Targeting the core program of metastasis with a novel drug combination.

BACKGROUND: We previously reported that metastases are generally characterized by a core program of gene expression that activates tissue remodeling/vascularization, alters ion homeostasis, induces the oxidative metabolism, and silences extracellular matrix interactions. This core program distinguishes metastases from their originating primary tumors as well as from their destination host tissues. Therefore, the gene products involved are potential targets for anti-metastasis drug treatment. METHODS: Because the silencing of extracellular matrix interactions predisposes to anoiks in the absence of active survival mechanisms, we tested inhibitors against the other three components. RESULTS: Individually, the low-specificity VEGFR blocker pazopanib (in vivo combined with marimastat), the antioxidant dimethyl sulfoxide (or the substitute atovaquone, which is approved for internal administration), and the ionic modulators bumetanide and tetrathiomolybdate inhibited soft agar colony formation by breast and pancreatic cancer cell lines. The individual candidate agents have a record of use in humans (with limited efficacy when administered individually) and are available for repurposing. In combination, the effects of these drugs were additive or synergistic. In two mouse models of cancer (utilizing 4T1 cells or B16-F10 cells), the combination treatment with these medications, applied immediately (to prevent metastasis formation) or after a delay (to suppress established metastases), dramatically reduced the occurrence of disseminated foci. CONCLUSIONS: The combination of tissue remodeling inhibitors, suppressors of the oxidative metabolism, and ion homeostasis modulators has very strong promise for the treatment of metastases by multiple cancers.

Niraparib enhances antitumor immunity and contributes to the efficacy of PD-L1 blockade in cervical cancer.

PURPOSE: With the development of immunotherapy research, the role of immune checkpoint blockade (ICB) in the treatment of cervical cancer has been emphasized, but many patients still can't receive long-term benefits from ICB. Poly ADP ribose polymerase inhibitor (PARPi) has been proved to exert significant antitumor effects in multiple solid tumors. Whether cervical cancer patients obtain better benefits from the treatment regimen of PARPi combined with ICB remains unclear. METHODS: The alteration of PD-L1 expression induced by niraparib in cervical cancer cells and its underlying mechanism were assessed by western blot and immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR).The regulation of PTEN by KDM5A was confirmed using Chromatin immunoprecipitation (ChIP) assay and RNA interference. Analyzing the relationship between PD-L1 and immune effector molecules through searching online databases. Therapeutic efficacy of niraparib, PD-L1 blockade or combination was assessed in syngeneic tumor model. The changes of immune cells and cytokines in vivo was detected by immunohistochemistry (IHC) and qRT-PCR. RESULTS: We found that niraparib upregulated PD-L1 expression and potentiated the antitumor effects of PD-L1 blockade in a murine cervical cancer model. Niraparib inhibited the Pten expression by increasing the abundance of KDM5A, which expanded PD-L1 abundance through activating the PI3K-AKT-S6K1 pathway. PD-L1 was positively correlated with immune effector molecules including TNF-α, IFN-γ, granzyme A and granzyme B based on biological information analysis. Niraparib increased the infiltration of CD8+ T cells and the level of IFN-γ, granzyme B in vivo. CONCLUSION: Our findings demonstrates the regulation of niraparib on local immune microenvironment of cervical cancer, and provides theoretical basis for supporting the combination of PARPi and PD-L1 blockade as a potential treatment for cervical cancer.

Effective Radiosensitization of HNSCC Cell Lines by DNA-PKcs Inhibitor AZD7648 and PARP Inhibitors Talazoparib and Niraparib.

(1) Head and neck squamous cell carcinoma (HNSCC) is common, while treatment is difficult, and mortality is high. Kinase inhibitors are promising to enhance the effects of radiotherapy. We compared the effects of the PARP inhibitors talazoparib and niraparib and that of the DNA-PKcs inhibitor AZD7648, combined with ionizing radiation. (2) Seven HNSCC cell lines, including Cal33, CLS-354, Detroit 562, HSC4, RPMI2650 (HPV-negative), UD-SCC-2 and UM-SCC-47 (HPV-positive), and two healthy fibroblast cell lines, SBLF8 and SBLF9, were studied. Flow cytometry was used to analyze apoptosis and necrosis induction (AnnexinV/7AAD) and cell cycle distribution (Hoechst). Cell inactivation was studied by the colony-forming assay. (3) AZD7648 had the strongest effects, radiosensitizing all HNSCC cell lines, almost always in a supra-additive manner. Talazoparib and niraparib were effective in both HPV-positive cell lines but only consistently in one and two HPV-negative cell lines, respectively. Healthy fibroblasts were not affected by any combined treatment in apoptosis and necrosis induction or G2/M-phase arrest. AZD7648 alone was not toxic to healthy fibroblasts, while the combination with ionizing radiation reduced clonogenicity. (4) In conclusion, talazoparib, niraparib and, most potently, AZD7648 could improve radiation therapy in HNSCC. Healthy fibroblasts tolerated AZD7648 alone extremely well, but irradiation-induced effects might occur. Our results justify in vivo studies.

