Mitochondrial complex II in intestinal epithelial cells regulates T cell-mediated immunopathology.

Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.

The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity.

The E3 ubiquitin ligase APC/C-Cdh1 maintains the G0/G1 state, and its inactivation is required for cell cycle entry. We reveal a novel role for Fas-associated protein with death domain (FADD) in the cell cycle through its function as an inhibitor of APC/C-Cdh1. Using real-time, single-cell imaging of live cells combined with biochemical analysis, we demonstrate that APC/C-Cdh1 hyperactivity in FADD-deficient cells leads to a G1 arrest despite persistent mitogenic signaling through oncogenic EGFR/KRAS. We further show that FADDWT interacts with Cdh1, while a mutant lacking a consensus KEN-box motif (FADDKEN) fails to interact with Cdh1 and results in a G1 arrest due to its inability to inhibit APC/C-Cdh1. Additionally, enhanced expression of FADDWT but not FADDKEN, in cells arrested in G1 upon CDK4/6 inhibition, leads to APC/C-Cdh1 inactivation and entry into the cell cycle in the absence of retinoblastoma protein phosphorylation. FADD's function in the cell cycle requires its phosphorylation by CK1α at Ser-194 which promotes its nuclear translocation. Overall, FADD provides a CDK4/6-Rb-E2F-independent "bypass" mechanism for cell cycle entry and thus a therapeutic opportunity for CDK4/6 inhibitor resistance.

The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle-dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells.

Tumor-initiating cells, also designated as cancer stem cells, are proposed to constitute a subpopulation of malignant cells central to tumorigenesis, metastasis, and treatment resistance. We analyzed the activity of the proteasome, the primary organelle for targeted protein degradation, as a marker of tumor- and metastasis-initiating cells. Using human and mouse breast cancer cells expressing a validated fluorescent reporter, we found a small subpopulation of cells with low proteasome activity that divided asymmetrically to produce daughter cells with low or high proteasome activity. Breast cancer cells with low proteasome activity had greater local tumor formation and metastasis in immunocompromised and immunocompetent mice. To allow flexible labeling of cells, we also developed a new proteasome substrate based on HaloTag technology. Patient-derived glioblastoma cells with low proteasome activity measured by the HaloTag reporter show key phenotypes associated with tumor-initiating cells, including expression of a stem cell transcription factor, reconstitution of the original starting population, and enhanced neurosphere formation. We also show that patient-derived glioblastoma cells with low proteasome activity have higher frequency of tumor formation in mouse xenografts. These studies support proteasome function as a tool to investigate tumor- and metastasis-initiating cancer cells and a potential biomarker for outcomes in patients with several different cancers.

Evaluation of therapeutic effects of natural killer (NK) cell-based immunotherapy in mice using in vivo apoptosis bioimaging with a caspase-3 sensor.

Natural killer (NK) cell-based immunotherapy is a promising strategy for cancer treatment, and caspase-3 is an important effector molecule in NK cell-mediated apoptosis in cancers. Here, we evaluated the antitumor effects of NK cell-based immunotherapy by serial noninvasive imaging of apoptosis using a caspase-3 sensor in mice with human glioma xenografts. Human glioma cells expressing both a caspase-3 sensor as a surrogate marker for caspase-3 activation and Renilla luciferase (Rluc) as a surrogate marker for cell viability were established and referred to as D54-CR cells. Human NK92 cells were used as effector cells. Treatment with NK92 cells resulted in a time- and effector number-dependent increase in bioluminescence imaging (BLI) activity of the caspase-3 sensor in D54-CR cells in vitro. Caspase-3 activation by NK92 treatment was blocked by Z-VAD treatment in D54-CR cells. Transfusion of NK92 cells induced an increase of the BLI signal by caspase-3 activation in a dose- and time-dependent manner in D54-CR tumor-bearing mice but not in PBS-treated mice. Accordingly, sequential BLI with the Rluc reporter gene revealed marked retardation of tumor growth in the NK92-treatment group but not in the PBS-treatment group. These data suggest that noninvasive imaging of apoptosis with a caspase-3 sensor can be used as an effective tool for evaluation of therapeutic efficacy as well as for optimization of NK cell-based immunotherapy.-Lee, H. W., Singh, T. D., Lee, S.-W., Ha, J.-H., Rehemtulla, A., Ahn, B.-C., Jeon, Y.-H., Lee, J. Evaluation of therapeutic effects of natural killer (NK) cell-based immunotherapy in mice using in vivo apoptosis bioimaging with a caspase-3 sensor.

