CAR27-28ζ NK cell serial killing against Raji cells.

(Related to Fig.6G) The list of differentially abundance proteins between CAR27-ζ vs. CAR27-28.

CAR27-28ζ NK cells show polyfunctionality.

Expression of CD70 at the protein and mRNA levels in different tumor types.

CD28 co-stimulation enhances phosphorylation of the CAR CD3ζ signaling endodomain.

CD70 is highly expressed in many cancers, including multiple myeloma. We show in two cohorts of patients with multiple myeloma that CD70 is elevated in several high-risk disease categories and correlates with poor survival. These findings were validated using single-cell RNA sequencing, flow cytometry, and IHC. Moreover, we demonstrate the feasibility of targeting CD70 in myeloma using NK cells engineered with a chimeric antigen receptor (CAR) incorporating the CD70 cognate receptor CD27 and IL-15 (CAR27/IL-15). CAR27/IL-15 NK cells exerted potent in vitro and in vivo cytotoxicity against CD70+ multiple myeloma cells, comparable with CAR27/IL-15 T cells, and remained effective in BCMA knockout models. Collectively, these results establish CD70 as a promising therapeutic target for high-risk multiple myeloma, particularly for patients who relapse after BCMA-directed therapy, providing preclinical support for the ongoing phase I/II clinical trial of CD70-targeting CAR NK cells (NCT05092451). SIGNIFICANCE:We demonstrate that CD70 expression is elevated in patients with high-risk multiple myeloma and in patients with t(4;14) translocation. CD70-targeting CAR NK cells exhibit potent cytotoxicity against CD70+ multiple myeloma cells and significantly improve survival in xenograft mouse models of multiple myeloma, even in the absence of BCMA expression. See related commentary by Benson Jr and Caligiuri, p. 166.

(Related to Fig.6F) The list of differentially abundance proteins between CAR27-ζ vs. CAR27-28ζ.

Introduction:This study investigated whether probiotics alleviate Endometriosis (EMs)-related inflammation by modulating the gut microbiota and short-chain fatty acids (SCFAs). Methods:An endometriosis model was established in SD rats, which were randomly divided into a normal diet group (NCD) and a probiotic group (NCD_Pro), with four rats per group. After a 4-week dietary intervention, serum and fecal samples were collected. Tumor Necrosis Factor (TNF)-α and Interleukin (IL)-6 levels were measured by ELISA, gut microbiota composition was analyzed via 16S rRNA sequencing, and fecal levels of nine SCFAs were quantified using GC-MS. Results:Probiotic supplementation significantly reduced serum levels of TNF-α and IL-6 (P < 0.05), but did not significantly affect body weight, body length, or lesion volume. Beta diversity analysis revealed significant structural differences in gut microbiota between the two groups (P < 0.05), while alpha diversity showed no significant difference. At the phylum level, probiotic intervention decreased the relative abundance of Firmicutes and increased that of Bacteroidota and Proteobacteria. At the family level, certain bacterial families showed opposite abundance patterns between the two groups. At the genus level, Bifidobacterium and Lactobacillus were significantly enriched in the probiotic group. Microbial co-occurrence network analysis indicated increased node number and connectivity along with enhanced network stability in the probiotic group. SCFA profiling showed decreased levels of butyric acid (BA) and caproic acid (CA), and a significant increase in isocaproic acid (4-MVA) in the probiotic group. Correlation analysis revealed a significant negative association between specific differential microbiota and 4-MVA (r < -0.6, P < 0.01). Conclusion:Probiotic intervention alleviates systemic inflammation in endometriosis by reshaping the gut microbiota structure, enhancing microbial network stability, and modulating the SCFA metabolism. Our findings underscore the role of the gut microbiota-metabolism-immunity axis in EMs pathophysiology and point to 4-MVA as a hypothesis-generating candidate metabolite that requires further validation.

In vivo antitumor activity of CAR27-expressing NK cells.

In vitro characterization of the CAR27 constructs.

