A modified mouse model of Friedreich's ataxia with conditional Fxn allele homozygosity delays onset of cardiomyopathy.

Friedreich's ataxia (FA) is an autosomal recessive disorder caused by a deficiency in frataxin (FXN), a mitochondrial protein that plays a critical role in the synthesis of iron-sulfur clusters (Fe-S), vital inorganic cofactors necessary for numerous cellular processes. FA is characterized by progressive ataxia and hypertrophic cardiomyopathy, with cardiac dysfunction as the most common cause of mortality in patients. Commonly used cardiac-specific mouse models of FA use the muscle creatine kinase (MCK) promoter to express Cre recombinase in cardiomyocytes and striated muscle cells in mice with one conditional Fxn allele and one floxed-out/null allele. These mice quickly develop cardiomyopathy that becomes fatal by 9-11 wk of age. Here, we generated a cardiac-specific model with floxed Fxn allele homozygosity (MCK-Fxnflox/flox). MCK-Fxnflox/flox mice were phenotypically normal at 9 wk of age, despite no detectable FXN protein expression. Between 13 and 15 wk of age, these mice began to display progressive cardiomyopathy, including decreased ejection fraction and fractional shortening and increased left ventricular mass. MCK-Fxnflox/flox mice began to lose weight around 16 wk of age, characteristically associated with heart failure in other cardiac-specific FA models. By 18 wk of age, MCK-Fxnflox/flox mice displayed elevated markers of Fe-S deficiency, cardiac stress and injury, and cardiac fibrosis. This modified model reproduced important pathophysiological and biochemical features of FA over a longer timescale than previous cardiac-specific mouse models, offering a larger window for studying potential therapeutics.NEW & NOTEWORTHY Previous cardiac-specific frataxin knockout models exhibit rapid and fatal cardiomyopathy by 9 wk of age. This severe phenotype poses challenges for the design and execution of intervention studies. We introduce an alternative cardiac-specific model, MCK-Fxnflox/flox, with increased longevity and delayed onset of all major phenotypes. These phenotypes develop to the same severity as previous models. Thus, this new model provides the same cardiomyopathy-associated mortality with a larger window for potential studies.

Fully automated mouse echocardiography analysis using deep convolutional neural networks.

Echocardiography (echo) is a translationally relevant ultrasound imaging modality widely used to assess cardiac structure and function in preclinical models of heart failure (HF) during research and drug development. Although echo is a very valuable tool, the image analysis is a time-consuming, resource-demanding process, and is susceptible to interreader variability. Recent advancements in deep learning have enabled researchers to automate image processing and reduce analysis time and interreader variability in the field of medical imaging. In the present study, we developed a fully automated tool, mouse-echocardiography neural net (MENN), for the analysis of both long-axis brightness (B)-mode and short-axis motion (M)-mode images of left ventricle. MENN is a series of fully convolutional neural networks that were trained and validated using manually segmented B-mode and M-mode echo images of the left ventricle. The segmented images were then used to compute cardiac structural and functional metrics. The performance of MENN was further validated in two preclinical models of HF. MENN achieved excellent correlations (Pearson's r = 0.85-0.99) and good-to-excellent agreement between automated and manual analyses. Further interreader variability analysis showed that MENN has better agreements with an expert analyst than both a trained analyst and a novice. Notably, the use of MENN reduced manual analysis time by >92%. In conclusion, we developed an automated echocardiography analysis tool that allows for fast and accurate analysis of B-mode and M-mode mouse echo data and mitigates the issue of interreader variability in manual analysis.NEW & NOTEWORTHY Echocardiography is commonly used in preclinical research to evaluate cardiac structure and function. Despite the broad applications across therapeutic areas, the analysis of echo data is laborious and susceptible to interreader variability. In this study, we developed a fully automated mouse-echocardiography neural net (MENN). Cardiac measurements from MENN showed excellent correlations with manual analysis. Furthermore, the use of MENN leads to >92% reduction in analysis time and potentially eliminates the interobserver variability issue.

Cabozantinib Unlocks Efficient In Vivo Targeted Delivery of Neutrophil-Loaded Nanoparticles into Murine Prostate Tumors.

