Neutrophil-activating secretome characterizes palbociclib-induced senescence of breast cancer cells.

Senescent cells have a profound impact on the surrounding microenvironment through the secretion of numerous bioactive molecules and inflammatory factors. The induction of therapy-induced senescence by anticancer drugs is known, but how senescent tumor cells influence the tumor immune landscape, particularly neutrophil activity, is still unclear. In this study, we investigate the induction of cellular senescence in breast cancer cells and the subsequent immunomodulatory effects on neutrophils using the CDK4/6 inhibitor palbociclib, which is approved for the treatment of breast cancer and is under intense investigation for additional malignancies. Our research demonstrates that palbociclib induces a reversible form of senescence endowed with an inflammatory secretome capable of recruiting and activating neutrophils, in part through the action of interleukin-8 and acute-phase serum amyloid A1. The activation of neutrophils is accompanied by the release of neutrophil extracellular trap and the phagocytic removal of senescent tumor cells. These findings may be relevant for the success of cancer therapy as neutrophils, and neutrophil-driven inflammation can differently affect tumor progression. Our results reveal that neutrophils, as already demonstrated for macrophages and natural killer cells, can be recruited and engaged by senescent tumor cells to participate in their clearance. Understanding the interplay between senescent cells and neutrophils may lead to innovative strategies to cope with chronic or tumor-associated inflammation.

Design, synthesis, and evaluation of formylpiperazine analogs of Ferrostatin-1 as novel improved ferroptosis inhibitors.

In this study, a series of new formylpiperazine-derived ferroptosis inhibitors were designed and synthesized based on the structure of a known ferroptosis inhibitor, ferrostatin-1 (Fer-1). The anti-ferroptosis activity of these synthetic compounds in human umbilical vein endothelial cells (HUVECs) induced by Erastin was evaluated. It was found that some of the new compounds, especially compound 26, showed potent anti-ferroptosis activity, as evidenced by its ability to restore cell viability, reduce iron accumulation, scavenge reactive oxygen species, maintain mitochondrial membrane potential, increase GSH levels, decrease LPO and MDA content, and upregulate GPX4 expression. Moreover, compound 26 exhibited superior microsomal stability than Fer-1. The present results suggest that compound 26 is a promising lead compound for the development of new ferroptosis inhibitors for the treatment of vascular diseases.

Inactivation of VRK1 sensitizes ovarian cancer to PARP inhibition through regulating DNA-PK stability.

Ovarian cancer is the leading cause of gynecologic cancer death. Among the most innovative anti-cancer approaches, the genetic concept of synthetic lethality is that mutations in multiple genes work synergistically to effect cell death. Previous studies found that although vaccinia-related kinase-1 (VRK1) associates with DNA damage repair proteins, its underlying mechanisms remain unclear. Here, we found high VRK1 expression in ovarian tumors, and that VRK1 depletion can significantly promote apoptosis and cell cycle arrest. The effect of VRK1 knockdown on apoptosis was manifested by increased DNA damage, genomic instability, and apoptosis, and also blocked non-homologous end joining (NHEJ) by destabilizing DNA-PK. Further, we verified that VRK1 depletion enhanced sensitivity to a PARP inhibitor (PARPi), olaparib, promoting apoptosis through DNA damage, especially in ovarian cancer cell lines with high VRK1 expression. Proteins implicated in DNA damage responses are suitable targets for the development of new anti-cancer therapeutic strategies, and their combination could represent an alternative form of synthetic lethality. Therefore, normal protective DNA damage responses are impaired by combining olaparib with elimination of VRK1 and could be used to reduce drug dose and its associated toxicity. In summary, VRK1 represents both a potential biomarker for PARPi sensitivity, and a new DDR-associated therapeutic target, in ovarian cancer.

Meningioma achieves malignancy and erastin-induced ferroptosis resistance through FOXM1-AURKA-NRF2 axis.

