Sergey Magnitsky

Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming

Background: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro. Methods: We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. Results: We found that a combination of the transforming growth factor-&bgr; inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-&bgr; and WNT inhibition and was achieved most efficiently with GMT plus myocardin. Conclusions: Transforming growth factor-&bgr; and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.

AN ESCHERICHIA COLI MUTANT QUINOL:FUMARATE REDUCTASE CONTAINS AN EPR-DETECTABLE SEMIQUINONE STABILIZED AT THE PROXIMAL QUINONE-BINDING SITE

The EPR and thermodynamic properties of semiquinone (SQ) species stabilized by mammalian succinate:quinone reductase (SQR) in situ in the mitochondrial membrane and in the isolated enzyme have been well documented. The equivalent semiquinones in bacterial membranes have not yet been characterized, either in SQR or quinol:fumarate reductase (QFR) in situ. In this work, we describe an EPR-detectable QFR semiquinone usingEscherichia coli mutant QFR (FrdC E29L) and the wild-type enzyme. The SQ exhibits a g = 2.005 signal with a peak-to-peak line width of ∼1.1 milliteslas at 150 K, has a midpoint potential (E m (pH 7.2)) of −56.6 mV, and has a stability constant of ∼1.2 × 10−2 at pH 7.2. It shows extremely fast spin relaxation behavior with aP 1/2 value of ≫500 milliwatts at 150 K, which closely resembles the previously described SQ species (SQs) in mitochondrial SQR. This SQ species seems to be present also in wild-type QFR, but its stability constant is much lower, and its signal intensity is near the EPR detection limit around neutral pH. In contrast to mammalian SQR, the membrane anchor ofE. coli QFR lacks heme; thus, this prosthetic group can be excluded as a spin relaxation enhancer. The trinuclear iron-sulfur cluster FR3 in the [3Fe-4S]1+ state is suggested as the dominant spin relaxation enhancer of the SQFR spins in this enzyme. E. coli QFR activity and the fast relaxing SQ species observed in the mutant enzyme are sensitive to the inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO). In wild-type E. coli QFR, HQNO causes EPR spectral line shape perturbations of the iron-sulfur cluster FR3. Similar spectral line shape changes of FR3 are caused by the FrdC E29L mutation, without addition of HQNO. This indicates that the SQ and the inhibitor-binding sites are located in close proximity to the trinuclear iron-sulfur cluster FR3. The data further suggest that this site corresponds to the proximal quinone-binding site in E. coli QFR.

Propionibacterium acnes infected intervertebral discs cause vertebral bone marrow lesions consistent with Modic changes.

Modic type I change (MC1) are vertebral bone marrow lesions adjacent to degenerated discs that are specific for discogenic low back pain. The etiopathogenesis is unknown, but occult discitis, in particular with Propionibacteria acnes (P. acnes), has been suggested as a possible etiology. If true, antibiotic therapy should be considered for patients with MC1. However, this hypothesis is controversial. While some studies report up to 40% infection rate in herniated discs, others fail to detect infected discs and attribute reports of positive cultures to contamination during sampling procedure. Irrespective of the clinical controversy, whether it is biologically plausible for P. acnes to cause MC1 has never been investigated. Therefore, the objective of this study was to test if P. acnes can proliferate within discs and cause reactive changes in the adjacent bone marrow. P. acnes was aseptically isolated from a symptomatic human L4/5 disc with MC1 and injected into rat tail discs. We demonstrate proliferation of P. acnes and up‐regulation of IL‐1 and IL‐6 within three days of inoculation. At day‐7, disc degeneration was apparent along with fibrotic endplate erosion. TNF‐α immunoreactivity was enhanced within the effected endplates along with cellular infiltrates. The bone marrow appeared normal. At day‐14, endplates and trabecular bone close to the disc were almost completely resorbed and fibrotic tissue extended into the bone marrow. T‐cells and TNF‐α immunoreactivity were identified at the disc/marrow junction. On MRI, bone marrow showed MC1‐like changes. In conclusion, P. acnes proliferate within the disc, induce degeneration, and cause MC1‐like changes in the adjacent bone marrow.

Kartogenin treatment prevented joint degeneration in a rodent model of osteoarthritis: A pilot study.

