Escape Of Cells Research Articles

The p38/MAPK pathway as a therapeutic target to prevent therapeutic escape of breast cancer stem cells.

Cancer stem cells (CSCs) play an important role in metastasis development, tumor recurrence, and treatment resistance, and are essential for the eradication of cancer. Currently, therapies fail to eradicate CSCs due to their therapeutic stress-induced cellular escape, which leads to enhanced aggressive behaviors compared with CSCs that have never been treated. However, the underlying mechanisms regulating the therapeutic escape remain unknown. To this end, we established a model to isolate the therapeutic escaped CSCs (TSCSCs) from breast CSCs and performed the transcription profile to reveal the mechanism. Mechanistically, we demonstrated that the behavior of therapeutic escape was regulated through the p38/MAPK signaling pathway, resulting in TSCSCs exhibiting enhanced motility and metastasis. Notably, blocking the p38/MAPK signaling pathway effectively reduced motility and metastasis ability both in vitro and in vivo, which were further supported by downregulated motility-related genes and epithelial-mesenchymal transition (EMT)-related proteins vimentin and N-cadherin. The obtained findings reveal the p38/MAPK pathway as a potential therapeutic target for TSCSCs and would provide profound implications for cancer therapy.

Porphyromonas gingivalis infection facilitates immune escape of esophageal cancer by enhancing YTHDF2-mediated Fas degradation

To investigate the effect of Porphyromonas gingivalis (Pg) infection on immune escape of oesophageal cancer cells and the role of YTHDF2 and Fas in this regulatory mechanism. We examined YTHDF2 and Fas protein expressions in esophageal squamous cell carcinoma (ESCC) tissues with and without Pg infection using immunohistochemistry and in Pg-infected KYSE150 cells using Western blotting. The interaction between YTHDF2 and Fas was investigated by co-immunoprecipitation (Co-IP). Pg-infected KYSE150 cells with lentivirus-mediated YTHDF2 knockdown were examined for changes in expression levels of YTHDF2, cathepsin B (CTSB), Fas and FasL proteins, and the effect of E64 (a cathepsin inhibitor) on these proteins were observed. After Pg infection and E64 treatment, KYSE150 cells were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the expressions of T cell-related effector molecules were detected by flow cytometry. ESCC tissues and cells with Pg infection showed significantly increased YTHDF2 expression and lowered Fas expression. The results of Co-IP demonstrated a direct interaction between YTHDF2 and Fas. In Pg-infected KYSE150 cells with YTHDF2 knockdown, the expression of CTSB was significantly reduced while Fas and FasL expressions were significantly increased. E64 treatment of KYSE150 cells significantly decreased the expression of CTSB without affecting YTHDF2 expression and obviously increased Fas and FasL expressions. Flow cytometry showed that in Pg-infected KYSE150 cells co-cultured with PBMCs, the expressions of Granzyme B and Ki67 were significantly decreased while PD-1 expression was significantly enhanced. Pg infection YTHDF2-dependently regulates the expression of Fas to facilitate immune escape of esophageal cancer and thus promoting cancer progression, suggesting the key role of YTHDF2 in regulating immune escape of esophageal cancer.

