Differentially Expressed Genes Involved in Primary Resistance to Immunotherapy in Patients with Advanced-Stage Pulmonary Cancer.

In the last few years, nivolumab has become the standard of care for advanced-stage lung cancer patients. Unfortunately, up to 60% of patients do not respond to this treatment. In our study, we identified variations in gene expression related to primary resistance to immunotherapy. Bronchoscopy biopsies were obtained from advanced non-small cell lung cancer (NSCLC) patients previously characterized as responders or non-responders after nivolumab treatment. Ten tumor biopsies (from three responders and seven non-responders) were analyzed by the differential expression of 760 genes using the NanoString nCounter platform. These genes are known to be involved in the response to anti-PD1/PD-L1 therapy. All the patients were treated with nivolumab. Examining the dysregulated expression of 24 genes made it possible to predict the response to nivolumab treatment. Supervised analysis of the gene expression profile (GEP) revealed that responder patients had significantly higher levels of expression of CXCL11, NT5E, KLRK1, CD3G, GZMA, IDO1, LCK, CXCL9, GNLY, ITGAL, HLA-DRB1, CXCR6, IFNG, CD8A, ITK, B2M, HLA-B, and HLA-A than did non-responder patients. In contrast, PNOC, CD19, TP73, ARG1, FCRL2, and PTGER1 genes had significantly lower expression levels than non-responder patients. These findings were validated as predictive biomarkers in an independent series of 201 patients treated with nivolumab (22 hepatocellular carcinomas, 14 non-squamous cell lung carcinomas, 5 head and neck squamous cell carcinomas, 1 ureter/renal pelvis carcinoma, 120 melanomas, 4 bladder carcinomas, 31 renal cell carcinomas, and 4 squamous cell lung carcinomas). ROC curve analysis showed that the expression levels of ITK, NT5E, ITGAL, and CD8A were the best predictors of response to nivolumab. Further, 13/24 genes showed an adverse impact on overall survival (OS) in an independent, large series of patients with NSCLC (2166 cases). In summary, we found a strong association between the global GEP of advanced NSCLC and the response to nivolumab. The classification of NSCLC patients based on GEP enabled us to identify those patients who genuinely benefited from treatment with immune checkpoint inhibitors (ICIs). We also demonstrated that abnormal expression of most of the markers comprising the genomic signature has an adverse influence on OS, making them significant markers for therapeutic decision-making. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these biomarkers.

Association between diastolic dysfunction and fluid balance in critically ill patients during weaning from mechanical ventilation / Asociación entre la disfunción diastólica y el balance de hídrico en pacientes críticos durante el destete de la ventilación mecánica

Objective: To assess the role of diastolic dysfunction and fluid balance in weaning failure. Design: Prospective, observational, single center. Setting: Intensive care unit of a university hospital. Patients: Adult patients on mechanical ventilation for more than 48 h who underwent a spontaneous breathing trial (SBT). Interventions: Echocardiography was performed immediately before and at the end of SBT. Patients were classified into two groups according to weaning outcome. Main variable of interest: Weaning failure. Results: Among 89 patients included, weaning failure occurred in 33 patients (37%). Isolated diastolic dysfunction at the end of the SBT was more frequent in the failure group (39.3% vs. 17.8%, p = 0.025). Average daily fluid balance from ICU admission until first SBT was less negative in patients who failed than in those who succeed in the weaning (−648 mL [−884 to -138] vs. −893 mL [−1284 to −501], p = 0.007). Average daily fluid balance from the first SBT until the ICU discharge was more negative in the weaning failure than in the success group (−973 mL [−1493 to −201] vs. −425 mL [−1065 to 12], p = 0.034). Cox regression analysis showed that diastolic dysfunction was not an independent factor related to weaning failure but needed the association of positive fluid balance and age. Conclusions: Weaning failure due to diastolic dysfunction is highly related to fluid balance, and the deleterious effect of fluid balance on diastolic function is associated with age The timing of fluid removal could play a key role in this scenario. (AU)

