Loss of Kmt2c in vivo leads to EMT, mitochondrial dysfunction and improved response to lapatinib in breast cancer.

Deep sequencing of human tumours has uncovered a previously unappreciated role for epigenetic regulators in tumorigenesis. H3K4 methyltransferase KMT2C/MLL3 is mutated in several solid malignancies, including more than 10% of breast tumours. To study the tumour suppressor role of KMT2C in breast cancer, we generated mouse models of Erbb2/Neu, Myc or PIK3CA-driven tumorigenesis, in which the Kmt2c locus is knocked out specifically in the luminal lineage of mouse mammary glands using the Cre recombinase. Kmt2c knock out mice develop tumours earlier, irrespective of the oncogene, assigning a bona fide tumour suppressor role for KMT2C in mammary tumorigenesis. Loss of Kmt2c induces extensive epigenetic and transcriptional changes, which lead to increased ERK1/2 activity, extracellular matrix re-organization, epithelial-to-mesenchymal transition and mitochondrial dysfunction, the latter associated with increased reactive oxygen species production. Loss of Kmt2c renders the Erbb2/Neu-driven tumours more responsive to lapatinib. Publicly available clinical datasets revealed an association of low Kmt2c gene expression and better long-term outcome. Collectively, our findings solidify the role of KMT2C as a tumour suppressor in breast cancer and identify dependencies that could be therapeutically amenable.

A Metabolomic Approach to Investigate the Effect of Phytonutrients on Proteostasis and Metabolic Pathways in Drosophila melanogaster.

The use of Drosophila melanogaster as a biological platform to study the effect of diet and food bioactives on the metabolome remains a highly unexplored subject. Aiming to establish alternative solutions for the investigation of nutritional interventions with bioactive natural products by employing LC-MS-based metabolomics approaches, we assessed the effect of a phytonutrient-rich extract from the endemic Mediterranean plant Cichorium spinosum (stamnagkàthi) on a Drosophila population. The extract's modulating effect on the proteostasis network and metabolism of young D. melanogaster flies was evaluated. Furthermore, an untargeted metabolomics approach, employing a C18 UPLC-ESI-Orbitrap-HRMS/MS platform, permitted the detection of several biomarkers in the metabolic profile of Drosophila's tissues; while targeted amino acid quantification in Drosophila tissue was simultaneously performed by employing aTRAQ labeling and an ion-pairing UPLC-ESI-SWATH-HRMS/MS platform. The detected metabolites belong to different chemical classes, and statistical analysis with chemometrics tools was utilized to reveal patterns and trends, as well as to uncover potential class-distinguishing features and possible biomarkers. Our findings suggest that Drosophila can serve as a valuable in vivo model for investigating the role of bioactive phytoconstituents, like those found in C. spinosum's decoction, on diverse metabolic processes. Additionally, the fruit fly represents a highly effective platform to investigate the molecular mechanisms underlying sex differences in diverse aspects of nutrition and physiology in higher metazoans.

Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming.

BACKGROUND: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized. METHODS: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model. RESULTS: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model. CONCLUSIONS: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.

Evaluation of the Small Heat Shock Protein Family Members HSPB2 and HSPB3 in Bladder Cancer Prognosis and Progression.

