BACKGROUND:Constipation-predominant irritable bowel syndrome (IBS-C) is a disorder of brain-gut axis dysfunction closely associated with gut microbiota dysbiosis and disruption of mucosal immune homeostasis. Fecal microbiota transplantation (FMT) has been shown to alleviate IBS symptoms; however, its underlying molecular mechanisms remain incompletely understood. MER proto-oncogene tyrosine kinase (Mertk), a member of the receptor tyrosine kinase family, plays an important role in macrophage polarization-related regulation and inflammation resolution. OBJECTIVE:To investigate the role of Mertk-mediated immune regulation in FMT-induced improvement of IBS-C and its underlying mechanisms. METHODS:IBS-C was induced in wild-type(WT) and Mertk conditional knockout(cKO) mice (Mertkflox/floxLyz2Cre/+) by ice-water gavage combined with tail-clamping stress, followed by FMT treatment. Defecation, fecal water content, intestinal transit, and visceral sensitivity were assessed. Colonic histopathology, macrophage polarization-related markers, inflammatory cytokines, tight junction proteins, AKT-GSK3β signaling, and gut microbiota composition were examined by HE staining, immunohistochemistry, qPCR, Western blotting, and 16S rRNA sequencing. RESULTS:In WT IBS-C mice, FMT improved constipation-like symptoms, intestinal transit, and visceral hypersensitivity, reduced colonic inflammation, restored Occludin and Claudin-1 expression, decreased CD86 and IL-1β, increased CD206 and IL-10, and activated AKT-GSK3β signaling. These beneficial effects were markedly attenuated in Mertk-deficient mice. However, FMT similarly remodeled gut microbiota composition in both WT and Mertk-deficient mice. CONCLUSION:FMT alleviates IBS-C partly through Mertk-dependent immune regulation and AKT-GSK3β activation. Gut microbiota remodeling alone is insufficient for full therapeutic efficacy in the absence of intact host Mertk signaling.

Innate lymphoid cells (ILCs) are crucial for cancer immunosurveillance. While mouse type 1 ILCs (ILC1s) control acute myeloid leukemia (AML) by targeting leukemia stem cells (LSCs), the role of human ILC1s in AML remains largely undefined. Here, we find that ILC1s in AML patients are impaired, with reduced total ILC1 numbers and diminished function. In contrast, healthy donor (HD) ILC1s-derived TNFα inhibits the leukemic transition from CD34+CD38+ to CD34-CD38+ cells and blocks the differentiation of LSCs (CD34+CD38-) into immunosuppressive, macrophage-like leukemia-supporting cells. HD ILC1-derived IFNγ partially suppresses the differentiation of CD34-CD38+ to CD34-CD38- cells. These combined effects limit human leukemogenesis in vivo. We also identify a human ILC1 subset as Lin-CD127+CD161-CRTH2-CD117- (CD161- ILC1s) that can be generated from umbilical cord blood CD34+ hematopoietic stem cells. This method could provide a reliable source of ILC1s for potential adoptive transfer therapies in AML, offering a therapeutic approach to prolong disease-free survival in AML.

Background: Hepatocellular carcinoma (HCC) continues to pose a major health concern for global public health, characterized by pronounced clinical heterogeneity in survival outcomes and therapeutic efficacies. The stromal senescence constitutes a major impediment in constructing robust prognostic models. This study aimed to develop and validate a robust prognostic prediction model to accurately assess the prognostic risk of HCC patients and facilitate personalized precision treatment. Methods: We obtained data from the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases. Area Under the Curve Cell (AUCell) was used to quantify senescence in single-cell RNA sequencing (scRNA-seq) of HCC. A stromal-senescence prognostic signature was then built by using Cox and least absolute shrinkage and selection operator (LASSO) analyses. High-and low-risk stratification was interrogated with clusterProfiler, maftools, immune-checkpoint analysis, and oncoPredict. Signature expression was cross-validated in scRNAseq, bulk RNA sequencing (RNA-seq), proteomic, and spatial transcriptomic datasets. Small-molecule ligands targeting DNASE1L3 were identified by virtual screening, and their binding was verified by molecular docking and dynamics simulations. Results: It was found that HCC-associated stromal cells exhibited the most pronounced levels of senescence among cell populations. Using machine-learning algorithms, we successfully established DNASE1L3, NDRG2, ADAM15, IGFBP3, MMP14, and ANGPT2 as a stromal-senescence prognostic signature in HCC. Subsequent comparisons between patient subgroups based on this signature revealed distinct differences in long-term survival outcomes, mutational landscapes, immune checkpoint expression, and responsiveness to chemotherapeutic agents. Further, the stromal-senescence prognostic signature is significantly associated with metabolic reprogramming, immune inflammation, cell proliferation, and metastasis. Multi-omics data analyses were employed to validate the expression levels of this signature in both tumor and normal tissues. In addition, virtual screening identified three potential DNASE1L3 candidates: STL167676, STK598132, and STK119225. Conclusions: This study developed a robust stromal senescence-associated gene signature that accurately predicts survival outcomes in HCC patients and identified candidate stromal senescence-related molecules for more effective personalized therapy.