Objetivo: Evaluar el papel de la disfunción diastólica (DD) y el balance hídrico en el fracaso del destete de la ventilación mecánica. Diseño: Prospectivo, observacional.Ámbito: Unidad de cuidados intensivos hospital universitario. Pacientes: Pacientes adultos con ventilación mecánica más de 48 h sometidos a una prueba de respiración espontánea (SBT).Intervenciones: Ecocardiografía antes y al final del SBT. Los pacientes se clasificaron en dos grupos según el resultado del destete. Variable de interés principal: Fracaso del destete. Resultados: Se incluyeron 89 pacientes, el fracaso del destete ocurrió en 33 (37%). La DD al final de la SBT fue más frecuente en el grupo de fracaso (39,3% vs 17,8%, p = 0,025). El balance hídrico diario desde el ingreso en UCI hasta la SBT fue menos negativo en los pacientes que fracasaron que en los que tuvieron éxito del destete (−648 mL [-884 a -138] vs −893 mL [-1284 a -501], p = 0,007). El balance hídrico diario desde el primer SBT hasta el alta de UCI fue más negativo en el grupo de fracaso que en el de éxito (−973 ml [−1493 a −201] vs. −425 ml [−1065 a 12], p = 0,034). La regresión de Cox mostró que la DD no era un factor independiente relacionado con el fracaso, necesitando la asociación del balance hídrico y la edad. Conclusiones: El fracaso del destete debido a DD se relaciona con el balance hídrico, incrementando su efecto nocivo con la edad. El momento de inicio del balance hídrico negativo puede jugar un papel clave en el fracaso. (AU)

Mapping density, diversity and species-richness of the Amazon tree flora.

Using 2.046 botanically-inventoried tree plots across the largest tropical forest on Earth, we mapped tree species-diversity and tree species-richness at 0.1-degree resolution, and investigated drivers for diversity and richness. Using only location, stratified by forest type, as predictor, our spatial model, to the best of our knowledge, provides the most accurate map of tree diversity in Amazonia to date, explaining approximately 70% of the tree diversity and species-richness. Large soil-forest combinations determine a significant percentage of the variation in tree species-richness and tree alpha-diversity in Amazonian forest-plots. We suggest that the size and fragmentation of these systems drive their large-scale diversity patterns and hence local diversity. A model not using location but cumulative water deficit, tree density, and temperature seasonality explains 47% of the tree species-richness in the terra-firme forest in Amazonia. Over large areas across Amazonia, residuals of this relationship are small and poorly spatially structured, suggesting that much of the residual variation may be local. The Guyana Shield area has consistently negative residuals, showing that this area has lower tree species-richness than expected by our models. We provide extensive plot meta-data, including tree density, tree alpha-diversity and tree species-richness results and gridded maps at 0.1-degree resolution.

An amino acid transporter subunit as an antibody-drug conjugate target in colorectal cancer.

BACKGROUND: Advanced colorectal cancer (CRC) is difficult to treat. For that reason, the development of novel therapeutics is necessary. Here we describe a potentially actionable plasma membrane target, the amino acid transporter protein subunit CD98hc. METHODS: Western blot and immunohistochemical analyses of CD98hc protein expression were carried out on paired normal and tumoral tissues from patients with CRC. Immunofluorescence and western studies were used to characterize the action of a DM1-based CD98hc-directed antibody-drug conjugate (ADC). MTT and Annexin V studies were performed to evaluate the effect of the anti-CD98hc-ADC on cell proliferation and apoptosis. CRISPR/Cas9 and shRNA were used to explore the specificity of the ADC. In vitro analyses of the antitumoral activity of the anti-CD98hc-ADC on 3D patient-derived normal as well as tumoral organoids were also carried out. Xenografted CRC cells and a PDX were used to analyze the antitumoral properties of the anti-CD98hc-ADC. RESULTS: Genomic as well proteomic analyses of paired normal and tumoral samples showed that CD98hc expression was significantly higher in tumoral tissues as compared to levels of CD98hc present in the normal colonic tissue. In human CRC cell lines, an ADC that recognized the CD98hc ectodomain, reached the lysosomes and exerted potent antitumoral activity. The specificity of the CD98hc-directed ADC was demonstrated using CRC cells in which CD98hc was decreased by shRNA or deleted using CRISPR/Cas9. Studies in patient-derived organoids verified the antitumoral action of the anti-CD98hc-ADC, which largely spared normal tissue-derived colon organoids. In vivo studies using xenografted CRC cells or patient-derived xenografts confirmed the antitumoral activity of the anti-CD98hc-ADC. CONCLUSIONS: The studies herewith reported indicate that CD98hc may represent a novel ADC target that, upon well-designed clinical trials, could be used to increase the therapeutic armamentarium against CRC.

