Multiple Cancer Cell Research Articles

Identification of resistance mechanisms to small-molecule inhibition of TEAD-regulated transcription

The Hippo tumor suppressor pathway controls transcription by regulating nuclear abundance of YAP and TAZ, which activate transcription with the TEAD1-TEAD4 DNA-binding proteins. Recently, several small-molecule inhibitors of YAP and TEADs have been reported, with some entering clinical trials for different cancers with Hippo pathway deregulation, most notably, mesothelioma. Using genome-wide CRISPR/Cas9 screens we reveal that mutations in genes from the Hippo, MAPK, and JAK-STAT signaling pathways all modulate the response of mesothelioma cell lines to TEAD palmitoylation inhibitors. By exploring gene expression programs of mutant cells, we find that MAPK pathway hyperactivation confers resistance to TEAD inhibition by reinstating expression of a subset of YAP/TAZ target genes. Consistent with this, combined inhibition of TEAD and the MAPK kinase MEK, synergistically blocks proliferation of multiple mesothelioma and lung cancer cell lines and more potently reduces the growth of patient-derived lung cancer xenografts in vivo. Collectively, we reveal mechanisms by which cells can overcome small-molecule inhibition of TEAD palmitoylation and potential strategies to enhance the anti-tumor activity of emerging Hippo pathway targeted therapies.

Bioactivity-driven fungal metabologenomics identifies antiproliferative stemphone analogs and their biosynthetic gene cluster

IntroductionFungi biosynthesize chemically diverse secondary metabolites with a wide range of biological activities. Natural product scientists have increasingly turned towards bioinformatics approaches, combining metabolomics and genomics to target secondary metabolites and their biosynthetic machinery. We recently applied an integrated metabologenomics workflow to 110 fungi and identified more than 230 high-confidence linkages between metabolites and their biosynthetic pathways.ObjectivesTo prioritize the discovery of bioactive natural products and their biosynthetic pathways from these hundreds of high-confidence linkages, we developed a bioactivity-driven metabologenomics workflow combining quantitative chemical information, antiproliferative bioactivity data, and genome sequences.MethodsThe 110 fungi from our metabologenomics study were tested against multiple cancer cell lines to identify which strains produced antiproliferative natural products. Three strains were selected for further study, fractionated using flash chromatography, and subjected to an additional round of bioactivity testing and mass spectral analysis. Data were overlaid using biochemometrics analysis to predict active constituents early in the fractionation process following which their biosynthetic pathways were identified using metabologenomics.ResultsWe isolated three new-to-nature stemphone analogs, 19-acetylstemphones G (1), B (2) and E (3), that demonstrated antiproliferative activity ranging from 3 to 5 µM against human melanoma (MDA-MB-435) and ovarian cancer (OVACR3) cells. We proposed a rational biosynthetic pathway for these compounds, highlighting the potential of using bioactivity as a filter for the analysis of integrated—Omics datasets.ConclusionsThis work demonstrates how the incorporation of biochemometrics as a third dimension into the metabologenomics workflow can identify bioactive metabolites and link them to their biosynthetic machinery.

Quality control and validation of extracellular vesicles isolated from cultured human breast cancer cells

ObjectiveExtracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles.ResultsTo isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFβ1, β-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies.

PARP4 interacts with hnRNPM to regulate splicing during lung cancer progression

BackgroundThe identification of cancer driver genes from sequencing data has been crucial in deepening our understanding of tumor biology and expanding targeted therapy options. However, apart from the most commonly altered genes, the mechanisms underlying the contribution of other mutations to cancer acquisition remain understudied. Leveraging on our whole-exome sequencing of the largest Asian lung adenocarcinoma (LUAD) cohort (n = 302), we now functionally assess the mechanistic role of a novel driver, PARP4.MethodsIn vitro and in vivo tumorigenicity assays were used to study the functional effects of PARP4 loss and mutation in multiple lung cancer cell lines. Interactomics analysis by quantitative mass spectrometry was conducted to identify PARP4’s interaction partners. Transcriptomic data from cell lines and patient tumors were used to investigate splicing alterations.ResultsPARP4 depletion or mutation (I1039T) promotes the tumorigenicity of KRAS- or EGFR-driven lung cancer cells. Disruption of the vault complex, with which PARP4 is commonly associated, did not alter tumorigenicity, indicating that PARP4’s tumor suppressive activity is mediated independently. The splicing regulator hnRNPM is a potentially novel PARP4 interaction partner, the loss of which likewise promotes tumor formation. hnRNPM loss results in splicing perturbations, with a propensity for dysregulated intronic splicing that was similarly observed in PARP4 knockdown cells and in LUAD cohort patients with PARP4 copy number loss.ConclusionsPARP4 is a novel modulator of lung adenocarcinoma, where its tumor suppressive activity is mediated not through the vault complex—unlike conventionally thought, but in association with its novel interaction partner hnRNPM, thus suggesting a role for splicing dysregulation in LUAD tumorigenesis.