Vorolanib, sunitinib, and axitinib: A comparative study of vascular endothelial growth factor receptor inhibitors and their anti-angiogenic effects.

PURPOSE: Pathological angiogenesis and vascular instability are observed in diabetic retinopathy (DR), diabetic macular edema (DME), and wet age-related macular degeneration (wAMD). Many receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptors (VEGFRs) contribute to angiogenesis, whereas the RTK TIE2 is important for vascular stability. Pan-VEGFR tyrosine kinase inhibitors (TKIs) such as vorolanib, sunitinib, and axitinib are of therapeutic interest over current antibody treatments that target only one or two ligands. This study compared the anti-angiogenic potential of these TKIs. METHODS: A kinase HotSpot™ assay was conducted to identify TKIs inhibiting RTKs associated with angiogenesis and vascular stability. Half-maximal inhibitory concentration (IC50) for VEGFRs and TIE2 was determined for each TKI. In vitro angiogenesis inhibition was investigated using a human umbilical vein endothelial cell sprouting assay, and in vivo angiogenesis was studied using the chorioallantoic membrane assay. Melanin binding was assessed using a melanin-binding assay. Computer modeling was conducted to understand the TIE2-axitinib complex as well as interactions between vorolanib and VEGFRs. RESULTS: Vorolanib, sunitinib, and axitinib inhibited RTKs of interest in angiogenesis and exhibited pan-VEGFR inhibition. HotSpot™ assay and TIE2 IC50 values showed that only axitinib potently inhibited TIE2 (up to 89%). All three TKIs effectively inhibited angiogenesis in vitro. In vivo, TKIs were more effective at inhibiting VEGF-induced angiogenesis than the anti-VEGF antibody bevacizumab. Of the three TKIs, only sunitinib bound melanin. TKIs differ in their classification and binding to VEGFRs, which is important because type II inhibitors have greater selectivity than type I TKIs. CONCLUSIONS: Vorolanib, sunitinib, and axitinib exhibited pan-VEGFR inhibition and inhibited RTKs associated with pathological angiogenesis. Of the three TKIs, only axitinib potently inhibited TIE2 which is an undesired trait as TIE2 is essential for vascular stability. The findings support the use of vorolanib for therapeutic inhibition of angiogenesis observed in DR, DME, and wAMD.

Lipid nephrotoxicity mediated by HIF-1α activation accelerates tubular injury in diabetic nephropathy.

This study is intended to explore the effect of hypoxia-inducible factor-1α (HIF-1α) activation on lipid accumulation in the diabetic kidney. A type 1 diabetic rat model was established by STZ intraperitoneal injection. Cobalt chloride (CoCl2) and YC-1 were used as the HIF-1α activator and antagonist, respectively. CoCl2 treatment significantly increased HIF-1α expression, accelerated lipid deposition, and accelerated tubular injury in diabetic kidneys. In vitro, CoCl2 effectively stabilized HIF-1α and increased its transportation from the cytoplasm to the nucleus, which was accompanied by significantly increased lipid accumulation in HK-2 cells. Furthermore, results obtained in vivo showed that HIF-1α protein expression in the renal tubules of diabetic rats was significantly downregulated by YC-1 treatment. Meanwhile, lipid accumulation in the tubules of the DM + YC-1 group was markedly decreased in comparison to the DM + DMSO group. Accordingly, PAS staining revealed that the pathological injury caused to the tubular epithelial cells was alleviated by YC-1 treatment. Furthermore, the blood glucose level, urine albumin creatinine ratio, and NAG creatinine ratio in the DM + YC-1 group were significantly decreased compared to the DM + DMSO group. Moreover, the protein expression levels of transforming growth factor ß1 (TGF-ß1) and connective tissue growth factor (CTGF) in diabetic kidneys were decreased by YC-1 treatment. Our findings demonstrate that the activation of HIF-1α contributed to interstitial injury in a rat model of diabetic nephropathy and that the underlying mechanism involved the induction of lipid accumulation.

