Journal of Hematology & Oncology

Chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 has transformed outcomes for children with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL), yet the influence of molecular subtype on outcomes remains unclear. We evaluated the impact of cytogenetic and molecular signatures on complete response (CR), overall survival (OS), and leukemia-free survival (LFS) after CD19 CAR-T therapy in eighty-six pediatric patients with R/R B-ALL treated with tisagenlecleucel. CR was assessed 30 days after infusion. Cytogenetic data were available for 84 patients and molecular profiling for 62. Survival analyses included 72 patients who received CD19 CAR-T as the sole cellular therapy. Seventy-seven patients achieved CR (89.5%). Pre-infusion bone marrow blasts of [&ge;]20% were associated with lower CR rates (53.8% vs 95.9%, p<0.0001) and significantly reduced OS and LFS (both p<0.0001). Among molecular markers, RAS mutations correlated with inferior OS (p=0.0222) and LFS (0.0402). In multivariate analysis, bone marrow blasts >20% and RAS mutations independently predicted inferior OS. Post CAR-T, CD19 negative relapses showed almost twice higher prevalence of RAS mutations (66% vs 37.5%). These findings highlight RAS mutations as a key molecular predictor of outcome after CD19 CAR-T therapy and suggest emergence of unique risk stratification for patients receiving CD19-targeting therapy.

Children with acute lymphoblastic leukemia (ALL) are treated with multiagent chemotherapy that causes profound changes to the immune system. There are limited data on how disease and therapy impact antigen-specific immune memory, leading to inconsistent guidelines on best practices for revaccination of this population. Here, to inform vaccine guidance, we investigated whether immunity derived from routine childhood measles and varicella zoster virus (VZV) vaccines is maintained during and after therapy for childhood ALL. We report that antibodies against measles and VZV were significantly reduced in children with ALL (n=45) compared to healthy controls (n=13), particularly in older children in whom a longer time had passed since their most recent vaccine dose. However, the avidity of the measles and VZV-specific antibodies was indistinguishable between groups. Despite changes to the composition of the T cell compartment, both overall and antigen-specific T cell function were preserved in children with ALL. These data provide compelling evidence for revaccination of children following ALL treatment. Intact T cell responses suggest that post-treatment revaccination would be effective.

CAR-T immunotherapy has achieved remarkable efficacy in hematologic malignancies. However, the widespread clinical adoption of autologous CAR-T products remains constrained by high costs, lengthy manufacturing process, and limited accessibility. Universal or off the shelf CAR-T (UCAR-T) cells derived from healthy donors offer a promising alternative, enabling immediate treatment at a lower cost. However, the allogeneic nature of UCAR-T cells triggers immune rejection by the host immune system after infusion, thereby compromising their persistence and therapeutic efficacy. Current strategies to circumvent this rejection focus on disrupting HLA class I expression. Although this modification allows UCAR-T cells to successfully evade T cell mediated elimination, the loss of HLA class I molecules renders them vulnerable to attack by host natural killer (NK) cells. In contrast to previous approaches that attempt to retain certain non-classical HLA molecules (such as HLA-E or HLA-G) to inhibit NK cells, we directly focused on editing the ligands that mediate NK cell rejection. Through transcriptomic and in vitro validation analyses, we found that UL16 binding proteins (ULBP) 2/5/6 were substantially upregulated in UCAR-T cells compared with nontransduced donor T cells. Elevated ULBP expression effectively activates the NKG2D receptor on allogeneic NK cells and leads to killing of UCAR-T cells, thereby impairing UCAR-T function. To test whether abrogating this NK activating signal could improve UCAR-T persistence and antitumor efficacy, we generated ULBP knockout UCAR-T cells using CRISPR-Cas9 editing. Deletion of ULBP2/5/6 significantly reduced NK cell mediated killing in vitro without affecting CAR expression or T cell effector function. Compared with wild type UCAR-T cells, ULBP deficient UCAR-T cells exhibited enhanced tumor killing efficacy in the presence of NK cells. Collectively, our findings identify ULBP upregulation as one of the mechanisms underlying NK cell mediated rejection of HLA deficient UCAR-T cells. Targeted ablation of ULBP molecules provides a novel strategy to confer resistance to host NK cells, thereby improving the therapeutic potential of universal CAR T products.