Dual inhibition of allosteric mitogen-activated protein kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) oncogenic targets with a bifunctional inhibitor.

The MAP kinase (Ras/MEK/ERK) and PI3K/Akt/mTOR oncogenic signaling pathways are central regulators of KRAS-mediated transformation. Molecular reciprocity between the Ras/MEK/ERK and PI3K/Akt/mTOR pathways provides cancer cells with the ability to evade treatment when targeting only one pathway with monotherapy. Multi-kinase targeting was explored through the development of a single bivalent chemical entity by covalent linking of high-affinity MEK and PI3K inhibitors. A prototype dual-acting agent (compound 8) designed using the PI3K inhibitor ZSTK474 and the Raf/MEK inhibitor RO5126766 as scaffolds displayed high in vitro inhibition of both PI3K (IC50=172nM) and MEK1 (IC50=473nM). Additionally, compound 8 demonstrated significant modulation of MEK and PI3K signaling pathway activity in human A549 human lung adenocarcinoma cells and pancreatic cancer cells (PANC-1) and also decreased cellular viability in these two cell lines.

Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization.

An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.

Noninvasive imaging of apoptosis induced by adenovirus-mediated cancer gene therapy using a caspase-3 biosensor in living subjects.

We attempted to visualize the serial induction of caspase-3-dependent apoptosis mediated by Fas ligand/tumor necrosis factor-related apoptosis-inducing ligand (FasL/TRAIL) adenoviral gene therapy in mice bearing human glioma xenografts using a caspase-3 biosensor and monitored its therapeutic effects. Human D54 glioma cells expressing both the caspase-3 sensor and the Renilla luciferase (Rluc) gene were established (referred to as D54-CR cells). The bioluminescence imaging (BLI) signals of the caspase-3 sensor in the D54-CR cells were increased in a time- and virus dose-dependent manner by Ad-TRAIL or Ad-FasL transduction. Fluorescence-activated cell sorting (FACS) analysis revealed an increase in both cleaved caspase-3 or poly(ADP-ribose) polymerase (PARP) and annexin V- and propidium iodide-positive cells depending on the dosage of administered virus. Ad-FasL treatment resulted in a significant increase in the BLI activity of the caspase-3 sensor in the D54-CR tumors, which were ≈ 8.2, ≈ 12.9, and ≈ 46.6 times higher than those of control at 12 hours, 24 hours, and 96 hours posttreatment, respectively. In contrast, a significant reduction in Rluc activity, as a surrogate marker of cell viability, was detected in the tumors treated with Ad-FasL but not in those treated with Ad-null. Overall, the activation of caspase-3-dependent apoptosis induced by Ad-FasL/Ad-TRAIL gene therapy was successfully monitored by a sensitive imaging platform for caspase-3 activation.

Parametric response mapping as an indicator of bronchiolitis obliterans syndrome after hematopoietic stem cell transplantation.

The management of bronchiolitis obliterans syndrome (BOS) after hematopoietic cell transplantation presents many challenges, both diagnostically and therapeutically. We developed a computed tomography (CT) voxel-wise methodology termed parametric response mapping (PRM) that quantifies normal parenchyma, functional small airway disease (PRM(fSAD)), emphysema, and parenchymal disease as relative lung volumes. We now investigate the use of PRM as an imaging biomarker in the diagnosis of BOS. PRM was applied to CT data from 4 patient cohorts: acute infection (n = 11), BOS at onset (n = 34), BOS plus infection (n = 9), and age-matched, nontransplant control subjects (n = 23). Pulmonary function tests and bronchoalveolar lavage were used for group classification. Mean values for PRM(fSAD) were significantly greater in patients with BOS (38% ± 2%) when compared with those with infection alone (17% ± 4%, P < .0001) and age-matched control subjects (8.4% ± 1%, P < .0001). Patients with BOS had similar PRM(fSAD) profiles, whether a concurrent infection was present or not. An optimal cut-point for PRM(fSAD) of 28% of the total lung volume was identified, with values >28% highly indicative of BOS occurrence. PRM may provide a major advance in our ability to identify the small airway obstruction that characterizes BOS, even in the presence of concurrent infection.