PiscesCSM: prediction of anticancer synergistic drug combinations

While drug combination therapies are of great importance, particularly in cancer treatment, identifying novel synergistic drug combinations has been a challenging venture. Computational methods have emerged in this context as a promising tool for prioritizing drug combinations for further evaluation, though they have presented limited performance, utility, and interpretability. Here, we propose a novel predictive tool, piscesCSM, that leverages graph-based representations to model small molecule chemical structures to accurately predict drug combinations with favourable anticancer synergistic effects against one or multiple cancer cell lines. Leveraging these insights, we developed a general supervised machine learning model to guide the prediction of anticancer synergistic drug combinations in over 30 cell lines. It achieved an area under the receiver operating characteristic curve (AUROC) of up to 0.89 on independent non-redundant blind tests, outperforming state-of-the-art approaches on both large-scale oncology screening data and an independent test set generated by AstraZeneca (with more than a 16% improvement in predictive accuracy). Moreover, by exploring the interpretability of our approach, we found that simple physicochemical properties and graph-based signatures are predictive of chemotherapy synergism. To provide a simple and integrated platform to rapidly screen potential candidate pairs with favourable synergistic anticancer effects, we made piscesCSM freely available online at https://biosig.lab.uq.edu.au/piscescsm/ as a web server and API. We believe that our predictive tool will provide a valuable resource for optimizing and augmenting combinatorial screening libraries to identify effective and safe synergistic anticancer drug combinations.Scientific contributionThis work proposes piscesCSM, a machine-learning-based framework that relies on well-established graph-based representations of small molecules to identify and provide better predictive accuracy of syngenetic drug combinations. Our model, piscesCSM, shows that combining physiochemical properties with graph-based signatures can outperform current architectures on classification prediction tasks. Furthermore, implementing our tool as a web server offers a user-friendly platform for researchers to screen for potential synergistic drug combinations with favorable anticancer effects against one or multiple cancer cell lines.

Dual inhibition of SUMOylation and MEK conquers MYC-expressing KRAS-mutant cancers by accumulating DNA damage

BackgroundKRAS mutations frequently occur in cancers, particularly pancreatic ductal adenocarcinoma, colorectal cancer, and non-small cell lung cancer. Although KRASG12C inhibitors have recently been approved, effective precision therapies have not yet been established for all KRAS-mutant cancers. Many treatments for KRAS-mutant cancers, including epigenome-targeted drugs, are currently under investigation. Small ubiquitin-like modifier (SUMO) proteins are a family of small proteins covalently attached to and detached from other proteins in cells via the processes called SUMOylation and de-SUMOylation. We assessed whether SUMOylation inhibition was effective in KRAS-mutant cancer cells.MethodsThe efficacy of the first-in-class SUMO-activating enzyme E inhibitor TAK-981 (subasumstat) was assessed in multiple human and mouse KRAS-mutated cancer cell lines. A gene expression assay using a TaqMan array was used to identify biomarkers of TAK-981 efficacy. The biological roles of SUMOylation inhibition and subsequent regulatory mechanisms were investigated using immunoblot analysis, immunofluorescence assays, and mouse models.ResultsWe discovered that TAK-981 downregulated the expression of the currently undruggable MYC and effectively suppressed the growth of MYC-expressing KRAS-mutant cancers across different tissue types. Moreover, TAK-981-resistant cells were sensitized to SUMOylation inhibition via MYC-overexpression. TAK-981 induced proteasomal degradation of MYC by altering the balance between SUMOylation and ubiquitination and promoting the binding of MYC and Fbxw7, a key factor in the ubiquitin–proteasome system. The efficacy of TAK-981 monotherapy in immunocompetent and immunodeficient mouse models using a mouse-derived CMT167 cell line was significant but modest. Since MAPK inhibition of the KRAS downstream pathway is crucial in KRAS-mutant cancer, we expected that co-inhibition of SUMOylation and MEK might be a good option. Surprisingly, combination treatment with TAK-981 and trametinib dramatically induced apoptosis in multiple cell lines and gene-engineered mouse-derived organoids. Moreover, combination therapy resulted in long-term tumor regression in mouse models using cell lines of different tissue types. Finally, we revealed that combination therapy complementally inhibited Rad51 and BRCA1 and accumulated DNA damage.ConclusionsWe found that MYC downregulation occurred via SUMOylation inhibition in KRAS-mutant cancer cells. Our findings indicate that dual inhibition of SUMOylation and MEK may be a promising treatment for MYC-expressing KRAS-mutant cancers by enhancing DNA damage accumulation.

