Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.

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

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

Kinetic Modeling and Constrained Reconstruction of Hyperpolarized [1-13C]-Pyruvate Offers Improved Metabolic Imaging of Tumors.

Hyperpolarized [1-(13)C]-pyruvate has shown tremendous promise as an agent for imaging tumor metabolism with unprecedented sensitivity and specificity. Imaging hyperpolarized substrates by magnetic resonance is unlike traditional MRI because signals are highly transient and their spatial distribution varies continuously over their observable lifetime. Therefore, new imaging approaches are needed to ensure optimal measurement under these circumstances. Constrained reconstruction algorithms can integrate prior information, including biophysical models of the substrate/target interaction, to reduce the amount of data that is required for image analysis and reconstruction. In this study, we show that metabolic MRI with hyperpolarized pyruvate is biased by tumor perfusion and present a new pharmacokinetic model for hyperpolarized substrates that accounts for these effects. The suitability of this model is confirmed by statistical comparison with alternates using data from 55 dynamic spectroscopic measurements in normal animals and murine models of anaplastic thyroid cancer, glioblastoma, and triple-negative breast cancer. The kinetic model was then integrated into a constrained reconstruction algorithm and feasibility was tested using significantly undersampled imaging data from tumor-bearing animals. Compared with naïve image reconstruction, this approach requires far fewer signal-depleting excitations and focuses analysis and reconstruction on new information that is uniquely available from hyperpolarized pyruvate and its metabolites, thus improving the reproducibility and accuracy of metabolic imaging measurements.

High-throughput hyperpolarized (13)C metabolic investigations using a multi-channel acquisition system.

Magnetic resonance imaging and spectroscopy of hyperpolarized (HP) compounds such as [1-(13)C]-pyruvate have shown tremendous potential for offering new insight into disease and response to therapy. New applications of this technology in clinical research and care will require extensive validation in cells and animal models, a process that may be limited by the high cost and modest throughput associated with dynamic nuclear polarization. Relatively wide spectral separation between [1-(13)C]-pyruvate and its chemical endpoints in vivo are conducive to simultaneous multi-sample measurements, even in the presence of a suboptimal global shim. Multi-channel acquisitions could conserve costs and accelerate experiments by allowing acquisition from multiple independent samples following a single dissolution. Unfortunately, many existing preclinical MRI systems are equipped with only a single channel for broadband acquisitions. In this work, we examine the feasibility of this concept using a broadband multi-channel digital receiver extension and detector arrays that allow concurrent measurement of dynamic spectroscopic data from ex vivo enzyme phantoms, in vitro anaplastic thyroid carcinoma cells, and in vivo in tumor-bearing mice. Throughput and the cost of consumables were improved by up to a factor of four. These preliminary results demonstrate the potential for efficient multi-sample studies employing hyperpolarized agents.

The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming.

Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumorigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programmes by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anticancer metabolism therapy development in future.

IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo.

TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the ß cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.

Feasibility of multianimal hyperpolarized (13) C MRS.

PURPOSE: There is great potential for real-time investigation of metabolism with MRS and hyperpolarized (HP) (13) C agents. Unfortunately, HP technology has high associated costs and efficiency limitations that may constrain in vivo studies involving many animals. To improve the throughput of preclinical investigations, we evaluate the feasibility of performing HP MRS on multiple animals simultaneously. METHODS: Simulations helped assess the viability of a dual-coil strategy for spatially localized multivolume MRS. A dual-mouse system was assembled and characterized with bench- and scanner-based experiments. Enzyme phantoms mixed with HP [1-(13) C] pyruvate emulated real-time metabolism and offered a controlled mechanism for evaluating system performance. Finally, a normal mouse and a mouse bearing a subcutaneous xenograft of colon cancer were simultaneously scanned in vivo using an agent containing HP [1-(13) C] pyruvate. RESULTS: Geometric separation/rotation, active decoupling, and use of low input impedance preamplifiers permitted an encode-by-channel approach for spatially localized MRS. A precalibrated shim allowed straightforward metabolite differentiation in enzyme phantom and in vivo experiments at 7 Tesla, with performance similar to conventional acquisitions. CONCLUSION: The initial feasibility of multi-animal HP (13) C MRS was established. Throughput scales with the number of simultaneously scanned animals, demonstrating the potential for significant improvements in study efficiency.

Evaluation of hyperpolarized [1-¹³C]-pyruvate by magnetic resonance to detect ionizing radiation effects in real time.