Abstract PO-028: A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma

Abstract Background. Small molecules targeting the B-cell receptor (BCR) signaling, including PI3K and BTK inhibitors, have represented a step forward for managing lymphoma patients. However, resistance often develops, negatively impacting the benefit of patients exposed to drugs. Thus, developing preclinical tools to identify modalities to overcome this phenomenon is important. The bone marrow is commonly involved in marginal zone lymphoma (MZL) patients, and it might represent a safe environment where lymphoma cells escape therapy. Bone marrow stromal cells (BMSCs) induce proliferation and drug resistance in several tumor entities, mainly through the release of cytokines. To study potential resistance mechanisms to BCR targeting agents, we aimed to develop a 3D co-culture system in which primary BMSCs were used to model the bone marrow tumor microenvironment while exposing an MZL cell line to the PI3K inhibitor copanlisib. Methods. The MZL cell line VL51 was cultured in 3D fibrin gel (10mg/mL), either alone or with primary BMSCs, in 96-well plates using the OT-2 liquid handler (Opentrons) for high-throughput seeding purposes. Copanlisib treatment started on day 3. The effects of the drug were assessed through morphological observations and quantitative measurements of images taken on days 3, 5, and 7 in the presence or absence of BMSCs. On day 7, supernatants were analyzed for 105 secreted factors using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems / Bio-Techne). Results. We observed differences in VL51 growth pattern in the different conditions. Most importantly, the co-culture with BMSCs significantly (p=0.025) decreased VL51 sensitivity to copanlisib. When not treated, VL51 cells formed significantly larger clusters (p<0.001) in the presence of BMSCs. However, upon copanlisib treatment, VL51 cells formed smaller and more distributed clusters comparable in size to those in the mono-culture. Subsequent analyses of the supernatants from the mono- or co-cultures following DMSO or copanlisib treatment identified 14 differentially secreted cytokines among the different conditions. Validation experiments were done using recombinant proteins in 2D cultures for 6 factors. Among these, recombinant IGFBP-3, serpin E1, and PTX-3 significantly decreased VL51 sensitivity to copanlisib in 2D cultures (p=0.05, 0.01, and 0.04, respectively). Validation experiments are ongoing with additional in vitro models and using BTK inhibitors. Conclusions. We created a high-throughput 3D co-culture MZL system and found that BMSCs reduce sensitivity to copanlisib in MZL models. Secretion of IGFBP-3, Serpin E1, and PTX-3 reduced response to copanlisib. This 3D-model will be used to assess resistance mechanisms to clinically relevant compounds and to evaluate alternative therapeutic approaches to improve the treatment of lymphoma patients. Citation Format: Alex Zadro, Alberto Arribas, Maria Vittoria Colombo, Eleonora Cannas, Filippo Spriano, Luciano Cascione, Chiara Arrigoni, Christian Candrian, Matteo Moretti, Francesco Bertoni. A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-028.

IMMU-18. ENHANCING GD2.CAR T CELL THERAPY FOR DIFFUSE MIDLINE GLIOMA WITH LSD1 AND HDAC INHIBITORS

Abstract BACKGROUND Chimeric antigen receptor T cells against the glycolipid GD2 (GD2.CARTs) have shown promise against Diffuse Midline Glioma (DMG) in clinical trials but are not yet curative. Previous studies in neuroblastoma demonstrate that histone deacetylase inhibitor (HDACi) treatment can increase GD2 on tumor cells and improve targeting by GD2.CARTs. Our lab previously found that simultaneous HDACi and lysine-demethylase 1 inhibitor (LSD1i) treatment in DMG cells causes synergistic alterations in gene expression and reduces DMG growth. Here, we find that treating GD2-low DMG cell lines with combinations of LSD1i and HDACi improves DMG response to GD2.CARTs. METHODS Surface expression of GD2 before and after LSD1i/HDACi treatment were analyzed using immunofluorescence microscopy and flow cytometry. The cytotoxicity of GD2.CARTs against DMG cells was determined from co-culture experiments and in patient-derived orthotopic xenograft models using flow cytometry and bioluminescent imaging. RESULTS Since low target antigen expression is one mechanism by which tumor cells can escape CAR T cell therapy, we profiled GD2 expression on 9 different H3K27M-mutant DMG cell lines. This analysis shows that only 5 of the 9 cell lines exhibit high GD2 expression while the remaining 4 lines have low GD2 expression. When treating these cell lines with GD2.CARTs, we find that low GD2 expression on DMG cells correlates with tumor cell escape and resistance to GD2.CARTs. Treating GD2-low DMG cells with LSD1i+HDACi increases GD2 expression and enhances tumor cell targeting by GD2.CARTs in subsequent co-culture assays. Further studies in mice bearing GD2-low DMG tumors were initially GD2.CART-insensitive but showed reduced tumor growth when pre-treated with LSD1i+HDACi before intravenous GD2.CART infusion. CONCLUSIONS These studies suggest that variable GD2 expression is a major driver of DMG resistance to GD2.CARTs and that LSD1i+HDACi pre-treatment may improve DMG patient responses to GD2.CART therapy by increasing target antigen expression.

Dehydroepiandrosterone attenuated the immune escape of oral squamous cell carcinoma through NF-κB p65/miR-15b-5p/B7-H4 axis