Osteoarthritis (OA) is a major degenerative joint disease characterized by progressive loss of articular cartilage, synovitis, subchondral bone changes, and osteophyte formation. Currently there is no treatment for OA except temporary pain relief and end‐stage joint replacement surgery. We performed a pilot study to determine the effect of kartogenin (KGN, a small molecule) on both cartilage and subchondral bone in a rat model of OA using multimodal imaging techniques. OA was induced in rats (OA and KGN treatment group) by anterior cruciate ligament transection (ACLT) surgery in the right knee joint. Sham surgery was performed on the right knee joint of control group rats. KGN group rats received weekly intra‐articular injection of 125 μM KGN 1 week after surgery until week 12. All rats underwent in vivo magnetic resonance imaging (MRI) at 3, 6, and 12 weeks after surgery. Quantitative MR relaxation measures (T1ρ and T2) were determined to evaluate changes in articular cartilage. Cartilage and bone turnover markers (COMP and CTX‐I) were determined at baseline, 3, 6, and 12 weeks. Animals were sacrificed at week 12 and the knee joints were removed for micro‐computed tomography (micro‐CT) and histology. KGN treatment significantly lowered the T1ρ and T2 relaxation times indicating decreased cartilage degradation. KGN treatment significantly decreased COMP and CTX‐I levels indicating decreased cartilage and bone turnover rate. KGN treatment also prevented subchondral bone changes in the ACLT rat model of OA. Thus, kartogenin is a potential drug to prevent joint deterioration in post‐traumatic OA.

Heterogeneous drug penetrance of veliparib and carboplatin measured in triple negative breast tumors

BackgroundPoly(ADP-ribose) polymerase inhibitors (PARPi), coupled to a DNA damaging agent is a promising approach to treating triple negative breast cancer (TNBC). However, not all patients respond; we hypothesize that non-response in some patients may be due to insufficient drug penetration. As a first step to testing this hypothesis, we quantified and visualized veliparib and carboplatin penetration in mouse xenograft TNBCs and patient blood samples.MethodsMDA-MB-231, HCC70 or MDA-MB-436 human TNBC cells were implanted in 41 beige SCID mice. Low dose (20 mg/kg) or high dose (60 mg/kg) veliparib was given three times daily for three days, with carboplatin (60 mg/kg) administered twice. In addition, blood samples were analyzed from 19 patients from a phase 1 study of carboplatin + PARPi talazoparib. Veliparib and carboplatin was quantified using liquid chromatography–mass spectrometry (LC-MS). Veliparib tissue penetration was visualized using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) and platinum adducts (covalent nuclear DNA-binding) were quantified using inductively coupled plasma–mass spectrometry (ICP-MS). Pharmacokinetic modeling and Pearson’s correlation were used to explore associations between concentrations in plasma, tumor cells and peripheral blood mononuclear cells (PBMCs).ResultsVeliparib penetration in xenograft tumors was highly heterogeneous between and within tumors. Only 35% (CI 95% 26–44%), 74% (40–97%) and 46% (9–37%) of veliparib observed in plasma penetrated into MDA-MB-231, HCC70 and MDA-MB-436 cell-based xenografts, respectively. Within tumors, penetration heterogeneity was larger with the 60 mg/kg compared to the 20 mg/kg dose (RSD 155% versus 255%, P = 0.001). These tumor concentrations were predicted similar to clinical dosing levels, but predicted tumor concentrations were below half maximal concentration values as threshold of response. Xenograft veliparib concentrations correlated positively with platinum adduct formation (R2 = 0.657), but no PARPi–platinum interaction was observed in patients’ PBMCs. Platinum adduct formation was significantly higher in five gBRCA carriers (ratio of platinum in DNA in PBMCs/plasma 0.64% (IQR 0.60–1.16%) compared to nine non-carriers (ratio 0.29% (IQR 0.21–0.66%, P < 0.0001).ConclusionsPARPi/platinum tumor penetration can be measured by MALDI-MSI and ICP-MS in PBMCs and fresh frozen, OCT embedded core needle biopsies. Large variability in platinum adduct formation and spatial heterogeneity in veliparib distribution may lead to insufficient drug exposure in select cell populations.

Positive contrast from cells labeled with iron oxide nanoparticles: Quantitation of imaging data

Conventional T2‐weighted MRI produces a hypointense signal from iron‐labeled cells, which renders quantification unfeasible. We tested a SWeep Imaging with Fourier Transformation (SWIFT) MRI pulse sequence to generate a quantifiable hyperintense signal from iron‐labeled cells.

