Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs.

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-an early step in NHEJ-yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.

CAR T cell-based immunotherapy and radiation therapy: potential, promises and risks.

CAR T cell-based therapies have revolutionized the treatment of hematological malignancies such as leukemia and lymphoma within the last years. In contrast to the success in hematological cancers, the treatment of solid tumors with CAR T cells is still a major challenge in the field and attempts to overcome these hurdles have not been successful yet. Radiation therapy is used for management of various malignancies for decades and its therapeutic role ranges from local therapy to a priming agent in cancer immunotherapy. Combinations of radiation with immune checkpoint inhibitors have already proven successful in clinical trials. Therefore, a combination of radiation therapy may have the potential to overcome the current limitations of CAR T cell therapy in solid tumor entities. So far, only limited research was conducted in the area of CAR T cells and radiation. In this review we will discuss the potential and risks of such a combination in the treatment of cancer patients.

An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1.

BACKGROUND: The inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure. RESULTS: The Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT. CONCLUSION: Modifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles.

An oncogene addiction phosphorylation signature and its derived scores inform tumor responsiveness to targeted therapies.

PURPOSE: Oncogene addiction provides important therapeutic opportunities for precision oncology treatment strategies. To date the cellular circuitries associated with driving oncoproteins, which eventually establish the phenotypic manifestation of oncogene addiction, remain largely unexplored. Data suggest the DNA damage response (DDR) as a central signaling network that intersects with pathways associated with deregulated addicting oncoproteins with kinase activity in cancer cells. EXPERIMENTAL: DESIGN: We employed a targeted mass spectrometry approach to systematically explore alterations in 116 phosphosites related to oncogene signaling and its intersection with the DDR following inhibition of the addicting oncogene alone or in combination with irradiation in MET-, EGFR-, ALK- or BRAF (V600)-positive cancer models. An NSCLC tissue pipeline combining patient-derived xenografts (PDXs) and ex vivo patient organotypic cultures has been established for treatment responsiveness assessment. RESULTS: We identified an 'oncogene addiction phosphorylation signature' (OAPS) consisting of 8 protein phosphorylations (ACLY S455, IF4B S422, IF4G1 S1231, LIMA1 S490, MYCN S62, NCBP1 S22, P3C2A S259 and TERF2 S365) that are significantly suppressed upon targeted oncogene inhibition solely in addicted cell line models and patient tissues. We show that the OAPS is present in patient tissues and the OAPS-derived score strongly correlates with the ex vivo responses to targeted treatments. CONCLUSIONS: We propose a score derived from OAPS as a quantitative measure to evaluate oncogene addiction of cancer cell samples. This work underlines the importance of protein phosphorylation assessment for patient stratification in precision oncology and corresponding identification of tumor subtypes sensitive to inhibition of a particular oncogene.

ProtRank: bypassing the imputation of missing values in differential expression analysis of proteomic data.

BACKGROUND: Data from discovery proteomic and phosphoproteomic experiments typically include missing values that correspond to proteins that have not been identified in the analyzed sample. Replacing the missing values with random numbers, a process known as "imputation", avoids apparent infinite fold-change values. However, the procedure comes at a cost: Imputing a large number of missing values has the potential to significantly impact the results of the subsequent differential expression analysis. RESULTS: We propose a method that identifies differentially expressed proteins by ranking their observed changes with respect to the changes observed for other proteins. Missing values are taken into account by this method directly, without the need to impute them. We illustrate the performance of the new method on two distinct datasets and show that it is robust to missing values and, at the same time, provides results that are otherwise similar to those obtained with edgeR which is a state-of-art differential expression analysis method. CONCLUSIONS: The new method for the differential expression analysis of proteomic data is available as an easy to use Python package.

The relevance of tyrosine kinase inhibitors for global metabolic pathways in cancer.