Association between diastolic dysfunction and fluid balance in critically ill patients during weaning from mechanical ventilation.

OBJECTIVE: To assess the role of diastolic dysfunction and fluid balance in weaning failure. DESIGN: Prospective, observational, single center. SETTING: Intensive care unit of a university hospital. PATIENTS: Adult patients on mechanical ventilation for more than 48 h who underwent a spontaneous breathing trial (SBT). INTERVENTIONS: Echocardiography was performed immediately before and at the end of SBT. Patients were classified into two groups according to weaning outcome. MAIN VARIABLE OF INTEREST: Weaning failure. RESULTS: Among 89 patients included, weaning failure occurred in 33 patients (37%). Isolated diastolic dysfunction at the end of the SBT was more frequent in the failure group (39.3% vs. 17.8%, p = 0.025). Average daily fluid balance from ICU admission until first SBT was less negative in patients who failed than in those who succeed in the weaning (-648 mL [-884 to -138] vs. -893 mL [-1284 to -501], p = 0.007). Average daily fluid balance from the first SBT until the ICU discharge was more negative in the weaning failure than in the success group (-973 mL [-1493 to -201] vs. -425 mL [-1065 to 12], p = 0.034). Cox regression analysis showed that diastolic dysfunction was not an independent factor related to weaning failure but needed the association of positive fluid balance and age. CONCLUSIONS: Weaning failure due to diastolic dysfunction is highly related to fluid balance, and the deleterious effect of fluid balance on diastolic function is associated with age The timing of fluid removal could play a key role in this scenario.

Analysis of Circulating Tumor DNA in Synchronous Metastatic Colorectal Cancer at Diagnosis Predicts Overall Patient Survival.

Sporadic colorectal cancer (sCRC) initially presents as metastatic tumors in 25-30% of patients. The 5-year overall survival (OS) in patients with metastatic sCRC is 50%, falling to 10% in patients presenting with synchronous metastatic disease (stage IV). In this study, we systematically analyzed the mutations of RAS, PIK3CA and BRAF genes in circulating tumor DNA (ctDNA) and tumoral tissue DNA (ttDNA) from 51 synchronous metastatic colorectal carcinoma (SMCC) patients by real-time PCR, and their relationship with the clinical, biological and histological features of disease at diagnosis. The highest frequency of mutations detected was in the KRAS gene, in tumor biopsies and plasma samples, followed by mutations of the PIK3CA, NRAS and BRAF genes. Overall, plasma systematically contained those genetic abnormalities observed in the tumor biopsy sample from the same subject, the largest discrepancies detected between the tumor biopsy and plasma from the same patient being for mutations in the KRAS and PIK3CA genes, with concordances of genotyping results between ttDNA and ctDNA at diagnosis of 75% and 84%, respectively. Of the 51 SMCC patients in the study, 25 (49%) showed mutations in at least 1 of the 4 genes analyzed in patient plasma. From the prognostic point of view, the presence and number of the most common mutations in the RAS, PIK3CA and BRAF genes in plasma from SMCC patients are independent prognostic factors for OS. Determination of the mutational status of ctDNA in SMCC could be a key tool for the clinical management of patients.