A major barrier to the successful application of nanotechnology for cancer treatment is the suboptimal delivery of therapeutic payloads to metastatic tumor deposits. We previously discovered that cabozantinib, a tyrosine kinase inhibitor, triggers neutrophil-mediated anticancer innate immunity, resulting in tumor regression in an aggressive PTEN/p53-deficient genetically engineered murine model of advanced prostate cancer. Here, we specifically investigated the potential of cabozantinib-induced neutrophil activation and recruitment to enhance delivery of BSA-coated polymeric nanoparticles (BSA-NPs) into murine PTEN/p53-deficient prostate tumors. On the basis of the observation that BSA coating of NPs enhanced association and internalization by activated neutrophils by approximately 6-fold in vitro, relative to uncoated NPs, we systemically injected BSA-coated, dye-loaded NPs into prostate-specific PTEN/p53-deficient mice that were pretreated with cabozantinib. Flow cytometric analysis revealed an approximately 4-fold increase of neutrophil-associated BSA-NPs and an approximately 32-fold increase in mean fluorescent dye uptake following 3 days of cabozantinib/BSA-NP administration, relative to BSA-NP alone. Strikingly, neutrophil depletion with Ly6G antibody abolished dye-loaded BSA-NP accumulation within tumors to baseline levels, demonstrating targeted neutrophil-mediated intratumoral NP delivery. Furthermore, we observed an approximately 13-fold decrease in accumulation of BSA-NPs in the liver, relative to uncoated NPs, post-cabozantinib treatment, suggesting that BSA coating of NPs can significantly enhance cabozantinib-induced, neutrophil-mediated targeted intratumoral drug delivery, while mitigating off-target toxicity. Collectively, we demonstrate a novel targeted nano-immunotherapeutic strategy for enhanced intratumoral delivery of BSA-NPs, with translational potential to significantly augment therapeutic indices of cancer medicines, thereby overcoming current pharmacologic barriers commonly encountered in preclinical/early-phase drug development.

Pericytes Elicit Resistance to Vemurafenib and Sorafenib Therapy in Thyroid Carcinoma via the TSP-1/TGFß1 Axis.

PURPOSE: The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown. EXPERIMENTAL DESIGN: We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment. RESULTS: Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFß1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFß1 expression evoke gene-regulatory networks and pathways (TGFß signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand-VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFß1 axis reduces tumor cell growth and overcomes drug resistance. CONCLUSIONS: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFß1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.

Synergy of radiotherapy and PD-1 blockade in Kras-mutant lung cancer.

Radiation therapy (RT), a critical modality in the treatment of lung cancer, induces direct tumor cell death and augments tumor-specific immunity. However, despite initial tumor control, most patients suffer from locoregional relapse and/or metastatic disease following RT. The use of immunotherapy in non-small-cell lung cancer (NSCLC) could potentially change this outcome by enhancing the effects of RT. Here, we report significant (up to 70% volume reduction of the target lesion) and durable (up to 12 weeks) tumor regressions in conditional Kras-driven genetically engineered mouse models (GEMMs) of NSCLC treated with radiotherapy and a programmed cell death 1 antibody (αPD-1). However, while αPD-1 therapy was beneficial when combined with RT in radiation-naive tumors, αPD-1 therapy had no antineoplastic efficacy in RT-relapsed tumors and further induced T cell inhibitory markers in this setting. Furthermore, there was differential efficacy of αPD-1 plus RT among Kras-driven GEMMs, with additional loss of the tumor suppressor serine/threonine kinase 11/liver kinase B1 (Stk11/Lkb1) resulting in no synergistic efficacy. Taken together, our data provide evidence for a close interaction among RT, T cells, and the PD-1/PD-L1 axis and underscore the rationale for clinical combinatorial therapy with immune modulators and radiotherapy.

Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion.

We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1(f/f)p53(f/f)), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors.

Phosphoinositide 3-Kinase Regulates Glycolysis through Mobilization of Aldolase from the Actin Cytoskeleton.

The phosphoinositide 3-kinase (PI3K) pathway regulates multiple steps in glucose metabolism and also cytoskeletal functions, such as cell movement and attachment. Here, we show that PI3K directly coordinates glycolysis with cytoskeletal dynamics in an AKT-independent manner. Growth factors or insulin stimulate the PI3K-dependent activation of Rac, leading to disruption of the actin cytoskeleton, release of filamentous actin-bound aldolase A, and an increase in aldolase activity. Consistently, PI3K inhibitors, but not AKT, SGK, or mTOR inhibitors, cause a significant decrease in glycolysis at the step catalyzed by aldolase, while activating PIK3CA mutations have the opposite effect. These results point toward a master regulatory function of PI3K that integrates an epithelial cell's metabolism and its form, shape, and function, coordinating glycolysis with the energy-intensive dynamics of actin remodeling.