In a PDX mouse model of metastatic breast cancer, CAR27-28ζ NK cells lead to a reduction in tumor size and increased NK cell infiltration in the lungs.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy characterized by rapid onset of chemoresistance and poor clinical outcomes. Following decades of, at best, modest clinical advances, the recent FDA approval of tarlatamab, a DLL3 targeting bispecific T-cell engager (BiTE), alongside unprecedented response rates observed with multiple antibody-drug conjugates (ADCs), have ushered in a paradigm shift towards surface targeting strategies in relapsed SCLC patients. These same agents are demonstrating similar efficacy in more rare high-grade neuroendocrine carcinomas, both pulmonary and extrapulmonary; however, they are being largely tested in unselected populations. While providing much-needed optimism for SCLC patients, resistance, both de novo and acquired, is common and must be better characterized to maximize the potential of these new therapeutic classes. We hypothesize that combinatorial targeting of multiple surface proteins using distinct strategies (i.e., BiTEs, ADCs, etc.) represents a novel way to overcome intratumoral heterogeneity common to relapsed SCLC and enhance antitumor immunity engendered by ADC payloads (i.e., topoisomerase 1 [TOP1] inhibitor). To better define the surfaceome of relapsed SCLC, we performed surfaceome mass spectrometry analysis of SCLC cell lines, naïve and relapsed patient derived xenografts (PDXs), and PDXs treated with frontline chemotherapy until relapse occurred, and identified a number of novel and known surface proteins (i.e., TROP2, HER2, B7H3). Surface targeting strategies against HER2, TROP2, and DLL3 are effective in preclinical models (i.e., cell lines and xenograft models) resistant to other common SCLC therapies (i.e., platinum chemotherapy). In particular, ADCs with TOP1 inhibitor payloads were more effective in models with high SLFN11 and target levels, suggesting that sensitivity requires both surface target expression and SLFN11 positivity for greatest response. Notably, single-cell transcriptional profiling of relapsed patient biopsies revealed mutually exclusive expression of surface genes in distinct cell populations, including senescent, drug tolerant persister cells (DTPCs), representing an unique opportunity to target heterogeneous populations. We show that combination targeting against different surface proteins (e.g., DLL3, TROP2, HER2) using both immune (i.e., chimeric antigen receptor T-cells, BiTEs) and payload-based modalities (i.e., ADCs) was more effective than single-agent targeting in resistant, neuroendocrine-low models. Therefore, intratumoral heterogeneity associated with relapsed SCLC, which limits efficacy of single-agent surface targeting strategies, may be exploited with combinatorial therapies to target resistant cell populations, including DTPCs, using payload and immune-based methods. C. Allison Stewart, Kavya Ramkumar, Runsheng Wang, Yan Yang, Bingnan Zhang, Yuanxin Xi, Lixia Diao, Qi Wang, Alberto Duarte, Ping Li, Azusa Tanimoto, Alejandra G. Serrano, Jody Vykoukal, Mukulika Bose, Loukia G. Karacosta, Luisa Solis Soto, Samir Hanash, Jing Wang, John V. Heymach, Lauren Averett Byers, Carl M. Gay. Combination surface targeting strategies in relapsed small cell lung cancer (SCLC) to overcome intratumoral heterogeneity associated with treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2928.

Chimeric antigen receptor (CAR) natural killer (NK) cell immunotherapy offers a promising approach against cancer1-3. However, the molecular mechanisms that regulate CAR-NK cell activity remain unclear. Here we identify the transcription factor cyclic AMP response element modulator (CREM) as a crucial regulator of NK cell function. Transcriptomic analysis revealed a significant induction of CREM in CAR-NK cells during the peak of effector function after adoptive transfer in a tumour mouse model, and this peak coincided with signatures of both activation and dysfunction. We demonstrate that both CAR activation and interleukin-15 signalling rapidly induce CREM upregulation in NK cells. Functionally, CREM deletion enhances CAR-NK cell effector function both in vitro and in vivo and increases resistance to tumour-induced immunosuppression after rechallenge. Mechanistically, we establish that induction of CREM is mediated by the PKA-CREB signalling pathway, which can be activated by immunoreceptor tyrosine-based activation motif signalling downstream of CAR activation or by interleukin-15. Finally, our findings reveal that CREM exerts its regulatory functions through epigenetic reprogramming of CAR-NK cells. Our results provide support for CREM as a therapeutic target to enhance the antitumour efficacy of CAR-NK cells.

