Selective Inhibitor Research Articles

Selpercatinib – a new option for a precision approach to the treatment of medullary thyroid cancer

Introduction. Multikinase inhibitors are used to treat nonresectable locally advanced and/or metastatic medullary thyroid cancer (MTC). However they are characterized by high toxicity associated with kinase inhibition. Selective RET inhibitor selpercatinib demonstrates high selectivity and tolerance which makes it a promising agent for MTC treatment.Aim. To evaluate selpercatinib effectiveness and tolerance in patients with metastatic MTC associated with a mutation in the RET gene.Materials and methods. The study included 9 patients with metastatic MTC and mutation in the RET gene who received treatment with selpercatinib 160 mg 2 times a day. The drug effectiveness was evaluated every 2–3 months based on the results of multispiral computed tomography of the whole body and tumor marker (calcitonin and carcinoembryonic antigen) levels.Results. Median duration of therapy was 29 months overall response rate was 78 %; complete response was observed in 56 % of patients. After 12 months of therapy progression-free survival was 100 %; after 24 months it was 89 %. Persistent decrease in calcitonin level (by more than 90 %) was achieved in all patients. The most common adverse events were arterial hypertension and insignificant creatinine increase.Conclusion. The results of therapy show significant improvement in the rate of objective response and progression[1]free survival which makes selpercatinib a preferential treatment choice in this category of patients.

Selective deficiency of mitochondrial respiratory complex I subunits Ndufs4/6 causes tumor immunogenicity.

Cancer cells frequently rewire their metabolism to support proliferation and evade immune surveillance, but little is known about metabolic targets that could increase immune surveillance. Here we show a specific means of mitochondrial respiratory complex I (CI) inhibition that improves tumor immunogenicity and sensitivity to immune checkpoint blockade (ICB). Targeted genetic deletion of either Ndufs4 or Ndufs6, but not other CI subunits, induces an immune-dependent growth attenuation in melanoma and breast cancer models. We show that deletion of Ndufs4 induces expression of the major histocompatibility complex (MHC) class I co-activator Nlrc5 and antigen presentation machinery components, most notably H2-K1. This induction of MHC-related genes is driven by a pyruvate dehydrogenase-dependent accumulation of mitochondrial acetyl-CoA, which leads to an increase in histone H3K27 acetylation within the Nlrc5 and H2-K1 promoters. Taken together, this work shows that selective CI inhibition restricts tumor growth and that specific targeting of Ndufs4 or Ndufs6 increases T cell surveillance and ICB responsiveness.

Coffee Pulp from Azores: A Novel Phytochemical-Rich Food with Potential Anti-Diabetic Properties.

Coffee pulp, a by-product of wet coffee processing, shows significant potential in the food and health domains, but its real applications remain underexplored. This work investigated the chemical composition and bioactive properties of coffee pulp from São Miguel Island (Azores, Portugal). The studied coffee pulp exhibited high fiber content (52% dw), mostly insoluble; notable mineral levels (10.6%), mainly K, Ca, and Mg; and 6% dw of total amino acids, with hydroxyproline, aspartic acid, glutamic acid, and leucine in higher amounts. Despite containing low fat (1.6% dw), mainly saturated, it also showed considerable amounts of polyunsaturated fatty acids with a favorable n6/n3 ratio (1.40) and vitamin E (α-, β-, and γ-tocopherols). Its antioxidant capacity can be partially explained by the chlorogenic acid content (9.2 mg/g dw), and caffeine (0.98%) was present in similar amounts to those observed in some arabica coffee beans. A decrease in glucose uptake in Caco-2 cells was found, but not in fructose, suggesting selective inhibition of SGLT1 and potential antidiabetic effects. These results show that Azorean coffee pulp has potential as a sustainable and bioactive ingredient for incorporation into functional foods or dietary supplements.

Targeting the mitotic kinase NEK2 enhances CDK4/6 inhibitor efficacy by potentiating genome instability.