Ionizing radiation (IR) cytotoxicity is primarily mediated through reactive oxygen species (ROS). Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP) magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC), an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential in vitro and [1-¹³C] pyruvate HP-MRS/MRSI imaging of ATC orthotopic xenografts was used to study in vivo effects of IR. IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.

Radial spectroscopic MRI of hyperpolarized [1-(13) C] pyruvate at 7 tesla.

PURPOSE: The transient and nonrenewable signal from hyperpolarized metabolites necessitates extensive sequence optimization for encoding spatial, spectral, and dynamic information. In this work, we evaluate the utility of radial single-timepoint and cumulative spectroscopic MRI of hyperpolarized [1-(13) C] pyruvate and its metabolic products at 7 Tesla (T). METHODS: Simulations of radial echo planar spectroscopic imaging (EPSI) and multiband frequency encoding (MBFE) acquisitions were performed to confirm feasibility and evaluate performance for HP (13) C imaging. Corresponding sequences were implemented on a 7T small-animal MRI system, tested in phantom, and demonstrated in a murine model of anaplastic thyroid cancer. RESULTS: MBFE provides excellent spectral separation but is susceptible to blurring and T2 * signal loss inherent to using low readout gradients. The higher readout gradients and more flexible spectral encoding for EPSI result in good spatial resolution and spectral separation. Radial acquisition throughout HP signal evolution offers the flexibility for reconstructing spatial maps of mean metabolite distribution and global dynamic time courses of multiple metabolites. CONCLUSION: Radial EPSI and MBFE acquisitions are well-suited for hyperpolarized (13) C MRI over short and long durations. Advantages to this approach include robustness to nonstationary magnetization, insensitivity to precise acquisition timing, and versatility for reconstructing dynamically acquired spectroscopic data.

A catalyzing phantom for reproducible dynamic conversion of hyperpolarized [1-¹³C]-pyruvate.

In vivo real time spectroscopic imaging of hyperpolarized ¹³C labeled metabolites shows substantial promise for the assessment of physiological processes that were previously inaccessible. However, reliable and reproducible methods of measurement are necessary to maximize the effectiveness of imaging biomarkers that may one day guide personalized care for diseases such as cancer. Animal models of human disease serve as poor reference standards due to the complexity, heterogeneity, and transient nature of advancing disease. In this study, we describe the reproducible conversion of hyperpolarized [1-¹³C]-pyruvate to [1-¹³C]-lactate using a novel synthetic enzyme phantom system. The rate of reaction can be controlled and tuned to mimic normal or pathologic conditions of varying degree. Variations observed in the use of this phantom compare favorably against within-group variations observed in recent animal studies. This novel phantom system provides crucial capabilities as a reference standard for the optimization, comparison, and certification of quantitative imaging strategies for hyperpolarized tracers.

A throughput-optimized array system for multiple-mouse MRI.

MRI is a versatile tool for the systematic assessment of anatomical and functional changes in small-animal models of human disease. Its noninvasive nature makes it an ideal candidate for longitudinal evaluations of disease progression, but relatively long scan times limit the number of observations that can be made in a given interval of time, imposing restrictions on experimental design and potentially compromising statistical power. Methods that reduce the overall time required to scan multiple cohorts of animals in distinct experimental groups are therefore highly desirable. Multiple-mouse MRI, in which several animals are simultaneously scanned in a common MRI system, has been successfully used to improve study throughput. However, to best utilize the next generation of small-animal MRI systems that will be equipped with an increased number of receive channels, a paradigm shift from the simultaneous scanning of as many animals as possible to the scanning of a more manageable number, at a faster rate, must be considered. This work explores the tradeoffs between the number of animals to scan at once and the number of array elements dedicated to each animal, to maximize throughput in systems with 16 receive channels. An array system consisting of 15 receive and five transmit coils allows acceleration by a combination of multi-animal and parallel imaging techniques. The array system was designed and fabricated for use on a 7.0-T/30-cm Bruker Biospec MRI system, and tested for high-throughput imaging performance in phantoms and live mice. Results indicate that up to a nine-fold throughput improvement of a single sequence is possible compared with an unaccelerated single-animal acquisition. True data throughput of a contrast-enhanced anatomical study is estimated to be improved by just over six-fold.

Multiple-mouse MRI with multiple arrays of receive coils.