ObjectivesWe aimed to explore the effects and mechanisms of action of dehydroepiandrosterone (DHEA) on immune evasion of oral squamous cell carcinoma (OSCC) to provide evidence for enhancing the effect of immunotherapy. Materials and MethodsA xenograft mouse model and immunohistochemistry were used to reveal the patterns of tumor-infiltrating lymphocytes (TILs). The CAL27 and SCC VII cell lines were used for the in vitro study. Western blotting, qPCR, immunofluorescence, and flow cytometry were used to evaluate the expression of B7-H4. Recombinant mouse B7-H4 protein (rmB7-H4) and PG490, an inhibitor of NF-κB p65 were used for the “rescue study.” Gain- and loss-of-function, luciferase reporter, and chromatin immunoprecipitation assays were performed to verify this mechanism. ResultsDHEA inhibited tumor growth in an OSCC xenograft mouse model, increased CD8 + cells, and decreased FOXP3 + cells in TILs. DHEA reduced the expression of B7-H4 in CAL27 and SCC VII cells RmB7-H4 reverses the effect of DHEA on tumor growth and TIL patterns. DHEA increased the expression of miR-15b-5p and activated its transcriptional factor NF-κB p65. Further experiments demonstrated that miR-15b-5p inhibited B7-H4 expression by binding to its 3′-UTR regions, and NF-κB p65 activated miR-15b transcription. PG490 reversed the effects of DHEA on tumor growth, antitumor immunity in the OSCC xenograft model, and the expression/phosphorylation of NF-κB p65, miR-15b-5p, and B7-H4. ConclusionsThis study indicates that DHEA attenuates the immune escape of OSCC cells by inhibiting B7-H4 expression, providing new insights for cancer immunotherapy.

Synergistic upregulation of PD‑L1 in tumor cells and CD39 in tumor‑infiltrating CD8+ T cells leads to poor prognosis in patients with hepatocellular carcinoma.

The immune escape of tumor cells and functional status of tumor-infiltrating T cells may serve pivotal roles in the tumor immune microenvironment and progression of hepatocellular carcinoma (HCC). The present study enrolled 91 patients with HCC and examined programmed cell death ligand 1 (PD-L1) expression in tumor cells and CD39 expression in tumor-infiltrating CD8+ T cells in patient samples using multiplex immunofluorescence assays. The impact of PD-L1 and CD39 expression levels on the prognosis of patients with HCC was investigated utilizing Kaplan-Meier analyses. The individual upregulation of PD-L1 in tumor cells, as well as the individual upregulation of CD39 expression in tumor-infiltrating CD8+ T cells did not significantly affect the prognosis of patients with HCC. However, the simultaneous upregulation of both PD-L1 in tumor cells and CD39 in tumor-infiltrating CD8+ T cells was associated with reduced overall survival in patients with HCC. Therefore, the results of the present study suggested that the interplay between tumor cell immune escape and tumor-infiltrating immune cell functional status within the tumor immune microenvironment may have had a substantial impact on the prognosis of patients with HCC. Mechanistically, increased expression levels of PD-L1 in tumor cells may improve the immune escape capacity of tumors, whilst upregulation of CD39 in tumor-infiltrating T cells may be associated with T cell exhaustion. Therefore, the upregulation of PD-L1 expression in tumor cells, in conjunction with the exhaustion of tumor-infiltrating CD8+ T cells, could serve as a future potential prognostic indicator of patients with HCC.

PD-L2 drives resistance to EGFR-TKIs: dynamic changes of the tumor immune environment and targeted therapy.

There is a lack of effective treatments to overcome resistance to EGFR-TKIs in EGFR mutant tumors. A deeper understanding of resistance mechanisms can provide insights into reducing or eliminating resistance, and can potentially deliver targeted treatment measures to overcome resistance. Here, we identified that the dynamic changes of the tumor immune environment were important extrinsic factors driving tumor resistance to EGFR-TKIs in EGFR mutant cell lines and syngeneic tumor-bearing mice. Our results demonstrate that the acquired resistance to EGFR-TKIs is accompanied by aberrant expression of PD-L2, leading a dynamic shift from an initially favorable tumor immune environment to an immunosuppressive phenotype. PD-L2 expression significantly affected EGFR mutant cell apoptosis that depended on the proportion and function of CD8+ T cells in the tumor immune environment. Combined with single-cell sequencing and experimental results, we demonstrated that PD-L2 specifically inhibited the proliferation of CD8+ T cells and the secretion of granzyme B and perforin, leading to reduced apoptosis mediated by CD8+ T cells and enhanced immune escape of tumor cells, which drives EGFR-TKIs resistance. Importantly, we have identified a potent natural small-molecule inhibitor of PD-L2, zinc undecylenate. In vitro, it selectively and potently blocks the PD-L2/PD-1 interaction. In vivo, it abolishes the suppressive effect of the PD-L2-overexpressing tumor immune microenvironment by blocking PD-L2/PD-1 signaling. Moreover, the combination of zinc undecylenate and EGFR-TKIs can synergistically reverse tumor resistance, which is dependent on CD8+ T cells mediating apoptosis. Our study uncovers the PD-L2/PD-1 signaling pathway as a driving factor to mediate EGFR-TKIs resistance, and identifies a new naturally-derived agent to reverse EGFR-TKIs resistance.