Selective inhibitors that target cyclin dependent kinases 4 and 6 (CDK4/6i) are FDA approved for treatment of a subset of breast cancers and are being evaluated in numerous clinical trials for other cancers. Despite this advance, a subset of tumors are intrinsically resistant to these drugs and acquired resistance is nearly inevitable. Recent mechanistic evidence suggests that in addition to stalling the cell cycle, the anti-tumor effects of CDK4/6i involve the induction of chromosomal instability (CIN). Here, we exploit this mechanism by combining CDK4/6i with other instability-promoting agents to induce maladaptive CIN and irreversible cell fates. Specifically, dual targeting of CDK4/6 and the mitotic kinase NEK2 in vitro drives centrosome amplification and the accumulation of CIN that induces catastrophic mitoses, cell cycle exit, and cell death. Dual targeting also induces CIN in vivo and significantly decreases mouse tumor volume to a greater extent than either drug alone, without inducing overt toxicity. Importantly, we provide evidence that breast cancer cells are selectively dependent on NEK2, but non-transformed cells are not, in contrast with other mitotic kinases that are commonly essential in all cell types. These findings implicate NEK2 as a potential therapeutic target for breast cancer that could circumvent the dose-limiting toxicities that are commonly observed when blocking other mitotic kinases. Moreover, these data suggest that NEK2 inhibitors could be used to sensitize tumors to FDA-approved CDK4/6i for the treatment of breast cancers, improving their efficacy and providing a foundation for expanding the patient population that could benefit from CDK4/6i.

Protein kinase A suppresses anti-proliferative effect of interferon-α in hepatocellular carcinoma by activation of protein tyrosine phosphatase SHP2.

Src homology-2-containing protein tyrosine phosphatase 2 (SHP2) plays a dual role in cancer initiation and progression. Identifying signals that modulate the function of SHP2 can improve current therapeutic approaches for IFN-α/β in HCC. We showed that cAMP-dependent protein kinase A (PKA) suppresses IFN-α/β-induced JAK/STAT signaling by increasing the phosphatase activity of SHP2, promoting the dissociation of SHP2 from the receptor for activated C-kinase 1 (RACK1) and binding to STAT1. Additionally, cAMP-degrading phosphodiesterase 4D5 (PDE4D) physically interacts with RACK1 to regulate PKA-mediated SHP2 activity and STAT1 phosphorylation. IFN-α activates PKA by inducing the expression of cyclooxygenase 2 (COX2) and the production of prostaglandin E2 (PGE2), which in turn stimulates the binding of SHP2 to IFNAR2 via RACK1. A COX inhibitor aspirin potently increases the antitumor effects of IFN-α in the suppression of HCC cell proliferation in vivo. Higher expression of COX2 and phosphorylated STAT3 is associated with poor development and prognosis in HCC patients by analyzing human HCC clinical samples. These observations suggest that a fundamental PKA/SHP2-dependent negative feedback loop acts on IFN signaling, and inhibition of this signaling by the selective COX2 inhibitors may enhance the clinical efficacy of type I IFNs in treating HCC.

Single-plate kinome screening in live-cells to enable highly cost-efficient kinase inhibitor profiling.

Cancer research, cancer treatment, and the field of chemical biology are examples which heavily rely on the discovery of selective kinase inhibitors. While determining on-target potency is often feasible for most laboratories, the equally critical but frequently neglected selectivity screening remains less accessible to the broader scientific community. This limitation can stem from various factors, such as the unavailability of a large number of purified kinases or the costs associated with commercial screening systems. To address these challenges and enable a wider range of scientists, this protocol leverages a commercial kinome selectivity screen to facilitate a low-cost, two-day, single-plate selectivity screen against 192 kinases.

Biomedical Effects of Protein Arginine Methyltransferase Inhibitors.

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of arginine residues in eukaryotic proteins, playing critical roles in modulating diverse cellular processes. The importance of PRMTs in the incidence and progression of a wide range of diseases, particularly cancers, such as breast, liver, lung, colorectal cancer, lymphoma, leukemia, and acute myeloid leukemia (AML) is increasingly recognized. This underscores the critical need for the development of effective PRMT inhibitors as therapeutic intervention. The field of PRMT inhibitors is in the rapidly growing phase and it is necessary to conduct a summative review of how the so-far developed inhibitors impact PRMT functions and cellular physiology. Our review aims to summarize molecular action mechanisms of these PRMT inhibitors and particularly elaborate their triggered biomedical effects. We delineate the cellular phenotype consequences of select PRMT inhibitors across various disease models, thereby providing an understanding of the pharmacological mechanisms underpinning PRMT inhibition. The promising effects of PRMT5 inhibitors in targeted therapy of MTAP-deleted cancers is particularly highlighted. At last, we provide a perspective on the challenges and further opportunities of developing and applying novel PRMT inhibitors for clinical advancement.