Molecular imaging of Akt kinase activity.

The serine/threonine kinase Akt mediates mitogenic and anti-apoptotic responses that result from activation of multiple signaling cascades. It is considered a key determinant of tumor aggressiveness and is a major target for anticancer drug development. Here, we describe a new reporter molecule whose bioluminescence activity within live cells and in mice can be used to measure Akt activity. Akt activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to activation or inhibition of receptor tyrosine kinase, inhibition of phosphoinositide 3-kinase, or direct inhibition of Akt. The results provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate Akt activity, revealing the usefulness of this reporter for rapid dose and schedule optimization in the drug development process.

A molecularly engineered lectin destroys EGFR and inhibits the growth of non-small cell lung cancer.

Survival rates for non-small cell lung cancer (NSCLC) remain low despite the advent of novel therapeutics. Tyrosine kinase inhibitors (TKIs) targeting mutant epidermal growth factor receptor (EGFR) in NSCLC have significantly improved mortality but are plagued with challenges--they can only be used in the small fraction of patients who have susceptible driver mutations, and resistance inevitably develops. Aberrant glycosylation on the surface of cancer cells is an attractive therapeutic target as these abnormal glycosylation patterns are typically specific to cancer cells and are not present on healthy cells. H84T BanLec (H84T), a lectin previously engineered by our group to separate its antiviral activity from its mitogenicity, exhibits precision binding of high mannose, an abnormal glycan present on the surface of many cancer cells, including NSCLC. Here, we show that H84T binds to and inhibits the growth of diverse NSCLC cell lines by inducing lysosomal degradation of EGFR and leading to cancer cell death through autophagy. This is a mechanism distinct from EGFR TKIs and is independent of EGFR mutation status; H84T inhibited proliferation of both cell lines expressing wild type EGFR and those expressing mutant EGFR that is resistant to all TKIs. Further, H84T binds strongly to multiple and diverse clinical samples of both pulmonary adenocarcinoma and squamous cell carcinoma. H84T is thus a promising potential therapeutic in NSCLC, with the ability to circumvent the challenges currently faced by EGFR TKIs.

NanoLuc reporter for dual luciferase imaging in living animals.

Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the 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, adenosine triphosphate­independent 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 the 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 transforming growth factor ß 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 a 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.

Multi-system repeatability and reproducibility of apparent diffusion coefficient measurement using an ice-water phantom.

PURPOSE: To determine quantitative quality control procedures 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 five b-values were collected on 35 clinical MRI systems from three vendors at two field strengths (1.5 and 3 Tesla [T]) and analyzed at a central processing site. RESULTS: 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%, although variability increased for off-center measurements. Significant (>10%) vendor-specific and system-specific spatial nonuniformity ADC bias was detected for the off-center measurement that was consistent with gradient nonlinearity. CONCLUSION: Standardization of DWI protocol has improved reproducibility of ADC measurements and allowed identifying spatial ADC nonuniformity as a source of error in multi-site clinical studies.

Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization.

EGFR signaling initiates upon ligand binding which leads to activation and internalization of the receptor-ligand complex. Here, we evaluated if BUB1 impacted EGFR signaling by regulating EGFR receptor internalization and activation. BUB1 was ablated genomically (siRNA) or biochemically (2OH-BNPP1) in cells. EGF ligand was used to initiate EGFR signaling while disuccinimidyl suberate (DSS) was used for cross linking cellular proteins. EGFR signaling was measured by western immunoblotting and receptor internalization was evaluated by fluorescent microscopy (pEGFR (pY1068) colocalization with early endosome marker EEA1). siRNA mediated BUB1 depletion led to an overall increase in total EGFR levels and more phospho-EGFR (Y845, Y1092, and Y1173) dimers while the amount of total EGFR (non-phospho) dimers remained unchanged. BUB1 inhibitor (BUB1i) decreased EGF mediated EGFR signaling including pEGFR Y845, pAKT S473 and pERK1/2 in a time dependent manner. Additionally, BUB1i also reduced EGF mediated pEGFR (Y845) dimers (asymmetric dimers) without affecting total EGFR dimers (symmetric dimers) indicating that dimerization of inactive EGFR is not affected by BUB1. Furthermore, BUB1i blocked EGF mediated EGFR degradation (increase in EGFR half-life) without impacting half-lives of HER2 or c-MET. BUB1i also reduced co-localization of pEGFR with EEA1 positive endosomes suggesting that BUB1 might modulate EGFR endocytosis. Our data provide evidence that BUB1 protein and its kinase activity may regulate EGFR activation, endocytosis, degradation, and downstream signaling without affecting other members of the receptor tyrosine kinase family.