Quantification of Propionic Acid in the Bovine Spinal Disk after Infection of the Tissue with P. Acnes Bacteria

Study Design. Research. Objective. The goal of this study was to investigate whether Propionibacteria acnes infection of the intervertebral disc can be detected noninvasively by nuclear magnetic resonance (NMR) spectroscopy. Summary of Background Data. Microbiological studies of surgical samples suggest that a significant subpopulation of back pain patients may have occult disc infection with P. acnes bacteria. This hypothesis is further supported by a double-blind clinical trial showing that back pain patients with Modic type 1 changes may respond to antibiotic treatment. Because significant side effects are associated with antibiotic treatment, there is a need for a noninvasive method to detect whether specific discs in back pain patients are infected with P acnes bacteria. Methods. P. acnes bacteria were obtained from human patients. NMR detection of a propionic acid (PA) in the bacteria extracts was conducted on 500 MHz high-resolution spectrometer, whereas in vivo NMR spectroscopy of an isolated bovine disk tissue infected with P. acnes was conducted on 7 T magnetic resonance imaging scanner. Results. NMR spectra of P. acnes metabolites revealed a distinct NMR signal with identical chemical shits (1.05 and 2.18 ppm) as PA (a primary P. acne metabolite). The 1.05 ppm signal does not overlap with other bacteria metabolites, and its intensity increases linearly with P. acnes concentration. Bovine disks injected with P. acnes bacteria revealed a very distinct NMR signal at 1.05 ppm, which linearly increased with P. acnes concentration. Conclusion. The 1.05 ppm NMR signal from PA can be used as a marker of P. acnes infection of discs. This signal does not overlap with other disc metabolites and linearly depends on P. acnes concentration. Consequently, NMR spectroscopy may provide a noninvasive method to detect disc infection in the clinical setting. Level of Evidence: N/A

Antisecretory Factor-mediated Inhibition of Cell Volume Dynamics Produces Anti-tumor Activity in Glioblastoma

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AFs ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777–90. ©2018 AACR.

Imaging of a high concentration of iron labeled cells with positive contrast in a rat knee

The sweep imaging with Fourier transformation (SWIFT) imaging technique has been shown to provide positive contrast from diluted cell suspensions labeled with super‐paramagnetic iron oxide (SPIO) in a tissue, as an alternative to T2*‐weighted imaging. Here we demonstrate a variation of the SWIFT technique that yields a hyperintense signal from a concentrated cell suspension. The proposed technique provides minimal background signal from host tissue and facilitates visualization of injected cells.

Abstract C61: Non-homogeneous drug penetrance of veliparib measured in triple negative breast tumors

Background: Veliparib, an inhibitor of Poly(ADP-ribose) polymerase (PARPi), in combination with carboplatin showed efficacy in triple negative breast cancer (TNBC) patients in the I-SPY 2 TRIAL. However ∼42% of TNBC did not achieve pathologic complete response. Insufficient uptake of drug in TNBC may lead to inadequate response to PARPi. As a first step toward testing this hypothesis in patients, we quantified veliparib penetration in mouse xenograft models of TNBC. Methods: MDA-MB-231, HCC70 or MDA-MB-436 human TNBC cells were implanted in 41 beige SCID mice. Veliparib at low dose (20mg/kg) or high dose (60mg/kg) and carboplatin (60mg/kg) was given three times daily for three days. MR images were taken at day 1. Plasma, fresh frozen and OCT embedded tissues were analyzed using Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI) Liquid chromatography–mass spectrometry (LC-MS). Drug penetration was compared among doses and cell lines. Results:Ex vivo veliparib concentrations quantified by LC-MS differed significantly among the tumors derived from the three cell lines. Liver and plasma concentrations were uniformly high in all mice compared to tumor and muscle tissues. Plasma pharmacokinetics in mice exhibited non-linear clearance resulting in prolonged high plasma levels at higher doses, while tumor and plasma concentrations were linearly correlated. MALDI-MSI images of tumor and muscle in 12 mice showed higher veliparib concentrations in necrotic areas compared to areas with viable tumor cells (P = 0.126, Table) and higher concentrations at the rim then in the center of the tumor (P = 0.046). Lower concentrations were found in MDA-MB-231 than in other cell lines (0.008). Contrast agent and veliparib accumulated near the rim of the tumors and a fast elimination of the contrast agent from the tumor correlated with relatively low veliparib tumor concentrations. Conclusions: The spatial distribution of veliparib in TNBC depends on the dose and tumor cell biology. We demonstrated that MALDI-MSI can be used to measure veliparib penetration tumor samples, which may have potential to monitor response to PARPi therapies. Table: Veliparib concentrations by LC-MS and its spatial distribution by MALDI varies by tissue, drug dose and TNBC cell line of origin. Citation Format: Imke H. Bartelink, Brendan Prideaux, Gregor Krings, Lisa Wilmes, Pei R.E. Lee, Byron Hann, Jean-Philippe Coppe, Diane Heditsian, Lamorna Swigart-Brown, Ella F. Jones, Sergey Magnitsky, Ron Keizer, Laura Esserman, Weiming Ruan, Alan Wu, Douglas Yee, Veronique Dartois, Denise Wolf, Rada Savic, Laura vantVeer. Non-homogeneous drug penetrance of veliparib measured in triple negative breast tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C61.