Multiple Kinase Small Molecule Inhibitor Tinengotinib (TT-00420) Alone or With Chemotherapy Inhibit the Growth of SCLC.

There is an urgent need to develop new targeted treatment agents for small cell lung cancer (SCLC). Tinengotinib (TT-00420) is a novel, multi-targeted, and spectrally selective small-molecule kinase inhibitor that has shown significant inhibitory effects on certain solid tumors in preclinical studies. However, its role and mechanism of action in SCLC remain unclear. In this study, we demonstrated that tinengotinib effectively inhibited SCLC cell proliferation, especially highly expressing NeuroD1 (SCLC-N), in the SCLC cell line-derived xenograft (CDX) model and the malignant pleural effusion cell model of patients with SCLC. When combined with etoposide/cisplatin, it synergistically inhibited SCLC growth. Tinengotinib regulates proliferation, apoptosis, migration, cell cycle and angiogenesis in SCLC cells. Mechanistic studies revealed that c-Myc expression may be a key factor influencing the effect of tinengotinib in SCLC-N. This study provides reliable preclinical data and a new direction for tinengotinib as a promising therapy for SCLC, either alone or in combination with chemotherapy.

A Simple and Efficient Stereoselective Synthesis of a 2,3-Difluorosialic Acid-Based Influenza Virus Neuraminidase Inhibitor.

3-Fluoroneuraminosyl fluorides are invaluable probes for studying the catalytic mechanism of sialidases (neuraminidases), and as sialidase inhibitors. Significantly, when a C-3 equatorial fluorine is installed on a C-4 functionalised N-acylneuraminic acid (Neu)-based template, the compounds are potent and selective inhibitors of both influenza and parainfluenza sialidases, and of virus replication. Typically, the reported syntheses of 3-fluoroneuraminosyl fluorides involve either an enzymatic or a chemical synthesis that have uncontrolled stereoselectivity in the introduction of fluorine at C-3 of Neu and consequently yield a mixture of C-3 ax and C-3 eq fluoro derivatives. We now report a simple approach for the exquisitely stereo-controlled introduction of the C-3 equatorial fluorine on Neu by incorporation of steric bulk at C-4. Through this method, we have elaborated a novel synthetic route that exclusively produces the potent anti-influenza drug candidate; 2,3-difluoro-zanamivir analogue with C-3 eq fluoride.

A physiologically-based quantitative systems pharmacology model for mechanistic understanding of the response to alogliptin and its application in patients with renal impairment.

Alogliptin is a highly selective inhibitor of dipeptidyl peptidase-4 and primarily excreted as unchanged drug in the urine, and differences in clinical outcomes in renal impairment patients increase the risk of serious adverse reactions. In this study, we developed a comprehensive physiologically-based quantitative systematic pharmacology model of the alogliptin-glucose control system to predict plasma exposure and use glucose as a clinical endpoint to prospectively understand its therapeutic outcomes with varying renal function. Our model incorporates a PBPK model for alogliptin, DPP-4 activity described by receptor occupancy theory, and the crosstalk and feedback loops for GLP-1-GIP-glucagon, insulin, and glucose. Based on the optimization of renal function-dependent parameters, the model was extrapolated to different stages renal impairment patients. Ultimately our model adequately describes the pharmacokinetics of alogliptin, the progression of DPP-4 inhibition over time and the dynamics of the glucose control system components. The extrapolation results endorse the dose adjustment regimen of 12.5mg once daily for moderate patients and 6.25mg once daily for severe and ESRD patients, while providing additional reflections and insights. In clinical practice, our model could provide additional information on the in vivo fate of DPP4 inhibitors and key regulators of the glucose control system.

Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags.

Protein kinases are prime targets for drug development due to their involvement in various cancers. However, selective inhibition of kinases, while avoiding off-target effects remains a significant challenge for the development of protein kinase inhibitors. Activity-based protein profiling (ABPP), in combination with pan-kinase activity-based probes (ABPs) and mass spectrometry-based proteomics, enables the identification of kinase drug targets. Here, we extend existing ABPP strategies for kinase profiling with a site-specific analysis, allowing for protein kinase inhibitor target engagement profiling with amino acid specificity. The site-specific approach involves highly efficient enrichment of ABP-labeled peptides, resulting in a less complex peptide matrix, straightforward data analysis, and the screening of over ∼100 kinase active sites in a single LC-MS analysis. The complementary use of both trypsin and pepsin in parallel to generate the ABP-labeled peptides considerably expanded the coverage of kinases and pinpoint the exact binding sites. Using the site-specific strategy to examine the on- and off-targets of the Ephrin receptor (Eph) B4 inhibitor NVP-BHG712 showed binding to EphA2 with an IC50 of 17 nM and EphB4 with an IC50 of 20 nM. Next to the known targets, EphA2 and EphB4, NVP-BHG712 bound to the discoidin domain-containing receptor 1 (DDR1) with an IC50 of 2.1 nM, suggesting that a DDR1-targeting regio-isomer of NVP-BHG712 was used. The promiscuity of XO44 toward ATP-binding pockets on non-kinase proteins facilitated the screening of additional off-target sites, revealing inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) as a putative off-target. Expanding the search to other amino acids revealed that XO44, in addition to 745 lysines, also covalently linked 715 tyrosines, which significantly expands the competitive ABPP search space and highlights the added value of the site-specific method. Therefore, the presented approach, which can be fully automated with liquid handling platforms, provides a straightforward, valuable new approach for competitive site-specific kinase inhibitor target profiling.

Resveratrol-Based Carbamates as Selective Butyrylcholinesterase Inhibitors: Design, Synthesis, Computational Study and Biometal Complexation Capability.

Considering our previous experience in the design of new cholinesterase inhibitors, especially resveratrol analogs, in this research, the basic stilbene skeleton was used as a structural unit for new carbamates designed as potentially highly selective butyrylcholinesterase (BChE) inhibitors with excellent absorption, distribution, metabolism, excretion and toxicity ADMET properties. The inhibitory activity of newly prepared carbamates 1-13 was tested toward the enzymes acetylcholinesterase (AChE) and BChE. In the tested group of compounds, the leading inhibitors were 1 and 7, which achieved excellent selective inhibitory activity for BChE with IC50 values of 0.12 ± 0.09 μM and 0.38 ± 0.01 μM, respectively. Both were much more active than the standard inhibitor galantamine against BChE. Molecular docking of the most promising inhibitor candidates, compounds 1 and 7, revealed that stabilizing interactions between the active site residues of BChE and the ligands involve π-stacking, alkyl-π interactions, and, when the carbamate orientation allows, H-bond formation. MD analysis confirmed the stability of the obtained complexes. Some bioactive resveratrol-based carbamates displayed complex-forming capabilities with Fe3+ ions as metal centers. Spectrophotometric investigation indicated that they coordinate one or two metal ions, which is in accordance with their chemical structure, offering two binding sites: an amine and a carboxylic group in the carbamate moiety. Based on the obtained in silico, experimental and computational results on biological activity in the present work, new carbamates 1 and 7 represent potential selective BChE inhibitors as new therapeutics for neurological disorders.

Small molecule inhibits KCNQ channels with a non-blocking mechanism.

Voltage-gated ion channels (VGICs) are crucial targets for neuropsychiatric therapeutics owing to their role in controlling neuronal excitability and the established link between their dysfunction and neurological diseases, highlighting the importance of identifying modulators with distinct mechanisms. Here we report two small-molecule modulators with the same chemical scaffold, Ebio2 and Ebio3, targeting a potassium channel KCNQ2, with opposite effects: Ebio2 acts as a potent activator, whereas Ebio3 serves as a potent and selective inhibitor. Guided by cryogenic electron microscopy, patch-clamp recordings and molecular dynamics simulations, we reveal that Ebio3 attaches to the outside of the inner gate, employing a unique non-blocking inhibitory mechanism that directly squeezes the S6 pore helix to inactivate the KCNQ2 channel. Ebio3 also showed efficacy in inhibiting currents of KCNQ2 pathogenic gain-of-function mutations, presenting an avenue for VGIC-targeted therapies. Overall, these findings contribute to the understanding of KCNQ2 inhibition and provide insights into developing selective, non-blocking VGIC inhibitors.

