A CD38-directed, single-chain T-cell engager targets leukemia stem cells through IFN-γ-induced CD38 expression.

ABSTRACT: Treatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), LSCs are frequently enriched in the CD34posCD38neg blast fraction. Here, we report that interferon gamma (IFN-γ) reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38pos and CD38neg LSC-enriched blasts. IFN-γ-induced CD38 upregulation depends on interferon regulatory factor 1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T-cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38pos AML cells and both CD8pos and CD4pos T cells. We demonstrate that BN-CD38 engages autologous CD4pos and CD8pos T cells and CD38pos AML blasts, leading to T-cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFN-γ, driving the expression of CD38 on CD34posCD38neg LSC-enriched blasts and their subsequent elimination. Critically, although BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient-derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multilineage human immune cells in CD34pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM).

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM. METHODS: KDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways. RESULTS: Levels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth. CONCLUSIONS: The results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.

Association between Clonal Hematopoiesis and Late Nonrelapse Mortality after Autologous Hematopoietic Cell Transplantation.

Clonal hematopoiesis (CH), characterized by the accumulation of acquired somatic mutations in the blood, is associated with an elevated risk of aging-related diseases and premature mortality in non-cancer populations. Patients who undergo autologous hematopoietic cell transplantation (HCT) are also at high risk of premature onset of aging-related conditions. Therefore, we examined the association between pretreatment CH and late-occurring (≥1 year) nonrelapse mortality (NRM) after HCT. We evaluated pathogenic and likely pathogenic CH variants (PVs) in 10 patients who developed NRM after HCT and in 29 HCT recipient controls matched by age at HCT ± 2 years (median, 64.6 years; range, 38.5 to 74.7 years), sex (79.5% male), diagnosis (61.5% with non-Hodgkin lymphoma, 18.0% with Hodgkin lymphoma, and 20.5% with multiple myeloma), and duration of follow-up. We analyzed mobilized hematopoietic stem cell DNA in samples collected before HCT using a custom panel of amplicons covering the coding exons of 79 myeloid-related genes associated with CH. PVs with allele fractions >2% were used for analyses. Cases were significantly more likely than controls to have CH (70% versus 24.1%; P = .002), to have ≥2 unique PVs (60% versus 6.9%; P < .001), and to have PVs with allelic fractions ≥10% (40% versus 3.4%; P = .003). Here we provide preliminary evidence of an association between pre-HCT CH and NRM after HCT independent of chronologic age. Integration of CH analyses may improve the accuracy of existing pre-HCT risk prediction models, setting the stage for personalized risk assessment strategies and targeted treatments to optimally prevent or manage late complications associated with HCT.

Towards microbiome transplant as a therapy for periodontitis: an exploratory study of periodontitis microbial signature contrasted by oral health, caries and edentulism.

BACKGROUND: Conventional periodontal therapy aims at controlling supra- and subgingival biofilms. Although periodontal therapy was shown to improve periodontal health, it does not completely arrest the disease. Almost all subjects compliant with periodontal maintenance continue to experience progressive clinical attachment loss and a fraction of them loses teeth. An oral microbial transplant may be a new alternative for treating periodontitis (inspired by fecal transplant). First, it must be established that microbiomes of oral health and periodontitis are distinct. In that case, the health-associated microbiome could be introduced into the oral cavity of periodontitis patients. This relates to the goals of our study: (i) to assess if microbial communities of the entire oral cavity of subjects with periodontitis were different from or oral health contrasted by microbiotas of caries and edentulism patients; (ii) to test in vitro if safe concentration of sodium hypochlorite could be used for initial eradication of the original oral microbiota followed by a safe neutralization of the hypochlorite prior transplantation. METHODS: Sixteen systemically healthy white adults with clinical signs of one of the following oral conditions were enrolled: periodontitis, established caries, edentulism, and oral health. Oral biofilm samples were collected from sub- and supra-gingival sites, and oral mucosae. DNA was extracted and 16S rRNA genes were amplified. Amplicons from the same patient were pooled, sequenced and quantified. Volunteer's oral plaque was treated with saline, 16 mM NaOCl and NaOCl neutralized by ascorbate buffer followed by plating on blood agar. RESULTS: Ordination plots of rRNA gene abundances revealed distinct groupings for the oral microbiomes of subjects with periodontitis, edentulism, or oral health. The oral microbiome in subjects with periodontitis showed the greatest diversity harboring 29 bacterial species at significantly higher abundance compared to subjects with the other assessed conditions. Healthy subjects had significantly higher abundance in 10 microbial species compared to the other conditions. NaOCl showed strong antimicrobial properties; nontoxic ascorbate was capable of neutralizing the hypochlorite. CONCLUSIONS: Distinct oral microbial signatures were found in subjects with periodontitis, edentulism, or oral health. This finding opens up a potential for a new therapy, whereby a health-related entire oral microbial community would be transplanted to the diseased patient.