Rapidly-manufactured CD276 CAR-T cells exhibit enhanced persistence and efficacy in pancreatic cancer

BackgroundPancreatic cancer is one of the most lethal malignancies and the lack of treatment options makes it more deadly. Chimeric Antigen Receptor T-cell (CAR-T) immunotherapy has revolutionized cancer treatment and made great breakthroughs in treating hematological malignancies, however its success in treating solid cancers remains limited mainly due to the lack of tumor-specific antigens. On the other hand, the prolonged traditional manufacturing process poses challenges, taking 2 to 6 weeks and impacting patient outcomes. CD276 has recently emerged as a potential therapeutic target for anti-solid cancer therapy. Here, we investigated the efficacy of CD276 CAR-T and rapidly-manufactured CAR-T against pancreatic cancer.MethodsIn the present study, CD276 CAR-T was prepared by CAR structure carrying 376.96 scFv sequence, CD8 hinge and transmembrane domain, 4-1BB and CD3ζ intracellular domains. Additionally, CD276 rapidly-manufactured CAR-T (named CD276 Dash CAR-T) was innovatively developed by shortening the duration of ex vitro culture to reduce CAR-T manufacturing time. We evaluated the anti-tumor efficacy of CD276 CAR-T and further compared the functional assessment of Dash CAR-T and conventional CAR-T in vitro and in vivo by detecting the immunophenotypes, killing ability, expansion capacity and tumor-eradicating effect of CAR-T.ResultsWe found that CD276 was strongly expressed in multiple solid cancer cell lines and that CD276 CAR-T could efficiently kill these solid cancer cells. Moreover, Dash CAR-T was successfully manufactured within 48–72 h and the functional validation was carried out subsequently. In vitro, CD276 Dash CAR-T possessed a less-differentiated phenotype and robust proliferative ability compared to conventional CAR-T. In vivo xenograft mouse model, CD276 Dash CAR-T showed enhanced anti-pancreatic cancer efficacy and T cell expansion. Besides, except for the high-dose group, the body weight of mice was maintained stable, and the state of mice was normal.ConclusionsIn this study, we proved CD276 CAR-T exhibited powerful activity against pancreatic cancer cells in vitro and in vivo. More importantly, we demonstrated the manufacturing feasibility, acceptable safety and superior anti-tumor efficacy of CD276 Dash CAR-T generated with reduced time. The results of the above studies indicated that CD276 Dash CAR-T immunotherapy might be a novel and promising strategy for pancreatic cancer treatment.

ESF1 positively regulates MDM2 and promotes tumorigenesis

Eighteen S rRNA factor 1 (ESF1) is a predominantly nucleolar protein essential for embryogenesis. Our previous studies have suggested that Esf1 is a negative regulator of the tumor suppressor protein p53. However, it remains unclear whether ESF1 contributes to tumorigenesis. In this current research, we find that increased ESF1 expression correlates with poor survival in multiple tumors including pancreatic cancer. ESF1 is able to regulate cell proliferation, migration, DNA damage-induced apoptosis, and tumorigenesis. Mechanistically, ESF1 physically interacts with MDM2 and is essential for maintaining the stability of MDM2 protein by inhibiting its ubiquitination. Additionally, ESF1 also prevented stress-induced stabilization of p53 in multiple cancer cells. Hence, our findings suggest that ESF1 is a potent regulator of the MDM2-p53 pathway and promotes tumor progression.

