control) and enhancing fraction (P < .05; P < .01 versus control), and an increase in R2* (P < .005; P < .05 versus control). CONCLUSION: The T1 relaxation time is a robust noninvasive imaging biomarker of response to therapy in tumors in TH-MYCN mice, which emulate high-risk neuroblastoma in children. T1 measurements can be readily implemented on clinical MR systems and should be investigated in translational clinical trials of new targeted therapies for pediatric neuroblastoma. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120128/-/DC1.

¹H NMR and hyperpolarized ¹³C NMR assays of pyruvate-lactate: a comparative study.

Pyruvate-lactate exchange is mediated by the enzyme lactate dehydrogenase (LDH) and is central to the altered energy metabolism in cancer cells. The measurement of exchange kinetics using hyperpolarized (13) C NMR has provided a biomarker of response to novel therapeutics. However, the observable signal is restricted to the exchanging hyperpolarized (13) C pools and the endogenous pools of (12) C-labelled metabolites are invisible in these measurements. In this study, we investigated an alternative in vitro (1) H NMR assay, using [3-(13) C]pyruvate, and compared the measured kinetics with a hyperpolarized (13) C NMR assay, using [1-(13) C]pyruvate, under the same conditions in human colorectal carcinoma SW1222 cells. The apparent forward reaction rate constants (kPL ) derived from the two assays showed no significant difference, and both assays had similar reproducibility (kPL = 0.506 ± 0.054 and kPL = 0.441 ± 0.090 nmol/s/10(6) cells; mean ± standard deviation; n = 3); (1) H, (13) C assays, respectively). The apparent backward reaction rate constant (kLP ) could only be measured with good reproducibility using the (1) H NMR assay (kLP = 0.376 ± 0.091 nmol/s/10(6) cells; mean ± standard deviation; n = 3). The (1) H NMR assay has adequate sensitivity to measure real-time pyruvate-lactate exchange kinetics in vitro, offering a complementary and accessible assay of apparent LDH activity.

Exploring ΔR(2) * and ΔR(1) as imaging biomarkers of tumor oxygenation.

PURPOSE: To investigate the combined use of hyperoxia-inducedΔR(2) * and ΔR(1) as a noninvasive imaging biomarker of tumor hypoxia. MATERIALS AND METHODS: MRI was performed on rat GH3 prolactinomas (n = 6) and human PC3 prostate xenografts (n = 6) propagated in nude mice. multiple gradient echo and inversion recovery truefisp images were acquired from identical transverse slices to quantify tumor R(2) * and R(1)before and during carbogen (95% O2 /5% CO2 ) challenge, and correlates of ΔR(2) * and ΔR(1) assessed. RESULTS: Mean baseline R(2) * and R(1) were 119 ± 7 s(-1) and 0.6 ± 0.03 s(-1) for GH3 prolactinomas and 77 ± 12 s(-1) and 0.7 ± 0.02 s(-1) for PC3 xenografts, respectively. During carbogen breathing, mean ΔR(2) * and ΔR(1) were -20 ± 8 s(-1) and 0.08 ± 0.03 s(-1) for GH3 and -0.5 ± 1 s(-1) and 0.2 ± 0.08 s(-1) for the PC3 tumors, respectively. A pronounced relationship betweenΔR(2) * and ΔR(1) was revealed. CONCLUSION: Considering the blood oxygen-hemoglobin dissociation curve, fast R2 * suggested that GH3 prolactinomas were more hypoxic at baseline, and their carbogen response dominated by increased hemoglobin oxygenation, evidenced by highly negative ΔR(2) *. PC3 tumors were less hypoxic at baseline, and their response to carbogen dominated by increased dissolved oxygen, evidenced by highly positive ΔR(1) . Because the two biomarkers are sensitive to different oxygenation ranges, the combination of ΔR(2) * and ΔR(1) may better characterize tumor hypoxia than each alone.

Investigating the contribution of hyaluronan to the breast tumour microenvironment using multiparametric MRI and MR elastography.