Case report: Selpercatinib in the treatment of RET fusion-positive advanced lung adenocarcinoma: a challenging clinical case

BackgroundRearranged during transfection (RET) fusions represent a distinct molecular subset of non-small cell lung cancer (NSCLC) with targeted therapeutic potential. Selpercatinib, a highly selective RET inhibitor, has demonstrated efficacy in various solid tumors harboring RET alterations. Here, we present a case highlighting the use and clinical outcomes of selpercatinib in a patient diagnosed with advanced lung adenocarcinoma harboring a RET fusion.Case presentationA 59-year-old woman with a history of stage IV lung adenocarcinoma harboring a KIF5B-RET fusion presented with disease progression following first-line chemo-immunotherapy. Selpercatinib was initiated as a targeted therapy, leading to a notable radiographic response and clinical improvement. The patient experienced a significant reduction in tumor burden and reported improved symptom control, with no significant adverse effects during the 21-month follow-up period.ConclusionsThis case highlights the efficacy and tolerability of selpercatinib in treating advanced lung adenocarcinoma with a RET fusion. The observed clinical response supports the early use of selpercatinib as a targeted therapy for RET fusion-positive NSCLC, including in patients with compromised general and respiratory conditions, especially in cases refractory to conventional treatments. Long-term follow-up studies are warranted to validate these findings and assess the durability of responses.

Selective Gamma-Secretase Inhibition by CHF5074 Attenuates Inflammation and Neovascularization in a Murine Model of Choroidal Neovascularization

Purpose Chronic inflammation plays an important role in the pathogenesis of choroidal neovascularization (CNV). This study aimed to investigate the effect of the CHF5074, a γ-secretase inhibitor, on angiogenesis in a laser-induced CNV model and elucidate its possible molecular mechanism. Methods Male C57/BL6J mice aged between 6 to 8 weeks were employed to set up a laser-induced model of CNV. Then, CHF5074 was injected intraperitoneally on the day after laser modeling, as well as on the second, third, and fourth days. Immunofluorescence staining was used to evaluate the retinal and choroidal complex. The markers used were CD31 for neovascularization and IBA1 for microglia staining in ocular tissue slices. Fundus fluorescein angiography on days 3d, 7d, and 14d analyzed neovascularization and leakage areas. Inflammatory indicators were examined by Western blot (WB) and enzyme-linked immunosorbent assay (ELISA). High-throughput whole-tissue sequencing of retinal choroids identified relevant cell pathways. Key regulatory factors modulated by CHF5074 were identified via WB. Co-culture of BV2 cells and human umbilical vein endothelial cells (HUVEC) were used to explore the function of CHF5074 on the inhibition of tube formation. Results CHF5074 significantly decreased CD31 expression in the choroid on 3d, 7d, and 14d post-laser modeling (p < 0.05) and decreased both neovascularization and leakage areas (p < 0.05). Additionally, CHF5074 significantly lowered TNF-α, IL-10, and IL-1β expression levels in the choroid (p < 0.05), as demonstrated by WB analysis and ELISA. High-throughput whole-tissue sequencing identified P38-MAPK, JNK, and Wnt signaling pathways associated with neovascularization. CHF5074 decreased P38 protein phosphorylation (p < 0.05) as confirmed by WB analysis. CHF5074 inhibited the tube formation of HUVECs co-cultured with LPS and ATP-treated BV2 cells. Conclusion CHF5074 significantly suppresses angiogenesis in laser-induced choroidal neovascularization models, suggesting its potential as a novel agent for preventing and treating CNV.