Colorectal cancer (CRC) is the most prevalent digestive system malignancy worldwide. The development of targeted therapeutics specifically effective for CRC is currently in dire need. Preclinical studies showed that CDK4/6 inhibitor palbociclib suppressed the growth of CRC, but whether this effect is durable is unclear. In this study, we aimed to evaluate the roles of palbociclib-induced senescence and find a new strategy to maximize its effectiveness in CRC treatment. Animal and cellular experiments revealed that palbociclib-induced senescence and the senescence-associated secretory phenotype (SASP) caused drug resistance, anti-apoptosis, PD-L1 upregulation and inhibition of CD8+ T cells’ function. Using CRISPR/Cas9 screening, we identified MCL1 as a senolytic target to eliminate palbociclib-induced senescent CRC cells in the presence of palbociclib. Mechanically, palbociclib-induced senescent cells upregulated ZHX2 and its transcriptional target MCL1, rendered their resistance to apoptosis and T cell-mediated cytotoxicity, whereases combining palbociclib with MCL1 inhibitor markedly induced apoptosis in senescent cells by activating both extrinsic and intrinsic apoptotic pathways. Lastly, we proposed a seno-therapy consisting of a palbociclib pre-treatment plus a combination treatment of palbociclib and MCL1 inhibitor and found it effectively inhibited tumor growth and improved the survival of CRC xenografted mice. Besides its senolytic effect, seno-therapy also reduced PD-L1-positive cells and enhancing the cytotoxic functions of CD8+ T cells. In conclusion, co-targeting CDK4/6 and MCL1 efficiently eliminates palbociclib-induced senescent CRC cells and offers a promising CDK4/6 inhibitor-based strategy for CRC treatment, ensuring prolonged tumor suppression and reducing the risk of progression or recurrence.

AIMS:Chemotherapy-induced intestinal mucositis (CIM) is a common and severe side effect linked to disrupted intestinal stem cell (ISC) balance, though its molecular regulation remains unclear. The principal objective of this research was to elucidate the role of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) in intestinal stem cell balance and mucosal repair during CIM, with a specific focus on the Wnt/β-catenin pathway. MATERIALS AND METHODS:This study used multi-omics and gene editing to elucidate ECSIT's role in intestinal stem cell balance and mucosal repair via the Wnt/β-catenin pathway. Multi-omics and Gene Set Enrichment Analyses (GSEA) identified ECSIT as a key CIM regulator. KEY FINDINGS:Chemotherapeutic drugs dose-dependently reduced ECSIT expression in intestinal epithelial cells. Clinical analysis revealed that ECSIT expression decreased with increasing pathological severity (normal > inflammation > adenocarcinoma). In intestinal epithelium-specific knockout mice, ECSIT deficiency worsened irinotecan (CPT11)-induced CIM and blocked β-catenin nuclear translocation. ECSIT stabilized the β-catenin complex, regulating Wnt target genes like Axis inhibition protein (AXIN) and Cyclin D1 (CCND1); its knockout reduced Wnt signaling. Single-cell sequencing (scRNA-seq) revealed that ECSIT knockout reduced Lgr5 stem cells and increased inflammatory cell infiltration. Lgr5-specific inducible knockout and ECSIT complementation demonstrated that restoring ECSIT reversed β-catenin inhibition and improved CIM pathology. SIGNIFICANCE:This study clarified ECSIT's dual role in stabilizing β-catenin and sustaining Wnt signaling-regulating Lgr5 intestinal stem cell proliferation and differentiation, epithelial renewal, and immune balance. These findings offer insights into CIM pathogenesis and establish the basis for developing targeted therapy through ECSIT-Wnt axis regulation.