Teriflunomide/leflunomide synergize with chemotherapeutics by decreasing mitochondrial fragmentation via DRP1 in SCLC.

Although up to 80% small cell lung cancer (SCLC) patients' response is good for first-line chemotherapy regimen, most patients develop recurrence of the disease within weeks to months. Here, we report cytostatic effect of leflunomide (Leflu) and teriflunomide (Teri) on SCLC cell proliferation through inhibition of DRP1 phosphorylation at Ser616 and decreased mitochondrial fragmentation. When administered together, Teri and carboplatin (Carbo) act synergistically to significantly inhibit cell proliferation and DRP1 phosphorylation, reduce abundance of intermediates in pyrimidine de novo pathway, and increase apoptosis and DNA damage. Combination of Leflu&Carbo has anti-tumorigenic effect in vivo. Additionally, lurbinectedin (Lur) and Teri potently and synergistically inhibited spheroid growth and depleted uridine and DRP1 phosphorylation in mouse tumors. Our results suggest combinations of Carbo and Lur with Teri or Leflu are efficacious and underscore how the relationship between DRP1/DHODH and mitochondrial plasticity serves as a potential therapeutic target to validate these treatment strategies in SCLC clinical trials.

Toward Elimination of Electrochemical Corrosion in Dental Implants: A Zirconia-Titanium Composite Prototype.

Background Titanium dental implants (e.g., Nobel Biocare, Switzerland) are routinely used as support for dental restoration. Titanium has been the material of choice due to its corrosion resistance and ability to integrate with bone. Nevertheless, corrosion and titanium dissolution do occur. Compared to control, peri-implantitis tissue biopsies have been shown to contain high concentrations of dissolved titanium as well as metal particles. Dissolved titanium species have been found to be associated with the structure/diversity of the subgingival plaque microbiome and the extent of global methylation. Of note, peri-implantitis and peri-implant mucositis are common biological complications of implant therapy. Microorganisms and local inflammation together with a gradient of oxygen have been proven to form an electrochemical fuel cell, which generates the current that flows through the body of the titanium implant. Effectively, the fuel cell reduces oxygen and oxidizes titanium that turns into a soluble form. We are proposing a new zirconia-titanium composite implant design whereby the electrical current is disrupted while other properties are still conducive to osseointegration. Methodology Biocompatible zirconia bolts were treated with hydrofluoric acid (HF) and coated with titanium in a vacuum evaporator. The coating was masked with nail polish, and unmasked areas were etched with HF followed by mask removal with a solvent. Microbial challenges were conducted with a volunteer's plaque. Regular implant (control) and the prototype were inserted into simulated peri-implant environments implemented as a fiberglass sleeve immersed into a growth medium. After a five-day growth, samples were taken and HNO3 digested. Dissolved titanium was evaluated by inductively coupled plasma mass spectrometry. Results Proof-of-concept implant prototypes were successfully created. Vacuum deposition results in reproducible stable titanium coating. The thickness of the titanium coating was estimated using atomic force microscopy. A microbial challenge revealed that compared to the commercial titanium implant, the new implant prototype showed decreased amounts of corrosion-leached titanium. Conclusions We demonstrate a path forward toward a new design of a dental implant, whereby corrosion-induced electrical currents are interrupted resulting in a decreased amount of dissolved titanium.