Bladder cancer (BlCa) represents the sixth most commonly diagnosed type of male malignancy. Due to the clinical heterogeneity of BlCa, novel markers would optimize treatment efficacy and improve prognosis. The small heat shock proteins (sHSP) family is one of the major groups of molecular chaperones responsible for the maintenance of proteome functionality and stability. However, the role of sHSPs in BlCa remains largely unknown. The present study aimed to examine the association between HSPB2 and HSPB3 expression and BlCa progression in patients, and to investigate their role in BlCa cells. For this purpose, a series of experiments including reverse transcription-quantitative PCR, Western blotting, MTT assay and flow cytometry were performed. Initial analyses revealed increased vs. human transitional carcinoma cells, expression levels of the HSPB2 and HSPB3 genes and proteins in high grade BlCa cell lines. Therefore, we then evaluated the clinical significance of the HSPB2 and HSPB3 genes expression levels in bladder tumor samples and matched adjusted normal bladder specimens. Total RNA from 100 bladder tumor samples and 49 paired non-cancerous bladder specimens were isolated, and an accurate SYBR-Green based real-time quantitative polymerase chain reaction (qPCR) protocol was developed to quantify HSPB2 and HSPB3 mRNA levels in the two cohorts of specimens. A significant downregulation of the HSPB2 and HSPB3 genes expression was observed in bladder tumors as compared to matched normal urothelium; yet, increased HSPB2 and HSPB3 levels were noted in muscle-invasive (T2-T4) vs. superficial tumors (TaT1), as well as in high-grade vs. low-grade tumors. Survival analyses highlighted the significantly higher risk for post-treatment disease relapse in TaT1 patients poorly expressing HSPB2 and HSPB3 genes; this effect tended to be inverted in advanced disease stages (muscle-invasive tumors) indicating the biphasic impact of HSPB2, HSPB3 genes in BlCa progression. The pro-survival role of HSPB2 and HSPB3 in advanced tumor cells was also evident by our finding that HSPB2, HSPB3 genes expression silencing in high grade BlCa cells enhanced doxorubicin toxicity. These findings indicate that the HSPB2, HSPB3 chaperone genes have a likely pro-survival role in advanced BlCa; thus, they can be targeted as novel molecular markers to optimize treatment efficacy in BlCa and to limit unnecessary interventions.

Immune response and adverse events after vaccination against SARS-CoV-2 in adult patients with transfusion-dependent thalassaemia.

Patients with transfusion-dependent thalassaemia (TDT) are considered an at increased-risk population for severe and/or morbid coronavirus disease 2019 (COVID-19) infection. Timely vaccination is the main preventive method for severe COVID-19. Different adverse events and reactions after vaccination have been reported, with severe ones being extremely rare. Patients with TDT may have altered immunity due to chronic transfusions, iron overload and chelation therapy, and splenic dysfunction. Here, we show that adult patients with TDT following vaccination with the novel messenger RNA vaccines have mild adverse events and can produce protective antibodies comparable to the healthy population.

Myeloma patients with COVID-19 have superior antibody responses compared to patients fully vaccinated with the BNT162b2 vaccine.

Patients with multiple myeloma (MM) have a suboptimal antibody response following vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and lower seroconversion rates following coronavirus disease 2019 (COVID-19) compared with healthy individuals. In this context, we evaluated the development of neutralising antibodies (NAbs) against SARS-CoV-2 in non-vaccinated patients with MM and COVID-19 compared with patients after vaccination with two doses of the BNT162b2 vaccine. Serum was collected either four weeks post confirmed diagnosis or four weeks post a second dose of BNT162b2. NAbs were measured with a Food and Drug Administration-approved enzyme-linked immunosorbent assay methodology. Thirty-five patients with COVID-19 and MM along with 35 matched patients were included. The two groups did not differ in age, sex, body mass index, prior lines of therapy, disease status, lymphocyte count, immunoglobulin levels and comorbidities. Patients with MM and COVID-19 showed a superior humoral response compared with vaccinated patients with MM. The median (interquartile range) NAb titre was 87·6% (71·6-94%) and 58·7% (21·4-91·8%) for COVID-19-positive and vaccinated patients, respectively (P = 0·01).Importantly, there was no difference in NAb production between COVID-19-positive and vaccinated patients who did not receive any treatment (median NAb 85·1% vs 91·7%, P = 0·14). In conclusion, our data indicate that vaccinated patients with MM on treatment without prior COVID-19 should be considered for booster vaccine doses.

SARS-CoV-2 humoral responses following booster BNT162b2 vaccination in patients with B-cell malignancies.

Patients with B-cell malignancies have suboptimal immune responses to SARS-CoV-2 vaccination and are a high-risk population for severe COVID19 disease. We evaluated the effect of a third booster BNT162b2 vaccine on the kinetics of anti- SARS-CoV-2 neutralizing antibody (NAbs) titers in patients with B-cell malignancies. Patients with NHL (n = 54) Waldenström's macroglobulinemia (n = 90) and chronic lymphocytic leukemia (n = 49) enrolled in the ongoing NCT04743388 study and compared against matched healthy controls. All patient groups had significantly lower NAbs compared to controls at all time points. 1 month post the third dose (M1P3D) NAbs increased significantly compared to previous time points (median NAbs 77.9%, p < .05 for all comparisons) in all patients. NAbs ≥ 50% were seen in 59.1% of patients, 34.5% of patients with suboptimal responses post-second dose, elicited a protective NAb titer ≥50%. Active treatment, rituximab, and BTKi treatment were the most important prognostic factors for a poor NAb response at 1MP3D; only 25.8% of patients on active treatment had NAbs ≥ 50%. No significant between-group differences were observed. Patients with B-cell malignancies have inferior humoral responses against SARS-CoV-2 and booster dose enhances the NAb response in a proportion of these patients.