Chemical-proteomics Identify Peroxiredoxin-1 as an Actionable Target in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) is difficult to treat; therefore, the development of drugs directed against its oncogenic vulnerabilities is a desirable goal. Herein, we report the antitumor effects of CM728, a novel quinone-fused oxazepine, against this malignancy. CM728 potently inhibited TNBC cell viability and decreased the growth of MDA-MB-231-induced orthotopic tumors. Furthermore, CM728 exerted a strong synergistic antiproliferative effect with docetaxel in vitro and this combination was more effective than the individual treatments in vivo. Chemical proteomic approaches revealed that CM728 bound to peroxiredoxin-1 (Prdx1), thereby inducing its oxidation. Molecular docking corroborated these findings. CM728 induced oxidative stress and a multi-signal response, including JNK/p38 MAPK activation and STAT3 inhibition. Interestingly, Prdx1 downregulation mimicked these effects. Finally, CM728 led to DNA damage, cell cycle blockage at the S and G2/M phases, and the activation of caspase-dependent apoptosis. Taken together, our results identify a novel compound with antitumoral properties against TNBC. In addition, we describe the mechanism of action of this drug and provide a rationale for the use of Prdx1 inhibitors, such as CM728, alone or in combination with other drugs, for the treatment of TNBC.

The WNK1-ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib.

BACKGROUND: The dismal prognosis of advanced ovarian cancer calls for the development of novel therapies to improve disease outcome. In this regard, we set out to discover new molecular entities and to assess the preclinical effectiveness of their targeting. METHODS: Cell lines, mice and human ovarian cancer samples were used. Proteome profiling of human phosphokinases, in silico genomic analyses, genetic (shRNA and CRISPR/Cas9) and pharmacological strategies as well as an ex vivo human preclinical model were performed. RESULTS: We identified WNK1 as a highly phosphorylated protein in ovarian cancer and found that its activation or high expression had a negative impact on patients' survival. Genomic analyses showed amplification of WNK1 in human ovarian tumours. Mechanistically, we demonstrate that WNK1 exerted its action through the MEK5-ERK5 signalling module in ovarian cancer. Loss of function, genetic or pharmacological experiments, demonstrated anti-proliferative and anti-tumoural effects of the targeting of the WNK1-MEK5-ERK5 route. Additional studies showed that this pathway modulated the anti-tumoural properties of the MEK1/2 inhibitor trametinib. Thus, treatment with trametinib activated the WNK1-MEK5-ERK5 route, raising the possibility that this effect may limit the therapeutic benefit of ERK1/2 targeting in ovarian cancer. Moreover, in different experimental settings, including an ex vivo patient-derived model consisting of ovarian cancer cells cultured with autologous patient sera, we show that inhibition of WNK1 or MEK5 increased the anti-proliferative and anti-tumour efficacy of trametinib. CONCLUSIONS: The present study uncovers the participation of WNK1-MEK5-ERK5 axis in ovarian cancer pathophysiology, opening the possibility of acting on this pathway with therapeutic purposes. Another important finding of the present study was the activation of that signalling axis by trametinib, bypassing the anti-tumoural efficacy of this drug. That fact should be considered in the context of the use of trametinib in ovarian cancer.

Study protocol for a randomized controlled trial of Proportional Assist Ventilation for Minimizing the Duration of Mechanical Ventilation: the PROMIZING study.

BACKGROUND: Proportional assist ventilation with load-adjustable gain factors (PAV+) is a mechanical ventilation mode that delivers assistance to breathe in proportion to the patient's effort. The proportional assistance, called the gain, can be adjusted by the clinician to maintain the patient's respiratory effort or workload within a normal range. Short-term and physiological benefits of this mode compared to pressure support ventilation (PSV) include better patient-ventilator synchrony and a more physiological response to changes in ventilatory demand. METHODS: The objective of this multi-centre randomized controlled trial (RCT) is to determine if, for patients with acute respiratory failure, ventilation with PAV+ will result in a shorter time to successful extubation than with PSV. This multi-centre open-label clinical trial plans to involve approximately 20 sites in several continents. Once eligibility is determined, patients must tolerate a short-term PSV trial and either (1) not meet general weaning criteria or (2) fail a 2-min Zero Continuous Positive Airway Pressure (CPAP) Trial using the rapid shallow breathing index, or (3) fail a spontaneous breathing trial (SBT), in this sequence. Then, participants in this study will be randomized to either PSV or PAV+ in a 1:1 ratio. PAV+ will be set according to a target of muscular pressure. The weaning process will be identical in the two arms. Time to liberation will be the primary outcome; ventilator-free days and other outcomes will be measured. DISCUSSION: Meta-analyses comparing PAV+ to PSV suggest PAV+ may benefit patients and decrease healthcare costs but no powered study to date has targeted the difficult to wean patient population most likely to benefit from the intervention, or used consistent timing for the implementation of PAV+. Our enrolment strategy, primary outcome measure, and liberation approaches may be useful for studying mechanical ventilation and weaning and can offer important results for patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT02447692 . Prospectively registered on May 19, 2015.