Identification of 5-Thiocyanatothiazol-2-amines Disrupting WDR5-MYC Protein-Protein Interactions.

MYC amplification is frequently observed in approximately 50% of human cancers, rendering it a highly desired anticancer target. Given the challenge of direct pharmacological inhibiting of MYC, impairing the interaction of MYC and its key cofactor WDR5 has been proposed as a promising strategy for MYC-driven cancer treatment. Herein, we report the discovery of 5-thiocyanatothiazol-2-amines that disrupt the WDR5-MYC interaction. Hit fragments were initially identified in a fluorescence polarization (FP)-based screening of an in-house library, and structural-activity relationship exploration resulted in the lead compounds 4m and 4o with potent inhibitory activities on WDR5-MYC interaction (K i = 2.4 μM for 4m; K i = 1.0 μM for 4o). These compounds were further validated via differential scanning fluorimetry (DSF) and coimmunoprecipitation (Co-IP). Moreover, 4m and 4o exhibited good cellular activities with the IC50 values at the micromolar level (IC50 = 0.71-7.40 μM) against multiple MYC-driven cancer cell lines. Our findings afforded a potential small molecule blocking the WDR5-MYC interaction.

CRISPR screens reveal convergent targeting strategies against evolutionarily distinct chemoresistance in cancer

Resistance to chemotherapy has been a major hurdle that limits therapeutic benefits for many types of cancer. Here we systematically identify genetic drivers underlying chemoresistance by performing 30 genome-scale CRISPR knockout screens for seven chemotherapeutic agents in multiple cancer cells. Chemoresistance genes vary between conditions primarily due to distinct genetic background and mechanism of action of drugs, manifesting heterogeneous and multiplexed routes towards chemoresistance. By focusing on oxaliplatin and irinotecan resistance in colorectal cancer, we unravel that evolutionarily distinct chemoresistance can share consensus vulnerabilities identified by 26 second-round CRISPR screens with druggable gene library. We further pinpoint PLK4 as a therapeutic target to overcome oxaliplatin resistance in various models via genetic ablation or pharmacological inhibition, highlighting a single-agent strategy to antagonize evolutionarily distinct chemoresistance. Our study not only provides resources and insights into the molecular basis of chemoresistance, but also proposes potential biomarkers and therapeutic strategies against such resistance.

Depolymerization of tubulin as the main molecular mechanism of the cytotoxic and antitumor activity of pyrrole-containing heterocyclic compounds

Introduction. Microtubules are highly dynamic polymers of α, β-tubulin dimers involves in a broad spectrum of the processes, such as intracellular transport and cell proliferation. This makes them an attractive molecular target for anti-cancer therapies. Substances that affect the dynamic state of tubulin microtubules are known as the mitotic poisons that are effectiveand widely used in the chemotherapy of various tumors. Mitotic poisons are able to interfere with polymerization (stabilization) or depolymerization of tubulin, which in turn leads to the arrest of cells in the M-phase (named as a mitotic catastrophe) and their subsequent death via activation of apoptotic mechanisms. However, the effectiveness of MP-based therapies is gradually decreasing over the time due to development of multiple drug resistance mechanisms in cancer cells. Thus, development of novel compounds selectively targeting tubulin and effectively overcoming multiple drugresistance phenotype in cancer is an urgent need in current oncology. Aim. To examine the cytotoxic and antitumor activities of several pyrrole-containing heterocyclic compounds (EPC-91, EPC-92 and PCA-93) against cancer cell lines with epithelial and mesenchymal origin, including those with multiple drug resistance phenotype. Materials and methods. Studies were performed on parental human cancer cell lines – triple-negative breast cancer HCC1806, gastrointestinal stromal tumor GIST T-1, osteosarcoma SaOS-2, – sensitive to chemotherapy (paclitaxel, doxorubicin) and their resistant sublines (HCC1806 Tx-R, GIST T-1 Tx-R, SaOS-2 Dox-R), as well as on murine colorectal adenocarcinoma cell line Colon-26, exhibiting primary resistance to the aforementioned chemotherapeutic agents. Results. The cytotoxic activities of EPC-91 and PCA-93 were due to their abilities to depolymerize tubulin. The results of immunofluorescence microscopy and Western blotting indicated that the compounds disrupt assembly of tubulin microtubules and prevent polymerization of α-tubulin in cancer cells. Inhibition of tubulin polymerizations led to significant increasein number of round-shaped and phospho-histone 3 (e. g. mitotic) cells, followed by their death through apoptosis. PCA-93 also exhibited potent anti-tumor effect against Colon-26 cells due to its anti-proliferative and proapoptotic activities. Conclusion. The data shown here illustrates potent cytotoxic activities of EPC-91 and PCA-93 against multiple cancer cell lines in vitro including those with multiple drug resistance phenotype. Similarly, PCA-93 was found to be highly effective against Colon-26 cell in vivo, thereby illustrating the attractive platform for the development of novel pyrrole-based agents exhibiting potent anti-tumor activities.