Hyaluronan (HA) is a key component of the dense extracellular matrix in breast cancer, and its accumulation is associated with poor prognosis and metastasis. Pegvorhyaluronidase alfa (PEGPH20) enzymatically degrades HA and can enhance drug delivery and treatment response in preclinical tumour models. Clinical development of stromal-targeted therapies would be accelerated by imaging biomarkers that inform on therapeutic efficacy in vivo. Here, PEGPH20 response was assessed by multiparametric magnetic resonance imaging (MRI) in three orthotopic breast tumour models. Treatment of 4T1/HAS3 tumours, the model with the highest HA accumulation, reduced T1 and T2 relaxation times and the apparent diffusion coefficient (ADC), and increased the magnetisation transfer ratio, consistent with lower tissue water content and collapse of the extracellular space. The transverse relaxation rate R2 * increased, consistent with greater erythrocyte accessibility following vascular decompression. Treatment of MDA-MB-231 LM2-4 tumours reduced ADC and dramatically increased tumour viscoelasticity measured by MR elastography. Correlation matrix analyses of data from all models identified ADC as having the strongest correlation with HA accumulation, suggesting that ADC is the most sensitive imaging biomarker of tumour response to PEGPH20.

Palbociclib releases the latent differentiation capacity of neuroblastoma cells.

Neuroblastoma is the most common extracranial solid tumor in infants, arising from developmentally stalled neural crest-derived cells. Driving tumor differentiation is a promising therapeutic approach for this devastating disease. Here, we show that the CDK4/6 inhibitor palbociclib not only inhibits proliferation but induces extensive neuronal differentiation of adrenergic neuroblastoma cells. Palbociclib-mediated differentiation is manifested by extensive phenotypic and transcriptional changes accompanied by the establishment of an epigenetic program driving expression of mature neuronal features. In vivo palbociclib significantly inhibits tumor growth in mouse neuroblastoma models. Furthermore, dual treatment with retinoic acid resets the oncogenic adrenergic core regulatory circuit of neuroblastoma cells, further suppresses proliferation, and can enhance differentiation, altering gene expression in ways that significantly correlate with improved patient survival. We therefore identify palbociclib as a therapeutic approach to dramatically enhance neuroblastoma differentiation efficacy that could be used in combination with retinoic acid to improve patient outcomes.

Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models.

PURPOSE: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.

Evaluation of novel combined carbogen USPIO (CUSPIO) imaging biomarkers in assessing the antiangiogenic effects of cediranib (AZD2171) in rat C6 gliomas.

The recently described combined carbogen USPIO (CUSPIO) magnetic resonance imaging (MRI) method uses spatial correlations in independent imaging biomarkers to assess specific components of tumor vascular structure and function. Our study aimed to evaluate CUSPIO biomarkers for the assessment of tumor response to antiangiogenic therapy. CUSPIO imaging was performed in subcutaneous rat C6 gliomas before and 2 days after treatment with the potent VEGF-signaling inhibitor cediranib (n = 12), or vehicle (n = 12). Histological validation of Hoechst 33342 uptake (perfusion), smooth muscle actin staining (maturation), pimonidazole adduct formation (hypoxia) and necrosis were sought. Following treatment, there was a significant decrease in fractional blood volume (-43%, p < 0.01) and a significant increase in hemodynamic vascular functionality (treatment altered ΔR(2) *(carbogen) from 1.2 to -0.2 s(-1) , p < 0.05). CUSPIO imaging revealed an overall significant decrease in plasma perfusion (-27%, p < 0.05) following cediranib treatment, that was associated with selective effects on immature blood vessels. The CUSPIO responses were associated with a significant 15% reduction in Hoechst 33342 uptake (p < 0.05), but no significant difference in vascular maturation or necrosis. Additionally, treatment with cediranib resulted in a significant 40% increase in tumor hypoxia (p < 0.05). The CUSPIO imaging method provides novel and more specific biomarkers of tumor vessel maturity and vascular hemodynamics, and their response to VEGF-signaling inhibition, compared to current MR imaging biomarkers utilized in the clinic. Such biomarkers may prove effective in longitudinally monitoring tumor vascular remodeling and/or evasive resistance in response to antiangiogenic therapy.