The origins of immunosuppression, neutropenia, and anemia in patients with chronic lymphocytic leukemia (CLL) are not fully understood. Because in patients with CLL, circulating exosomes, which participate in cell-to-cell interactions, are CLL cell-derived, we examined whether those exosomes contribute to abnormal features of this disease. Our data revealed that CLL cell-derived exosomes engulfed by healthy donors’ monocytes, fibrocytes, and lymphocytes altered target-cell gene and protein expression and suppressed normal hematopoiesis. CLL cell-derived exosomes increased normal monocytes’ CD14 and CD16 expression such that it mimicked the accessory-cell profile and upregulated T cells’ checkpoint PD-1 and CD160 protein levels, potentially reducing T-cell-mediated anti-CLL activity. In normal B cells, CLL cell-derived exosomes induced apoptosis and CD5 expression, suggesting that CLL cell-derived exosomes eliminate B cells and not all CD19+/CD5+ cells in CLL patients are clonal. RNA sequencing and quantitative real-time PCR revealed that CLL cell-derived exosomes harbored RNAs of pro-apoptotic genes and genes that increase metabolism, induce proliferation, and induce constitutive PI3K-mTOR pathway activation. CLL cell-derived exosomes inhibited hematopoietic progenitor proliferation, hindering the supportive effect of monocyte-derived fibrocytes. Together, our findings suggest that CLL cell-derived exosomes disrupt the immune and hematopoietic systems and contribute to disease progression in patients with CLL.

Background Hyperosmolarity, a key pathological feature of intervertebral disc degeneration (IVDD), significantly contributes to nucleus pulposus cell (NPC) apoptosis. Aquaporin 3 (AQP3), an osmolyte channel, is markedly downregulated in degenerative discs. However, its role in hyperosmotic stress-induced NPC apoptosis remains unclear. Methods Rat NPCs were exposed to hyperosmotic conditions in vitro. Lentiviral vectors were employed to overexpress AQP3, while a selective inhibitor was used to block its function. Western blotting, flow cytometry, and mitochondrial membrane potential assays were performed to assess PI3K/Akt/mTOR signaling, apoptosis, and oxidative stress. An in vivo rat IVDD model was established, and disc integrity was evaluated histologically. Results Hyperosmolarity suppressed AQP3 expression, inhibited PI3K/Akt/mTOR signaling, induced mitochondrial dysfunction, and increased ROS production, thereby promoting NPC apoptosis. AQP3 overexpression restored PI3K/Akt/mTOR activation, attenuated oxidative stress, and reduced apoptosis. In rat models, AQP3 overexpression alleviated IVDD progression and preserved disc structure, whereas AQP3 inhibition exacerbated extracellular matrix degradation and tissue destruction. Conclusions AQP3 deficiency contributes to hyperosmolarity-induced NPC apoptosis through suppression of PI3K/Akt/mTOR signaling, forming a pathological cycle that accelerates IVDD progression. These findings suggest that AQP3 may serve as a promising therapeutic target for delaying IVDD.

Despite extensive investigation into estrogen's role in pulmonary hypertension (PH) development, its effects, whether beneficial or detrimental, remain contentious. This study aimed to elucidate estrogen's potential role in PH under normoxic and hypoxic conditions. Using norfenfluramine- and hypoxia-induced rat models of PH, the study evaluated the impact of 17β-estradiol (E2) on PH progression. E2 promoted PH development under normoxia while providing protection under hypoxia. Mechanistically, under normoxia, E2 upregulated METTL3 (methyltransferase-like 3) gene transcription and protein via an estrogen response element-dependent pathway, which in turn increased the N6-methyladenosine methylation and translational efficiency of PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) mRNA, leading to increased PFKFB3 protein levels and enhanced proliferation and migration of pulmonary artery smooth muscle cells. Conversely, under hypoxia, E2 downregulated METTL3 transcription through a hypoxia response element-dependent mechanism driven by increased HIF-1α (hypoxia-inducible factor 1α) levels, resulting in reduced PFKFB3 protein expression and diminished pulmonary artery smooth muscle cell proliferation and migration. METTL3 and PFKFB3 proteins are upregulated in the pulmonary arteries of patients with pulmonary arterial hypertension. Collectively, these findings suggest that E2 exerts differential effects on PH progression via dual regulation of the METTL3/PFKFB3 protein under normoxic and hypoxic conditions, positioning the METTL3/PFKFB3 protein as a potential therapeutic target for PH treatment.