Tumor metabolism is a thrilling discipline that focuses on mechanisms used by cancer cells to earn crucial building blocks and energy to preserve growth and overcome resistance to various treatment modalities. At the same time, therapies directed specifically against aberrant signalling pathways driven by protein tyrosine kinases (TKs) involved in proliferation, metastasis and growth count for several years to promising anti-cancer approaches. In this respect, small molecule inhibitors are the most widely used clinically relevant means for targeted therapy, with a rising number of approvals for TKs inhibitors. In this review, we discuss recent observations related to TKs-associated metabolism and to metabolic feedback that is initialized as cellular response to particular TK-targeted therapies. These observations provide collective evidence that therapeutic responses are primarily linked to such pathways as regulation of lipid and amino acid metabolism, TCA cycle and glycolysis, advocating therefore the development of further effective targeted therapies against a broader spectrum of TKs to treat patients whose tumors display deregulated signalling driven by these proteins.

PIK3CA hotspot mutations differentially impact responses to MET targeting in MET-driven and non-driven preclinical cancer models.

BACKGROUND: The MET receptor tyrosine kinase represents a promising target in cancer. PIK3CA activating mutations are common in several tumor types and can potentially confer resistance to anti-receptor tyrosine kinase therapy. METHODS: MET and/or PI3K pathway inhibition was assessed in NIH3T3 cells harboring MET-activating point mutation with or without ectopic expression of PIK3CAE545K and PIK3CAH1047R, as well as in MET-expressing head and neck cancer cells with endogenous PIK3CA mutations. Endpoints included PI3K pathway activation, cell proliferation, colony-forming ability, cell death, wound-healing, and an in vivo model. RESULTS: PIK3CAE545K and PIK3CAH1047R confer resistance to MET inhibition in MET-driven models. PIK3CAH1047R was more potent than PIK3CAE545K at inducing resistance in PI3K pathway activation, cell proliferation, colony-forming ability, induction of cell death and wound-healing upon MET inhibition. Resistance to MET inhibition could be synergistically overcome by co-targeting PI3K. Furthermore, combined MET/PI3K inhibition led to enhanced anti-tumor activity in vivo in tumors harboring PIK3CAH1047R. In head and neck cancer cells the combination of MET/PI3K inhibitors led to more-than-additive effects. CONCLUSIONS: PIK3CA mutations can lead to resistance to MET inhibition, supporting future clinical evaluation of combinations of PI3K and MET inhibitors in common scenarios of malignant neoplasms featuring aberrant MET expression and PIK3CA mutations.

E2F1-Associated Purine Synthesis Pathway Is a Major Component of the MET-DNA Damage Response Network.

Various lines of investigation support a signaling interphase shared by receptor tyrosine kinases and the DNA damage response. However, the underlying network nodes and their contribution to the maintenance of DNA integrity remain unknown. We explored MET-related metabolic pathways in which interruption compromises proper resolution of DNA damage. Discovery metabolomics combined with transcriptomics identified changes in pathways relevant to DNA repair following MET inhibition (METi). METi by tepotinib was associated with the formation of γH2AX foci and with significant alterations in major metabolic circuits such as glycolysis, gluconeogenesis, and purine, pyrimidine, amino acid, and lipid metabolism. 5'-Phosphoribosyl-N-formylglycinamide, a de novo purine synthesis pathway metabolite, was consistently decreased in in vitro and in vivo MET-dependent models, and METi-related depletion of dNTPs was observed. METi instigated the downregulation of critical purine synthesis enzymes including phosphoribosylglycinamide formyltransferase, which catalyzes 5'-phosphoribosyl-N-formylglycinamide synthesis. Genes encoding these enzymes are regulated through E2F1, whose levels decrease upon METi in MET-driven cells and xenografts. Transient E2F1 overexpression prevented dNTP depletion and the concomitant METi-associated DNA damage in MET-driven cells. We conclude that DNA damage following METi results from dNTP reduction via downregulation of E2F1 and a consequent decline of de novo purine synthesis. SIGNIFICANCE: Maintenance of genome stability prevents disease and affiliates with growth factor receptor tyrosine kinases. We identified de novo purine synthesis as a pathway in which key enzymatic players are regulated through MET receptor and whose depletion via MET targeting explains MET inhibition-associated formation of DNA double-strand breaks. The mechanistic importance of MET inhibition-dependent E2F1 downregulation for interference with DNA integrity has translational implications for MET-targeting-based treatment of malignancies.