ABSTRACT:We report a phase 1 study assessing the safety and efficacy of CD19 chimeric antigen receptor (CAR) T cells as definitive consolidation in older adults (aged ≥55 years) with B-cell acute lymphoblastic leukemia (B-ALL) in first complete remission (CR1). Eighteen patients received lymphodepletion followed by infusion of memory-enriched CD19 CAR T cells. The median age was 64 years, and all patients were measurable residual disease (MRD)-negative before lymphodepletion. There were no dose-limiting toxicities, grade ≥2 cytokine release syndrome, or any grade immune effector cell-associated neurotoxicity syndrome. Estimated 18-month event-free and overall survival were 84% and 100%, respectively. CAR T cells expanded in the blood and cerebrospinal fluid despite patients' MRD-negative status. Comparing clinical samples from patients with relapsed/refractory (R/R) B-ALL from our historical trial (ClinicalTrials.gov identifier: NCT02146924) and patients in CR1, we found that the blood and CAR T-cell products from patients with R/R B-ALL were hyperinflammatory and hyperimmunometabolic, respectively. First-line CAR T-cell therapy was safe and well tolerated and potentially extended remission in patients in MRD-negative CR1. These findings support further investigation of the early use of CAR T-cell therapy for B-ALL. This trial was registered at www.clinicaltrials.gov as NCT05707273.

BackgroundFusobacterium nucleatum (Fn), a gram-negative oral bacterium, has been implicated in promoting colorectal cancer (CRC) progression by modulating the inflammatory microenvironment. Recent studies have shown that gut microbiota imbalance plays a crucial role in CRC development, and Fn, specifically, has been found to exist in the intestine and exert pro-tumorigenic effects. However, the precise mechanisms underlying Fn-induced CRC progression remain elusive.MethodsA systematic experimental strategy was adopted to explore the function of Fusobacterium nucleatum (Fn) in colorectal cancer (CRC) progression. Tissue specimens were collected from healthy volunteers, patients with ulcerative colitis (UC), colorectal adenoma (CRA) and CRC at the First Affiliated Hospital of Henan University of Science and Technology via colonoscopy and surgical resection. Two CRC mouse models, subcutaneous MC38 model and AOM-DSS orthotopic model, were constructed, followed by intratumoral injection or oral gavage of Fn, Streptococcus mutans (S.M.) or normal saline. qPCR was used to quantify Fn abundance in colorectal tissues. IHC and FISH detected inflammatory cytokines and immune cells in tumor tissues. 16S rRNA amplicon sequencing analyzed Fn-mediated alterations in mouse gut microbiota. Flow cytometry and ELISA evaluated immune cell subsets and inflammatory cytokine levels. Western blot examined NLRP3 inflammasome and other proteins expression.ResultsIn this study, we employed two mouse models of CRC (MC38 subcutaneous and AOM-DSS orthotopic models) to investigate the role of Fn in CRC progression. Our findings demonstrated that Fn administration significantly increased tumor growth compared to controls. Quantitative PCR confirmed elevated Fn abundance in CRC tissues, and microbiome analysis revealed distinct bacterial clades in Fn-treated mice. Immunofluorescence analysis showed enrichment of myeloid cells (CD45/CD11b, CD11C, F4/80,MHCI,MHCII) in tumors, suggesting that Fn reshapes the immune landscape towards a proinflammatory state. In vitro and in vivo experiments further revealed that Fn selectively expands myeloid-derived immune cells. Critically, Fn failed to induce tumor growth in NLRP3-/- mice, confirming that NLRP3 inflammasome activation is a key mechanism in Fn-induced CRC progression.ConclusionOur results establish Fn as a driver of CRC progression through bone marrow-derived immune cell recruitment and NLRP3-dependent inflammatory microenvironment induction. These findings provide new insights into the mechanisms by which Fn contributes to CRC development and suggest potential therapeutic targets for CRC prevention and treatment.