The oncogene Aurora kinase A (AURKA) has been implicated in various tumor, yet its role in meningioma remains unexplored. Recent studies have suggested a potential link between AURKA and ferroptosis, although the underlying mechanisms are unclear. This study presented evidence of AURKA upregulation in high grade meningioma and its ability to enhance malignant characteristics. We identified AURKA as a suppressor of erastin-induced ferroptosis in meningioma. Mechanistically, AURKA directly interacted with and phosphorylated kelch-like ECH-associated protein 1 (KEAP1), thereby activating nuclear factor erythroid 2 related factor 2 (NFE2L2/NRF2) and target genes transcription. Additionally, forkhead box protein M1 (FOXM1) facilitated the transcription of AURKA. Suppression of AURKA, in conjunction with erastin, yields significant enhancements in the prognosis of a murine model of meningioma. Our study elucidates an unidentified mechanism by which AURKA governs ferroptosis, and strongly suggests that the combination of AURKA inhibition and ferroptosis-inducing agents could potentially provide therapeutic benefits for meningioma treatment.

Comprehensive Transcriptomic Analysis of EWSR1::WT1 Targets Identifies CDK4/6 Inhibitors as an Effective Therapy for Desmoplastic Small Round Cell Tumors.

Desmoplastic small round cell tumors (DSRCT) are a type of aggressive, pediatric sarcoma characterized by the EWSR1::WT1 fusion oncogene. Targeted therapies for DSRCT have not been developed, and standard multimodal therapy is insufficient, leading to a 5-year survival rate of only 15% to 25%. Here, we depleted EWSR1::WT1 in DSRCT and established its essentiality in vivo. Transcriptomic analysis revealed that EWSR1::WT1 induces unique transcriptional alterations compared with WT1 and other fusion oncoproteins and that EWSR1::WT1 binding directly mediates gene upregulation. The E-KTS isoform of EWSR1::WT1 played a dominant role in transcription, and it bound to the CCND1 promoter and stimulated DSRCT growth through the cyclin D-CDK4/6-RB axis. Treatment with the CDK4/6 inhibitor palbociclib successfully reduced growth in two DSRCT xenograft models. As palbociclib has been approved by the FDA for the treatment of breast cancer, these findings demonstrate the sensitivity of DSRCT to palbociclib and support immediate clinical investigation of palbociclib for treating this aggressive pediatric cancer. SIGNIFICANCE: EWSR1::WT1 is essential for desmoplastic small round cell tumors and upregulates the cyclin D-CDK4/6-RB axis that can be targeted with palbociclib, providing a targeted therapeutic strategy for treating this deadly tumor type.

Targeting cellular senescence as a therapeutic vulnerability in gastric cancer.

AIMS: Cellular senescence (CS) represents an intracellular defense mechanism responding to stress signals and can be leveraged as a "vulnerability" in cancer treatment. This study aims to construct a CS atlas for gastric cancer (GC) and uncover potential therapeutics for GC patients. MATERIALS AND METHODS: 38 senescence-associated regulators with prognostic significance in GC were obtained from the CellAge database to construct Gastric cancer-specific Senescence Score (GSS). Using eXtreme Sum algorism, GSS-based drug repositioning was conducted to identify drugs that could antagonize GSS in CMap database. In vitro experiments were conducted to test the effect of combination of palbociclib and exisulind in eliminating GC cells. KEY FINDINGS: Patients with high GSS exhibited CS-related features, such as CS markers upregulation, adverse clinical outcomes and hypomethylation status. scRNA-seq data showed malignant cells with high GSS exhibited enhanced senescence state and more immunosuppressive signals such as PVR-CD96 compared with malignant cells with low GSS. In addition, the GSS-High cancer associated fibroblasts might secrete cytokines and chemokines such as IL-6, CXCL1, CXCL12, and CCL2 to from an immunosuppressive microenvironment, and GSS could serve as an indicator for immunotherapy resistance. Exisulind exhibited the greatest potential to reverse GSS. In vitro experiments demonstrated that exisulind could induce apoptosis and suppress the proliferation of palbociclib-induced senescent GC cells. SIGNIFICANCE: Overall, GSS offers a framework for better understanding of correlation between senescence and GC, which might provide new insights into the development of novel therapeutics in GC.

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

The coupling between healthspan and lifespan in Caenorhabditis depends on complex interactions between compound intervention and genetic background.