MRI measurements of vessel calibre in tumour xenografts: comparison with vascular corrosion casting.

Vessel size index (R(v), µm) has been proposed as a quantitative magnetic resonance imaging (MRI) derived imaging biomarker in oncology, for the non-invasive assessment of tumour blood vessel architecture and vascular targeted therapies. Appropriate pre-clinical evaluation of R(v) in animal tumour models will improve the interpretation and guide the introduction of the biomarker into clinical studies. The objective of this study was to compare R(v) measured in vivo with vessel size measurements from high-resolution X-ray computed tomography (µCT) of vascular corrosion casts measured post mortem from the same tumours, with and without vascular targeted therapy. MRI measurements were first acquired from subcutaneous SW1222 colorectal xenografts in mice following treatment with 0 (n=6), 30 (n=6) or 200 mg/kg (n=3) of the vascular disrupting agent ZD6126. The mice were then immediately infused with a low viscosity resin and, following polymerisation and maceration of surrounding tissues, the resulting tumour vascular casts were dissected and subsequently imaged using an optimised µCT imaging approach. Vessel diameters were not measurable by µCT in the 200 mg/kg group as the high dose of ZD6126 precluded delivery of the resin to the tumour vascular bed. The mean R(v) for the three treatment groups was 24, 23 and 23.5 µm respectively; the corresponding µCT measurements from corrosion casts from the 0 and 30 mg/kg cohorts were 25 and 28 µm. The strong association between the in vivo MRI and post mortem µCT values supports the use of R(v) as an imaging biomarker in clinical trials of investigational vascular targeted therapies.

Active site mutant dimethylarginine dimethylaminohydrolase 1 expression confers an intermediate tumour phenotype in C6 gliomas.

Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes the endogenous inhibitor of nitric oxide synthesis, asymmetric dimethylarginine (ADMA). Constitutive over-expression of DDAH1, the isoform primarily associated with neuronal nitric oxide synthase (nNOS) results in increased tumour growth and vascularization, and elevated VEGF secretion. To address whether DDAH1-mediated tumour growth is reliant upon the enzymatic activity of DDAH1, cell lines expressing an active site mutant of DDAH1 incapable of metabolizing ADMA were created. Xenografts derived from these cell lines grew significantly faster than those derived from control cells, yet not as fast as those over-expressing wild-type DDAH1. VEGF expression in DDAH1 mutant-expressing tumours did not differ from control tumours but was significantly lower than that of wild-type DDAH1-over-expressing tumours. Fluorescence microscopy for CD31 and pimonidazole adduct formation demonstrated that DDAH1 mutant-expressing tumours had a lower endothelial content and demonstrated less hypoxia, respectively, than wild-type DDAH1-expressing tumours. However, there was no difference in uptake of the perfusion marker Hoechst 33342. Non-invasive multiparametric quantitative MRI, including the measurement of native T(1) and T(2) relaxation times and apparent water diffusion coefficient, was indicative of higher cellularity in DDAH1-expressing xenografts, which was confirmed by histological quantification of necrosis. C6 xenografts expressing active site mutant DDAH1 displayed an intermediate phenotype between tumours over-expressing wild-type DDAH1 and control tumours. These data suggest that enhanced VEGF expression downstream of DDAH1 was dependent upon ADMA metabolism, but that the DDAH1-mediated increase in tumour growth was only partially dependent upon its enzymatic activity, and therefore must involve an as-yet unidentified mechanism. DDAH1 is an important mediator of tumour progression, but appears to have addition roles independent of its metabolism of ADMA, which need to be considered in therapeutic strategies targeted against the NO/DDAH pathway in cancer.

Preclinical Three-Dimensional Vibrational Shear Wave Elastography for Mapping of Tumour Biomechanical Properties In Vivo.