INTRODUCTION:Esophageal squamous cell carcinoma (ESCC) is a highly fatal cancer with unclear molecular underpinnings. This study utilized bioinformatics to uncover key genes and pathways associated with ESCC and to identify prognostic markers. METHODS:We identified the differentially expressed genes (DEGs) using three datasets (GSE53625, GSE67269, and GSE23400-GPL96). Meanwhile, weighted gene co-expression network analysis (WGCNA) constructed gene co-expression networks based on the GSE23400-GLP97 dataset. Machine-learning algorithms further identified the most critical genes. Additionally, we validated the expression and diagnostic potential of the hub genes using the GSE161533 and GSE38129 datasets. Survival analysis and Gene Set Enrichment Analysis (GSEA) revealed the prognostic value and potential functions of the hub genes, respectively. RESULTS:The study identified 240 DGEs (103 upregulated and 137 downregulated). Concurrently, WGCNA pinpointed 209 genes associated with ESCC. Subsequently, machine-learning algorithms identify four hub genes, including KIF14, GALNT12, MGLL, and EMP1. Moreover, their expression differences and potential as diagnostic biomarkers for ESCC were validated. Survival analysis indicated that elevated GALNT12 expression was associated with a poor prognosis for ESCC patients. GSEA delineated the involvement of GALNT12 in critical biological pathways. CONCLUSION:Our results identified GALNT12 as a novel potential diagnostic and prognostic marker for ESCC.

Background: Lysosomal free cholesterol efflux plays a crucial role in foam cell formation and atherogenesis. In addition to its lipid-modifying functions, whether the direct effect of niacin on lysosomal cholesterol efflux contributes to its anti-atherosclerotic actions remains unknown. In this study, we investigated the role of niacin in atherosclerotic lesion formation in low-density lipoprotein (LDL) receptor knockout (LDLr-/-) mice, focusing on the egress of lysosomal free cholesterol in macrophages. Methods: Aortic atherosclerotic lesions were assessed in LDLr-/- mice administered with niacin. Bone marrow-derived macrophages were cultured as a cell model. The influence of niacin on lysosomal free cholesterol efflux and the protein expression of liver X receptor alpha (LXR alpha), Niemann-Pick type C1 (NPC1), ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) were evaluated. ELISA was conducted to assess the role of niacin in interleukin (IL)-1 beta secretion. Results: Niacin inhibited atherosclerotic lesion formation in LDLr-/- mice; promoted lysosomal free cholesterol efflux; increased CD38 ADP-ribosylcyclase (CD38), LXR alpha, NPC1, ABCA1, and ABCG1 expression; enhanced nicotinic acid adenine dinucleotide phosphate synthesis; and decreased IL-1 beta secretion. Silencing CD38 and LXR alpha attenuated niacin-promoted NPC1, ABCA1, and ABCG1 expression. Silencing LXR alpha or NPC1 abolished the promoting effect of niacin on lysosomal free cholesterol efflux. Conclusion: The promoting effect of niacin on lysosomal free cholesterol efflux via LXR alpha-mediated signaling pathways may contribute to its pleiotropic anti-atherosclerotic actions.

The incidence of atherosclerosis markedly rises following menopause. Our previous findings demonstrated that elevated follicle-stimulating hormone (FSH) levels in postmenopausal women accelerate atherosclerosis progression. Plaque instability, the fundamental pathological factor in acute coronary syndrome, primarily results from vascular embolism due to plaque rupture. Recent evidence highlights that endothelial-to-mesenchymal transition (EndMT) exacerbates plaque instability, although the link between FSH and EndMT has not been fully established. This investigation sought to explore the possible influence of FSH in modulating EndMT. In this study, apolipoprotein E-deficient (ApoE−/−) mice served as an atherosclerosis model, while human umbilical vascular endothelial cells (HUVECs) were used as cellular models. Protein levels were assessed through immunochemical techniques, gene expression was quantified via RT-qPCR, and nucleic acid–protein interactions were evaluated using immunoprecipitation. The m6A modification status was determined by MeRIP, and cellular behaviors were analyzed through standard biochemical assays. Our results indicate that FSH induces EndMT both in vitro and in vivo. Additional investigation suggested that FSH upregulates the transcription factor Forkhead box protein M1 (FOXM1) at both protein and mRNA levels by enhancing the expression of AlkB homolog 5, RNA demethylase (ALKBH5). FSH reduces m6A modifications on FOXM1 through ALKBH5, leading to increased nascent transcript levels and mRNA stability of FOXM1. Dual-luciferase reporter assays highlighted cAMP-response element binding protein (CREB)’s essential function in facilitating the FSH-induced upregulation of ALKBH5. These findings suggest that FSH promotes ALKBH5 expression, facilitates N6-methyladenosine (m6A) demethylation on FOXM1, and consequently, induces EndMT. This study elucidates the impact of FSH on plaque instability and provides insights into potential strategies to prevent acute coronary syndrome in postmenopausal women.