Aging is characterized by declining health that results in decreased cellular resilience and neuromuscular function. The relationship between lifespan and health, and the influence of genetic background on that relationship, has important implications in the development of pharmacological anti-aging interventions. Here we assessed swimming performance as well as survival under thermal and oxidative stress across a nematode genetic diversity test panel to evaluate health effects for three compounds previously studied in the Caenorhabditis Intervention Testing Program and thought to promote longevity in different ways - NP1 (nitrophenyl piperazine-containing compound 1), propyl gallate, and resveratrol. Overall, we find the relationships among median lifespan, oxidative stress resistance, thermotolerance, and mobility vigor to be complex. We show that oxidative stress resistance and thermotolerance vary with compound intervention, genetic background, and age. The effects of tested compounds on swimming locomotion, in contrast, are largely species-specific. In this study, thermotolerance, but not oxidative stress or swimming ability, correlates with lifespan. Notably, some compounds exert strong impact on some health measures without an equally strong impact on lifespan. Our results demonstrate the importance of assessing health and lifespan across genetic backgrounds in the effort to identify reproducible anti-aging interventions, with data underscoring how personalized treatments might be required to optimize health benefits.

Alpha-synuclein interaction with mitochondria is the final mechanism of ferroptotic death induced by erastin in SH-SY5Y cells.

Ferroptosis has been characterized as a form of iron-dependent regulated cell death accompanied by an accumulation of reactive oxygen species and lipid oxidation products along with typical morphological alterations in mitochondria. Ferroptosis is activated by diverse triggers and inhibited by ferrostatin-1 and liproxstatin-1, apart from iron chelators and several antioxidants, and the process is implicated in multiple pathological conditions. There are, however, certain ambiguities about ferroptosis, especially regarding the final executioner of cell death subsequent to the accumulation of ROS. This study uses a typical inducer of ferroptosis such as erastin on SH-SY5Y cells, and shows clearly that ferroptotic death of cells is accompanied by the loss of mitochondrial membrane potential and intracellular ATP content along with an accumulation of oxidative stress markers. All these are prevented by ferrostatin-1 and liproxstatin-1. Additionally, cyclosporine A prevents mitochondrial alterations and cell death induced by erastin implying the crucial role of mitochondrial permeability transition pore (mPTP) activation in ferroptotic death. Furthermore, an accumulation of α-synuclein occurs during erastin induced ferroptosis which can be inhibited by ferrostatin-1 and liproxstatin-1. When the knock-down of α-synuclein expression is performed by specific siRNA treatment of SH-SY5Y cells, the mitochondrial impairment and ferroptotic death of the cells induced by erastin are markedly prevented. Thus, α-synuclein through the involvement of mPTP appears to be the key executioner protein of ferroptosis induced by erastin, but it needs to be verified if it is a generalized mechanism of ferroptosis by using other inducers and cell lines.

Discovery of Dolutegravir Derivative against Liver Cancer via Inducing Autophagy and DNA Damage.

We introduced a terminal alkyne into the core structure of dolutegravir, resulting in the synthesis of 34 novel dolutegravir-1,2,3-triazole compounds through click chemistry. These compounds exhibited remarkable inhibitory activities against two hepatocellular carcinoma cell lines, Huh7 and HepG2. Notably, compounds 5e and 5p demonstrated exceptional efficacy, particularly against Huh7 cells, with IC50 values of 2.64 and 5.42 µM. Additionally, both compounds induced apoptosis in Huh7 cells, suppressed tumor cell clone formation, and elevated reactive oxygen species (ROS) levels, further promoting tumor cell apoptosis. Furthermore, compounds 5e and 5p activated the LC3 signaling pathway, inducing autophagy, and triggered the γ-H2AX signaling pathway, resulting in DNA damage in tumor cells. Compound 5e exhibited low toxicity, highlighting its potential as a promising anti-tumor drug.

Multitarget action of Benzothiazole-piperazine small hybrid molecule against Alzheimer's disease: In silico, In vitro, and In vivo investigation.

A novel small molecule based on benzothiazole-piperazine has been identified as an effective multi-target-directed ligand (MTDL) against Alzheimer's disease (AD). Employing a medicinal chemistry approach, combined with molecular docking, MD simulation, and binding free energy estimation, compound 1 emerged as a potent MTDL against AD. Notably, compound 1 demonstrated efficient binding to both AChE and Aß1-42, involving crucial molecular interactions within their active sites. It displayed a binding free energy (ΔGbind) -18.64± 0.16 and -16.10 ± 0.18 kcal/mol against AChE and Aß1-42, respectively. In-silico findings were substantiated through rigorous in vitro and in vivo studies. In vitro analysis confirmed compound 1 (IC50=0.42 µM) as an effective, mixed-type, and selective AChE inhibitor, binding at both the enzyme's catalytic and peripheral anionic sites. Furthermore, compound 1 demonstrated a remarkable ability to reduce the aggregation propensity of Aß, as evidenced by Confocal laser scanning microscopy and TEM studies. Remarkably, in vivo studies exhibited the promising therapeutic potential of compound 1. In a scopolamine-induced memory deficit mouse model of AD, compound 1 showed significantly improved spatial memory and cognition. These findings collectively underscore the potential of compound 1 as a promising therapeutic candidate for the treatment of AD.