Ferroptosis Inducers Erastin and RSL3 Enhance Adriamycin and Topotecan Sensitivity in ABCB1/ABCG2-Expressing Tumor Cells.

Acquired resistance to chemotherapeutic drugs is the primary cause of treatment failure in the clinic. While multiple factors contribute to this resistance, increased expression of ABC transporters-such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance proteins-play significant roles in the development of resistance to various chemotherapeutics. We found that Erastin, a ferroptosis inducer, was significantly cytotoxic to NCI/ADR-RES, a P-gp-expressing human ovarian cancer cell line. Here, we examined the effects of both Erastin and RSL3 (Ras-Selected Ligand 3) on reversing Adriamycin resistance in these cell lines. Our results show that Erastin significantly enhanced Adriamycin uptake in NCI/ADR-RES cells without affecting sensitive cells. Furthermore, we observed that Erastin enhanced Adriamycin cytotoxicity in a time-dependent manner. The selective iNOS inhibitor, 1400W, reduced both uptake and cytotoxicity of Adriamycin in P-gp-expressing NCI/ADR-RES cells only. These findings were also confirmed in a BCRP-expressing human breast cancer cell line (MCF-7/MXR), which was selected for resistance to Mitoxantrone. Both Erastin and RSL3 were found to be cytotoxic to MCF-7/MXR cells. Erastin significantly enhanced the uptake of Hoechst dye, a well-characterized BCRP substrate, sensitizing MCF-7/MXR cells to Topotecan. The effect of Erastin was inhibited by 1400W, indicating that iNOS is involved in Erastin-mediated enhancement of Topotecan cytotoxicity. RSL3 also significantly increased Topotecan cytotoxicity. Our findings-demonstrating increased cytotoxicity of Adriamycin and Topotecan in P-gp- and BCRP-expressing cells-suggest that ferroptosis inducers may be highly valuable in combination with other chemotherapeutics to manage patients' cancer burden in the clinical setting.

The Selective WEE1 Inhibitor Azenosertib Shows Synergistic Antitumor Activity with KRASG12C Inhibitors in Preclinical Models.

KRAS is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. KRASG12C mutations are common and targetable alterations. Therapeutic inhibition of KRASG12C and eventual resistance to these inhibitors are also known to drive RS and DNA damage through adaptive mechanisms that maintain addiction to high MAPK signaling. High levels of RS result in a stronger reliance on cell cycle checkpoints, thereby introducing a vulnerability to inhibition of cell cycle checkpoint regulators such as the WEE1 kinase. This provides rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with KRASG12C inhibitors. In vitro combination of azenosertib with multiple KRASG12C inhibitors demonstrated synergistic cell growth inhibition across a panel of KRASG12C cell lines in both 2D and 3D assays. In vivo studies demonstrated azenosertib exhibited significant monotherapy activity as well as synergistic tumor growth inhibition (TGI) when combined with KRASG12C inhibitors, including tumor regression in CDX models of NSCLC, CRC, and PDAC. Importantly, KRASG12C inhibitor-resistant CDX and PDX models demonstrated synergistic TGI in combination arms. Finally, analysis of biomarkers from in vitro and in vivo tumor samples displayed increases in protein markers of RS, DNA damage, and apoptosis after combination treatment. Taken together, our results suggest that the combination of azenosertib with KRASG12C inhibitors enhances tumor growth inhibition over single agent therapy and may be an effective treatment strategy for patients with KRASG12C tumors.

Adenine nucleotide translocator and ATP synthase cooperate in mediating the mitochondrial permeability transition.