Unraveling Amazon tree community assembly using Maximum Information Entropy: a quantitative analysis of tropical forest ecology.

In a time of rapid global change, the question of what determines patterns in species abundance distribution remains a priority for understanding the complex dynamics of ecosystems. The constrained maximization of information entropy provides a framework for the understanding of such complex systems dynamics by a quantitative analysis of important constraints via predictions using least biased probability distributions. We apply it to over two thousand hectares of Amazonian tree inventories across seven forest types and thirteen functional traits, representing major global axes of plant strategies. Results show that constraints formed by regional relative abundances of genera explain eight times more of local relative abundances than constraints based on directional selection for specific functional traits, although the latter does show clear signals of environmental dependency. These results provide a quantitative insight by inference from large-scale data using cross-disciplinary methods, furthering our understanding of ecological dynamics.

Neuregulin modulates hormone receptor levels in breast cancer through concerted action on multiple signaling pathways.

The Neuregulins (NRGs) are growth factors that bind and activate ErbB/HER receptor tyrosine kinases. Some reports have described an interplay between this ligand-receptor system and hormonal receptors in breast cancer. However, the mechanisms by which NRGs regulate hormonal receptor signaling have not been sufficiently described. Here, we show that in breast cancer cells the activation of NRG receptors down-regulated ERα through a double mechanism that included post-transcriptional and transcriptional effects. This regulation required the concerted participation of three signaling routes: the PI3K/AKT/mTOR, ERK1/2, and ERK5 pathways. Moreover, these three routes were also involved in the phosphorylation of ERα at serines 118 and 167, two residues implicated in resistance to endocrine therapies. On the other hand, NRGs conferred resistance to fulvestrant in breast cancer cells and this resistance could be reversed when the three pathways activated by NRGs were simultaneously inhibited. Our results indicate that estrogen receptor-positive (ER+) breast tumors that can have access to NRGs may be resistant to fulvestrant. This resistance could be overcome if strategies to target the three main pathways involved in the interplay between NRG receptors and ERα could be developed.

Analysis of the Frequency and Correlated Factors of Midpalatal Suture Maturation Stages in Young Adults, Based on Cone Beam Computed Tomography Imaging.

BACKGROUND: The choice of whether to perform a palatal disjunction in constricted maxilla has traditionally been decided based on the age of the patients, although there are gradually increasing references to the fact that this is not a determining factor. The main goal of this study was to evaluate the frequency of the different stages of midpalatal suture maturation in a sample of young adults between 15 and 30 years of age. Other objectives also included analyzing the possible correlation the maturation stages could maintain with sex and age groups. METHODS: 142 Cone Beam Computed Tomography (CBCT) scans of young adults were performed. The images were divided into four age groups based on age ranges of 15-18, 19-22, 23-26, and 27-30 years. Each group consisted of 26, 41, 39, and 36 patients, respectively, which were classified using Angelieri's method. In addition, sex and age groups were considered as variables, and the possible correlation of the prevalence of each one, according to age and sex, was studied. RESULTS: the sample was classified into 4.9% stage B; 52.1% stage C; 27.5% stage D; and 15.5% stage E. In addition, no statistically significant correlation between sex and the maturation stages was found, but more advanced stages did appear to be related to the chronological age of the subjects. CONCLUSIONS: The frequency of maturational stages where the suture is shown to be consolidated did not appear to be as high as expected; therefore, the idea of rejecting transverse plane treatment in a conventional manner in an out-of-growth patient should be discarded.

Dysregulated Expression of Three Genes in Colorectal Cancer Stratifies Patients into Three Risk Groups.