New Metabolites from the Marine Sponge Scopalina hapalia Collected in Mayotte Lagoon.

The biological screening of 44 marine sponge extracts for the research of bioactive molecules, with potential application in the treatment of age-related diseases (cancer and Alzheimer's disease) and skin aging, resulted in the selection of Scopalina hapalia extract for chemical study. As no reports of secondary metabolites of S. hapalia were found in the literature, we undertook this research to further extend current knowledge of Scopalina chemistry. The investigation of this species led to the discovery of four new compounds: two butenolides sinularone J (1) and sinularone K (2), one phospholipid 1-O-octadecyl-2-pentanoyl-sn-glycero-3-phosphocholine (3) and one lysophospholipid 1-O-(3-methoxy-tetradecanoyl)-sn-glycero-3-phosphocholine (4) alongside with known lysophospholipids (5 and 6), alkylglycerols (7-10), epidioxysterols (11 and 12) and diketopiperazines (13 and 14). The structure elucidation of the new metabolites (1-4) was determined by detailed spectroscopic analysis, including 1D and 2D NMR as well as mass spectrometry. Molecular networking was also explored to complement classical investigation and unravel the chemical classes within this species. GNPS analysis provided further information on potential metabolites with additional bioactive natural compounds predicted.

High mRNA Expression Levels of Heat Shock Protein Family B Member 2 (HSPB2) Are Associated with Breast Cancer Patients' Relapse and Poor Survival.

Small heat shock proteins (sHSPs) are ubiquitous ATP-independent chaperones that contribute to the maintenance of proteome integrity and functionality. Recent evidence suggests that sHSPs are ubiquitously expressed in numerous types of tumors and have been proposed to be implicated in oncogenesis and malignant progression. Heat shock protein family B member 2 (HSPB2) is a member of the sHSPs, which is found to be expressed, among others, in human breast cancer cell lines and constitutes an inhibitor of apical caspase activation in the extrinsic apoptotic pathway. In this study, we investigated the potential prognostic significance of HSPB2 mRNA expression levels in breast cancer, which represents the most frequent malignancy in females and one of the three most common cancer types worldwide. To this end, malignant breast tumors along with paired non-cancerous breast tissue specimens were used. HSPB2 expression levels were quantified in these two cohorts using a sensitive and accurate SYBR green-based quantitative real-time polymerase chain reaction (q-RT-PCR). Extensive biostatistical analyses were performed including Kaplan-Meier and Cox regression survival analyses for the assessment of the results. The significant downregulation of HSPB2 gene expression was revealed in breast tumors compared to their adjacent non-cancerous breast tissues. Notably, high HSPB2 mRNA expression predicts poor disease-free survival and overall survival of breast cancer patients. Multivariate Cox regression analysis revealed that HSPB2 mRNA overexpression is a significant predictor of poor prognosis in breast cancer, independent of other clinicopathological factors. In conclusion, high HSPB2 mRNA expression levels are associated with breast cancer patients' relapse and poor survival.

Isolation of an Extract from the Soft Coral Symbiotic Microorganism Salinispora arenicola Exerting Cytoprotective and Anti-Aging Effects.

Cells have developed a highly integrated system responsible for proteome stability, namely the proteostasis network (PN). As loss of proteostasis is a hallmark of aging and age-related diseases, the activation of PN modules can likely extend healthspan. Here, we present data on the bioactivity of an extract (SA223-S2BM) purified from the strain Salinispora arenicola TM223-S2 that was isolated from the soft coral Scleronephthya lewinsohni; this coral was collected at a depth of 65 m from the mesophotic Red Sea ecosystem EAPC (south Eilat, Israel). Treatment of human cells with SA223-S2BM activated proteostatic modules, decreased oxidative load, and conferred protection against oxidative and genotoxic stress. Furthermore, SA223-S2BM enhanced proteasome and lysosomal-cathepsins activities in Drosophila flies and exhibited skin protective effects as evidenced by effective inhibition of the skin aging-related enzymes, elastase and tyrosinase. We suggest that the SA223-S2BM extract constitutes a likely promising source for prioritizing molecules with anti-aging properties.