Compared to traditional single-animal imaging methods, multiple-mouse MRI has been shown to dramatically improve imaging throughput and reduce the potentially prohibitive cost for instrument access. To date, up to a single radiofrequency coil has been dedicated to each animal being simultaneously scanned, thus limiting the sensitivity, flexibility, and ultimate throughput. The purpose of this study was to investigate the feasibility of multiple-mouse MRI with a phased-array coil dedicated to each animal. A dual-mouse imaging system, consisting of a pair of two-element phased-array coils, was developed and used to achieve acceleration factors greater than the number of animals scanned at once. By simultaneously scanning two mice with a retrospectively gated cardiac cine MRI sequence, a 3-fold acceleration was achieved with signal-to-noise ratio in the heart that is equivalent to that achieved with an unaccelerated scan using a commercial mouse birdcage coil.

Monitoring histone deacetylase inhibition in vivo: noninvasive magnetic resonance spectroscopy method.

Histone deacetylase inhibitors (HDACis) are emerging as promising and selective antitumor agents. However, HDACis can lead to tumor stasis rather than shrinkage, in which case, traditional imaging methods are not adequate to monitor response. Consequently, novel approaches are needed. We have shown in cells that (19)F magnetic resonance spectroscopy (MRS)-detectable levels of the HDAC substrate Boc-Lys-TFA-OH (BLT) are inversely correlated with HDAC activity. We extended our investigations to a tumor xenograft model. Following intraperitoneal injection of BLT, its accumulation within the tumor was monitored by in vivo (19)F MRS. In animals treated with the HDACi suberoylanilide hydroxamic acid (SAHA), tumoral BLT levels were higher by 77% and 132% on days 2 and 7 of treatment compared with pretreatment levels (n = 6; p < .05). In contrast, tumoral BLT levels remained unchanged in control animals and in normal tissue. Thus, (19)F MRS of BLT detected the effect of HDACi treatment as early as day 2 of treatment. Importantly, tumor size confirmed that SAHA treatment leads to inhibition of tumor growth. However, difference in tumor size reached significance only on day 6 of treatment. Thus, this work identifies BLT as a potential molecular imaging agent for the early noninvasive MRS detection of HDAC inhibition in vivo.

Wireless self-gated multiple-mouse cardiac cine MRI.

Despite the excellent image-contrast capability of MRI and the ability to synchronize MRI with the murine cardiac cycle, this technique is underused for assessing mouse models of cardiovascular disease because of its perceived cost and complexity. This perception stems, in part, from complications associated with the placement and adjustment of electrocardiographic leads that may interact with gradient pulses and the relatively long acquisition times required with traditional gating schemes. To improve the efficiency and reduce the cost and complexity of using cardiac MRI in mice, we combined wireless self-gating techniques (with which we derived cardiac synchronization signals from acquired data) with an imaging technique that acquires multislice cardiac cine images from four mice simultaneously. As a result, the wireless self-gated acquisitions minimized animal preparation time and improved image quality. The simultaneous acquisition of cardiac cine data from multiple animals greatly increased throughput and reduced costs associated with instrument access.

Feasibility of multiple-mouse dynamic contrast-enhanced MRI.

Dynamic contrast-enhanced (DCE-) MRI is often used to evaluate the response to experimental antiangiogenic therapies in small animal models of cancer. Unfortunately, DCE-MRI studies often require a substantial investment of both time and money to achieve the desired level of statistical significance. Multiple-mouse MRI has previously been used to improve imaging efficiency, but its feasibility for DCE-MRI has not been investigated. The purpose of this work was to determine if multiple-mouse DCE-MRI is feasible when using gadolinium-based contrast agents with a low molecular weight. A population of tumor-bearing mice was scanned using two four-element arrays and a single-coil configuration on a 4.7T, 40 cm bore Bruker Biospec MRI scanner. Pharmacokinetic parameters were calculated and compared to determine if a significant difference between methodologies existed. With both four-animal imaging configurations, animal throughput accelerations of just less than three were achieved and quantitative data were not significantly different than from single-animal acquisitions.

A practical method for 2D multiple-animal MRI.

PURPOSE: To investigate practical methods for achieving routine simultaneous 2D MRI of multiple animals in large-bore experimental scanners. MATERIALS AND METHODS: Three four-element array geometries were compared against a standard single-coil configuration in terms of image quality, ease of use, and data efficiency using a four-channel, 4.7 T small animal imaging system. RESULTS: A linear arrangement of volume resonators permits unobstructed animal preparation and use of an imaging protocol that is almost identical to the single-coil configuration without requiring any image correction or other additional postprocessing. Resulting in vivo images were visually indistinguishable from those acquired through the single-coil configuration. CONCLUSION: The efficiency of animal studies employing 2D MRI techniques can be substantially improved by using a linear array of commercially available resonators.