Machine Learning Algorithm to Perform the American Society of Anesthesiologists Physical Status Classification.

OBJECTIVE: The American Society of Anesthesiologists (ASA) Physical Status (PS) Classification System defines perioperative patient scores ranging from 1 to 6 (healthy to brain dead, respectively). The scoring is performed and used by physician anesthesiologists and providers to classify surgical patients based on co-morbidities and various clinical characteristics. There is potentially a variability in scoring stemming from individual biases. The biases impact the prediction of operating times, length of stay in the hospital, anesthetic management, and billing. This study's purpose was to develop an automated system to achieve reproducible scoring. METHODS: A machine learning (ML) model was trained on already assigned ASA PS scores of 12,064 patients. The ML algorithm was automatically selected by Wolfram Mathematica (Wolfram Research, Champaign, IL) and tested with retrospective records not used in training. Manual scoring was performed by the anesthesiologist as part of the standard preoperative evaluation. Intraclass correlation coefficient (ICC) in R (version 4.2.2; R Development Core Team, Vienna, Austria) was calculated to assess the consistency of scoring. RESULTS: An ML model was trained on the data corresponding to 12,064 patients. Logistic regression was chosen automatically, with an accuracy of 70.3±1.0% against the training dataset. The accuracy against 1,999 patients (the test dataset) was 69.6±1.0%. The ICC for the comparison between ML and the anesthesiologists' ASA PS scores was greater than 0.4 ("fair to good"). CONCLUSIONS: We have shown the feasibility of applying ML to assess the ASA PS score within an oncology patient population. Though our accuracy was not very good, we feel that, as more data are mined, a valid foundation for refinement to ML will emerge.

Leveraging IFNγ/CD38 regulation to unmask and target leukemia stem cells in acute myelogenous leukemia.

Elimination of drug-resistant leukemia stem cells (LSCs) represents a major challenge to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), the presence of CD34 and lack of CD38 expression (CD34posCD38neg) are immunophenotypic features of both LSC-enriched AML blasts and normal hematopoietic stem cells (HSCs). We report that IFN-γ induces CD38 upregulation in LSC-enriched CD34posCD38neg AML blasts, but not in CD34posCD38neg HSCs. To leverage the IFN-γ mediated CD38 up-regulation in LSCs for clinical application, we created a compact, single-chain CD38-CD3-T cell engager (CD38-BIONIC) able to direct T cells against CD38pos blasts. Activated CD4pos and CD8pos T cells not only kill AML blasts but also produce IFNγ, which leads to CD38 expression on CD34posCD38neg LSC-enriched blasts. These cells then become CD38-BIONIC targets. The net result is an immune-mediated killing of both CD38neg and CD38pos AML blasts, which culminates in LSC depletion.

Beyond Affymetrix arrays: expanding the set of known hybridization isotherms and observing pre-wash signal intensities.

Microarray hybridization studies have attributed the nonlinearity of hybridization isotherms to probe saturation and post-hybridization washing. Both processes are thought to distort 'true' target abundance because immobilized probes are saturated with excess target and stringent washing removes loosely bound targets. Yet the paucity of studies aimed at understanding hybridization and dissociation makes it difficult to align physicochemical theory to microarray results. To fill the void, we first examined hybridization isotherms generated on different microarray platforms using a ribosomal RNA target and then investigated hybridization signals at equilibrium and after stringent wash. Hybridization signal at equilibrium was achieved by treating the microarray with isopropanol, which prevents nucleic acids from dissolving into solution. Our results suggest that (i) the shape of hybridization isotherms varied by microarray platform with some being hyperbolic or linear, and others following a power-law; (ii) at equilibrium, fluorescent signal of different probes hybridized to the same target were not similar even with excess of target and (iii) the amount of target removed by stringent washing depended upon the hybridization time, the probe sequence and the presence/absence of nonspecific targets. Possible physicochemical interpretations of the results and future studies are discussed.