Multifunctional fluorescent probe for simultaneous revealing Cys and ONOO- dynamic correlation in the ferroptosis.

Ferroptosis is a type of lipid peroxidation-induced apoptosis brought on by imbalances in iron metabolism and redox. It involves both the thiol-associated anti-ferroptosis pathway and the excessive buildup of reactive oxygen species (ROS), which stimulates the ferroptosis pathway. Determining the precise control mechanism of ferroptosis requires examining the dynamic connection between reactive sulfur species (RSS) and ROS. Cysteine (Cys) and peroxynitrite (ONOO-) are highly active redox species in organisms and play dynamic roles in the ferroptosis process. In this study, a coumarin dye was conjugated with specific response sites for Cys and ONOO-, enabling the simultaneous detection of Cys and ONOO- through the green and red fluorescence channels, respectively (λem = 498 nm for Cys and λem = 565 nm for ONOO-). Using the probe LXB, we monitored the changes in Cys and ONOO- levels in the ferroptosis pathway induced by erastin. The results demonstrate a significant generation of ONOO- and a noticeable decrease in intracellular Cys levels at the beginning upon erastin treatment and finally maintains a relatively low level. This study presents the first probe to investigate the intracellular redox modulation and control between Cys and ONOO- during ferroptosis, providing valuable insights into the potential mutual correlation between Cys and ONOO- in this process.

Design, synthesis, and antiviral activity of 1-aryl-4-arylmethylpiperazine derivatives as Zika virus inhibitors with broad antiviral spectrum.

Zika virus (ZIKV) disease has been given attention due to the risk of congenital microcephaly and neurodevelopmental disorders after ZIKV infection in pregnancy, but no vaccine or antiviral drug is available. Based on a previously reported ZIKV inhibitor ZK22, a series of novel 1-aryl-4-arylmethylpiperazine derivatives was designed, synthesized, and investigated for antiviral activity by quantify cellular ZIKV RNA amount using RT-qPCR method in ZIKV-infected human venous endothelial cells (HUVECs) assay. Structure-activity relationship (SAR) analysis demonstrated that anti-ZIKV activity of 1-aryl-4-arylmethylpiperazine derivatives is not correlated with molecular hydrophobicity, multiple new derivatives with pyridine group to replace the benzonitrile moiety of ZK22 showed stronger antiviral activity, higher ligand lipophilicity efficiency as well as lower cytotoxicity. Two active compounds 13 and 33 were further identified as novel ZIKV entry inhibitors with the potential of oral available. Moreover, both ZK22 and newly active derivatives also possess of obvious inhibition on the viral replication of coronavirus and influenza A virus at low micromolar level. In summary, this work provided better candidates of ZIKV inhibitor for preclinical study and revealed the promise of 1-aryl-4-arylmethylpiperazine chemotype in the development of broad-spectrum antiviral agents.

Cryptic piperazine derivatives activated by knocking out the global regulator LaeA in Aspergillus flavipes.

Genome sequencing on an intertidal zone-derived Aspergillus flavipes strain revealed its great potential to produce secondary metabolites. To activate the cryptic compounds of A. flavipes, the global regulator flLaeA was knocked out, leading to substantial up-regulation of the expression of two NRPS-like biosynthetic gene clusters in the ΔflLaeA mutant. With a scaled-up fermentation of the ΔflLaeA strain, five compounds, including two previously undescribed piperazine derivatives flavipamides A and B (1 and 2), along with three known compounds (3-5), were obtained by LC-MS guided isolation. The new compounds were elucidated by spectroscopic analysis and electronic circular dichroism (ECD) calculations, and the biosynthetic pathway was proposed on the bias of bioinformatic analysis and 13C isotope labeling evidence. This is the first report to access cryptic fungi secondary metabolites by inactivating global regulator LaeA and may provide a new approach to discovering new secondary metabolites by such genetic manipulation.

Inhibitor Trapping in Kinases.