Preclinical investigation of the biomechanical properties of tissues and their treatment-induced changes are essential to support drug-discovery, clinical translation of biomarkers of treatment response, and studies of mechanobiology. Here we describe the first use of preclinical 3D elastography to map the shear wave speed (cs), which is related to tissue stiffness, in vivo and demonstrate the ability of our novel 3D vibrational shear wave elastography (3D-VSWE) system to detect tumour response to a therapeutic challenge. We investigate the use of one or two vibrational sources at vibrational frequencies of 700, 1000 and 1200 Hz. The within-subject coefficients of variation of our system were found to be excellent for 700 and 1000 Hz and 5.4 and 6.2%, respectively. The relative change in cs measured with our 3D-VSWE upon treatment with an anti-vascular therapy ZD6126 in two tumour xenografts reflected changes in tumour necrosis. U-87 MG drug vs vehicle: Δcs = −24.7 ± 2.5 % vs 7.5 ± 7.1%, (p = 0.002) and MDA-MB-231 drug vs vehicle: Δcs = −12.3 ± 2.7 % vs 4.5 ± 4.7%, (p = 0.02). Our system enables rapid (<5 min were required for a scan length of 15 mm and three vibrational frequencies) 3D mapping of quantitative tumour viscoelastic properties in vivo, allowing exploration of regional heterogeneity within tumours and speedy recovery of animals from anaesthesia so that longitudinal studies (e.g., during tumour growth or following treatment) may be conducted frequently.

Indisulam targets RNA splicing and metabolism to serve as a therapeutic strategy for high-risk neuroblastoma.

Neuroblastoma is the most common paediatric solid tumour and prognosis remains poor for high-risk cases despite the use of multimodal treatment. Analysis of public drug sensitivity data showed neuroblastoma lines to be sensitive to indisulam, a molecular glue that selectively targets RNA splicing factor RBM39 for proteosomal degradation via DCAF15-E3-ubiquitin ligase. In neuroblastoma models, indisulam induces rapid loss of RBM39, accumulation of splicing errors and growth inhibition in a DCAF15-dependent manner. Integrative analysis of RNAseq and proteomics data highlight a distinct disruption to cell cycle and metabolism. Metabolic profiling demonstrates metabolome perturbations and mitochondrial dysfunction resulting from indisulam. Complete tumour regression without relapse was observed in both xenograft and the Th-MYCN transgenic model of neuroblastoma after indisulam treatment, with RBM39 loss, RNA splicing and metabolic changes confirmed in vivo. Our data show that dual-targeting of metabolism and RNA splicing with anticancer indisulam is a promising therapeutic approach for high-risk neuroblastoma.

Noninvasive detection of carboxypeptidase G2 activity in vivo.

The pseudomonad protein, carboxypeptidase G2 (CPG2), is a prodrug-activating enzyme utilized in the targeted chemotherapy strategies of antibody- and gene-directed enzyme prodrug therapy (ADEPT and GDEPT). We have developed a noninvasive imaging approach to monitor CPG2 activity in vivo that will facilitate the preclinical and clinical development of CPG2-based ADEPT and GDEPT strategies. Cleavage of the novel reporter probe, 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu), by CPG2, in human colon adenocarcinoma WiDr xenografts engineered to stably express CPG2, was monitored using (19)F MRSI. The high signal-to-noise ratio afforded by the two MR-equivalent (19)F nuclei of 3,5-DFBGlu, and the 1.4 ppm (19)F chemical shift difference on CPG2-mediated cleavage, enabled the dynamics and quantification of the apparent pharmacokinetics of 3,5-DFBGlu and its CPG2-mediated cleavage in the tumor to be evaluated. In addition, the apparent rate of increase of 3,5-difluorobenzoic acid concentration could also provide a biomarker of CPG2 activity levels in tumors of patients undergoing CPG2-based therapies, as well as a biomarker of treatment response. The addition of in vivo reporter probes, such as 3,5-DFBGlu, to the armamentarium of prodrugs cleaved by CPG2 affords new applications for CPG2 as a gene reporter of transgene expression.

Accelerating Whole-Body Diffusion-weighted MRI with Deep Learning-based Denoising Image Filters.