Blinatumomab (blina), a bispecific anti-CD19/CD3 T cell engaging antibody (Ab), provides improved outcomes in patients (pts) with r/r B-ALL but over half fail to respond (CR/CRh, 44%) and most relapse without consolidation therapy. We hypothesized that pembrolizumab (pembro), a PD-1 Ab, would enhance T cell effector function and augment blina anti-leukemia activity. Here we report the primary analysis from a single arm, phase 1/2 trial (NCT03512405) evaluating blina-pembro in r/r B-ALL. Patients ≥18 years with CD19+ r/r B-ALL received standard blina 9 µg/day on days 1-7 and 28 µg/day on days 8-28 in cycle 1 and on days 1-28 in subsequent cycles. Pembro 200 mg was given on day 15 in cycle 1 and on days 1 and 22 in subsequent cycles which was defined as schedule A. Blood and bone Marrow (BM) biopsy were collected at prespecified timepoints for disease assessment and coorelative analysis. High parameter immunohistochemistry (IHC) was used to evaluate T cell infiltration and phenotypic profiles using a supervised machine learning cell classifier. As of July 14, 2025, 7 pts enrolled to phase 1 (1 unevaluable) and 19 patients enrolled to phase 2 (1 unevaluable). All were treated at Schedule A dosing which was determined as recommended phase 2 dose. Median age was 49 years (range, 24-74), with 1 median prior regimens (range 1-5), and 49% (range, 10-95%) median BM blasts with 50% of evaluable patients with more than 50% BM blast. Poor risk features were common in 87% (21/24) including 50% Philadelphia chromosome like ALL. The study met its prespecified primary endpoint. Of 24 evaluable pts, 17 (71%) achieved CR/CRi after a median of 1 (range, 1-2 cycles). The CR/CRi rate was 71.4% (15/21) in cytogenetic poor-risk group, 58% (7/12) in high disease burden group, and 66.7% (9/12) in Ph-like group. Sixteen pts were measurable residual disease not detectable (MRD-) by flow cytometry, one achieved CR after cycle 2 (refractory after cycle 1). Thirteen CR pts underwent allogeneic transplant (alloHCT) with post-transplant cyclophosphamide (PTCy) as graft versus host disease (GVHD) prophylaxis. The median treatment cycles were 2 for responders who received alloHCT vs 3 for those who did not (P=0.237). Both groups received 3 median doses of pembro (P=0.442). Among 4 CR pts not undergoing alloHCT, 3 relapsed with median progression free survival (PFS) of 3.0 (range, 2.3-12.4) months; one remained in remission at month 28 after CAR T consolidation. Three pts developed ≥gr3 acute GVHD and all resolved with treatment, and 5 had NIH defined mild cGVHD. With a median follow-up of 14.7 months (1.8-20.9), 11 (65%) CRs are ongoing (10 post-transplant and 1 post-CAR-T). Sixteen of 24 evaluable pts were alive at data cut-off, with a 1-year OS rate of 74% (95% CI: 58-94%). In all cycles of treatment, cytokine release syndrome (CRS) occured in 50% patients (13/26) including 2 gr 3 events. All except 3 occured prior to first pembro dose. Neurologic toxicities were observed in 50% (13/26) patients, with 2 ≥ gr3 events; all reversible. Five pts (19%) experienced pembro-related immune AEs. All-non-hematologic gr3 toxicity were reversible. No dose limiting toxicity, ≥ gr4 non-hematologic toxicity, or treatment related deaths were reported. Immunoprofiling of tumor immune microenvironment (TIME) using high-dimensional IHC revealed significantly increased densities of memory CD45RO+CD8+ T cells from baseline to end of first treatment cycle (p=0.028, 48.36 vs 66.65 cells/mm2), and Tim-3+ CD4+ T cells (p=0.03, 86.89 vs 504.2 cells/mm2), with a significant rise in Tim-3+ cell frequency amongst total CD4+ T cells (p= 0.0006, 6.2 vs 26.5%). A trend towards higher densities of memory CD4+ T cell at baseline was seen in pts with CR/CRi compared to refractory pts (p= 0.0548, 735.4 vs 128.8 cells/mm2). In conclusion, our study showed that blina combined with pembro demonstrated a manageable safety and encouraging high efficacy in patients with poor risk r/r B-ALL, supporting its potential as a successful bridging therapy to alloHCT with high rate of durable remissions among those receiving alloHCT, without increasing the GVHD rate. Immune profiling revealed significant remodeling of the TME in the BM, suggesting mechanisms of response involving CD4+ and CD8+ T cell activation and infiltration.

BackgroundTriple-negative breast cancer (TNBC) is clinically aggressive. CF33-hNIS-antiPDL1, an oncolytic orthopoxvirus, shows robust anti-cancer activity in TNBC xenografts in mice. CF33-hNIS-antiPDL1-infected tumor cells express functional human sodium iodide symporter (hNIS) and are visible by single-photon emission computed tomography (SPECT) or positron emission tomography (PET). We evaluated the ability of virus-encoded hNIS to track OV in mice using PET imaging and in a phase I study in TNBC patients using SPECT. The aim of this first-in-human study was to determine imageability and safety of intratumoral (IT) CF33-hNIS-antiPDL1 injections.MethodsImageability of CF33-hNIS-antiPDL1 was first assessed in mice bearing human xenografts. Virus or PBS-treated mice were imaged using a PET scanner. For the first-in-human trial, 9 patients were enrolled in this phase I, single-center, single-arm trial from October 2021 to August 2023. Key eligibility criteria included unresectable/metastatic TNBC; progressed on at least 2 prior chemotherapies; ECOG 0-2; RECIST 1.1 measurable disease; and at least one tumor amenable to repeated IT injections. Eligible patients received CF33-hNIS-antiPDL1 IT at 1 of 6 assigned dose levels (ranging from 1 × 105 PFU to 3 x 108 PFU) on days 1 and 15 of each 28-day cycle for 3 treatment cycles. SPECT whole-body imaging was performed using technetium-99 at cycle 1 day 8.ResultsAll mice treated with the virus showed clear PET signal from tumors whereas no signal was observed in PBS-treated mice. In the phase I study, 7 of 9 patients (78%) showed uptake at the injection site on SPECT imaging at C1D8. Five of 5 patients (100%) with injection sites at metastatic subcutaneous nodules, intramuscular masses, or axillary lymph nodes, and 2/4 patients (50%) with injection sites at matted dermal metastatic lesions had uptake at injected lesions.ConclusionSPECT imaging successfully showed enhancement at the injected lesions in 78% of patients treated with CF33-hNIS-antiPDL1, even at low doses of the oncolytic virus (OV), suggesting local viral replication and hNIS expression. This is the first report of hNIS-based imaging to track oncolytic poxvirus replication in humans. This technology holds promise for noninvasive tracking of systemically administered OVs and other therapies.Clinical trial registrationhttps://www.clinicaltrials.gov/study/NCT05081492, identifier NCT05081492.