New insights into immune cells in cancer immunotherapy: from epigenetic modification, metabolic modulation to cell communication.

Cancer is one of the leading causes of death worldwide, and more effective ways of attacking cancer are being sought. Cancer immunotherapy is a new and effective therapeutic method after surgery, radiotherapy, chemotherapy, and targeted therapy. Cancer immunotherapy aims to kill tumor cells by stimulating or rebuilding the body's immune system, with specific efficiency and high safety. However, only few tumor patients respond to immunotherapy and due to the complex and variable characters of cancer immune escape, the behavior and regulatory mechanisms of immune cells need to be deeply explored from more dimensions. Epigenetic modifications, metabolic modulation, and cell-to-cell communication are key factors in immune cell adaptation and response to the complex tumor microenvironment. They collectively determine the state and function of immune cells through modulating gene expression, changing in energy and nutrient demands. In addition, immune cells engage in complex communication networks with other immune components, which are mediated by exosomes, cytokines, and chemokines, and are pivotal in shaping the tumor progression and therapeutic response. Understanding the interactions and combined effects of such multidimensions mechanisms in immune cell modulation is important for revealing the mechanisms of immunotherapy failure and developing new therapeutic targets and strategies.

Hsa_circ_0004872 mitigates proliferation, metastasis and immune escape of meningioma cells by suppressing PD-L1

Meningioma is a prevalent intracranial malignancy known for its aggressive growth. Circular RNAs (circRNAs) play a crucial role in the development of various cancers. However, their involvement in meningioma remains understudied. This study aimed to investigate the function and underlying mechanism of hsa_circ_0004872 in meningioma. The molecular expression of hsa_circ_0004872, PD-L1 and EIF4A3 was identified by RT-qPCR and/or western blot assays. Cell viability, migration, and invasion were assessed through CCK-8 and Transwell assays, respectively. Cytotoxicity was determined using an LDH assay, and cell apoptosis was monitored by flow cytometry. The RNA and protein interactions were assessed through RNA-protein immunoprecipitation (RIP) and RNA pull down analyses. Our findings revealed that hsa_circ_0004872 expression was significantly downregulated in both meningioma tissue samples and cells. Overexpression of hsa_circ_0004872 inhibited the proliferation, metastasis, and immune escape of meningioma cells, as well as enhanced the cytotoxicity of CD8+ T cells by suppressing PD-L1. Furthermore, hsa_circ_0004872 directly interacted with EIF4A3, leading to the degradation of PD-L1 mRNA. Finally, inhibiting EIF4A3 improved the proliferation, metastasis, and immune escape of meningioma cells, as well as the cytotoxicity of CD8+ T cells. Our study demonstrated that hsa_circ_0004872 mitigated the proliferation, metastasis,and immune escape of meningioma cells by targeting the EIF4A3/PD-L1 axis. These findings suggested that hsa_circ_0004872 and EIF4A3 might serve as promising biological markers and therapeutic targets for meningioma treatment.

Different contractility modes control cell escape from multicellular spheroids and tumor explants.

Cells can adapt their active contractile properties to switch between dynamical migratory states and static homeostasis. Collective tissue surface tension, generated among others by the cortical contractility of single cells, can keep cell clusters compact, while a more bipolar, anisotropic contractility is predominantly used by mesenchymal cells to pull themselves into the extracellular matrix (ECM). Here, we investigate how these two contractility modes relate to cancer cell escape into the ECM. We compare multicellular spheroids from a panel of breast cancer cell lines with primary tumor explants from breast and cervical cancer patients by measuring matrix contraction and cellular spreading into ECM mimicking collagen matrices. Our results in spheroids suggest that tumor aggressiveness is associated with elevated contractile traction and reduced active tissue surface tension, allowing cancer cell escape. We show that it is not a binary switch but rather the interplay between these two contractility modes that is essential during this process. We provide further evidence in patient-derived tumor explants that these two contractility modes impact cancer cells' ability to leave cell clusters within a primary tumor. Our results indicate that cellular contractility is an essential factor during the formation of metastases and thus may be suitable as a prognostic criterion for the assessment of tumor aggressiveness.

MyD88 and Its Inhibitors in Cancer: Prospects and Challenges.