Despite advances in recent years in the study of the molecular profile of sporadic colorectal cancer (sCRC), the specific genetic events that lead to increased aggressiveness or the development of the metastatic process of tumours are not yet clear. In previous studies of the gene expression profile (GEP) using a high-density array (50,000 genes and 6000 miRNAs in a single assay) in sCRC tumours, we identified a 28-gene signature that was found to be associated with an adverse prognostic value for predicting patient survival. Here, we analyse the differential expression of these 28 genes for their possible association with tumour local aggressiveness and metastatic processes in 66 consecutive sCRC patients, followed for >5 years, using the NanoString nCounter platform. The global transcription profile (expression levels of the 28 genes studied simultaneously) allowed us to discriminate between sCRC tumours and nontumoral colonic tissues. Analysis of the biological and functional significance of the dysregulated GEPs observed in our sCRC tumours revealed 31 significantly altered canonical pathways. Among the most commonly altered pathways, we observed the increased expression of genes involved in signalling pathways and cellular processes, such as the PI3K-Akt pathway, the interaction with the extracellular matrix (ECM), and other functions related to cell signalling processes (SRPX2). From a prognostic viewpoint, the altered expression of BST2 and SRPX2 genes were the only independent variables predicting for disease-free survival (DFS). In addition to the pT stage at diagnosis, dysregulated transcripts of ADH1B, BST2, and FER1L4 genes showed a prognostic impact on OS in the multivariate analysis. Based on the altered expression of these three genes, a scoring system was built to stratify patients into low-, intermediate-, and high-risk groups with significantly different 5-year OS rates: 91%, 83%, and 52%, respectively. The prognostic impact was validated in two independent series of sCRC patients from the public GEO database (n = 562 patients). In summary, we show a strong association between the altered expression of three genes and the clinical outcome of sCRC patients, making them potential markers of suitability for adjuvant therapy after complete tumour resection. Additional prospective studies in larger series of patients are required to confirm the clinical utility of the newly identified biomarkers because the number of patients analysed remains small.

The PROMIZING trial enrollment algorithm for early identification of patients ready for unassisted breathing.

BACKGROUND: Liberating patients from mechanical ventilation (MV) requires a systematic approach. In the context of a clinical trial, we developed a simple algorithm to identify patients who tolerate assisted ventilation but still require ongoing MV to be randomized. We report on the use of this algorithm to screen potential trial participants for enrollment and subsequent randomization in the Proportional Assist Ventilation for Minimizing the Duration of MV (PROMIZING) study. METHODS: The algorithm included five steps: enrollment criteria, pressure support ventilation (PSV) tolerance trial, weaning criteria, continuous positive airway pressure (CPAP) tolerance trial (0 cmH2O during 2 min) and spontaneous breathing trial (SBT): on fraction of inspired oxygen (FiO2) 40% for 30-120 min. Patients who failed the weaning criteria, CPAP Zero trial, or SBT were randomized. We describe the characteristics of patients who were initially enrolled, but passed all steps in the algorithm and consequently were not randomized. RESULTS: Among the 374 enrolled patients, 93 (25%) patients passed all five steps. At time of enrollment, most patients were on PSV (87%) with a mean (± standard deviation) FiO2 of 34 (± 6) %, PSV of 8.7 (± 2.9) cmH2O, and positive end-expiratory pressure of 6.1 (± 1.6) cmH2O. Minute ventilation was 9.0 (± 3.1) L/min with a respiratory rate of 17.4 (± 4.4) breaths/min. Patients were liberated from MV with a median [interquartile range] delay between initial screening and extubation of 5 [1-49] hours. Only 7 (8%) patients required reintubation. CONCLUSION: The trial algorithm permitted identification of 93 (25%) patients who were ready to extubate, while their clinicians predicted a duration of ventilation higher than 24 h.

Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer.