Background and Significance: Pegaspargase-based therapy for acute lymphoblastic leukemia (ALL) offers well-established benefits but treatment-related toxicity precludes its widespread adoption. Obesity (defined as body mass index [BMI] >30 mg/m²) and older age (≥40 years) are risk factors linked to high-grade asparaginase toxicity, with hepatotoxicity often emerging early in induction, following the initial dose of pegaspargase. Beyond hepatotoxicity, advanced age and/or elevated BMI are also associated with increased risk of asparaginase-induced thrombosis and pancreatitis. Given the rising national prevalence of obesity, this patient subgroup presents a unique and increasingly relevant clinical concern, warranting the development of alternative asparaginase formulations to mitigate toxicity risks. Erwinia-based asparaginase is approved for patients who experience hypersensitivity to E. coli-derived formulations. Recombinantly produced asparaginase Erwinia (Rylaze) has a short half-life compared to pegaspargase, and it has demonstrated efficacy in depleting asparagine among hypersensitive patients. Studies examining Erwinia-based asparaginase as a second-line therapy reveal a low incidence of high-grade hepatotoxicity and generally reduced toxicity compared to other formulations. We propose evaluating recombinant Erwinia asparaginase during induction in newly diagnosed adults with ALL who are at high risk for asparaginase-related toxicities. This approach aims to enhance safety and broaden the use and tolerability of pediatric-inspired regimens. Eligible patients will include obese or older AYA individuals who are often under-dosed or not offered asparaginase containing regimens due to toxicity concerns. Given its short half-life, recombinant Erwinia asparaginase may result in shorter biochemical toxicity duration. As newly diagnosed ALL patients are typically hospitalized during early induction, frequent dosing is unlikely to pose a significant burden. We hypothesize that this regimen will offer a more favorable toxicity profile than pegaspargase, while maintaining comparable efficacy. Study Design and Methods: This study (NCT06918431) is a prospective, non-randomized, two-stages, single arm, multi-center (City of Hope, University of Chicago, Memorial Sloan Kettering, Cleveland Clinic, University of Cincinnati) phase 2 clinical trial. We will utilize recombinant Erwinia asparaginase (7 doses, 25 mg/m2 per dose every 48 hours intramuscular) as part of the C10403 induction regimen in order to assess safety, tolerability and response in high-risk newly diagnosed adults with B/T ALL and lymphoblastic lymphoma (LBL) who are either obese (age 18-39 and BMI ≥30) or older (age 40-54, regardless of BMI). A single dose level and a single cycle (28-day induction cycle 1) safety lead-in (SLI) phase will ensure there are no above-threshold unacceptable toxicities (UT) encountered at the FDA approved dose. The SLI phase will enroll 6-12 patients. If the number of UTs is below the rule-based threshold, then an expansion cohort will assess the primary endpoint - grade 3 or higher hepatotoxicity at least possibly related to the study drug and not recovered to grade 1 within 14 days of occurrence. The study will evaluate key secondary endpoints including rates of complete remission (CR)/CR with incomplete count recovery (CRi), undetected minimal residual disease, other asparaginase-related toxicities and maintaining adequate 48 hours nadir serum asparaginase activity. Enrolled patients who achieve CR after induction may receive recombinant Erwinia asparaginase during consolidation at the discretion of the treating physician. The study aims to accrue a total of 50 patients assuming 40% hepatotoxicity (grade 3 or higher) rate and 80% average CR/CRi rate for the tested therapy. If our study demonstrates the tolerability and safety of using frontline recombinant Erwinia asparaginase during induction in high-risk adults with ALL, this approach could potentially establish it as the first-line asparaginase in this population and support its use in subsequent treatment cycles. Acknowledgements:This study is financially supported by Jazz Pharmaceuticals, which also conducted a courtesy medical review of this abstract.