Comparative kinetics of SARS-CoV-2 anti-spike protein RBD IgGs and neutralizing antibodies in convalescent and naïve recipients of the BNT162b2 mRNA vaccine versus COVID-19 patients.

BACKGROUND: Coronavirus SARS-CoV-2, the causative agent of COVID-19, has caused a still evolving global pandemic. Given the worldwide vaccination campaign, the understanding of the vaccine-induced versus COVID-19-induced immunity will contribute to adjusting vaccine dosing strategies and speeding-up vaccination efforts. METHODS: Anti-spike-RBD IgGs and neutralizing antibodies (NAbs) titers were measured in BNT162b2 mRNA vaccinated participants (n = 250); we also investigated humoral and cellular immune responses in vaccinated individuals (n = 21) of this cohort 5 months post-vaccination and assayed NAbs levels in COVID-19 hospitalized patients (n = 60) with moderate or severe disease, as well as in COVID-19 recovered patients (n = 34). RESULTS: We found that one (boosting) dose of the BNT162b2 vaccine triggers robust immune (i.e., anti-spike-RBD IgGs and NAbs) responses in COVID-19 convalescent healthy recipients, while naïve recipients require both priming and boosting shots to acquire high antibody titers. Severe COVID-19 triggers an earlier and more intense (versus moderate disease) immune response in hospitalized patients; in all cases, however, antibody titers remain at high levels in COVID-19 recovered patients. Although virus infection promotes an earlier and more intense, versus priming vaccination, immune response, boosting vaccination induces antibody titers significantly higher and likely more durable versus COVID-19. In support, high anti-spike-RBD IgGs/NAbs titers along with spike (vaccine encoded antigen) specific T cell clones were found in the serum and peripheral blood mononuclear cells, respectively, of vaccinated individuals 5 months post-vaccination. CONCLUSIONS: These findings support vaccination efficacy, also suggesting that vaccination likely offers more protection than natural infection.

Molecular mechanisms of carfilzomib-induced cardiotoxicity in mice and the emerging cardioprotective role of metformin.

Carfilzomib (Cfz), an irreversible proteasome inhibitor licensed for relapsed/refractory myeloma, is associated with cardiotoxicity in humans. We sought to establish the optimal protocol of Cfz-induced cardiac dysfunction, to investigate the underlying molecular-signaling and, based on the findings, to evaluate the cardioprotective potency of metformin (Met). Mice were randomized into protocols 1 and 2 (control and Cfz for 1 and 2 consecutive days, respectively); protocols 3 and 4 (control and alternate doses of Cfz for 6 and 14 days, respectively); protocols 5A and 5B (control and Cfz, intermittent doses on days 0, 1 [5A] and 0, 1, 7, and 8 [5B] for 13 days); protocols 6A and 6B (pharmacological intervention; control, Cfz, Cfz+Met and Met for 2 and 6 days, respectively); and protocol 7 (bortezomib). Cfz was administered at 8 mg/kg (IP) and Met at 140 mg/kg (per os). Cfz resulted in significant reduction of proteasomal activity in heart and peripheral blood mononuclear cells in all protocols except protocols 5A and 5B. Echocardiography demonstrated that Cfz led to a significant fractional shortening (FS) depression in protocols 2 and 3, a borderline dysfunction in protocols 1 and 4, and had no detrimental effect on protocols 5A and 5B. Molecular analysis revealed that Cfz inhibited AMPKα/mTORC1 pathways derived from increased PP2A activity in protocol 2, whereas it additionally inhibited phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase pathway in protocol 3. Coadministration of Met prevented Cfz-induced FS reduction and restored AMPKα phosphorylation and autophagic signaling. Conclusively, Cfz decreased left ventricular function through increased PP2A activity and inhibition of AMPKα and its downstream autophagic targets, whereas Met represents a novel promising intervention against Cfz-induced cardiotoxicity.

Insights to SARS-CoV-2 life cycle, pathophysiology, and rationalized treatments that target COVID-19 clinical complications.