BACKGROUND: Despite the incorporation of novel therapeutics, advanced triple negative breast cancer (TNBC) still represents a relevant clinical problem. Considering this, as well as the clinical efficacy of antibody-drug conjugates (ADCs), we aimed at identifying novel ADC targets that could be used to treat TNBC. METHODS: Transcriptomic analyses were performed on TNBC and normal samples from three different studies. Plasma membrane proteins of three cell lines representative of the TNBC subtype were identified by cell surface biotinylation or plasma membrane isolation, followed by analyses of cell surface proteins using the Surfaceome online tool. Immunofluorescence and western studies were used to characterize the action of a CD98hc-directed ADC, which was prepared by in house coupling of emtansine to an antibody that recognized the ectodomain of CD98hc. Xenografted TNBC cells were used to analyze the antitumoral properties of the anti-CD98hc ADC. RESULTS: Comparative genomic studies between normal breast and TNBC tissues, together with proteomic and bioinformatic analyses resulted in the elaboration of a catalog of potential ADC targets. One of them, the CD98hc transmembrane protein, was validated as an ADC target. An antibody recognizing the ectodomain of CD98hc efficiently internalized and reached the lysosomal compartment. An emtansine-based ADC derived from such antibody was prepared and showed antitumoral properties in TNBC in vitro and in vivo models. Mechanistically, the anti-CD98hc ADC blocked cell cycle progression, that was followed by cell death caused by mitotic catastrophe. CONCLUSIONS: This work describes a list of potential ADC targets in TNBC and validates one of them, the transmembrane protein CD98hc. The studies presented here also demonstrate the robustness of the multiomic approach herewith described to identify novel potential ADC targets.

JKST6, a novel multikinase modulator of the BCR-ABL1/STAT5 signaling pathway that potentiates direct BCR-ABL1 inhibition and overcomes imatinib resistance in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a hematological malignancy that highly depends on the BCR-ABL1/STAT5 signaling pathway for cell survival. First-line treatments for CML consist of tyrosine kinase inhibitors that efficiently target BCR-ABL1 activity. However, drug resistance and intolerance are still therapeutic limitations in Ph+ cells. Therefore, the development of new anti-CML drugs that exhibit alternative mechanisms to overcome these limitations is a desirable goal. In this work, the antitumoral activity of JKST6, a naphthoquinone-pyrone hybrid, was assessed in imatinib-sensitive and imatinib-resistant human CML cells. Live-cell imaging analysis revealed JKST6 potent antiproliferative activity in 2D and 3D CML cultures. JKST6 provoked cell increase in the subG1 phase along with a reduction in the G0/G1 phase and altered the expression of key proteins involved in the control of mitosis and DNA damage. Rapid increases in Annexin V staining and activation/cleavage of caspases 8, 9 and 3 were observed after JKST6 treatment in CML cells. Of interest, JKST6 inhibited BCR-ABL1/STAT5 signaling through oncokinase downregulation that was preceded by rapid polyubiquitination. In addition, JKST6 caused a transient increase in JNK and AKT phosphorylation, whereas the phosphorylation of P38-MAPK and Src was reduced. Combinatory treatment unveiled synergistic effects between imatinib and JKST6. Notably, JKST6 maintained its antitumor efficacy in BCR-ABL1-T315I-positive cells and CML cells that overexpress BCR-ABL and even restored imatinib efficacy after a short exposure time. These findings, together with the observed low toxicity of JKST6, reveal a novel multikinase modulator that might overcome the limitations of BCR-ABL1 inhibitors in CML therapy.

Preclinical and Clinical Characterization of Fibroblast-derived Neuregulin-1 on Trastuzumab and Pertuzumab Activity in HER2-positive Breast Cancer.

PURPOSE: To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab. EXPERIMENTAL DESIGN: Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2-based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial. RESULTS: NRG1 was expressed in HER2-positive breast cancer-derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of NRG1 from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the NRG1-high group. CONCLUSIONS: CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab.

Altered proTGFα/cleaved TGFα ratios offer new therapeutic strategies in renal carcinoma.