Recently, we identified a novel mechanism of enzyme inhibition in N-myristoyltransferases (NMTs), which we have named 'inhibitor trapping'. Inhibitor trapping occurs when the protein captures the small molecule within its structural confines, thereby preventing its free dissociation and resulting in a dramatic increase in inhibitor affinity and potency. Here, we demonstrate that inhibitor trapping also occurs in the kinases. Remarkably, the drug imatinib, which has revolutionized targeted cancer therapy, is entrapped in the structure of the Abl kinase. This effect is also observed in p38α kinase, where inhibitor trapping was found to depend on a 'magic' methyl group, which stabilizes the protein conformation and increases the affinity of the compound dramatically. Altogether, these results suggest that inhibitor trapping is not exclusive to N-myristoyltransferases, as it also occurs in the kinase family. Inhibitor trapping could enhance the binding affinity of an inhibitor by thousands of times and is as a key mechanism that plays a critical role in determining drug affinity and potency.

Serotonin Signaling in Hippocampus during Initial Cocaine Abstinence Drives Persistent Drug Seeking.

The initiation of abstinence after chronic drug self-administration is stressful. Cocaine-seeking behavior on the first day of the absence of the expected drug (Extinction Day 1, ED1) is reduced by blocking 5-HT signaling in dorsal hippocampal cornu ammonis 1 (CA1) in both male and female rats. We hypothesized that the experience of ED1 can substantially influence later relapse behavior and that dorsal raphe (DR) serotonin (5-HT) input to CA1 may be involved. We inhibited 5-HT1A/1B receptors (WAY-100635 plus GR-127935), or DR input (chemogenetics), in CA1 on ED1 to test the role of this pathway on cocaine-seeking persistence 2 weeks later. We also inhibited 5-HT1A or 5-HT1B receptors in CA1 during conditioned place preference (CPP) for cocaine, to examine mechanisms involved in the persistent effects of ED1 manipulations. Inhibition of DR inputs, or 5-HT1A/1B signaling, in CA1 decreased drug seeking on ED1 and decreased cocaine seeking 2 weeks later revealing that 5-HT signaling in CA1 during ED1 contributes to persistent drug seeking during abstinence. In addition, 5-HT1B antagonism alone transiently decreased drug-associated memory performance when given prior to a CPP test, whereas similar antagonism of 5-HT1A alone had no such effect but blocked CPP retrieval on a test 24 h later. These CPP findings are consistent with prior work showing that DR inputs to CA1 augment recall of the drug-associated context and drug seeking via 5-HT1B receptors and prevent consolidation of the updated nondrug context via 5-HT1A receptors. Thus, treatments that modulate 5-HT-dependent memory mechanisms in CA1 during initial abstinence may facilitate later maintenance of abstinence.

Cannabinoids induce cell death in leukaemic cells through Parthanatos and PARP-related metabolic disruptions.

BACKGROUND: Several studies have described a potential anti-tumour effect of cannabinoids (CNB). CNB receptor 2 (CB2) is mostly present in hematopoietic stem cells (HSC). The present study evaluates the anti-leukaemic effect of CNB. METHODS: Cell lines and primary cells from acute myeloid leukaemia (AML) patients were used and the effect of the CNB derivative WIN-55 was evaluated in vitro, ex vivo and in vivo. RESULTS: We demonstrate a potent antileukemic effect of WIN-55 which is abolished with CB antagonists. WIN-treated mice, xenografted with AML cells, had better survival as compared to vehicle or cytarabine. DNA damage-related genes were affected upon exposure to WIN. Co-incubation with the PARP inhibitor Olaparib prevented WIN-induced cell death, suggesting PARP-mediated apoptosis which was further confirmed with the translocation of AIF to the nucleus observed in WIN-treated cells. Nicotinamide prevented WIN-related apoptosis, indicating NAD+ depletion. Finally, WIN altered glycolytic enzymes levels as well as the activity of G6PDH. These effects are reversed through PARP1 inhibition. CONCLUSIONS: WIN-55 exerts an antileukemic effect through Parthanatos, leading to translocation of AIF to the nucleus and depletion of NAD+, which are reversed through PARP1 inhibition. It also induces metabolic disruptions. These effects are not observed in normal HSC.

Senolytic drugs dasatinib and quercetin combined with Carboplatin or Olaparib reduced the peritoneal and adipose tissue metastasis of ovarian cancer.