Structure-Activity Relationships, Deuteration, and Fluorination of Synthetic Cannabinoid Receptor Agonists Related to AKB48, 5F-AKB-48, and AFUBIATA.

Synthetic cannabinoid receptor agonists (SCRAs) are a growing class of new psychoactive substances (NPS) commonly derived from an N-alkylated indole, indazole, or 7-azaindole scaffold. Diversification of this core (at the 3-position) with amide-linked pendant amino acid groups and modular N-alkylation (of the indole/indazole/7-azaindole core) ensures that novel SCRAs continue to enter the illicit drug market rapidly. In response to the large number of SCRAs that have been detected, pharmacological evaluation of this NPS class has become increasingly common. Adamantane-derived SCRAs have consistently appeared throughout the market since 2011, and as such, a systematic set of these derivatives was synthesized and pharmacologically evaluated. Deuterated and fluorinated adamantane derivatives were prepared to evaluate typical hydrogen bioisosteres, as well as evaluation of the newly detected AFUBIATA.

A new framework for novel analogues of pazopanib as potent and selective human carbonic anhydrase inhibitors: Design, repurposing rational, synthesis, crystallographic, in vivo and in vitro biological assessments.

Herein, we describe the design and synthesis of novel aryl pyrimidine benzenesulfonamides APBSs 5a-n, 6a-c, 7a-b, and 8 as pazopanib analogues to explore new potent and selective inhibitors for the CA IX. All APBSs were examined in vitro for their promising inhibition activity against a small panel of hCAs (isoforms I, II, IX, and XII). The X-ray crystal structure of CA I in adduct with a representative APBS analogue was solved. APBS-5m, endowed with the best hCA IX inhibitory efficacy and selectivity, was evaluated for antiproliferative activity against a small panel of different cancer cell lines, SK-MEL-173, MDA-MB-231, A549, HCT-116, and HeLa, and it demonstrated one-digit IC50 values range from 2.93 µM (MDA-MB-231) to 5.86 µM (A549). Furthermore, compound APBS-5m was evaluated for its influence on hypoxia-inducible factor (HIF-1α) production, apoptosis induction, and colony formation in MDA-MB-231 cancer cells. The in vivo efficacy of APBS-5m as an antitumor agent was additionally investigated in an animal model of Solid Ehrlich Carcinoma (SEC). In order to offer perceptions into the conveyed hCA IX inhibitory efficacy and selectivity in silico, a molecular docking investigation was also carried out.

QbD Enabled Development and Evaluation of Pazopanib Loaded Nanoliposomes for PDAC Treatment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the highly fatal types of cancer with high mortality/incidence. Considering the crucial role of vascular endothelial growth factor (VEGF) in PDAC progression, its inhibition can be a viable strategy for the treatment. Pazopanib, a second-generation VEGF inhibitor, is approved for the treatment of various oncological conditions. However, due to associated limitations like low oral bioavailability (14-39%), high inter/intra-subject variability, stability issues, etc., high doses (800 mg) are required, which further lead to non-specific toxicities and also contribute toward cancer resistance. Thus, to overcome these challenges, pazopanib-loaded PEGylated nanoliposomes were developed and evaluated against pancreatic cancer cell lines. The nanoliposomes were prepared by thin-film hydration method, followed by characterization and stability studies. This QbD-enabled process design successfully led to the development of a suitable pazopanib liposomal formulation with desirable properties. The % entrapment of PZP-loaded non-PEGylated and PEGylated nanoliposomes was found to be 75.2% and 84.9%, respectively, whereas their particle size was found to be 129.7 nm and 182.0 nm, respectively. The developed liposomal formulations exhibited a prolonged release and showed desirable physicochemical properties. Furthermore, these liposomal formulations were also assessed for in vitro cell lines, such as cell cytotoxicity assay and cell uptake. These studies confirm the effectiveness of developed liposomal formulations against pancreatic cancer cell lines. The outcomes of this work provide encouraging results and a way forward to thoroughly investigate its potential for PDAC treatment.