SUMOylation inhibition enhances dexamethasone sensitivity in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable plasma cell malignancy. Although Dexamethasone (Dex) is the most widely used therapeutic drug in MM treatment, patients develop Dex resistance leading to progressive disease, demanding an urgent need to investigate the mechanisms driving Dex resistance and develop new reagents to address this problem. We propose SUMOylation as a potential mechanism regulating Dex resistance and SUMOylation inhibition can enhance Dex sensitivity in MM. METHODS: Using MM cell lines and primary MM samples from relapsing MM patients, we evaluated the effects of knockdown of SUMO E1 (SAE2) or using TAK-981, a novel and specific SUMO E1 inhibitor, on Dex sensitivity. Xenograft mouse models were generated to determine the in vivo anti-MM effects of TAK-981 as a single agent and in combination with Dex. miRNA-seq, RNA-seq and GSEA analysis were utilized for evaluating key factors mediating Dex resistance. Chromatin immunoprecipitation (ChIP) assay was performed to determine the binding occupancy of c-Myc on promoter region of miRs. RESULTS: We observed a significant negative correlation between SUMO E1 (SAE2) expression and Dex sensitivity in primary MM samples. Knockdown of SAE2 or using TAK-981 significantly enhances myeloma sensitivity to Dex in MM cell lines. Moreover, the enhanced anti-MM activity by TAK-981 and Dex combination has been validated using primary relapsing MM patient samples and xenograft mouse models. SUMOylation inhibition increased glucocorticoid receptor (GR) expression via downregulation miR-130b. Using RNA and microRNA sequencing, we identified miR-551b and miR-25 as important miRs mediating Dex resistance in MM. Overexpression of miR-551b and miR-25 caused resistance to Dex, however, knockdown of miR-551b and miR-25 significantly enhanced Dex sensitivity in MM. SAE2 knockdown or TAK-981 treatment downregulated the expression of miR-551b and miR-25, leading to induction of miR targets ZFP36, ULK1 and p27, resulting in apoptosis and autophagy. We demonstrated c-Myc as a major transcriptional activator of miR-130b, miR-551b and miR-25 and SUMOylation inhibition downregulates these miRs level by decreasing c-Myc level. CONCLUSION: Our study proves SUMOylation plays a crucial role in Dex resistance in MM and SUMOylation inhibition appears to be an attractive strategy to advance to the clinic for MM patients.

A competitive hybridization model predicts probe signal intensity on high density DNA microarrays.

A central, unresolved problem of DNA microarray technology is the interpretation of different signal intensities from multiple probes targeting the same transcript. We propose a competitive hybridization model for DNA microarray hybridization. Our model uses a probe-specific dissociation constant that is computed with current nearest neighbor model and existing parameters, and only four global parameters that are fitted to Affymetrix Latin Square data. This model can successfully predict signal intensities of individual probes, therefore makes it possible to quantify the absolute concentration of targets. Our results offer critical insights into the design and data interpretation of DNA microarrays.

A Non-genetic Mechanism Involving the Integrin ß4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer.

Tumor heterogeneity and cisplatin resistance are major causes of tumor relapse and poor survival. Here, we show that in lung cancer, interaction between paxillin (PXN) and integrin ß4 (ITGB4), components of the focal adhesion (FA) complex, contributes to cisplatin resistance. Knocking down PXN and ITGB4 attenuated cell growth and improved cisplatin sensitivity, both in 2D and 3D cultures. PXN and ITGB4 independently regulated expression of several genes. In addition, they also regulated expression of common genes including USP1 and VDAC1, which are required for maintaining genomic stability and mitochondrial function, respectively. Mathematical modeling suggested that bistability could lead to stochastic phenotypic switching between cisplatin-sensitive and resistant states in these cells. Consistently, purified subpopulations of sensitive and resistant cells re-created the mixed parental population when cultured separately. Altogether, these data point to an unexpected role of the FA complex in cisplatin resistance and highlight a novel non-genetic mechanism.