The interplay between the immune system and cancer underscores the central role of immunotherapy in cancer treatment. In this context, the innate immune system plays a critical role in preventing tumor invasion. Myeloid differentiation factor 88 (MyD88) is crucial for innate immunity, and activation of MyD88 promotes the production of inflammatory cytokines and induces infiltration, polarization, and immune escape of immune cells in the tumor microenvironment. Additionally, abnormal MyD88 signaling induces tumor cell proliferation and metastasis, which are closely associated with poor prognosis. Therefore, MyD88 could serve as a novel tumor biomarker and is a promising target for cancer therapy. Current strategies targeting MyD88 including inhibition of signaling pathways and protein multimerization, have made substantial progress, especially in inflammatory diseases and chronic inflammation-induced cancers. However, the specific role of MyD88 in regulating tumor immunity and tumorigenic mechanisms remains unclear. Therefore, this review describes the involvement of MyD88 in tumor immune escape and disease therapy. In addition, classical and non-classical MyD88 inhibitors were collated to provide insights into potential cancer treatment strategies. Despite several challenges and complexities, targeting MyD88 is a promising avenue for improving cancer treatment and has the potential to revolutionize patient outcomes.

Brain Metastasis from EGFR-Mutated Non-Small Cell Lung Cancer: Secretion of IL11 from Astrocytes Up-Regulates PDL1 and Promotes Immune Escape.

Patients who have non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations are more prone to brain metastasis (BM) and poor prognosis. Previous studies showed that the tumor microenvironment of BM in these patients is immunosuppressed, as indicated by reduced T-cell abundance and activity, although the mechanism of this immunosuppression requires further study. This study shows that reactive astrocytes play a critical role in promoting the immune escape of BM from EGFR-mutated NSCLC by increasing the apoptosis of CD8+ T lymphocytes. The increased secretion of interleukin 11(IL11) by astrocytes promotes the expression of PDL1 in BM, and this is responsible for the increased apoptosis of T lymphocytes. IL11 functions as a ligand of EGFR, and this binding activates EGFR and downstream signaling to increase the expression of PDL1, culminating in the immune escape of tumor cells. IL11 also promotes immune escape by binding to its intrinsic receptor (IL11Rα/glycoprotein 130 [gp130]). Additional in vivo studies show that the targeted inhibition of gp130 and EGFR suppresses the growth of BM and prolongs the survival time of mice. These results suggest a novel therapeutic strategy for treatment of NSCLC patients with EGFR mutations.

Downregulation of lncRNA MIR17HG reduced tumorigenicity and Treg-mediated immune escape of non-small-cell lung cancer cells through targeting the miR-17-5p/RUNX3 axis.

Long noncoding RNA MIR17HG was involved with the progression of non-small-cell lung cancer (NSCLC), but specific mechanisms of MIR17HG-mediated immune escape of NSCLC cells were still unknown. The present study investigated the function of MIR17HG on regulatory T cell (Treg)-mediated immune escape and the underlying mechanisms in NSCLC. Expression of MIR17HG and miR-17-5p in NSCLC tissue samples were detected using quantitative real-time PCR (qRT-PCR). A549 and H1299 cells were transfected with sh-MIR17HG, miR-17-5p inhibitor, or sh-MIR17HG + miR-17-5p inhibitor, followed by cocultured with Tregs. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine (Edu) staining assay and cell counting kit-8 (CCK-8) assay. Flow cytometry was used for determining positive numbers of FOXP3+CD4+/CD25+/CD8+ Tregs. Through subcutaneous injection with transfected A549 cells, a xenograft nude mouse model was established. Weights and volumes of xenograft tumors were evaluated. Additionally, the expressions of immune-related factors includingtransforming growth factor beta (TGF-β), vascular endothelial growth factor A (VEGF-A), interleukin-10 (IL-10), IL-4, and interferon-gamma (IFN-γ) in cultured cells, were evaluated by enzyme-linked immunosorbent assay and western blot analysis. Then, miR-17-5p was decreased and MIR17HG was enhanced in both NSCLC tissues and cell lines. MIR17HG knockdown significantly suppressed cell proliferation, tumorigenicity, and immune capacity of Tregs in A549 and H1299 cells, whereas sh-MIR17HG significantly reduced expression levels of VEGF-A, TGF-β, IL-4, and IL-10 but promoted the IFN-γ level in vitro and in vivo. Moreover, downregulation of miR-17-5p significantly reversed the effects of sh-MIR17HG. Additionally, we identified that runt- related transcription factor 3 (RUNX3) was a target of miR-17-5p, and sh-MIR17HG and miR-17-5p mimics downregulated RUNX3 expression. In conclusion, downregulation of MIR17HG suppresses tumorigenicity and Treg-mediated immune escape in NSCLC through downregulating the miR-17-5p/RUNX3 axis, indicating that this axis contains potential biomarkers for NSCLC.