A lipid-associated macrophage lineage rewires the spatial landscape of adipose tissue in early obesity.

Adipose tissue macrophage (ATM) infiltration is associated with adipose tissue dysfunction and insulin resistance in mice and humans. Recent single-cell data highlight increased ATM heterogeneity in obesity but do not provide a spatial context for ATM phenotype dynamics. We integrated single-cell RNA-Seq, spatial transcriptomics, and imaging of murine adipose tissue in a time course study of diet-induced obesity. Overall, proinflammatory immune cells were predominant in early obesity, whereas nonresident antiinflammatory ATMs predominated in chronic obesity. A subset of these antiinflammatory ATMs were transcriptomically intermediate between monocytes and mature lipid-associated macrophages (LAMs) and were consistent with a LAM precursor (pre-LAM). Pre-LAMs were spatially associated with early obesity crown-like structures (CLSs), which indicate adipose tissue dysfunction. Spatial data showed colocalization of ligand-receptor transcripts related to lipid signaling among monocytes, pre-LAMs, and LAMs, including Apoe, Lrp1, Lpl, and App. Pre-LAM expression of these ligands in early obesity suggested signaling to LAMs in the CLS microenvironment. Our results refine understanding of ATM diversity and provide insight into the dynamics of the LAM lineage during development of metabolic disease.

Lung cancer lesion detection in histopathology images using graph-based sparse PCA network.

Early detection of lung cancer is critical for improvement of patient survival. To address the clinical need for efficacious treatments, genetically engineered mouse models (GEMM) have become integral in identifying and evaluating the molecular underpinnings of this complex disease that may be exploited as therapeutic targets. Assessment of GEMM tumor burden on histopathological sections performed by manual inspection is both time consuming and prone to subjective bias. Therefore, an interplay of needs and challenges exists for computer-aided diagnostic tools, for accurate and efficient analysis of these histopathology images. In this paper, we propose a simple machine learning approach called the graph-based sparse principal component analysis (GS-PCA) network, for automated detection of cancerous lesions on histological lung slides stained by hematoxylin and eosin (H&E). Our method comprises four steps: 1) cascaded graph-based sparse PCA, 2) PCA binary hashing, 3) block-wise histograms, and 4) support vector machine (SVM) classification. In our proposed architecture, graph-based sparse PCA is employed to learn the filter banks of the multiple stages of a convolutional network. This is followed by PCA hashing and block histograms for indexing and pooling. The meaningful features extracted from this GS-PCA are then fed to an SVM classifier. We evaluate the performance of the proposed algorithm on H&E slides obtained from an inducible K-rasG12D lung cancer mouse model using precision/recall rates, Fß-score, Tanimoto coefficient, and area under the curve (AUC) of the receiver operator characteristic (ROC) and show that our algorithm is efficient and provides improved detection accuracy compared to existing algorithms.

Subclonal evolution and expansion of spatially distinct THY1-positive cells is associated with recurrence in glioblastoma.