Synergistic effects of photodynamic therapy and chemotherapy: Activating the intrinsic/extrinsic apoptotic pathway of anoikis for triple-negative breast cancer treatment.

Triple-negative breast cancer (TNBC) is a highly invasive and metastatic subtype of breast cancer that often recurs after surgery. Herein, we developed a cyclodextrin-based tumor-targeted nano delivery system that incorporated the photosensitizer chlorin e6 (Ce6) and the chemotherapeutic agent lonidamine (LND) to form the R6RGD-CMßCD-se-se-Ce6/LND nanoparticles (RCC/LND NPS). This nanosystem could target cancer cells, avoid lysosomal degradation and further localize within the mitochondria. The RCC/LND NPS had pH and redox-responsive to control the release of Ce6 and LND. Consequently, the nanosystem had a synergistic effect by effectively alleviating hypoxia, enhancing the production of cytotoxic reactive oxygen species (ROS) and amplifying the efficacy of photodynamic therapy (PDT). Furthermore, the RCC/LND NPS + light weakened anoikis resistance, disrupted extracellular matrix (ECM), activated both the intrinsic apoptotic pathway (mitochondrial pathway) and extrinsic apoptotic pathway (receptor death pathway) of anoikis. In addition, the nanosystem showed significant anti-TNBC efficacy in vivo. These findings collectively demonstrated that RCC/LND NPS + light enhanced the anticancer effects, induced anoikis and inhibited tumor cell migration and invasion through a synergistic effect of chemotherapy and PDT. Overall, this study highlighted the promising potential of the RCC/LND NPS + light for the treatment of TNBC.

TYM-3-98, a novel selective inhibitor of PI3Kδ, demonstrates promising preclinical antitumor activity in B-cell lymphomas.

AIMS: PI3Kδ is expressed predominately in leukocytes and is commonly found to be aberrantly activated in human B-cell lymphomas. Although PI3Kδ has been intensively targeted for discovering anti-lymphoma drugs, the application of currently approved PI3Kδ inhibitors has been limited due to unwanted systemic toxicities, thus warranting the development of novel PI3Kδ inhibitors with new scaffolds. MAIN METHODS: We designed TYM-3-98, an indazole derivative, and evaluated its selectivity for all four PI3K isoforms, as well as its efficacy against various B-cell lymphomas both in vitro and in vivo. KEY FINDINGS: We identified TYM-3-98 as a highly selective PI3Kδ inhibitor over other PI3K isoforms at both molecular and cellular levels. It showed superior antiproliferative activity in several B-lymphoma cell lines compared with the approved first-generation PI3Kδ inhibitor idelalisib. TYM-3-98 demonstrated a concentration-dependent PI3K/AKT/mTOR signaling blockage followed by apoptosis induction. In vivo, TYM-3-98 showed good pharmaceutical properties and remarkably reduced tumor growth in a human lymphoma xenograft model and a mouse lymphoma model. SIGNIFICANCE: Our findings establish TYM-3-98 as a promising PI3Kδ inhibitor for the treatment of B-cell lymphoma.

Zymosan and lipopolysaccharide decrease gene expression of neuronal nitric oxide synthase in peripheral organs in chicks.

Nitric oxide (NO) is gaseous bioactive molecule that is synthesized by NO synthase (NOS). Inducible NOS (iNOS) expression occurs in response to pathogenic challenges, resulting in the production of large amounts of NO. However, there is a lack of knowledge regarding neuronal NOS (nNOS) and endothelial NOS (eNOS) in birds during pathogenic challenge. Therefore, the present study was conducted to determine the influence of intraperitoneal (IP) injection of zymosan (cell wall component of yeast) and lipopolysaccharide (LPS, a cell wall component of gram-negative bacteria) on NOS expression in chicks (Gallus gallus). Furthermore, the effect of NOS inhibitors on the corresponding behavioral and physiological parameters was investigated. Zymosan and LPS injections induced iNOS mRNA expression in several organs. Zymosan had no effect on eNOS mRNA expression in the organs investigated, whereas LPS increased its expression in the pancreas. Zymosan and LPS decreased nNOS mRNA expression in the lung, heart, kidney, and pancreas. The decreased nNOS mRNA expression in pancreas was probably associated with the NO from iNOS provided that such effect was reproduced by IP injection of sodium nitroprusside, which is a NO donor. Furthermore, pancreatic nNOS mRNA expression decreased following subcutaneous injection of corticosterone. Furthermore, IP injections of a nonspecific NOS inhibitor, NG-nitro-L-arginine methyl ester, and an nNOS-specific inhibitor, 7-nitroindazole, resulted in the significant decreases in food intake, cloacal temperature, and feed passage via the digestive tract in chicks. Collectively, the current findings imply the decreased nNOS expression because of fungal and bacterial infections, which affects food intake, body temperature, and the digestive function in birds.