PURPOSE: To use deep learning to improve the image quality of subsampled images (number of acquisitions = 1 [NOA1]) to reduce whole-body diffusion-weighted MRI (WBDWI) acquisition times. MATERIALS AND METHODS: Both retrospective and prospective patient groups were used to develop a deep learning-based denoising image filter (DNIF) model. For initial model training and validation, 17 patients with metastatic prostate cancer with acquired WBDWI NOA1 and NOA9 images (acquisition period, 2015-2017) were retrospectively included. An additional 22 prospective patients with advanced prostate cancer, myeloma, and advanced breast cancer were used for model testing (2019), and the radiologic quality of DNIF-processed NOA1 (NOA1-DNIF) images were compared with NOA1 images and clinical NOA16 images by using a three-point Likert scale (good, average, or poor; statistical significance was calculated by using a Wilcoxon signed ranked test). The model was also retrained and tested in 28 patients with malignant pleural mesothelioma (MPM) who underwent lung MRI (2015-2017) to demonstrate feasibility in other body regions. RESULTS: The model visually improved the quality of NOA1 images in all test patients, with the majority of NOA1-DNIF and NOA16 images being graded as either "average" or "good" across all image-quality criteria. From validation data, the mean apparent diffusion coefficient (ADC) values within NOA1-DNIF images of bone disease deviated from those within NOA9 images by an average of 1.9% (range, 1.1%-2.6%). The model was also successfully applied in the context of MPM; the mean ADCs from NOA1-DNIF images of MPM deviated from those measured by using clinical-standard images (NOA12) by 3.7% (range, 0.2%-10.6%). CONCLUSION: Clinical-standard images were generated from subsampled images by using a DNIF.Keywords: Image Postprocessing, MR-Diffusion-weighted Imaging, Neural Networks, Oncology, Whole-Body Imaging, Supervised Learning, MR-Functional Imaging, Metastases, Prostate, Lung Supplemental material is available for this article. Published under a CC BY 4.0 license.

Combined inhibition of Aurora-A and ATR kinase results in regression of MYCN-amplified neuroblastoma.

Amplification of MYCN is the driving oncogene in a subset of high-risk neuroblastoma. The MYCN protein and the Aurora-A kinase form a complex during S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription-replication conflicts and activates the Ataxia telangiectasia and Rad3 related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription-replication conflicts is an effective therapy for MYCN-driven neuroblastoma (141 words).

SuperHistopath: A Deep Learning Pipeline for Mapping Tumor Heterogeneity on Low-Resolution Whole-Slide Digital Histopathology Images.

High computational cost associated with digital pathology image analysis approaches is a challenge towards their translation in routine pathology clinic. Here, we propose a computationally efficient framework (SuperHistopath), designed to map global context features reflecting the rich tumor morphological heterogeneity. SuperHistopath efficiently combines i) a segmentation approach using the linear iterative clustering (SLIC) superpixels algorithm applied directly on the whole-slide images at low resolution (5x magnification) to adhere to region boundaries and form homogeneous spatial units at tissue-level, followed by ii) classification of superpixels using a convolution neural network (CNN). To demonstrate how versatile SuperHistopath was in accomplishing histopathology tasks, we classified tumor tissue, stroma, necrosis, lymphocytes clusters, differentiating regions, fat, hemorrhage and normal tissue, in 127 melanomas, 23 triple-negative breast cancers, and 73 samples from transgenic mouse models of high-risk childhood neuroblastoma with high accuracy (98.8%, 93.1% and 98.3% respectively). Furthermore, SuperHistopath enabled discovery of significant differences in tumor phenotype of neuroblastoma mouse models emulating genomic variants of high-risk disease, and stratification of melanoma patients (high ratio of lymphocyte-to-tumor superpixels (p = 0.015) and low stroma-to-tumor ratio (p = 0.028) were associated with a favorable prognosis). Finally, SuperHistopath is efficient for annotation of ground-truth datasets (as there is no need of boundary delineation), training and application (~5 min for classifying a whole-slide image and as low as ~30 min for network training). These attributes make SuperHistopath particularly attractive for research in rich datasets and could also facilitate its adoption in the clinic to accelerate pathologist workflow with the quantification of phenotypes, predictive/prognosis markers.