PURPOSE: Glioblastoma(GBM) is a lethal disease characterized by inevitable recurrence. Here we investigate the molecular pathways mediating resistance, with the goal of identifying novel therapeutic opportunities. EXPERIMENTAL DESIGN: We developed a longitudinal in vivo recurrence model utilizing patient-derived explants to produce paired specimens(pre- and post-recurrence) following temozolomide(TMZ) and radiation(IR). These specimens were evaluated for treatment response and to identify gene expression pathways driving treatment resistance. Findings were clinically validated using spatial transcriptomics of human GBMs. RESULTS: These studies reveal in replicate cohorts, a gene expression profile characterized by upregulation of mesenchymal and stem-like genes at recurrence. Analyses of clinical databases revealed significant association of this transcriptional profile with worse overall survival and upregulation at recurrence. Notably, gene expression analyses identified upregulation of TGFß signaling, and more than one-hundred-fold increase in THY1 levels at recurrence. Furthermore, THY1-positive cells represented <10% of cells in treatment-naïve tumors, compared to 75-96% in recurrent tumors. We then isolated THY1-positive cells from treatment-naïve patient samples and determined that they were inherently resistant to chemoradiation in orthotopic models. Additionally, using image-guided biopsies from treatment-naïve human GBM, we conducted spatial transcriptomic analyses. This revealed rare THY1+ regions characterized by mesenchymal/stem-like gene expression, analogous to our recurrent mouse model, which co-localized with macrophages within the perivascular niche. We then inhibited TGFBRI activity in vivo which decreased mesenchymal/stem-like protein levels, including THY1, and restored sensitivity to TMZ/IR in recurrent tumors. CONCLUSIONS: These findings reveal that GBM recurrence may result from tumor repopulation by pre-existing, therapy-resistant, THY1-positive, mesenchymal cells within the perivascular niche.

DCE and DW-MRI monitoring of vascular disruption following VEGF-Trap treatment of a rat glioma model.

Vascular-targeted therapies have shown promise as adjuvant cancer treatment. As these agents undergo clinical evaluation, sensitive imaging biomarkers are needed 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 9 L 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 9 L 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 (K(trans), volume transfer constant; k(ep), efflux rate constant from extravascular/extracellular space to plasma; and v(p), 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-Trap in 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-Trap anti-vascular targeted therapy.

AGTR1 overexpression defines a subset of breast cancer and confers sensitivity to losartan, an AGTR1 antagonist.

Breast cancer patients have benefited from the use of targeted therapies directed at specific molecular alterations. To identify additional opportunities for targeted therapy, we searched for genes with marked overexpression in subsets of tumors across a panel of breast cancer profiling studies comprising 3,200 microarray experiments. In addition to prioritizing ERBB2, we found AGTR1, the angiotensin II receptor type I, to be markedly overexpressed in 10-20% of breast cancer cases across multiple independent patient cohorts. Validation experiments confirmed that AGTR1 is highly overexpressed, in several cases more than 100-fold. AGTR1 overexpression was restricted to estrogen receptor-positive tumors and was mutually exclusive with ERBB2 overexpression across all samples. Ectopic overexpression of AGTR1 in primary mammary epithelial cells, combined with angiotensin II stimulation, led to a highly invasive phenotype that was attenuated by the AGTR1 antagonist losartan. Similarly, losartan reduced tumor growth by 30% in AGTR1-positive breast cancer xenografts. Taken together, these observations indicate that marked AGTR1 overexpression defines a subpopulation of ER-positive, ERBB2-negative breast cancer that may benefit from targeted therapy with AGTR1 antagonists, such as losartan.

Why All the Fury over Furin?

Analysis of the SARS-CoV-2 sequence revealed a multibasic furin cleavage site at the S1/S2 boundary of the spike protein distinguishing this virus from SARS-CoV. Furin, the best-characterized member of the mammalian proprotein convertases, is an ubiquitously expressed single pass type 1 transmembrane protein. Cleavage of SARS-CoV-2 spike protein by furin promotes viral entry into lung cells. While furin knockout is embryonically lethal, its knockout in differentiated somatic cells is not, thus furin provides an exciting therapeutic target for viral pathogens including SARS-CoV-2 and bacterial infections. Several peptide-based and small-molecule inhibitors of furin have been recently reported, and select cocrystal structures have been solved, paving the way for further optimization and selection of clinical candidates. This perspective highlights furin structure, substrates, recent inhibitors, and crystal structures with emphasis on furin's role in SARS-CoV-2 infection, where the current data strongly suggest its inhibition as a promising therapeutic intervention for SARS-CoV-2.