Erratum: A Non-genetic Mechanism Involving the Integrin ß4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer.

[This corrects the article DOI: 10.1016/j.isci.2020.101496.].

Use of hidden correlations in short oligonucleotide array data are insufficient for accurate quantification of nucleic acid targets in complex target mixtures.

Nonspecific target binding (i.e., cross-hybridization) is a major challenge for interpreting oligonucleotide microarray results because it is difficult to determine what portion of the signal is due to binding of complementary (specific) targets to a probe versus that due to binding of nonspecific targets. Solving this challenge would be a major accomplishment in microarray research potentially allowing quantification of targets in biological samples. Marcelino et al. recently described a new approach that reportedly solves this challenge by iteratively deconvoluting 'true' specific signal from raw signal, and quantifying ribosomal (rRNA) sequences in artificial and natural communities (i.e., "Accurately quantifying low-abundant targets amid similar sequences by revealing hidden correlations in oligonucleotide microarray data", Proc. Natl. Acad. Sci. 103, 13629-13634). We evaluated their approach using high-density oligonucleotide microarrays and Latin-square designed experiments consisting of 6 and 8 rRNA targets in 16 different artificial mixtures. Our results show that contrary to the claims in the article, the hidden correlations in the microarray data are insufficient for accurate quantification of nucleic acid targets in complex artificial target mixtures.

Revision of the nonequilibrium thermal dissociation and stringent washing approaches for identification of mixed nucleic acid targets by microarrays.

Microarray experiments typically involve washing steps that remove hybridized nonspecific targets with the purpose of improving the signal-to-noise ratio. The quality of washing ultimately affects downstream analysis of the microarray and interpretation. The paucity of fundamental studies directed towards understanding the dissociation of mixed targets from microarrays makes the development of meaningful washing/dissociation protocols difficult. To fill the void, we examined activation energies and preexponential coefficients of 47 perfect match (PM) and double-mismatch (MM) duplex pairs to discover that there was no statistical difference between the kinetics of the PM and MM duplexes. Based on these findings, we evaluated the nonequilibrium thermal dissociation (NTD) approach, which has been used to identify specific microbial targets in mixed target samples. We found that the major premises for various washing protocols and the NTD approach might be seriously compromised because: (i) nonspecific duplexes do not always dissociate before specific ones, and (ii) the relationship between dissociation rates of the PM and MM duplexes depends on temperature and duplex sequence. Specifically for the NTD, we show that previously suggested use of reference curves, indices of curves and temperature ramps lead to erroneous conclusions.

MiR-16 regulates crosstalk in NF-κB tolerogenic inflammatory signaling between myeloma cells and bone marrow macrophages.

High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/ß complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR-15a-16-1-KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.

Simultaneous quantification of multiple nucleic acid targets in complex rRNA mixtures using high density microarrays and nonspecific hybridization as a source of information.

To date, it has been problematic to accurately quantify multiple nucleic acid sequences, representing microbial targets, in multi-target mixtures using oligonucleotide microarrays, primarily due to nonspecific target binding (i.e., cross-hybridization). While some studies ignore the effects of nonspecific binding, other studies have developed approaches to minimize nonspecific binding, such as physical modeling to design highly specific probes, subtracting nonspecific signal using mismatch probes, and/or removing nonspecific duplexes by scanning through a range of wash stringencies. We have developed an alternative approach that, in contrast to previous approaches, uses nonspecific target binding as a source of information. Specifically, the new approach uses hybridization patterns (fingerprints) to quantify specific nucleic acid targets in complex target mixtures. We evaluated the approach by mixing together in vitro transcribed 28S rRNA targets at varying concentrations (up to 1.0 nM), and hybridizing the 24 mixtures to microarrays (n=3160 probes, in duplicate). Three independent Latin-square-designed experiments revealed accurate quantification of the targets. The regression between actual concentration of targets and those determined by the approach were highly positively correlated with high R(2) values (e.g., R(2)=0.90, n=6 targets; R(2)=0.84, n=8 targets; R(2)=0.82, n=10 targets).