The permeability transition (PT) is a permeability increase of the mitochondrial inner membrane causing mitochondrial swelling in response to matrix Ca2+. The PT is mediated by regulated channel(s), the PT pore(s) (PTP), which can be generated by at least two components, adenine nucleotide translocator (ANT) and ATP synthase. Whether these provide independent permeation pathways remains to be established. Here, we assessed the contribution of ANT to the PT based on the effects of the selective ANT inhibitors atractylate (ATR) and bongkrekate (BKA), which trigger and inhibit channel formation by ANT, respectively. BKA partially inhibited Ca2+-dependent PT and did not prevent the inducing effect of phenylarsine oxide, which was still present in mouse embryonic fibroblasts deleted for all ANT isoforms. The contribution of ANT to the PT emerged at pH 6.5 (a condition that inhibits ATP synthase channel opening) in the presence of ATR, which triggered mitochondrial swelling and elicited currents in patch-clamped mitoplasts. Unexpectedly, ANT-dependent PT at pH 6.5 could also be stimulated by benzodiazepine-423 [a selective ligand of the oligomycin sensitivity conferral protein (OSCP) subunit of ATP synthase], suggesting that the ANT channel is regulated by the peripheral stalk of ATP synthase. In keeping with docking simulations, ANT could be co-immunoprecipitated with ATP synthase subunits c and g, and oligomycin (which binds adjacent c subunits) decreased the association of ANT with subunit c. These results reveal a close cooperation between ANT and ATP synthase in the PT and open new perspectives in the study of this process. KEY POINTS: We have assessed the relative role of adenine nucleotide translocator (ANT) and ATP synthase in generating the mitochondrial permeability transition (PT). At pH 7.4, bongkrekate had little effect on Ca2+-dependent PT, and did not prevent the inducing effect of phenylarsine oxide, which was still present in mouse embryonic fibroblasts deleted for all ANT isoforms. The contribution of ANT emerged at pH 6.5 (which inhibits ATP synthase channel opening) in the presence of atractylate, which triggered mitochondrial swelling and elicited currents in patch-clamped mitoplasts. Benzodiazepine-423, a selective ligand of the oligomycin sensitivity conferral protein subunit of ATP synthase, stimulated ANT-dependent PT at pH 6.5, suggesting that the ANT channel is regulated by the peripheral stalk of ATP synthase. ANT could be co-immunoprecipitated with ATP synthase subunits c and g; oligomycin, which binds adjacent c subunits, decreased the association with subunit c, in keeping with docking simulations.

INOS-Produced Nitric Oxide from Cancer Cells as an Intermediate of Stemness Regulation by PARP-1 in Colorectal Cancer.

PARP-1 has been linked to the progression of several types of cancer. We have recently reported that PARP-1 influences tumor progression in CRC through the regulation of CSCs in a p53-dependent manner. In this study, we propose that nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) could act as a mediator. We evaluated the expression of iNOS in a cohort of patients previously used to analyze the effects of PARP-1 on CRC in relation to p53 status. We also developed an in vitro model in which PARP-1 was stably overexpressed. In CRC patients, iNOS expression correlated with the differentiation grade, and with a high expression of CSC markers, although only in wild-type p53 tumors, as previously found for PARP-1. In vitro, overexpression of PARP-1 induced increased growth and stemness in wild-type p53 cells, while exerting the opposite effect on mutated ones, as expected. Treatment with 1400 W, a selective inhibitor of iNOS, or gene silencing of the gene counteracted the effects of PARP-1 in both p53 wild-type and p53 mutated cells. Given that the development of resistance has been demonstrated after treatment with PARP-1 inhibitors, iNOS could be considered a new therapeutic target in CRC, although only in patients with wild-type p53 tumors.

Precision Medicine for High-Risk Gene Fusions in Paediatric AML: a focus on KMT2A, NUP98, and GLIS2 Rearrangements.

Robust genetic characterization of paediatric AML has demonstrated that fusion oncogenes are highly prevalent drivers of AML leukemogenesis in young children. Identification of fusion oncogenes associated with adverse outcomes has facilitated risk stratification of patients, although successful development of precision medicine approaches for most fusion-driven AML subtypes have been historically challenging. This knowledge gap has been in part due to difficulties in targeting structural alterations involving transcription factors and in identification of a therapeutic window for selective inhibition of the oncofusion without deleterious effects upon essential wild-type proteins. Herein, we discuss the current molecular landscape and functional characterisation of three of the most lethal childhood AML fusion-oncogene driven subtypes harbouring KMT2A, NUP98, or CBFA2T3::GLIS2 rearrangements. We further review early-phase clinical trial data of novel targeted inhibitors and immunotherapies that have demonstrated initial success specifically for children with these poor-prognosis genetic subtypes of AML and provide appreciable optimism to improve clinical outcomes in the future.