Protective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells.

MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer cells.

Beyond keratinocyte differentiation: emerging new biology of small proline-rich proteins.

Small proline-rich proteins (SPRRPs) are traditionally known for their function in keratinocyte homeostasis. Recent evidence demonstrates their involvement in additional diverse physiological processes ranging from p53 signaling and direct prevention of DNA damage to bactericidal activities. We highlight these novel, intriguing roles of SPRRPs and discuss them in the context of relevant pathological conditions.

Parameters of the Lyman Model for Calculation of Normal-Tissue Complication Probability: A Systematic Literature Review.

PURPOSE: The Lyman model is one of the most used radiobiological models for calculation of normal-tissue complication probability (NTCP). Since its introduction in 1985, many authors have published parameter values for the model based on clinical data of different radiotherapeutic situations. This study attempted to collect the entirety of radiobiological parameter sets published to date and provide an overview of the data basis for different variations of the model. Furthermore, it sought to compare the parameter values and calculated NTCPs for selected endpoints with sufficient data available. METHODS AND MATERIALS: A systematic literature analysis was performed, searching for publications that provided parameters for the different variations of the Lyman model in the Medline database using PubMed. Parameter sets were grouped into 13 toxicity-related endpoint groups. For 3 selected endpoint groups (≤25% reduction of saliva 12 months after irradiation of the parotid, symptomatic pneumonitis after irradiation of the lung, and bleeding of grade 2 or less after irradiation of the rectum), parameter values were compared and differences in calculated NTCP values were analyzed. RESULTS: A total of 509 parameter sets from 130 publications were identified. Considerable heterogeneities were detected regarding the number of parameters available for different radio-oncological situations. Furthermore, for the 3 selected endpoints, large differences in published parameter values were found. These translated into great variations of calculated NTCPs, with maximum ranges of 35.2% to 93.4% for the saliva endpoint, of 39.4% to 90.4% for the pneumonitis endpoint, and of 5.4% to 99.3% for the rectal bleeding endpoint. CONCLUSIONS: The detected heterogeneity of the data as well as the large variations of published radiobiological parameters underline the necessity for careful interpretation when using such parameters for NTCP calculations. Appropriate selection of parameters and validation of values are essential when using the Lyman model.

A DNA-PK phosphorylation site on MET regulates its signaling interface with the DNA damage response.

The DNA damage response (DDR) is intertwined with signaling pathways downstream of oncogenic receptor tyrosine kinases (RTKs). To drive research into the application of targeted therapies as radiosensitizers, a better understanding of this molecular crosstalk is necessary. We present here the characterization of a previously unreported MET RTK phosphosite, Serine 1016 (S1016) that represents a potential DDR-MET interface. MET S1016 phosphorylation increases in response to irradiation and is mainly targeted by DNA-dependent protein kinase (DNA-PK). Phosphoproteomics unveils an impact of the S1016A substitution on the overall long-term cell cycle regulation following DNA damage. Accordingly, the abrogation of this phosphosite strongly perturbs the phosphorylation of proteins involved in the cell cycle and formation of the mitotic spindle, enabling cells to bypass a G2 arrest upon irradiation and leading to the entry into mitosis despite compromised genome integrity. This results in the formation of abnormal mitotic spindles and a lower proliferation rate. Altogether, the current data uncover a novel signaling mechanism through which the DDR uses a growth factor receptor system for regulating and maintaining genome stability.

Tumour mutations in long noncoding RNAs enhance cell fitness.