CAR-T cells are a powerful yet expensive tool in cancer immunotherapy. Although their use in targeting hematological malignancies is well established, using a single CAR-T cell therapy to treat both hematological and solid tumors, which can reduce cost, remains largely unexplored. In this study, we identified CD155, an adhesion molecule that is upregulated during tumor progression, as a target for CAR-T cell therapy in both leukemia and solid tumors. We engineered CAR-T cells using human and mouse anti-CD155 antibodies generated from a Berkeley Lights' Beacon platform. These CAR-T cells demonstrated potent antitumor activity, significantly reducing tumor burden in preclinical models of acute myeloid leukemia, non-small cell lung cancer, and pancreatic cancer. To reduce potential allogeneic rejection, we generated CAR-T cells using humanized anti-CD155 antibody sequences that retained efficacy. Additionally, murine CAR-T cells targeting mouse CD155 exhibited limited toxic side effects in immunocompetent mice, highlighting the favorable safety profile of this therapy. These findings suggest that CD155 can be targeted by CD155 CAR-T cells safely and effectively, representing an innovative cellular therapeutic strategy that has the potential to expand its scope across both AML and multiple solid tumors, thereby lowering the cost of cellular immunotherapy, especially as allogenic, universal, and off-the-shelf CAR-T cell therapies advance to the clinic.

Blinatumomab is approved for the treatment of relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). Studies have correlated pre-blinatumomab high disease burden (HDB) [> 50% bone marrow blasts (BMB)] with lower response rates and increased risk for toxicities, including cytokine release syndrome (CRS) and neurotoxicity (NT). While the administration of pre-blinatumomab cytoreductive therapy is an appealing approach, larger studies validating the beneficial effect of this strategy in patients with HDB are lacking. We retrospectively analyzed 148 adult patients with R/R B-ALL treated with blinatumomab. Patients were grouped as low disease burden (LDB, n = 55), HDB without cytoreduction (n = 41), and HDB with cytoreduction (n = 52). The median age for the cohort was 40 years; the majority were males (63%) and Hispanic (73%), and the most common ALL subtype was Ph-like (40%). Patients with HDB with cytoreduction had a significantly higher rate of prior alloHCT (p = 0.041) and more lines of prior therapy (p = 0.006) compared to the other cohorts. Compared to patients with HDB, those with LDB had significantly higher response rates to blinatumomab (p < 0.0001), while rates of CRS and NT were not significantly different. Among patients with HDB, cytoreductive therapy was associated with a significantly lower rate of CRS compared to those who did not receive cytoreduction (p = 0.038). However, cytoreduction had no significant impact on treatment response, MRD negativity, or NT. Patients with B-ALL and HDB remain a challenging population for blinatumomab therapy; while cytoreduction may improve safety by reducing CRS, novel strategies are needed to enhance treatment efficacy.

NKp46 is a critical regulator of natural killer (NK) cell immunity, but its function in non-NK innate immune cells remains unclear. Here, we show that NKp46 is indispensable for expressing IL-2 receptor-α (IL-2Rα) by non-NK liver-resident type-1 innate lymphoid cells (ILC1s). Deletion of NKp46 reduces IL-2Rα on ILC1s by downregulating NF-κB signaling, thus impairing ILC1 proliferation and cytotoxicity in vitro and in vivo. The binding of anti-NKp46 antibody to NKp46 triggers the activation of NF-κB, the expression of IL-2Rα, interferon-γ (IFN-γ), tumor necrosis factor (TNF), proliferation, and cytotoxicity. Functionally, NKp46 expressed on mouse ILC1s interacts with tumor cells through cell-cell contact, increasing ILC1 production of IFN-γ and TNF, and enhancing cytotoxicity. In a mouse model of acute myeloid leukemia, deletion of NKp46 impairs the ability of ILC1s to control tumor growth and reduces survival. This can be reversed by injecting NKp46+ ILC1s into NKp46 knock-out mice. Human NKp46+ ILC1s exhibit stronger cytokine production and cytotoxicity than their NKp46- counterparts, suggesting that NKp46 plays a similar role in humans. These findings identify an NKp46-NF-κB-IL-2Rα axis and suggest that activating NKp46 with an anti-NKp46 antibody may provide a potential strategy for anti-tumor innate immunity.