Concentration dependency of nonequilibrium thermal dissociation curves in complex target samples.

The nonequilibrium thermal dissociation (NTD) methodology has been proposed to provide a superior discrimination between specific and nonspecific hybridizations than the commonly used array techniques involving hybridization followed by a single stringent wash. Multiple studies have used this method on gel-pad, planar, and nylon membrane arrays to identify specific microbial targets in complex target mixtures. A recent physicochemical study revealed several problems, particularly when the method was used to examine complex target samples. In the present study, we investigated the effect of target concentration on NTD of complex target samples obtained from an anaerobic bioreactor. Our purpose was to experimentally demonstrate that variation in the concentrations of both specific and nonspecific targets determines the course of dissociation, which was not evaluated in initial microbiological studies. We also present an approach for analyzing the dissociation curves that is less error prone compared to those used in the previous studies. Our results show that: (i) a specific target in a mixture, at a certain concentration, may have a higher dissociation temperature/time than that of the same pure target, and (ii) the concentration dependence of the dissociation precludes usage of reference curves for identifying a target. Contrary to the previous studies, an explicit calibration is required, which makes the NTD approach impractical for high throughput analysis.

Tests of rRNA hybridization to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be predicted.

Hybridization of rRNAs to microarrays is a promising approach for prokaryotic and eukaryotic species identification. Typically, the amount of bound target is measured by fluorescent intensity and it is assumed that the signal intensity is directly related to the target concentration. Using thirteen different eukaryotic LSU rRNA target sequences and 7693 short perfect match oligonucleotide probes, we have assessed current approaches for predicting signal intensities by comparing Gibbs free energy (DeltaG degrees) calculations to experimental results. Our evaluation revealed a poor statistical relationship between predicted and actual intensities. Although signal intensities for a given target varied up to 70-fold, none of the predictors were able to fully explain this variation. Also, no combination of different free energy terms, as assessed by principal component and neural network analyses, provided a reliable predictor of hybridization efficiency. We also examined the effects of single-base pair mismatch (MM) (all possible types and positions) on signal intensities of duplexes. We found that the MM effects differ from those that were predicted from solution-based hybridizations. These results recommend against the application of probe design software tools that use thermodynamic parameters to assess probe quality for species identification. Our results imply that the thermodynamic properties of oligonucleotide hybridization are by far not yet understood.

Inhibiting crosstalk between MET signaling and mitochondrial dynamics and morphology: a novel therapeutic approach for lung cancer and mesothelioma.

The receptor tyrosine kinase MET is frequently involved in malignant transformation and inhibiting its activity in MET-dependent cancers is associated with improved clinical outcomes. Emerging evidence also suggests that mitochondria play an essential role in tumorigenesis and Dynamin Related Protein (DRP1), a key component of the mitochondrial fission machinery, has emerged as an attractive therapeutic target. Here, we report that inhibiting MET activity with the tyrosine kinase inhibitor MGCD516 attenuates viability, migration, and invasion of non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM) cell lines in vitro, and significantly retards tumor growth in vivo. Interestingly, MGCD516 treatment also results in altered mitochondrial morphology in these cell lines. Furthermore, inhibiting MET pharmacologically or knocking down its expression using siRNA, decreases DRP1 activity alluding to possible crosstalk between them in these two cancers. Consistently, a combination of MGCD516 and mdivi-1, a quinazolinone reported to inhibit mitochondrial fission, is more effective in attenuating proliferation of NSCLC and MPM cell lines than either drug alone. Considered together, the present study has uncovered a novel mechanism underlying mitochondrial regulation by MET that involves crosstalk with DRP1, and suggests that a combination therapy targeting both MET and DRP1 could be a novel strategy for NSCLC and MPM.