Long noncoding RNAs (lncRNAs) are linked to cancer via pathogenic changes in their expression levels. Yet, it remains unclear whether lncRNAs can also impact tumour cell fitness via function-altering somatic "driver" mutations. To search for such driver-lncRNAs, we here perform a genome-wide analysis of fitness-altering single nucleotide variants (SNVs) across a cohort of 2583 primary and 3527 metastatic tumours. The resulting 54 mutated and positively-selected lncRNAs are significantly enriched for previously-reported cancer genes and a range of clinical and genomic features. A number of these lncRNAs promote tumour cell proliferation when overexpressed in in vitro models. Our results also highlight a dense SNV hotspot in the widely-studied NEAT1 oncogene. To directly evaluate the functional significance of NEAT1 SNVs, we use in cellulo mutagenesis to introduce tumour-like mutations in the gene and observe a significant and reproducible increase in cell fitness, both in vitro and in a mouse model. Mechanistic studies reveal that SNVs remodel the NEAT1 ribonucleoprotein and boost subnuclear paraspeckles. In summary, this work demonstrates the utility of driver analysis for mapping cancer-promoting lncRNAs, and provides experimental evidence that somatic mutations can act through lncRNAs to enhance pathological cancer cell fitness.

MET inhibition in tumor cells by PHA665752 impairs homologous recombination repair of DNA double strand breaks.

Abnormal activation of cellular DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems has broad implications for both cancer biology and treatment. Recent studies suggest a potential link between DNA repair and aberrant activation of the hepatocyte growth factor receptor Mesenchymal-Epithelial Transition (MET), an oncogene that is overexpressed in numerous types of human tumors and considered a prime target in clinical oncology. Using the homologous recombination (HR) direct-repeat direct-repeat green fluorescent protein ((DR)-GFP) system, we show that MET inhibition in tumor cells with deregulated MET activity by the small molecule PHA665752 significantly impairs in a dose-dependent manner HR. Using cells that express MET-mutated variants that respond differentially to PHA665752, we confirm that the observed HR inhibition is indeed MET-dependent. Furthermore, our data also suggest that decline in HR-dependent DNA repair activity is not a secondary effect due to cell cycle alterations caused by PHA665752. Mechanistically, we show that MET inhibition affects the formation of the RAD51-BRCA2 complex, which is crucial for error-free HR repair of double strand DNA lesions, presumably via downregulation and impaired translocation of RAD51 into the nucleus. Taken together, these findings assist to further support the role of MET in the cellular DNA damage response and highlight the potential future benefit of MET inhibitors for the sensitization of tumor cells to DNA damaging agents.

Prognostic and Predictive Biomarkers in Head and Neck Squamous Cell Carcinoma Treated with Radiotherapy-A Systematic Review.

Background: Radiotherapy is a mainstay in head and neck squamous cell carcinoma (HNSCC) treatment but is mostly applied without stratification by molecular diagnostics. Development of reliable biomarkers may have the potential to improve radiotherapy (RT) efficacy and reduce toxicity. We conducted a systematic review to summarize the field of biomarkers in HNSCC treated by RT. Methods: Pubmed and EMBASE were searched independently by two researchers following pre-defined inclusion and exclusion criteria. Z curves were generated to investigate publication bias. OncoKB was used for identification of druggable targets. Results: 134 manuscripts remained for data extraction. 12% of tumors were AJCC/UICC stage I-II and 82% were stage III-IV. The most common biomarkers were proteins (39%), DNA (14%) and mRNA (9%). Limiting analysis to prospective data and statistically significant results, we found three potentially druggable targets: ERCC2, PTCH1 and EGFR. Regarding data quality, AJCC/UICC stage was missing in 32% of manuscripts. 73% of studies were retrospective and only 7% were based on prospective randomized trials. Z-curves indicated the presence of publication bias. Conclusion: An abundance of potential biomarkers in HNSCC is available but data quality is limited by retrospective collection, lack of validation and publication bias. Improved study design and reporting quality might accelerate successful development of personalized treatments in HNSCC.