BACKGROUND: Gaining further insights into SARS-CoV-2 routes of infection and the underlying pathobiology of COVID-19 will support the design of rational treatments targeting the life cycle of the virus and/or the adverse effects (e.g., multi-organ collapse) that are triggered by COVID-19-mediated adult respiratory distress syndrome (ARDS) and/or other pathologies. MAIN BODY: COVID-19 is a two-phase disease being marked by (phase 1) increased virus transmission and infection rates due to the wide expression of the main infection-related ACE2, TMPRSS2 and CTSB/L human genes in tissues of the respiratory and gastrointestinal tract, as well as by (phase 2) host- and probably sex- and/or age-specific uncontrolled inflammatory immune responses which drive hyper-cytokinemia, aggressive inflammation and (due to broad organotropism of SARS-CoV-2) collateral tissue damage and systemic failure likely because of imbalanced ACE/ANGII/AT1R and ACE2/ANG(1-7)/MASR axes signaling. CONCLUSION: Here we discuss SARS-CoV-2 life cycle and a number of approaches aiming to suppress viral infection rates or propagation; increase virus antigen presentation in order to activate a robust and durable adaptive immune response from the host, and/or mitigate the ARDS-related "cytokine storm" and collateral tissue damage that triggers the severe life-threatening complications of COVID-19.

Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater.

Considering the lack of effective treatments against COVID-19, wastewater-based epidemiology (WBE) is emerging as a cost-effective approach for real-time population-wide SARS-CoV-2 monitoring. Here, we report novel molecular assays for sensitive detection and mutational/variant analysis of SARS-CoV-2 in wastewater. Highly stable regions of SARS-CoV-2 RNA were identified by RNA stability analysis and targeted for the development of novel nested PCR assays. Targeted DNA sequencing (DNA-seq) was applied for the analysis and quantification of SARS-CoV-2 mutations/variants, following hexamers-based reverse transcription and nested PCR-based amplification of targeted regions. Three-dimensional (3D) structure models were generated to examine the predicted structural modification caused by genomic variants. WBE of SARS-CoV-2 revealed to be assay dependent, and significantly improved sensitivity achieved by assay combination (94%) vs. single-assay screening (30%-60%). Targeted DNA-seq allowed the quantification of SARS-CoV-2 mutations/variants in wastewater, which agreed with COVID-19 patients' sequencing data. A mutational analysis indicated the prevalence of D614G (S) and P323L (RdRP) variants, as well as of the Β.1.1.7/alpha variant of concern, in agreement with the frequency of Β.1.1.7/alpha variant in clinical samples of the same period of the third pandemic wave at the national level. Our assays provide an innovative cost-effective platform for real-time monitoring and early-identification of SARS-CoV-2 variants at community/population levels.

Elucidating Carfilzomib's Induced Cardiotoxicity in an In Vivo Model of Aging: Prophylactic Potential of Metformin.

BACKGROUND: Carfilzomib is a first-line proteasome inhibitor indicated for relapsed/refractory multiple myeloma (MM), with its clinical use being hampered by cardiotoxic phenomena. We have previously established a translational model of carfilzomib cardiotoxicity in young adult mice, in which metformin emerged as a prophylactic therapy. Considering that MM is an elderly disease and that age is an independent risk factor for cardiotoxicity, herein, we sought to validate carfilzomib's cardiotoxicity in an in vivo model of aging. METHODS: Aged mice underwent the translational two- and four-dose protocols without and with metformin. Mice underwent echocardiography and were subsequently sacrificed for molecular analyses in the blood and cardiac tissue. RESULTS: Carfilzomib decreased proteasomal activity both in PBMCs and myocardium in both protocols. Carfilzomib induced mild cardiotoxicity after two doses and more pronounced cardiomyopathy in the four-dose protocol, while metformin maintained cardiac function. Carfilzomib led to an increased Bip expression and decreased AMPKα phosphorylation, while metformin coadministration partially decreased Bip expression and induced AMPKα phosphorylation, leading to enhanced myocardial LC3B-dependent autophagy. CONCLUSION: Carfilzomib induced cardiotoxicity in aged mice, an effect significantly reversed by metformin. The latter possesses translational importance as it further supports the clinical use of metformin as a potent prophylactic therapy.