Emerging advances in delivery systems for mRNA cancer vaccines

The success of lipid nanoparticles (LNPs) in treating COVID-19 promotes further research of mRNA vaccines for cancer vaccination. Aiming at overcoming the constraints of currently available mRNA carriers, various alternative nano-vectors have been developed for delivering tumor antigen encoding mRNA and showed versatility to induce potent anti-tumor immunity. The rationally designed nano-vaccines increase the immune activation capacity of the mRNA vaccines by promoting crucial aspects including mRNA stability, cellular uptake, endosomal escape and targeting of immune cells or organs. Herein, we summarized the research progress of various mRNA based nano-vaccines that have been reported for cancer vaccination, including LNPs, lipid enveloped hybrid nanoparticles, polymeric nanoparticles etc. Several strategies that have been reported for further enhancing the immune stimulation efficacy of mRNA nano-vaccines, including developing nano-vaccines for co-delivering adjuvants, combination of immune checkpoint inhibitors, and optimizing the injection routes for boosting immune responses, have been reviewed. The progress of mRNA nano-vaccines in clinical trials and the prospect of the mRNA vaccines for cancer vaccination are also discussed.

Cyclin-dependent kinase 12 deficiency reprogrammes cellular metabolism to alleviate ferroptosis potential and promote the progression of castration-resistant prostate cancer.

Cyclin-dependent kinase 12 (CDK12)-deficient prostate cancer defines a subtype of castration-resistant prostate cancer (CRPC) with a poor prognosis. Current therapy, including PARP inhibitors, shows minimal treatment efficacy for this subtype of CRPC, and the underlying mechanism remains elusive. Based on bioinformatics analysis, we evaluated the relationship between CDK12 deficiency and prostate cancer patient's prognosis and treatment resistance. Furthermore, we used CRISPR-Cas9 technology and mass spectrometry-based metabolomic profiling to reveal the metabolic characteristics of CDK12-deficient CRPC. To elucidate the specific mechanisms of CDK12 deficiency-mediated CRPC metabolic reprogramming, we utilized cell RNA-seq profiling and other molecular biology techniques, including cellular reactive oxygen species probes, mitochondrial function assays, ChIP-qPCR and RNA stability analyses, to clarify the role of CDK12 in regulating mitochondrial function and its contribution to ferroptosis. Finally, through in vitro drug sensitivity testing and in vivo experiments in mice, we identified the therapeutic effects of the electron transport chain (ETC) inhibitor IACS-010759 on CDK12-deficient CRPC. CDK12-deficient prostate cancers reprogramme cellular energy metabolism to support their aggressive progression. In particular, CDK12 deficiency enhanced the mitochondrial respiratory chain for electronic transfer and ATP synthesis to create a ferroptosis potential in CRPC cells. However, CDK12 deficiency downregulated ACSL4 expression, which counteracts the lipid oxidation stress, leading to the escape of CRPC cells from ferroptosis. Furthermore, targeting the ETC substantially inhibited the proliferation of CDK12-deficient CRPC cells in vitro and in vivo, suggesting a potential new target for the therapy of CDK12-deficient prostate cancer. Our findings show that energy and lipid metabolism in CDK12-deficient CRPC work together to drive CRPC progression and provide a metabolic insight into the worse prognosis of CDK12-deficient prostate cancer patients. CDK12 deficiency promotes castration-resistant prostate cancer (CRPC) progression by reprogramming cellular metabolism. CDK12 deficiency in CRPC leads to a more active mitochondrial electron transport chain (ETC), ensuring efficient cell energy supply. CDK12 phosphorylates RNA Pol II to ensure the transcription of ACSL4 to regulate ferroptosis. Mitochondrial ETC inhibitors exhibit better selectivity for CDK12-deficient CRPC cells, offering a promising new therapeutic approach for this subtype of CRPC patients.

Aldehyde Dehydrogenese-1 High Cancer Stem-like Cells/Cancer-initiating Cells Escape from Cytotoxic T Lymphocytes due to Lower Expression of Human Leukocyte Antigen Class 1.

Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDHhigh) cells. Cancer immunotherapy employing immune checkpoint blockade has been approved for advanced gastric cancer cases. However, the effectiveness of cancer immunotherapy against gastric CSCs/CICs remains unclear. This study aimed to investigate the susceptibility of gastric CSCs/CICs to immunotherapy. Gastric CSCs/CICs were isolated as ALDHhigh cells using the human gastric cancer cell line, MKN-45. ALDHhigh clone cells and ALDHlow clone cells were isolated using the ALDEFLUOR assay. ALDH1A1 expression was assessed via qRT-PCR. Sphere-forming ability was evaluated to confirm the presence of CSCs/CICs. A model neoantigen, AP2S1, was over-expressed in ALDHhigh clone cells and ALDHlow clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay. Three ALDHhigh clone cells were isolated from MKN-45 cells. ALDHhigh clone cells exhibited a stable phenotype in in vitro culture for more than 2 months. The High-36 clone cells demonstrated the highest sphere-forming ability, whereas the Low-8 cells showed the lowest sphere-forming ability. High-36 cells exhibited lower expression of HLA-A24 compared to Low-8 cells. TCR-T cells specific for AP2S1 showed lower reactivity to High-36 cells compared to Low-8 cells. High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDHhigh CSCs/CICs evade T cells due to lower expression of HLA class 1.

Immune escape of multiple myeloma cells results from low miR29b and the ensuing epigenetic silencing of proteasome genes

BackgroundActivation of CD28 on multiple myeloma (MM) plasma cells, by binding to CD80 and CD86 on dendritic cells, decreases proteasome subunit expression in the tumor cells and thereby helps them evade being killed by CD8+ T cells. Understanding how CD28 activation leads to proteasome subunit downregulation is needed to design new MM therapies.MethodsThis study investigates the molecular pathway downstream of CD28 activation, using an in vitro model consisting of myeloma cell lines stimulated with anti-CD28-coated beads.ResultsWe show that CD28 engagement on U266 and RPMI 8226 cells activates the PI3K/AKT pathway, reduces miR29b expression, increases the expression of DNA methyltransferase 3B (DNMT3B, a target of miR29b), and decreases immunoproteasome subunit expression. In vitro transfection of U266 and RPMI 8226 cells with a miR29b mimic downregulates the PI3K/AKT pathway and DNMT3B expression, restores proteasome subunit levels, and promotes myeloma cell killing by bone marrow CD8+ T cells from MM patients. Freshly purified bone marrow plasma cells (CD138+) from MM patients have lower miR29b and higher DNMT3B (mRNA and protein) than do cells from patients with monoclonal gammopathy of undetermined significance. Finally, in MM patients, high DNMT3B levels associate with shorter overall survival.ConclusionsAltogether, this study describes a novel molecular pathway in MM. This pathway starts from CD28 expressed on tumor plasma cells and, through the PI3K-miR29b-DNMT3B axis, leads to epigenetic silencing of immunoproteasome subunits, allowing MM plasma cells to elude immunosurveillance. This discovery has implications for the design of innovative miR29b-based therapies for MM.

PTPN7 mediates macrophage-polarization and determines immunotherapy in gliomas: A single-cell sequencing analysis.

Protein tyrosine phosphatase non-receptor type 7 (PTPN7) is a signaling molecule that regulates a multitude of cellular processes, spanning cell proliferation, cellular differentiation, the mitotic cycle, and oncogenic metamorphosis. However, the characteristic of PTPN7 in the glioma microenvironment has yet to be elucidated. The prognostic value, genomic features, immune characteristics, chemotherapy prediction, and immunotherapy prediction of PTPN7 were systematically explored at the bulk sequencing level. The cell evolution trajectory, cell communication pattern, and cell metabolic activity related to PTPN7 were systematically explored at the single-cell sequencing level. HMC3 and M0 cells were cocultured with U251 and T98G cells, and flow cytometry was carried out to investigate the polarization of HMC3 and M0. Transwell assay and CCK-8 assay were performed to explore the migration and proliferation activity of U251 and T98G. The expression level of PTPN7 is significantly elevated in glioma and indicates malignant features. PTPN7 expression predicts worse prognosis of glioma patients. PTPN7 is associated with genome alteration and immune infiltration. Besides, PTPN7 plays a crucial role in modulating metabolic and immunogenic processes, particularly by influencing the activity of microglia and macrophages through multiple signaling pathways involved in cellular communication. Specifically, PTPN7 actively mediates inflammation-resolving-polarization of macrophages and microglia and protects glioma from immune attack. PTPN7 could also predict the response of immunotherapy. PTPN7 is critically involved in inflammation-resolving-polarization mediated by macrophage and microglia and promotes the immune escape of glioma cells.