Detecting ovarian cancer (OC) early using existing biomarkers, for example, cancer antigen 125 (CA125), is challenging due to its ubiquitous expression in many tissues. Doppel, a prion-like protein, expresses in the male reproductive organ but is absent in female reproductive systems and healthy tissues, but plays an important role in neo-angiogenesis. Here, we have shown two platforms, soluble Doppel in sera/ascites and Doppel expressed in circulating tumor cells (Dpl+CTC) in the whole blood, to detect subsets of epithelial OC (EOC). Increased levels of Doppel in the sera of OC patients, in three different cohorts, confirm Doppel as an OC-specific biomarker. Serum Doppel levels can distinguish OC with high sensitivity and specificity (sensitivity = 0.91 and specificity = 0.89) and can also detect early-stage HGSOCs (FIGO stages I and II) from non-cancerous conditions with high sensitivity and specificity (sensitivity = 0.94 and specificity = 0.83). Moreover, significantly higher Doppel expression is observed in all EOC subtypes except clear cell OC. Stratifying the EOCs based on Doppel levels, we categorized them into Doppel-high (Dplhi) and Doppel-low (Dpllow) groups. Using ascites-derived organoids, made from Dplhi and Dpllow patients, we identify that Doppel induces epithelial-mesenchymal transition (EMT). Doppel levels in the sera/ascites correlate with the changes in Dpl+CTC number in whole blood, highlighting the association of Doppel-induced EMT with CTC dissemination in the circulation. Thus, Doppel-based detection of EOC subtypes could be a promising platform as a clinical biomarker and link the Doppel axis with OC dissemination.

Probiotics interventions may contribute to eradicate Helicobacter pylori infection and reduce gastrointestinal symptom, but their mechanisms require more research. This study involved 100H. pylori-positive adults, randomly assigned to either Weizmannia coagulans BC99 group or Placebo group. Both groups were received bismuth quadruple therapy for 4 weeks and took either BC99 or placebo for 6 weeks. After 6 weeks, BC99 significantly reduced eradication rate and happening of adverse events. BC99 reduced GSRS score, particularly the severity of stomach-abdominal pain during hunger symptoms. Additionally, BC99 reduced the disruption of bismuth quadruple therapy on the gut microbiota, particularly significantly increasing the abundance of Prevotella, Bacillus, Candidatus_Saccharimonas and reducing the abundance of Lachnoclostridium, Intestinibacter, Holdemania, Desulfovibrio. These findings supported the use of W. coagulans BC99 in combination with BQT in the clinical management of patients with H. pylori infection.

The heat shock factors (HSFs) play important roles in activating heat stress responses in plants. Cerasus humilis (Ch) is a nutrient-rich fruit tree that can resist various abiotic and biotic stressors. However, the HSFs in C. humilis have not yet been characterized and their roles remain unclear. In this study, 21 ChHSF gene members were identified after searching the entire genome of C. humilis. Gene structure and motif composition analysis revealed that 16 ChHSF genes had only one intron and the motif3 was highly conserved in family of ChHSFs. Furthermore, the cis-acting elements analysis indicated that they most ChHSFs participate in plant growth and development, abiotic stress responses, and plant hormone regulations. By analyzing the tissue specific transcriptomes, it was found that most ChHSF genes had higher expression levels in leaves than in other tissues of C.humilis. Notably, the ChHSF04 gene exhibited a striking 115.5-, 14.4-, and 16.0-fold higher expression in leaves relative to seeds, roots, and fruits, respectively. The high temperature (40 °C) treated C. humilis seedlings quantitative real-time polymerase chain reaction (qRT-PCR) was conducted on all ChHSF gene members. The results show that the expression of most ChHSF genes in the leaves was significantly upregulated and peaked at 12 h under the heat stress and the expression levels of ChHSF04, ChHSF05, ChHSF12, ChHSF13, ChHSF15 and ChHSF16 exhibited 53-, 33-, 24-, 22-, 43- and 65-fold upregulation, indicating that these genes may play important roles in early response to heat stress in C. humilis. These results provide valuable insights into the evolutionary relationship of the ChHSF gene family and its role in high temperature stress responses.