Chemotherapy and targeted drugs-induced senescent ovarian cancer cells that accumulate in peritoneal adipose tissue contribute significantly to chronic inflammation, disrupt homeostasis, and may fuel various aspects of cancer progression. However, the pro-senescence effects of chemotherapy and targeted drugs on adipose derived stem cells (ADSCs) within peritoneal adipose tissue remain poorly understood. In this study, we show that the first-line chemotherapy and targeted drugs can induce the cellular senescence of ADSCs in vitro and increase the aging of peritoneal adipose tissue in vivo. These treatments significantly promoted the dysregulation of glucose and lipid metabolism, including insulin resistance and liver lipid accumulation. Our study shows that dasatinib and quercetin, as senolytics, effectively restore glucose homeostasis in mice with ovarian cancer and significantly reduce adipose tissue aging. Importantly, combining these drugs with Carboplatin or Olaparib results in a marked decrease in both peritoneal and adipose tissue metastasis of ovarian cancer cells. Mechanistically, we revealed that there is crosstalk between ovarian cancer cells and senescent ADSCs. The crosstalk increases inflammatory cytokines and chemokines production in ADSCs and notably upregulates chemokine receptors on cancer cells. Collectively, these data indicate that senescent ADSCs induced by chemotherapy and targeted therapy drugs impair adipose tissue function. However, the senolytic drugs dasatinib and quercetin, can significantly ameliorate organ aging and damage induced by these treatments. Notably, dasatinib and quercetin combined with Carboplatin or Olaparib reduced the peritoneal and adipose tissue metastasis of ovarian cancer, ultimately benefiting the mice undergoing chemotherapy and targeted therapy.

Identification of a Panel of miRNAs Associated with Resistance to Palbociclib and Endocrine Therapy.

We investigated whether we could identify a panel of miRNAs associated with response to treatment in tumor tissues of patients with Hormone Receptor-positive/HER2-negative metastatic breast cancer treated with endocrine therapy (ET) and the CDK4/6 inhibitor (CDK4/6i)i palbociclib. In total, 52 patients were evaluated, with 41 receiving treatment as the first line. The overall median PFS was 20.8 months (range 2.5-66.6). In total, 23% of patients experienced early progression (<6 months). Seven miRNAs (miR-378e, miR-1233, miR-99b-5p, miR-1260b, miR-448, -miR-1252-5p, miR-324-3p, miR-1233-3p) showed a statistically significant negative association with PFS. When we considered PFS < 6 months, miR-378e, miR-99b-5p, miR-877-5p, miR-1297, miR-455-5p, and miR-4536-5p were statistically associated with a poor outcome. In the multivariate analysis, the first three miRNAs confirmed a significant and independent impact on PFS. The literature data and bioinformatic tools provide an underlying molecular rationale for most of these miRNAs, mainly involving the PI3K/AKT/mTOR pathway and cell-cycle machinery as cyclin D1, CDKN1B, and protein p27Kip1 and autophagy. Our findings propose a novel panel of miRNAs associated with a higher likelihood of early progression in patients treated with ET and Palbociclib and may contribute to shed some light on the mechanisms of de novo resistance to CDK4/6i, but this should be considered exploratory and evaluated in larger cohorts.

Light-Activatable Photocaged UNC2025 for Triggering TAM Kinase Inhibition in Bladder Cancer.

Photopharmacology is an emerging field that utilizes photo-responsive molecules to enable control over the activity of a drug using light. The aim is to limit the therapeutic action of a drug at the level of diseased tissues and organs. Considering the well-known implications of protein kinases in cancer and the therapeutic issues associated with protein kinase inhibitors, the photopharmacology is seen as an innovative and alternative solution with great potential in oncology. In this context, we developed the first photocaged TAM kinase inhibitors based on UNC2025, a first-in-class small molecule kinase inhibitor. These prodrugs showed good stability in biologically relevant buffer and rapid photorelease of the photoremovable protecting group upon UV-light irradiation (<10 min.). These light-activatable prodrugs led to a 16-fold decrease to a complete loss of kinase inhibition, depending on the protein and the position at which the coumarin-type phototrigger was introduced. The most promising candidate was the N,O-dicaged compound, showing the superiority of having two photolabile protecting groups on UNC2025 for being entirely inactive on TAM kinases. Under UV-light irradiation, the N,O-dicaged compound recovered its inhibitory potency in enzymatic assays and displayed excellent antiproliferative activity in RT112 cell lines.