Discovery of bivalent small molecule degraders of cyclin-dependent kinase 7 (CDK7)

Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.

Cell State Transition Models Stratify Breast Cancer Cell Phenotypes and Reveal New Therapeutic Targets.

Understanding signaling patterns of transformation and controlling cell phenotypes is a challenge of current biology. Here we applied a cell State Transition Assessment and Regulation (cSTAR) approach to a perturbation dataset of single cell phosphoproteomic patterns of multiple breast cancer (BC) and normal breast tissue-derived cell lines. Following a separation of luminal, basal, and normal cell states, we identified signaling nodes within core control networks, delineated causal connections, and determined the primary drivers underlying oncogenic transformation and transitions across distinct BC subtypes. Whereas cell lines within the same BC subtype have different mutational and expression profiles, the architecture of the core network was similar for all luminal BC cells, and mTOR was a main oncogenic driver. In contrast, core networks of basal BC were heterogeneous and segregated into roughly four major subclasses with distinct oncogenic and BC subtype drivers. Likewise, normal breast tissue cells were separated into two different subclasses. Based on the data and quantified network topologies, we derived mechanistic cSTAR models that serve as digital cell twins and allow the deliberate control of cell movements within a Waddington landscape across different cell states. These cSTAR models suggested strategies of normalizing phosphorylation networks of BC cell lines using small molecule inhibitors.

Synthesis, biological evaluation, and molecular docking of novel 1,3,4-substituted-thiadiazole derivatives as potential anticancer agent

In an attempt to develop potent anti-cancer agents, a new 1,3,4-substituted-thiadiazole derivatives (8b-g), starting from 4-substituted-thiazol-2-chloroacetamides (4b-g), were synthesized and evaluated for their cytotoxic effects on multiple human cancer cell lines, including the hepatocellular carcinoma (HEPG-2), human lung carcinoma (A549), human breast carcinoma (MCF-7) and pseudo-normal human embryonic liver (L02) cancer cell lines by an MTT assay. Among all synthesized compounds, compound 8d showed the potent anti-cancer activities with GI50 values of 2.98, 2.85 and 2.53 μM against MCF-7, A549 and HepG-2 cell lines respectively as compared to standard drug Doxorubicin. Furthermore, molecular modelling studies have spotlighted the anchoring role of 1,3,4-substituted-thiadiazole moiety in bonding and hydrophobic interaction with the key amino acid residues. Therefore, these results can provide promising starting points for further development of best anti-cancer agents.Graphical

UM171 suppresses breast cancer progression by inducing KLF2

PurposeBreast cancer is the most frequent cancer in women with significant death rate. Morbidity is associated with drug resistance and metastasis. Development of novel drugs is unmet need. The aim of this study is to show potent anti-neoplastic activity of the UM171 compound on breast cancer cells and its mechanism of action.MethodsThe inhibitory effect of UM171 on several breast cancer (BC) cell lines was examined using MTT and colony-forming assays. Cell cycle and apoptosis assays were utilized to determine the effect of UM171 on BC cell proliferation and survival. Wound healing scratch and transwell migration assays were used to examine the migration of BC cell lines in culture. Xenograft of mouse model with 4T1 cells was used to determine inhibitory effect of UM171 in vivo. Q-RT-PCR and western blotting were used to determine the expression level of genes effected by UM171. Lentivirus-mediated shRNAs were used to knockdown the expression of KLF2 in BC cells.ResultsUM171 was previously identified as a potent agonist of human hematopoietic stem cell renewal and inhibitor of leukemia. In this study, UM171 was shown to inhibit the growth of multiple breast cancer cell lines in culture. UM171-mediated growth inhibition was associated with the induction of apoptosis, G2/M cell cycle arrest, lower colony-forming capacity, and reduced motility. In a xenotransplantation model of mouse triple-negative breast cancer 4T1 cells injected into syngeneic BALB/c mice, UM171 strongly inhibited tumor growth at a level comparable to control paclitaxel. UM171 increased the expression of the three PIM genes (PIM1-3) in breast cancer cells. Moreover, UM171 strongly induced the expression of the tumor suppressor gene KLF2 and cell cycle inhibitor P21CIP1. Accordingly, knockdown of KLF2 using lentivirus-mediated shRNA significantly attenuated the growth suppressor activity of UM171. As PIM1-3 act as oncogenes and are involved in breast cancer progression, induction of these kinases likely impedes the inhibitory effect of KLF2 induction by UM171. Accordingly, combination of UM171 with a PAN-PIM inhibitor LGH447 significantly reduced tumor growth in culture.ConclusionThese results suggested that UM171 inhibited breast cancer progression in part through activation of KLF2 and P21. Combination of UM171 with a PAN-PIM inhibitor offer a novel therapy for aggressive forms of breast cancer.