BACKGROUND: Treatment of renal cancer has significantly improved with the arrival to the clinic of kinase inhibitors and immunotherapies. However, the disease is still incurable in advanced stages. The fact that several approved inhibitors for kidney cancer target receptor tyrosine kinases (RTKs) suggests that these proteins play a critical role in the pathophysiology of the disease. Based on these precedents, we decided to explore whether RTKs other than those targeted by approved drugs, contribute to the development of kidney cancer. METHODS: The activation status of 49 RTKs in 44 paired samples of normal and tumor kidney tissue was explored using antibody arrays, with validation by western blotting. Genetic and pharmacologic approaches were followed to study the biological implications of targeting the epidermal growth factor receptor (EGFR) and its ligand Transforming Growth Factor-α (TGFα). RESULTS: Activation of the EGFR was found in a substantial number of tumors. Moreover, kidney tumors expressed elevated levels of TGFα. Down-regulation of EGFR or TGFα using RNAi or their pharmacological targeting with blocking antibodies resulted in inhibition of the proliferation of in vitro cellular models of renal cancer. Importantly, differences in the molecular forms of TGFα expressed by tumors and normal tissues were found. In fact, tumor TGFα was membrane anchored, while that expressed by normal kidney tissue was proteolytically processed. CONCLUSIONS: The EGFR-TGFα axis plays a relevant role in the pathophysiology of kidney cancer. This study unveils a distinctive feature in renal cell carcinomas, which is the presence of membrane-anchored TGFα. That characteristic could be exploited therapeutically to act on tumors expressing transmembrane TGFα, for example, with antibody drug conjugates that could recognize the extracellular region of that protein.

MZ1 co-operates with trastuzumab in HER2 positive breast cancer.

BACKGROUND: Although the anti-HER2 antibody trastuzumab augments patient survival in HER2+ breast cancer, a relevant number of patients progress to this treatment. In this context, novel drug combinations are needed to increase its antitumor activity. In this work, we have evaluated the efficacy of proteolysis targeting chimera (PROTAC) compounds based on BET inhibitors (BETi) to augment the activity of trastuzumab in HER2+ breast cancer models. METHODS: BT474 and SKBR3 HER2+ breast cancer cell lines were used. The effects of trastuzumab and the BET-PROTAC MZ1 either alone or in combination, were evaluated using MTT proliferation assays, three-dimensional invasion and adhesion cultures, flow cytometry, qPCR and Western blot. In vivo studies were carried out in a xenografted model in mice. Finally, a Clariom_S_Human transcriptomic array was applied to identify deregulated genes after treatments. RESULTS: MZ1 induced a higher antiproliferative effect compared to the BETi JQ1. The combination of MZ1 and -trastuzumab significantly decreased cell proliferation, the formation of three-dimensional structures and cellular invasion compared to either of the drugs alone. Evaluation of apoptosis resulted in an increase of cell death following treatment with the combination, and biochemical studies displayed modifications of apoptosis and DNA damage components. In vivo administration of agents alone or combined, to tumors orthotopically xenografted in mice, resulted in a decrease of the tumor volume only after MZ1-Trastuzumab combination treatment. Results from a transcriptomic array indicated a series of newly described transcription factors including HOXB7, MEIS2, TCERG1, and DNAJC2, that were associated to poor outcome in HER2+ breast cancer subtype and downregulated by the MZ1-trastuzumab combination. CONCLUSIONS: We describe an active novel combination that includes the BET-PROTAC MZ1 and trastuzumab, in HER2+ tumors. Further studies should be performed to confirm these findings and pave the way for their future clinical development.

PDCD4 limits prooncogenic neuregulin-ErbB signaling.

The neuregulins and their ErbB/HER receptors play essential roles in mammalian development and tissue homeostasis. In addition, deregulation of their function has been linked to the pathogenesis of diseases such as cancer or schizophrenia. These circumstances have stimulated research into the biology of this ligand-receptor system. Here we show the identification of programmed cell death protein-4 (PDCD4) as a novel neuregulin-ErbB signaling mediator. Phosphoproteomic analyses identified PDCD4 as protein whose phosphorylation increased in cells treated with neuregulin. Mutagenesis experiments defined serine 67 of PDCD4 as a site whose phosphorylation increased upon activation of neuregulin receptors. Phosphorylation of that site promoted degradation of PDCD4 by the proteasome, which depended on exit of PDCD4 from the nucleus to the cytosol. Mechanistic studies defined mTORC1 and ERK1/2 as routes implicated in neuregulin-induced serine 67 phosphorylation and PDCD4 degradation. Functionally, PDCD4 regulated several important biological functions of neuregulin, such as proliferation, migration, or invasion.