Purpose
OTS514 is a highly specific inhibitor targeting lymphokine-activated killer T cell-originated protein kinase (TOPK). A fluorescently labeled TOPK inhibitor could be used for tumor delineation ...or intraoperative imaging, potentially improving patient care.
Methods
Fluorescently labeled OTS514 was obtained by conjugating the fluorescent small molecule NBD to the TOPK inhibitor. HCT116 colorectal cancer cells were used to generate tumors in NSG mice for in vivo studies. Images were generated in vitro using confocal microscopy and ex vivo using an IVIS Spectrum.
Results
OTS514 was successfully conjugated to a fluorescent sensor and validated in vitro, in vivo, and ex vivo. The labeling reaction led to TOPKi-NBD with 67% yield and 97% purity after purification. We were able to test binding properties of TOPKi-NBD to its target, TOPK, and compared them to the precursor inhibitor. EC
50
s showed similar target affinities for TOPKi-NBD and the unlabeled OTS514. TOPKi-NBD showed specific tumor uptake after systemic administration and was microscopically detectable inside cancer cells ex vivo. Blocking controls performed with an excess of the unlabeled OTS514 confirmed specificity of the compound. Overall, the results represent a first step toward the development of a class of TOPK-specific fluorescent inhibitors for in vivo imaging and tumor delineation.
Conclusions
TOPK has the potential to be a new molecular target for cancer-specific imaging in a large variety of tumors. This could lead to broad applications in vitro and in vivo.
Glioblastoma multiforme is a highly aggressive form of brain cancer whose location, tendency to infiltrate healthy surrounding tissue, and heterogeneity significantly limit survival, with scant ...progress having been made in recent decades.
I-MAPi (Iodine-123 Meitner-Auger PARP1 inhibitor) is a precise therapeutic tool composed of a PARP1 inhibitor radiolabeled with an Auger- and gamma-emitting iodine isotope. Here, the PARP inhibitor, which binds to the DNA repair enzyme PARP1, specifically targets cancer cells, sparing healthy tissue, and carries a radioactive payload within reach of the cancer cells' DNA.
The high relative biological efficacy of Auger electrons within their short range of action is leveraged to inflict DNA damage and cell death with high precision. The gamma ray emission of
I-MAPi allows for the imaging of tumor progression and therapy response, and for patient dosimetry calculation. Here we demonstrated the efficacy and specificity of this small-molecule radiotheranostic in a complex preclinical model.
and
studies demonstrate high tumor uptake and a prolonged survival in mice treated with
I-MAPi when compared with vehicle controls. Different methods of drug delivery were investigated to develop this technology for clinical applications, including convection enhanced delivery and intrathecal injection.
Taken together, these results represent the first full characterization of an Auger-emitting PARP inhibitor which demonstrate a survival benefit in mouse models of GBM and confirm the high potential of
I-MAPi for clinical translation.
Tumour hypoxia renders cancer cells resistant to cancer therapy, resulting in markedly worse clinical outcomes. To find clinical candidate compounds that reduce hypoxia in tumours, we conduct a ...high-throughput screen for oxygen consumption rate (OCR) reduction and identify a number of drugs with this property. For this study we focus on the anti-malarial, atovaquone. Atovaquone rapidly decreases the OCR by more than 80% in a wide range of cancer cell lines at pharmacological concentrations. In addition, atovaquone eradicates hypoxia in FaDu, HCT116 and H1299 spheroids. Similarly, it reduces hypoxia in FaDu and HCT116 xenografts in nude mice, and causes a significant tumour growth delay when combined with radiation. Atovaquone is a ubiquinone analogue, and decreases the OCR by inhibiting mitochondrial complex III. We are now undertaking clinical studies to assess whether atovaquone reduces tumour hypoxia in patients, thereby increasing the efficacy of radiotherapy.
Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour ...cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.
DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end ...joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2-6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.
Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific ...anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner.
Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.