Combining EHMT and PARP Inhibition: A Strategy to Diminish Therapy-Resistant Ovarian Cancer Tumor Growth while Stimulating Immune Activation.

Despite the success of Poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers Euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory dsRNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi resistant ovarian tumor growth in vivo, and promotes anti-tumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.

NK-92 cells labeled with Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobes for the targeted treatment and noninvasive therapeutic evaluation of breast cancer

Adoptive cellular immunotherapy as a promising and alternative cancer therapy platform is critical for future clinical applications. Natural killer (NK) cells have attracted attention as an important type of innate immune regulatory cells that can rapidly kill multiple adjacent cancer cells. However, these cells are significantly less effective in treating solid tumors than in treating hematological tumors. Herein, we report the synthesis of a Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobe labeled with NK-92 cells that can be used for adoptive cellular immunotherapy, photodynamic therapy and dual-modality imaging-based in vivo fate tracking. The labeled NK-92 cells specifically target the tumor cells, which increases the amount of cancer cell apoptosis in vitro. Furthermore, the in vivo results indicate that the labeled NK-92 cells can be used for tumor magnetic resonance imaging and fluorescence imaging, adoptive cellular immunotherapy, and photodynamic therapy after tail vein injection. These data show that the developed multifunctional nanostructure is a promising platform for efficient innate immunotherapy, photodynamic treatment and noninvasive therapeutic evaluation of breast cancer.

Crebanine, an aporphine alkaloid, induces cancer cell apoptosis through PI3K-Akt pathway in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is one of the most prevalent and lethal primary central nervous system malignancies. GBM is notorious for its high rates of recurrence and therapy resistance and the PI3K/Akt pathway plays a pivotal role in its malignant behavior. Crebanine (CB), an alkaloid capable of penetrating the blood-brain barrier (BBB), has been shown to have inhibitory effects on proinflammatory molecules and multiple cancer cell lines via pathways such as PI3K/Akt. This study aims to investigate the efficacy and mechanisms of CB treatment on GBM. It is the first study to elucidate the anti-tumor role of CB in GBM, providing new possibilities for GBM therapy. Through a series of experiments, we demonstrate the significant anti-survival, anti-clonogenicity, and proapoptotic effects of CB treatment on GBM cell lines. Next-generation sequencing (NGS) is also conducted and provides a complete list of significant changes in gene expression after treatment, including genes related to apoptosis, the cell cycle, FoxO, and autophagy. The subsequent protein expressions of the upregulation of apoptosis and downregulation of PI3K/Akt are further proved. The clinical applicability of CB to GBM treatment could be high for its BBB-penetrating feature, significant induction of apoptosis, and blockage of the PI3K/Akt pathway. Future research is needed using in vivo experiments and other therapeutic pathways shown in NGS for further clinical or in vivo studies.

A phase Ib study of PI3Kα inhibitor risovalisib mesylate in combination with fulvestrant in patients with PIK3CA-mutated, HR+/HER2- advanced breast cancer.