Exploring novel indazole derivatives as ASK1 inhibitors: Design, synthesis, biological evaluation and docking studies.

Apoptosis signal regulated kinase 1 (ASK1, MAP3K5) is a member of the mitogen activated protein kinase (MAPK) signaling pathway, involved in cell survival, differentiation, stress response, and apoptosis. ASK1 kinase inhibition has become a promising strategy for the treatment of Non-alcoholic steatohepatitis (NASH) disease. A series of novel ASK1 inhibitors with indazole scaffolds were designed and synthesized, and their ASK1 kinase activities were evaluated. The System Structure Activity Relationship (SAR) study discovered a promising compound 33c, which has a strong inhibitory effect on ASK1. Noteworthy observations included a discernible reduction in lipid droplets within LO2 cells stained with Oil Red O, coupled with a decrease in LDL, CHO, and TG content within the NASH model cell group. Mechanistic inquiries revealed that compound 33c could inhibit the protein expression levels of the upregulated ASK1-p38/JNK signaling pathway in TNF-α treated HGC-27 cells and regulate apoptotic proteins. In summary, these findings suggest that compound 33c may be valuable for further research as a potential candidate compound against NASH.

Inhibition of human UDP-glucuronosyltransferase enzyme by entrectinib: Implications for drug-drug interactions.

As a new type of oral tyrosine kinase inhibitor, entrectinib can act on multiple targets and exert efficacy and has been approved for the treatment of non-small cell lung cancer (NSCLC) and solid tumors. However, whether entrectinib affects the activities of recombinant human UDP-glucuronosyltransferases (UGTs) remains unclear. Herein, we aimed to investigate the inhibitory effects of entrectinib on human UGTs and to assess the potential risk of causing drug-drug interactions (DDIs) based on the inhibition against UGTs. High-performance liquid chromatography (HPLC) was used to evaluate the inhibitory effects of entrectinib on UGTs according to the product formation rate of UGT substrate with or without entrectinib, and the inhibition kinetics experiment was conducted to assess the inhibitory type of entrectinib on UGTs. Our results showed that entrectinib exhibited extensive inhibitory effects on most human UGTs, and especially inhibited the activities of UGT1A7, UGT1A8, and UGT2B15 with Ki (Inhibition constant) of lower than 5 µM (0.95-4.38 µM). Furthermore, the results from quantitative prediction research suggested that the combination of entrectinib at 600 mg/day with substrates primarily metabolized by hepatic UGT2B15 or intestinal UGT1A7 and UGT1A8 might cause clinical DDIs. Thus, special attention should be paid to avoid adverse reactions induced by DDIs when co-administration of entrectinib and drugs metabolized by UGTs.

Discovery of 4-phenyl-1H-indazole derivatives as novel small-molecule inhibitors targeting the PD-1/PD-L1 interaction.

The inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway with small molecules is a promising approach for cancer immunotherapy. Herein, novel small molecules compounds bearing various scaffolds including thiophene, thiazole, tetrahydroquinoline, benzimidazole and indazole were designed, synthesized and evaluated for their inhibitory activity against the PD-1/PD-L1 interaction. Among them, compound Z13 exhibited the most potent activity with IC50 of 189.6 nM in the homogeneous time-resolved fluorescence (HTRF) binding assay. Surface plasmon resonance (SPR) assay demonstrated that Z13 bound to PD-L1 with high affinity (KD values of 231 nM and 311 nM for hPD-L1 and mPD-L1, respectively). In the HepG2/Jurkat T co-culture cell model, Z13 decreased the viability rate of HepG2 cells in a concentration-dependent manner. In addition, Z13 showed significant in vivo antitumor efficacy (TGI = 52.6 % at 40 mg/kg) without obvious toxicity in the B16-F10 melanoma model. Furthermore, flow cytometry analysis demonstrated that Z13 inhibited tumor growth in vivo by activating the tumor immune microenvironment. These findings indicate that Z13 is a promising PD-1/PD-L1 inhibitor deserving further investigation.