Synergistic effect of the TLR5 agonist CBLB502 and its downstream effector IL-22 against liver injury.

The toll-like receptor 5 (TLR5) agonist, CBLB502/Entolimod, is a peptide derived from bacterial flagellin and has been shown to protect against radiation-induced tissue damage in animal models. Here we investigated the protective mechanism of CBLB502 in the liver using models of ischemia-reperfusion injury and concanavalin A (ConA) induced immuno-hepatitis. We report that pretreatment of mice with CBLB502 provoked a concomitant activation of NF-κB and STAT3 signaling in the liver and reduced hepatic damage in both models. To understand the underlying mechanism, we screened for cytokines in the serum of CBLB502 treated animals and detected high levels of IL-22. There was no transcriptional upregulation of IL-22 in the liver, rather it was found in extrahepatic tissues, mainly the colon, mesenteric lymph nodes (MLN), and spleen. RNA-seq analysis on isolated hepatocytes demonstrated that the concomitant activation of NF-κB signaling by CBLB502 and STAT3 signaling by IL-22 produced a synergistic cytoprotective transcriptional signature. In IL-22 knockout mice, the loss of IL-22 resulted in a decrease of hepatic STAT3 activation, a reduction in the cytoprotective signature, and a loss of hepatoprotection following ischemia-reperfusion-induced liver injury. Taken together, these findings suggest that CBLB502 protects the liver by increasing hepatocyte resistance to acute liver injury through the cooperation of TLR5-NF-κB and IL-22-STAT3 signaling pathways.

Hsp90 transcriptionally and post-translationally regulates the expression of NDRG1 and maintains the stability of its modifying kinase GSK3beta.

N-myc downstream-regulated gene 1 (NRDG1) is a stress-induced protein whose putative function is suppression of tumor metastasis. A recent proteonomic study showed NDRG1 interacts with the molecular chaperone heat shock protein 90 (Hsp90). From their reported association, we investigated if NDRG1 is dependent on Hsp90 for its stability and is therefore a yet unidentified Hsp90 client protein. Here, we demonstrate that endogenous NDRG1 and Hsp90 physically associate in hepatocellular cancer cell lines. However, geldanamycin (GA)-mediated inhibition of Hsp90 did not disrupt their interaction or result in NDRG1 protein destabilization. On the contrary, inhibition of Hsp90 led to a transcriptional increase of NDRG1 protein which was associated with cell growth arrest. We also observed that GA inhibited the phosphorylation of NDRG1 by targeting its regulating kinases, serum- and glucocorticoid-induced kinase 1 (SGK1) and glycogen synthase kinase 3 beta (GSK3beta). We demonstrate that in the presence of GA, GSK3beta protein and activity were decreased thus indicating that Hsp90 is necessary for GSK3beta stability. Taken together, our data demonstrate that NDRG1 is not a classic client protein but interacts with Hsp90 and is still dually regulated by Hsp90 at a transcriptional and post-translational level. Finally, we suggest for the first time GSK3beta as a new client protein of Hsp90.

MET targeting: time for a rematch.

MET, the receptor tyrosine kinase (RTK) for hepatocyte growth factor, is a proto-oncogene involved in embryonic development and throughout life in homeostasis and tissue regeneration. Deregulation of MET signaling has been reported in numerous malignancies, prompting great interest in MET targeting for cancer therapy. The present review offers a summary of the biology of MET and its known functions in normal physiology and carcinogenesis, followed by an overview of the most relevant MET-targeting strategies and corresponding clinical trials, highlighting both past setbacks and promising future prospects. By placing their efforts on a more precise stratification strategy through the genetic analysis of tumors, modern trials such as the NCI-MATCH trial could revive the past enthusiasm for MET-targeted therapy.