1047 Background: PIK3CAmutations represent a common mechanism of resistance to endocrine therapy (ET) ± CDK4/6 inhibitors (CDK4/6i) in patients (pts) with HR+/HER2– advanced breast cancer (ABC). Alpelisib (PI3Kα inhibitor) + fulvestrant has been approved for pts with PIK3CA-mutated HR+/HER2- ABC following progression on/after an ET-based regimen. Risovalisib Mesylate (CYH33) is a novel PI3Kα inhibitor with superior inhibitory activity to alpelisib in multiple cancer cell lines and tumor xenograft models, and it exhibited encouraging anti-tumor efficacy in solid tumors in the first-in-human study (NCT03544905). A multicenter, open-label, phase Ib study of CYH33 in combination with ET ± Palbociclib (CDK4/6i) is ongoing (NCT04856371). Here we only present data from the CYH33 + fulvestrant arm (Cohort 1). Methods: The safety, pharmacokinetics, pharmacodynamics and preliminary efficacy (assessed every 8 weeks by RECIST v1.1) of CYH33 30/40mg administered orally once daily in combination with intramuscular fulvestrant 500mg on Day1 of every 28-day cycle with 1 additional dose on Day15 of Cycle 1 were assessed until disease progression, unacceptable toxicity, or withdrawal of consent in pts with PIK3CA-mutated, HR+/ HER2- ABC who progressed on/after ET ± CDK4/6i treatments. Results: From Jul 29, 2021 to Nov17, 2023 (data cutoff), a total of 52 pts were enrolled into Cohort 1 and followed up for at least 8 months. The recommended phase 2 dose (RP2D) was identified as CYH33 30mg QD + Fulvestrant 500mg. For all pts, no grade 4/5 treatment-related AEs (TRAEs) were observed and the most common grade 3 TRAEs (≥5%) were hyperglycaemia (17.3%), rash (15.4%), stomatitis (13.5%), weight decreased (7.7%), decreased appetite (5.8%), diarrhoea (5.8%) and hypokalaemia (5.8%). For pts receiving RP2D, the incidence of grade 3 TRAEs were lower, and the most common grade 3 TRAEs (≥5%) were rash (13.6%) and stomatitis (9.1%). In addition, the most common grade 1-2 TRAEs were hyperglycaemia, nausea, decreased appetite, rash, weight decreased, diarrhoea, vomiting, stomatitis, AST increased and ALT increased. There were 39 pts harboring PIK3CA-mutations without prior fulvestrant treatment, 90% had received ≥1 prior lines of systemic therapy, 41% were premenopausal, 74% had visceral metastases, 49% had received a prior CDK4/6i, and 31% had received systemic chemotherapy. Among these 39 pts, confirmed ORR was 17.9%, CBR was 41.0%, and median PFS (mPFS) was 10.91 months. For pts receiving RP2D (n=21), confirmed ORR was 9.5%, CBR was 47.6% and mPFS was 10.97 months. For pts with prior CDK4/6i treatment at RP2D, the mPFS was 7.92 months. Conclusions: CYH33 + fulvestrant exhibited a manageable safety profile and encouraging preliminary anti-tumor efficacy in pts with PIK3CA-mutated, HR+/HER2- ABC who progressed on/after ET ± CDK4/6i treatments. Clinical trial information: NCT04856371 .

2-Aryl-1H-imidazo[4,5-f][1,10]phenanthroline-Based Binuclear Ru(II)/Ir(III)/Re(I) Complexes as Mitochondria Targeting Cancer Stem Cell Therapeutic Agents.

A series of novel Ru(II)/Ir(III)/Re(I)-based organometallic complexes [Ru2L1, Ru2L2, Ir2L1, Ir2L2, Re2L1, and Re2L2] have been synthesized to assess their potency and selectivity against multiple cancer cells A549, HCT-116, and HCT-116 colon CSCs. The cytotoxic screening of the synthesized complexes has revealed that complex Ru2L1 and Ir2L2 are two proficient complexes among all, but Ru2L1 is the most potent complex. A significant binding constant value was observed for DNA and BSA in all complexes. Significant lipophilic properties allow them to penetrate cancer cell membranes, and substantial quantum yield (ϕf) values support bioimaging potential. Again, these complexes are particular for mitochondrial localization and produce a profuse amount of ROS to damage the mitochondrial DNA and then G1 phase cell-cycle arrest. Protein expression analysis unveiled that pro-apoptotic Bax protein overexpressed in Ru2L1-treated cells, whereas antiapoptotic Bcl-2 protein was expressed twofold in Ir2L2-treated cells, which correlated with autophagy reticence.