In-silico design of novel potential HDAC inhibitors from indazole derivatives targeting breast cancer through QSAR, molecular docking and pharmacokinetics studies.

Latest studies confirmed that abnormal function of histone deacetylase (HDAC) plays a pivotal role in formation of tumors and is a potential therapeutic target for treating breast cancer. In this research, in-silico drug discovery approaches via quantitative structure activity relationship (QSAR) and molecular docking simulations were adapted to 43 compounds of indazole derivatives with HDAC inhibition for anticancer activity against breast cancer. The QSAR models were built from multiple linear regression (MLR), and models predictability was cross-validated by leave-one-out (LOO) method. Based on these results, compounds C32, C26 and C31 from model 3 showed superior inhibitory activity with pIC50 of 9.30103, 9.1549 and 9.1549. We designed 10 novel compounds with molecular docking scores ranging from -7.9 to -9.3 kcal/mol. The molecular docking simulation results reveal that amino acid residues ILE1122 and PRO1123 play a significant role in bonding with 6CE6 protein. Furthermore, newly designed compounds P5, P2 and P7 with high docking scores of -9.3 kcal/mol, -8.9 kcal/mol and -8.8 kcal/mol than FDA-approved drug Raloxifene (-8.5 kcal/mol) and aid in establishment of potential drug candidate for HDAC inhibitors. The in-silico ADME functionality is used in the final phase to evaluate newly designed inhibitors as potential drug candidates. The results suggest that newly designed compounds P5, P2 and P7 can be used as a potential anti-breast cancer drug candidate.

N1-Benzoylated 5-(4-pyridinyl)indazole-based kinase inhibitors: Attaining haspin and Clk4 selectivity via modulation of the benzoyl substituents.

Haspin and Clk4 are both understudied protein kinases (PKs), offering potential targets for the development of new anticancer agents. Thus, the identification of new inhibitors targeting these PKs is of high interest. However, the inhibitors targeting haspin or Clk4 developed to date show a poor selectivity profile over other closely related PKs, increasing the risk of side effects. Herein, we present two newly developed N1-benzyolated 5-(4-pyridinyl)indazole-based inhibitors (18 and 19), derived from a newly identified indazole hit. These inhibitors exhibit an exceptional inhibitory profile toward haspin and/or Clk4. Compound 18 (2-acetyl benzoyl) showed a preference to inhibit Clk4 and haspin over a panel of closely related kinases, with sixfold selectivity for Clk4 (IC50 = 0.088 and 0.542 µM, respectively). Compound 19 (4-acetyl benzoyl) showed high selectivity against haspin over the common off-target kinases (Dyrks and Clks) with an IC50 of 0.155 µM for haspin. Molecular docking studies explained the remarkable selectivity of 18 and 19, elucidating how the new scaffold can be modified to toggle between inhibition of haspin or Clk4, despite the high homology of the ATP-binding sites. Their distinguished profile allows these compounds to be marked as interesting chemical probes to assess the selective inhibition of haspin and/or Clk4.

Synthesis and evaluation of novel N1-acylated 5-(4-pyridinyl)indazole derivatives as potent and selective haspin inhibitors.

Protein kinase dysregulation was strongly linked to cancer pathogenesis. Moreover, histone alterations were found to be among the most important post-translational modifications that could contribute to cancer growth and development. In this context, haspin, an atypical serine/threonine kinase, phosphorylates histone H3 at threonine-3 and is notably overexpressed in various common cancer types. Herein, we report novel 5-(4-pyridinyl)indazole derivatives as potent and selective haspin inhibitors. Amide coupling at N1 of the indazole ring with m-hydroxyphenyl acetic acid yielded compound 21 with an IC50 value of 78 nM against haspin. This compound showed a meaningful selectivity over 15 of the most common off-targets, including Clk 1-3 and Dyrk1A, 1B, and 2. The most potent haspin inhibitors 5 and 21 effectively inhibited the growth of the NCI-60 cancer cell lines, further emphasizing the success of our scaffold as a new selective lead for the development of anti-cancer therapeutic agents.

Single-cell RNA sequencing reveals recruitment of the M2-like CCL8high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy.