Background. Human papillomavirus- (HPV-) associated oropharyngeal squamous cell carcinomas (OPSCCs) are clinically and pathologically distinct from HPV-negative tumors. Here, we explore whether HPV ...affects functional biomarkers, including γH2AX, RAD51, and PARP1. Moreover, the role of 18FPARPi as a broadly applicable imaging tool for head and neck carcinomas is investigated. Methods. HPV-positive and HPV-negative cell lines were used to evaluate the γH2AX, RAD51, and PARP1 expression with immunoblotting and immunofluorescence. Effects of external beam ionizing radiation were investigated in vitro, and survival was investigated via colony-formation assay. 18FPARPi uptake experiments were performed on HPV-negative and HPV-positive cell lines to quantify PARP1 expression. PARP1 IHC and γH2AX foci were quantified using patient-derived oropharyngeal tumor specimens. Results. Differences in DNA repair were detected, showing higher RAD51 and γH2AX expression in HPV-positive cell lines. Clonogenic assays confirm HPV-positive cell lines to be significantly more radiosensitive. PARP1 expression levels were similar, irrespective of HPV status. Consequently, 18FPARPi uptake assays demonstrated that this tracer is internalized in cell lines independently from their HPV status. Conclusion. The HPV status, often used clinically to stratify patients, did not affect PARP1 levels, suggesting that PARP imaging can be performed in both HPV-positive and HPV-negative patients. This study confirms that the PET imaging agent 18FPARPi could serve as a general clinical tool for oropharyngeal cancer patients.
Background
Accidental peripheral nerve injury during surgical intervention results in a broad spectrum of potentially debilitating side effects. Tissue distortion and poor visibility can ...significantly increase the risk of nerve injury with long-lasting consequences for the patient. We developed and characterized Hs1a-FL, a fluorescent near-infrared molecule for nerve visualization in the operating theater with the aim of helping physicians to visualize nerves during surgery. Hs1a was derived from the venom of the Chinese bird spider,
Haplopelma schmidti
, and conjugated to Cy7.5 dye. Hs1a-FL was injected intravenously in mice, and harvested nerves were imaged microscopically and with epifluorescence.
Results
Hs1a-FL showed specific and stable binding to the sodium channel Na
V
1.7, present on the surface of human and mouse nerves. Hs1a-FL allowed epifluorescence visualization of sciatic mouse nerves with favorable nerve-to-muscle contrast.
Conclusions
Fluorescent Na
V
1.7-targeted tracers have the potential to be adopted clinically for the intraoperative visualization of peripheral nerves during surgery, providing guidance for the surgeon and potentially improving the standard of care.
For oral, oropharyngeal and oesophageal cancer, the early detection of tumours and of residual tumour after surgery are prognostic factors of recurrence rates and patient survival. Here, we report ...the validation, in animal models and a human, of the use of a previously described fluorescently labelled small-molecule inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP1) for the detection of cancers of the oral cavity, pharynx and oesophagus. We show that the fluorescent contrast agent can be used to quantify the expression levels of PARP1 and to detect oral, oropharyngeal and oesophageal tumours in mice, pigs and fresh human biospecimens when delivered topically or intravenously. The fluorescent PARP1 inhibitor can also detect oral carcinoma in a patient when applied as a mouthwash, and discriminate between fresh biopsied samples of the oral tumour and the surgical resection margin with more than 95% sensitivity and specificity. The PARP1 inhibitor could serve as the basis of a rapid and sensitive assay for the early detection and for the surgical-margin assessment of epithelial cancers of the upper intestinal tract.
Platinum chemotherapies are highly effective cytotoxic agents but often induce resistance when used as monotherapies. Combinatorial strategies limit this risk and provide effective treatment options ...for many cancers. Here, we repurpose atovaquone (ATQ), a well-tolerated & FDA-approved anti-malarial agent by demonstrating that it potentiates cancer cell death of a subset of platinums. We show that ATQ in combination with carboplatin or cisplatin induces striking and repeatable concentration- and time-dependent cell death sensitization in vitro across a variety of cancer cell lines. ATQ induces mitochondrial reactive oxygen species (mROS), depleting intracellular glutathione (GSH) pools in a concentration-dependent manner. The superoxide dismutase mimetic MnTBAP rescues ATQ-induced mROS production and pre-loading cells with the GSH prodrug N-acetyl cysteine (NAC) abrogates the sensitization. Together, these findings implicate ATQ-induced oxidative stress as key mediator of the sensitizing effect. At physiologically achievable concentrations, ATQ and carboplatin furthermore synergistically delay the growth of three-dimensional avascular spheroids. Clinically, ATQ is a safe and specific inhibitor of the electron transport chain (ETC) and is concurrently being repurposed as a candidate tumor hypoxia modifier. Together, these findings suggest that ATQ is deserving of further study as a candidate platinum sensitizing agent.
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