Chimeric antigen receptor (CAR) natural killer (NK) cell immunotherapy offers a promising approach against cancer. However, the molecular mechanisms governing CAR NK cell activity remain poorly understood. In this study, we identified the transcription factor cAMP response element modulator (CREM) as a pivotal regulator of CAR NK cell function. Using a Raji model, single-cell RNA sequencing revealed a marked upregulation of CREM in CAR NK cells during peak anti-tumor activity after adoptive transfer. CREM expression correlated with a transcriptional profile indicative of both activation and regulatory functions.In a CD70-targeting CAR NK cell model, CREM was induced following CAR ligation to its cognate antigen CD70, but only when CAR signaling via CD3ζ ITAMs was functional, underscoring its dependence on intact CAR signaling. Further experiments demonstrated that IL-15 induced CREM in a dose-dependent manner, with CREM expression abolished upon IL-15 blockade. The combination of CAR signaling and IL-15 stimulation synergistically enhanced CREM expression, highlighting the interplay between these pathways. Mass cytometry analysis revealed that CREM-high CAR/IL-15 NK cell clusters exhibited elevated activation markers (e.g., NKG2D, granzyme B) and inhibitory molecules (e.g., NKG2A, LAG3), suggesting an activation-induced exhaustion state.Mechanistically, we demonstrated that CREM is induced via the PKA-CREB axis, activated downstream of CAR and IL-15 signaling. Phosphorylated CREB, driven by PKA and calcium mobilization, directly promoted CREM transcription, as confirmed by ChIP-qPCR. IL-15 further amplified this pathway by upregulating PKA catalytic subunits.Notably, CRISPR/Cas9-mediated knockout (KO) of CREM in CAR NK cells significantly enhanced their cytotoxicity, cytokine production, resistance to tumor-induced immunosuppression across hematologic and solid tumor models, including CD70- and TROP2-targeting CAR NK cells. In vivo, CREM KO improved NK cell proliferation, persistence, tumor infiltration, tumor control, and survival of mice in multiple aggressive solid and hematologic tumor models including metastatic breast cancer, orthotopic pancreatic cancer, and Raji Burkitt lymphoma.Transcriptional and chromatin accessibility analyses (ChIP-seq and ATAC-seq) revealed that CREM acts as a dual-function transcriptional regulator. It represses critical genes involved in NK cell activation and cytotoxicity while upregulating exhaustion-associated genes. CREM KO enhanced chromatin accessibility at effector gene loci, enriching motifs for AP-1, CEBP, and STAT transcription factors, thereby promoting NK cell activation and function.These findings establish CREM as a critical inhibitory checkpoint in CAR NK cells. CREM KO boosts NK cell activation and cytotoxicity, positioning CREM as a compelling target for improving CAR NK cell-based cancer immunotherapies. Hind Rafei, Rafet Basar, Sunil Acharya, Yu-Sung Hsu, Pinghua Liu, Deqiang Zhang, Qingnan Liang, Vakul Mohanty, Ranjan Upadhyay, Ping Li, Pravin Phadatare, Merve Dede, Donghai Xiong, Corry Jones, May Daher, Ana Karen Nunez Cortes, Mayra Shanley, Bin Liu, Sadie M. Moseley, Patrick Zhang, Dexing Fang, Pinaki Banerjee, Nadima Uprety, Ye Li, Rejeena Shrestha, Xinhai Wan, Hong Shen, Vernikka Woods, April Gilbert, Seema Rawal, Jinzhuang Dou, Yukun Tan, Jeong-Min Park, Francia Reyes Silva, Alexander Biederstädt, Mecit Kaplan, Xin Ru Jiang, Inci Biederstädt, Silvia Tiberti, Madison Moore, Jingling Jin, Luis Muniz-Feliciano, Paul Lin, Gary M. Deyter, Natalie W. Fowlkes, Abhinav Jain, David Marin, Ken Chen, Elizabeth Shpall, Katayoun Rezvani. CREM is a regulatory checkpoint of CAR and IL-15 signaling in NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6396.