Heat shock protein beta 3 (HSPB3) is an unfavorable molecular biomarker in colorectal adenocarcinoma.

Small heat shock proteins (sHSPs) participate in numerous cellular functions including cell signaling, differentiation, and apoptosis. Deregulation of the physiological expression level of sHSPs has been associated with several malignancies. Heat shock protein beta 3 (HSPB3) is the third member of the sHSP family in human and is mainly expressed in skeletal and smooth muscles. In this study, we investigated the potential prognostic significance of HSPB3 expression in colorectal adenocarcinoma, the most frequent type of colorectal cancer. For this purpose, we isolated total RNA from 188 colorectal adenocarcinoma specimens and 68 paired noncancerous ones. After reverse transcription of 2 µg total RNA, we quantified HSPB3 levels by using an in-house-developed real-time quantitative polymerase chain reaction method, based on the SYBR Green chemistry. Comparison of HSPB3 levels among 68 pairs of colorectal tumors and their adjacent noncancerous mucosae uncovered the downregulation of HSPB3 expression in the majority of malignant colorectal tumors. More importantly, high HSPB3 expression is associated with poor relapse-free survival (RFS) and overall survival (OS) of patients with colorectal adenocarcinoma. Multivariable Cox regression analysis revealed that HSPB3 overexpression could serve as an adverse prognostic biomarker in colorectal adenocarcinoma, independent of tumor location, histological grade, and TNM stage. Patients' stratification according to tumor location, histological grade, and TNM stage revealed that high HSPB3 messenger RNA expression retains its unfavorable prognostic potential regarding OS, in particular groups of patients with substantially different prognosis. In conclusion, high HSPB3 expression is associated with poor RFS and OS of patients with colorectal adenocarcioma, independently of clinicopathological prognosticators.

Inhibition of jasmonate-mediated plant defences by the fungal metabolite higginsianin B.

Infection of Arabidopsis thaliana by the ascomycete fungus Colletotrichum higginsianum is characterized by an early symptomless biotrophic phase followed by a destructive necrotrophic phase. The fungal genome contains 77 secondary metabolism-related biosynthetic gene clusters, whose expression during the infection process is tightly regulated. Deleting CclA, a chromatin regulator involved in the repression of some biosynthetic gene clusters through H3K4 trimethylation, allowed overproduction of three families of terpenoids and isolation of 12 different molecules. These natural products were tested in combination with methyl jasmonate, an elicitor of jasmonate responses, for their capacity to alter defence gene induction in Arabidopsis. Higginsianin B inhibited methyl jasmonate-triggered expression of the defence reporter VSP1p:GUS, suggesting it may block bioactive jasmonoyl isoleucine (JA-Ile) synthesis or signalling in planta. Using the JA-Ile sensor Jas9-VENUS, we found that higginsianin B, but not three other structurally related molecules, suppressed JA-Ile signalling by preventing the degradation of JAZ proteins, the repressors of jasmonate responses. Higginsianin B likely blocks the 26S proteasome-dependent degradation of JAZ proteins because it inhibited chymotrypsin- and caspase-like protease activities. The inhibition of target degradation by higginsianin B also extended to auxin signalling, as higginsianin B treatment reduced auxin-dependent expression of DR5p:GUS. Overall, our data indicate that specific fungal secondary metabolites can act similarly to protein effectors to subvert plant immune and developmental responses.

Screening for tyrosinase inhibitors from actinomycetes; identification of trichostatin derivatives from Streptomyces sp. CA-129531 and scale up production in bioreactor.

In the course of a primary screening of 614 microbial actinomycete extracts for the discovery of tyrosinase inhibitors, the EtOAc extract of the fermentation broth of the strain Streptomyces sp. CA-129531 isolated from a Martinique sample, exhibited in cell free and cell-based assays the most promising activity (IC50 value of 63 µg/mL). Scaled-up production in a bioreactor led to the isolation of one new trichostatic acid analogue, namely trichostatic acid B (1), along with six known trichostatin derivatives (2-7), four diketopiperazines (8-11), two butyrolactones (12-13) and one hydroxamic acid siderophore (14). Among them, trichostatin A (4) showed a Ki value of 6.1 µM and six times stronger anti-tyrosinase activity (IC50 2.18 µΜ) than kojic acid (IC50 14.07 µΜ) used as a positive control. Deoxytrichostatin A (6) displayed also strong inhibitory activity against tyrosinase (IC50 19.18 µΜ). Trichostatin A production in bioreactor started together with the exponential phase of growth (day 4) and the maximum concentration was reached at day 9 (2.67 ± 0.13 µg/mL). Despite the cytotoxicity of some individual components, the EtOAc extract showed no cytotoxic effect on HepG2, A2058, A549, MCF-7 and MIA PaCa-2 cell lines, (IC50 >2.84 mg/mL) and against BG fibroblasts at the concentrations where the whitening effect was exerted, reassuring its safety and great tyrosinase inhibitory potential.