Background: CF33-hNIS-anti-PD-L1 is a novel chimeric orthopoxvirus with robust anti-cancer activity in TNBC xenografts. Previously, we showed that CF33-hNIS-anti-PD-L1 increased CD8+ T cell infiltration and favorably altered the tumor microenvironment (TME) in xenograft models. We have observed similar effects in our first-in-human clinical trial of CF33-hNIS-anti-PD-L1 intratumoral (IT) injections in patients with mTNBC. Here we present updated immune analyses of nine patients treated in the phase I, single center, dose escalation trial of CF33-hNIS-anti-PD-L1. Methods: Eligibility criteria included patients with mTNBC that progressed on 2 or more chemotherapy regimens and had at least 1 tumor site amenable to IT injections. Formalin fixed tissue from baseline and on-treatment biopsies were assessed by multiplex immunofluorescence for CD3, CD4, CD8, PD-L1, PD-1 and pan-cytokeratin and digitally quantified for densities of immune phenotypes. Peripheral blood samples were analyzed by 30 marker flow cytometry panels on a Cytek Aurora. The Nanostring GeoMx Immune Profiling Panel of protein markers is being used for ongoing analysis of TME changes over the course of treatment. Results: From October 2021 to August 2023, 9 patients received at least 1 dose of IT CF33-hNIS-antiPDL1 at 1 of 3 dose levels (1x105, 3x105, or 1x106 PFU). The sites injected were axillary lymph node (LN) metastasis in 1 patient, intramuscular mass in 1 patient, matted dermal metastasis in 4 patients, and dermal/subcutaneous nodules in 3 patients. Seven patients had evaluable pre-and post-treatment tumor tissues from intradermal or subcutaneous injections. From baseline to cycle 1 day 2, CD8+ T cells in the TME increased in 5/7 patients, CD4+ T cells increased in 4/7 patients, and PD-L1 expression increased in 5/7 patients. Moreover, 4/7 patients had increased frequencies of PD-1-positive CD8+ T cells in the TME. Two patients had evaluable post-treatment tissues following 2 cycles of treatment (C2D2). These patients had increased PD-L1 expression in the TME, increased tumor-infiltrating T cells, and increased CD8+ T cells within the tumor islands of the TME. Digital spatial profiling of other immune features are ongoing. Additionally, 1 patient had 2 cycles of CF33-hNIS-anti-PD-L1 IT injection into a LN containing mTNBC. This patient had increased PD-L1 expression in the TME, increased PD-1-positive CD4+ and CD8+ T cells within the LN, and increased frequencies of circulating PD-1-positive CD39-positive exhausted CD8+ T cells in the peripheral blood over the course of therapy. Conclusions: CF33-hNIS-anti-PD-L1 induces tumor inflammation and immune activation as marked by increased tumor infiltration of CD4+ and CD8+ T cells, increased PD-L1 expression, and increased PD-1-positive T cells. Combination trials with CF33-hNIS and immune checkpoint inhibitor for the treatment of advanced solid tumors are ongoing. Additionally, more studies are needed to evaluate the use of OV in patients with high-risk stage II-III breast cancers. Citation Format: Colt Egelston, Shyambabu Chaurasiya, Jianying Zhang, Raju Pillai, Leslie Chong, Amanda Seiz, W. Hans Meisen, Yuan Yua, Yuman Fong, Jamie G. Rand. Induction of an anti-tumor immune response following intratumoral injection of oncolytic virus CF33-hNIS-antiPD-L1 in patients with metastatic triple negative breast cancer (mTNBC) [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A105.

Despite the success of the CD19xCD3 T cell engager blinatumomab in B-cell acute lymphoblastic leukemia (B-ALL), treatment failure is common and can manifest with antigen loss and extramedullary disease (EMD) relapse. To understand the impact of leukemia genetics on outcomes, we reviewed 267 adult patients with B-ALL treated with blinatumomab and used next generation sequencing to identify molecular alterations. Patients received blinatumomab for relapsed/refractory (R/R) disease (n=150), minimal residual disease (MRD+) (n=88), upfront as induction (n=10), or as consolidation in MRD- state (n=19). In the overall cohort, 50 (19%) patients had Philadelphia chromosome (Ph)-positive ALL, 80 (30%) had Ph-like ALL, 35 (13%) had TP53 mutations (TP53m), 7 (3%) had KMT2A-rearrangement, and 8 (3%) had PAX5-alterations. For patients treated for R/R ALL, the overall complete remission (CR)/CR with incomplete hematological recovery (CRi) rate was 59%. Only pretreatment high disease burden (p<0.01) and active EMD (p<0.01) were associated with inferior CR/CRi rate. Of 169 patients in CR/CRi post-blinatumomab, 79 (47%) patients relapsed, including 22 (28%) with CD19- and 54 (68%) with CD19+ relapse. In multivariable analysis, TP53m was associated with increased risk of CD19- relapse (HR=6.84; 95%CI: 2.68-17.45, p<0.01). Post blinatumomab allogenic stem cell transplantation consolidation was associated with lower risk of CD19- relapse (HR=0.10; 95%CI: 0.03-0.37, p<0.01) and EMD relapse (HR=0.36; 95%CI: 0.18-0.73, p<0.01). In conclusion, leukemia genetics may predict patterns of blinatumomab failure, with TP53m associated with CD19- relapse.