Regeneration of Thyroid Function by Transplantation of Differentiated Pluripotent Stem Cells.

Differentiation of functional thyroid epithelia from pluripotent stem cells (PSCs) holds the potential for application in regenerative medicine. However, progress toward this goal is hampered by incomplete understanding of the signaling pathways needed for directed differentiation without forced overexpression of exogenous transgenes. Here we use mouse PSCs to identify key conserved roles for BMP and FGF signaling in regulating thyroid lineage specification from foregut endoderm in mouse and Xenopus. Thyroid progenitors derived from mouse PSCs can be matured into thyroid follicular organoids that provide functional secretion of thyroid hormones in vivo and rescue hypothyroid mice after transplantation. Moreover, by stimulating the same pathways, we were also able to derive human thyroid progenitors from normal and disease-specific iPSCs generated from patients with hypothyroidism resulting from NKX2-1 haploinsufficiency. Our studies have therefore uncovered the regulatory mechanisms that underlie early thyroid organogenesis and provide a significant step toward cell-based regenerative therapy for hypothyroidism.

Tumor priming by Apo2L/TRAIL reduces interstitial fluid pressure and enhances efficacy of liposomal gemcitabine in a patient derived xenograft tumor model.

Interstitial fluid pressure (IFP) is elevated in tumors and high IFP, a negative cancer prognosticator, is known to limit the uptake and efficacy of anti-tumor therapeutics. Approaches that alter the tumor microenvironment and enhance uptake of therapeutics are collectively referred to as tumor "priming". Here we show that the cytotoxic biological therapy Apo2L/TRAIL can prime the tumor microenvironment and significantly lower IFP in three different human tumor xenograft models (Colo205, MiaPaca-2 and a patient gastrointestinal adenocarcinoma tumor xenograft). We found that a single dose of Apo2L/TRAIL resulted in a wave of apoptosis which reached a maximum at 8h post-treatment. Apoptotic debris subsequently disappeared concurrent with an increase in macrophage infiltration. By 24h post-treatment, treated tumors appeared less condensed with widening of the stromal areas which increased at 48 and 72h. Analysis of tumor vasculature demonstrated a significant increase in overall vessel size at 48 and 72h although the number of vessels did not change. Notably, IFP was significantly reduced in these tumors by 48h after Apo2L/TRAIL treatment. Administration of gemcitabine at this time resulted in increased tumor uptake of both gemcitabine and liposomal gemcitabine and significantly improved anti-tumor efficacy of liposomal gemcitabine. These results suggest that Apo2L/TRAIL has a potential as a tumor priming agent and provides a rationale for developing a sequencing schema for combination therapy such that an initial dose of Apo2L/TRAIL would precede administration of gemcitabine or other therapies.

A pilot study of the effects of mild systemic heating on human head and neck tumour xenografts: Analysis of tumour perfusion, interstitial fluid pressure, hypoxia and efficacy of radiation therapy.

PURPOSE: The tumour microenvironment is frequently hypoxic, poorly perfused, and exhibits abnormally high interstitial fluid pressure. These factors can significantly reduce efficacy of chemo and radiation therapies. The present study aims to determine whether mild systemic heating alters these parameters and improves response to radiation in human head and neck tumour xenografts in SCID mice. MATERIALS AND METHODS: SCID mice were injected with FaDu cells (a human head and neck carcinoma cell line), or implanted with a resected patient head and neck squamous cell carcinoma grown as a xenograft, followed by mild systemic heating. Body temperature during heating was maintained at 39.5 ± 0.5 °C for 4 h. Interstitial fluid pressure (IFP), hypoxia and relative tumour perfusion in the tumours were measured at 2 and 24 h post-heating. Tumour vessel perfusion was measured 24 h post-heating, coinciding with the first dose of fractionated radiotherapy. RESULTS: Heating tumour-bearing mice resulted in significant decrease in intratumoural IFP, increased the number of perfused tumour blood vessels as well as relative tumour perfusion in both tumour models. Intratumoural hypoxia was also reduced in tumours of mice that received heat treatment. Mice bearing FaDu tumours heated 24 h prior to five daily radiation treatments exhibited significantly enhanced tumour response compared to tumours in control mice. CONCLUSIONS: Mild systemic heating can significantly alter the tumour microenvironment of human head and neck tumour xenograft models, decreasing IFP and hypoxia while increasing microvascular perfusion. Collectively, these effects could be responsible for the improved response to radiotherapy.