BACKGROUND: Tumor-associated macrophages (TAMs) play a pivotal role in reshaping the tumor microenvironment following radiotherapy. The mechanisms underlying this reprogramming process remain to be elucidated. METHODS: Subcutaneous Lewis lung carcinoma (LLC) murine model was treated with hypofrationated radiotherapy (8 Gy × 3F). Single-cell RNA sequencing was utilized to identify subclusters and functions of TAMs. Multiplex assay and enzyme-linked immunosorbent assay (ELISA) were employed to measure serum chemokine levels. Bindarit was used to inhibit CCL8, CCL7, and CCL2. The infiltration of TAMs after combination treatment with hypofractionated radiotherapy and Bindarit was quantified with flow cytometry, while the influx of CD206 and CCL8 was assessed by immunostaining. RESULTS: Transcriptome analysis identified a distinct subset of M2-like macrophages characterized by elevated Ccl8 expression level following hypofractionated radiotherapy in LLC-bearing mice. Remarkbly, hypofractionated radiotherapy not only promoted CCL8high macrophages infiltration but also reprogrammed them by upregulating immunosuppressive genes, thereby fostering an immunosuppressive tumor microenvironment. Additioinally, hypofractionated radiotherapy enhanced the CCL signaling pathway, augmenting the pro-tumorigenic functions of CCL8high macrophages and boosting TAMs recruitment. The adjunctive treatment combining hypofractionated radiotherapy with Bindarit effectively reduced M2 macrophages infiltration and prolonged the duration of local tumor control. CONCLUSIONS: Hypofractionated radiotherapy enhances the infiltration of CCL8high macrophages and amplifies their roles in macrophage recruitment through the CCL signaling pathway, leading to an immunosuppressive tumor microenvironment. These findings highlight the potential of targeting TAMs and introduces a novel combination to improve the efficacy of hypofractionated radiotherapy.

Downregulation of UBB potentiates SP1/VEGFA-dependent angiogenesis in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) presents a unique profile characterized by high levels of angiogenesis and robust vascularization. Understanding the underlying mechanisms driving this heterogeneity is essential for developing effective therapeutic strategies. This study revealed that ubiquitin B (UBB) is downregulated in ccRCC, which adversely affects the survival of ccRCC patients. UBB exerts regulatory control over vascular endothelial growth factor A (VEGFA) by directly interacting with specificity protein 1 (SP1), consequently exerting significant influence on angiogenic processes. Subsequently, we validated that DNA methyltransferase 3 alpha (DNMT3A) is located in the promoter of UBB to epigenetically inhibit UBB transcription. Additionally, we found that an unharmonious UBB/VEGFA ratio mediates pazopanib resistance in ccRCC. These findings underscore the critical involvement of UBB in antiangiogenic therapy and unveil a novel therapeutic strategy for ccRCC.

Screening for Synergistic Reagents With Pazopanib Against Osteosarcoma Using a Compound Library.

BACKGROUND/AIM: Osteosarcoma (OS) is the most common malignant bone tumor. As the same agents have been in use since the mid-1970s, new therapeutic approaches are needed to improve prognosis. Pazopanib (PZP) has already demonstrated marked antitumor activity clinically and can be effective in patients with metastatic OS. We investigated the combination treatment of candidate agents with PZP and examined effects on tumor growth using an in vivo model. MATERIALS AND METHODS: A library of 324 compounds was used. MG63 OS cells were treated with PZP and each compound. Cell viability was measured. The antiproliferative effects of compound combination on four OS cell lines was tested. Cell signaling was evaluated by western blot analysis. In vivo antitumor testing was performed using 143B-bearing mice. RESULTS: The screening process identified crizotinib (CRZ) as the most effective drug for combination with PZP. The combination of PZP and CRZ demonstrated effects compared to control or single therapy. Cell signal investigation showed that dual therapy down-regulated c-MYC, p-AKT, p-STAT3, p-cyclin D1 and survivin and up-regulated cleaved caspase-3 and cleaved PARP compared to control or single therapy. In vivo analysis showed dual therapy achieved synergic effects for tumor growth compared to control or single-treatment groups. No significant difference in the change in body weight was observed among groups. CONCLUSION: Combined use of PZP and CRZ offers synergic anti-tumor effects against OS, inducing apoptosis in vitro and in vivo by down-regulating AKT and STAT3. Our data suggest that these agents can be used for patients clinically.