What sustains the multidrug resistance phenotype beyond ABC efflux transporters? Looking beyond the tip of the iceberg.

Identification of multidrug (MDR) efflux transporters that belong to the ATP-Binding Cassette (ABC) superfamily, represented an important breakthrough for understanding cancer multidrug resistance (MDR) and its possible overcoming. However, recent data indicate that drug resistant cells have a complex intracellular physiology that involves constant changes in energetic and oxidative-reductive metabolic pathways, as well as in the molecular circuitries connecting mitochondria, endoplasmic reticulum (ER) and lysosomes. The aim of this review is to discuss the key molecular mechanisms of cellular reprogramming that induce and maintain MDR, beyond the presence of MDR efflux transporters. We specifically highlight how cancer cells characterized by high metabolic plasticity - i.e. cells able to shift the energy metabolism between glycolysis and oxidative phosphorylation, to survive both the normoxic and hypoxic conditions, to modify the cytosolic and mitochondrial oxidative-reductive metabolism, are more prone to adapt to exogenous stressors such as anti-cancer drugs and acquire a MDR phenotype. Similarly, we discuss how changes in mitochondria dynamics and mitophagy rates, changes in proteome stability ensuring non-oncogenic proteostatic mechanisms, changes in ubiquitin/proteasome- and autophagy/lysosome-related pathways, promote the cellular survival under stress conditions, along with the acquisition or maintenance of MDR. After dissecting the complex intracellular crosstalk that takes place during the development of MDR, we suggest that mapping the specific adaptation pathways underlying cell survival in response to stress and targeting these pathways with potent pharmacologic agents may be a new approach to enhance therapeutic efficacy against MDR tumors.

The emergence of drug resistance to targeted cancer therapies: Clinical evidence.

For many decades classical anti-tumor therapies included chemotherapy, radiation and surgery; however, in the last two decades, following the identification of the genomic drivers and main hallmarks of cancer, the introduction of therapies that target specific tumor-promoting oncogenic or non-oncogenic pathways, has revolutionized cancer therapeutics. Despite the significant progress in cancer therapy, clinical oncologists are often facing the primary impediment of anticancer drug resistance, as many cancer patients display either intrinsic chemoresistance from the very beginning of the therapy or after initial responses and upon repeated drug treatment cycles, acquired drug resistance develops and thus relapse emerges, resulting in increased mortality. Our attempts to understand the molecular basis underlying these drug resistance phenotypes in pre-clinical models and patient specimens revealed the extreme plasticity and adaptive pathways employed by tumor cells, being under sustained stress and extensive genomic/proteomic instability due to the applied therapeutic regimens. Subsequent efforts have yielded more effective inhibitors and combinatorial approaches (e.g. the use of specific pharmacologic inhibitors with immunotherapy) that exhibit synergistic effects against tumor cells, hence enhancing therapeutic indices. Furthermore, new advanced methodologies that allow for the early detection of genetic/epigenetic alterations that lead to drug chemoresistance and prospective validation of biomarkers which identify patients that will benefit from certain drug classes, have started to improve the clinical outcome. This review discusses emerging principles of drug resistance to cancer therapies targeting a wide array of oncogenic kinases, along with hedgehog pathway and the proteasome and apoptotic inducers, as well as epigenetic and metabolic modulators. We further discuss mechanisms of resistance to monoclonal antibodies, immunomodulators and immune checkpoint inhibitors, potential biomarkers of drug response/drug resistance, along with possible new therapeutic avenues for the clinicians to combat devastating drug resistant malignancies. It is foreseen that these topics will be major areas of focused multidisciplinary translational research in the years to come.