Abstract CT151: Trial in progress:A phase 1 trial of TSC-100 and TSC-101, engineered T cell therapies that target minor histocompatibility antigens to eliminate residual disease after hematopoietic cell transplantation

Abstract Engineered T cell therapies such as CAR-T cell therapies have transformed the treatment of B-cell but not non-B cell hematologic malignancies. Allogeneic hematopoietic cell transplantation (HCT) remains the best curative option for hematologic malignancies but ~40% of patients relapse post-HCT with up to 90% mortality due to residual disease post-HCT. A key challenge in non-B cell malignancies is identifying the right antigens for T cell targeting. Cancer-associated antigens are heterogenous, enabling rapid escape of malignant cells with low antigens, while targeting lineage-specific antigens without distinct expression in malignant versus normal myeloid cells can cause prolonged neutropenia. A potential solution is targeting minor histocompatibility antigens (MiHAs) that are homogenously expressed on all hematopoietic cells and are genetically mismatched between donors and patients undergoing HCT. These mismatches enable T cells to selectively eliminate residual patient hematopoietic cells, normal or malignant, leaving donor cells untouched. TScan has developed allogeneic donor derived T-cell products TSC-100 and TSC-101, targeting MiHAs HA-1 and HA-2 respectively, both presented on HLA-A*02:01. By choosing HCT patients who are HLA-A*02:01 positive (>98% of whom are either HA-1 or HA-2 positive) and donors who are either HLA-A*02:01 or MiHA negative, TSC-100 or TSC-101 can potentially eliminate all residual patient-derived hematopoietic cells after HCT, to prevent disease relapse.Study NCT05473910 is a multi-center, multi-arm, non-randomized controlled Phase 1 umbrella study evaluating the feasibility, safety and preliminary efficacy of TSC-100 and TSC-101. Inclusion criteria (Figure 1) include adults with AML, MDS or ALL eligible for reduced intensity conditioning-based haploidentical donor transplantation from HLA or MiHA mismatched donors. HLA-A*02:01-positive patients undergo HA-1/HA-2 testing and are assigned to either TSC-100 or TSC-101 treatment arms in addition to HCT. HLA-A*02:01-negative patients in the control arm receive HCT alone. Upon count recovery after HCT, patients in treatment arms receive either TSC-100 or TSC-101, administered as single or two doses. Primary endpoints include adverse event profiles and dose limiting toxicities. Secondary endpoints include relapse rates, disease-free survival and overall survival. Exploratory endpoints include surrogates of efficacy such as minimal residual disease (MRD) rates and donor chimerism rates and kinetics. MRD is measured before and after HCT using flow cytometry, NGS and ddPCR. Donor chimerism is measured by standard STR-based and novel high-sensitivity NGS-based assays to quantify residual patient-derived hematopoietic cells. Together, these assays measure elimination of residual patient hematopoietic cells, malignant or normal, and could provide early evidence of biological activity. Citation Format: Monzr Al Malki, Alla Keyzner, Hyung Suh, Uday Popat, Chen Yi-Bin, Saar Gill, Melhem Solh, Joseph Uberti, Iris Isufi, Erica Buonomo, Yun Wang, Jim Murray, Gavin MacBeath, Debora Barton, Shrikanta Chattopadhyay, Ran Reshef. Trial in progress:A phase 1 trial of TSC-100 and TSC-101, engineered T cell therapies that target minor histocompatibility antigens to eliminate residual disease after hematopoietic cell transplantation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT151.

E2F1-EP300 co-activator complex potentiates immune escape in nasopharyngeal carcinoma through the mediation of MELK.

Nasopharyngeal carcinoma (NPC) is characterized by a highly suppressive microenvironment that protects tumor cells against immune attack and facilitates tumor progression. MELK is upregulated in various tumors, whereas its function in the immune escape remains largely unknown. In this study, we investigated the role of MELK during immune escape in NPC. Differentially expressed genes were filtered using GEO datasets and PPI network analysis. NPC cell colony formation and motility were examined, and the impact of CD8⁺ T cells on NPC cells was evaluated. A xenograft model was constructed to detect the growth of tumor cells and the T-cell phenotype of tumor infiltration. ChIP-qPCR and dual-luciferase assays were used to verify the transcriptional regulation of MELK by EP300/E2F1. MELK was overexpressed in NPC, and sh-MELK suppressed the clonogenic ability, migration, and invasion of NPC cells and promoted the killing effects of CD8⁺ T cells. These in vitro findings were reproduced in vivo. EP300 synergized E2F1 to regulate the transcription of MELK in NPC cells. Loss of EP300 or E2F1 reverted the malignant phenotype of NPC cells and promoted the immune effect of CD8⁺ T cells. MELK further suppressed the immune effect of CD8⁺ T cells in the presence of sh-E2F1. EP300 coordinated with E2F1 to promote the transcription of MELK which promoted the growth of NPC cells and repressed the killing effect of CD8⁺ T cells. Blockage of MELK may be a potential way to suppress the immune escape of NPC cells.