Chimeric antigen receptor (CAR) T-cell therapy has transformed treatment for relapsed /refractory(r/r) lymphoid malignancies. Yet, these cellular immunotherapies are often associated with immune-related adverse events (irAEs), namely cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), that pose significant risks to patient safety and limit broader clinical implementation of CAR T-cell therapies. In the current study, we used proteomics technology to establish circulating protein signatures that would predict severe CRS and ICANS in r/r lymphoma patients that subsequently received CAR T-cell therapy. Initial discovery was performed using plasma samples collected preceding CAR T-cell infusion from 39 r/r lymphoma patients at MD Anderson Cancer Center. A 5-marker and 8-marker protein panel was developed for predicting Grade [&ge;] 2 CRS and ICANS respectively, yielding respective AUCs of 0.85 [95% CI: 0.72-0.98] and 0.91 [95% CI: 0.81-1.00]. Independent testing of the CRS and ICANS panel was performed in a cohort of 59 r/r lymphoma patients from the Moffitt Cancer Center, with resultant AUCs of 0.76 [95% CI: 0.63-0.89] and 0.67 [95% CI: 0.51-0.84] for the CRS and ICANS panel, respectively. Patients were further classified into low-, intermediate-, and high-risk groups based on panel score tertiles. In the combined dataset (MDACC + Moffitt), compared to patients in the low-risk group (reference), patients in the intermediate- and high-risk groups were 3.15 [95% CI: 0.92-12.71] and 13.84 [95% CI: 4.21-56.26] more likely to have Grade [&ge;] 2 CRS, and 1.21 [95% CI: 0.36-4.23] and 8.59 [95% CI: 2.87-29.09] more likely to have Grade [&ge;]2 ICANS. The protein biomarker panels provide a means to risk stratify patients who are at high risk for developing severe CRS and ICANS, to inform on the need for prophylactic interventions and improve patient outcomes.

Children with relapsed or refractory acute lymphoblastic leukemia (ALL) require more effective and less toxic therapies. We established a prospective, multicenter Drug Response Profiling (DRP) registry (NCT06550102) integrating functional testing into precision-guided treatment. DRP was performed for 340 patients from 17 European countries with a turn-around time of two-weeks. Image-based drug screening with over 135000 unique perturbations revealed a heterogeneous landscape of ex vivo responses to 88 drugs on average. Ranking drug responses across the patient cohort defined individual drug fingerprints, identifying "DRP twins" by similarity in sensitivity and resistance independent of genetic ALL subtypes. Of 239 high-risk patients with follow-up, DRP-informed interventions were reported for 63 patients (26%). Patients received combination therapies based on venetoclax, tyrosine kinase inhibitors, trametinib, bortezomib or selinexor, resulting in objective clinical responses in 43 cases (68%). Precision-guided treatments allowed bridging to cellular therapies in 42 patients among whom 28 (67%) were still alive with a median follow-up of 21 months after DRP (IQR: 14.7-26.6 months). Top responders to venetoclax, ranked within the first tertile of the cohort, had superior 1-year event-survival compared to venetoclax non-responders (0.57 [95% CI, 0.39-0.85] vs. 0.25 [95% CI, 0.11-0.58]). Collectively, these findings demonstrate the feasibility and clinical relevance of functional profiling within an international network. This scalable framework enables individualized therapy selection for enrolment in adaptive precision trials for high-risk pediatric ALL.

Long-term follow-up of the CASSIOPEIA trial (NCT02541383) demonstrated superior progression-free survival (PFS) with daratumumab, both in combination with bortezomib, thalidomide, and dexamethasone during induction and consolidation, and during maintenance therapy, in transplant- eligible patients newly diagnosed with multiple myeloma (MM). However, outcomes among CASSIOPEIA patients remain heterogeneous across treatment groups. Measurable residual disease (MRD) is a strong indicator of the depth and duration of therapeutic response and is independently associated with both PFS and overall survival (OS), but it does not fully capture the biological diversity of MM. We performed a risk prediction analysis based on transcriptomic subgroups in CASSIOPEIA patients. A subset of 628 patients was characterized using RNA sequencing and consensus clustering identified five transcriptomic subtypes of MM. Long-term follow-up allowed the definition of three transcriptomic risk categories, with estimated 72-month PFS rates of 70%, 51%, and 27% for low, intermediate, and high-risk groups, respectively, among patients who received daratumumab in at least one treatment phase. In these patients, MRD negativity rates after consolidation and six months later were significantly higher in the low and high-risk groups compared with the intermediate-risk group. In the high-risk group, MRD status was not associated with PFS or OS. This suggests that, although daratumumab administered during both the induction/consolidation and maintenance phases improves the clinical outcomes of patients with activation of NSD2 or overexpressing members of the MAF family, highly aggressive minor clones may rapidly expand. These findings emphasize the need for novel therapeutic strategies in this high-risk population.

Carbohydrates are classically catabolized by fermentation or oxidation, a choice that impacts many cellular functions including proliferation. Proliferating cells including somatic stem and progenitor cells are thought to favor fermentation over oxidation, and most proliferating cells in vitro depend on lactate production. However, it has not been tested if fermentation and oxidation are the universal obligatory terminal fates for carbohydrates in vivo because the key enzymes, lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH), have not been simultaneously deleted in any cell type. Here we show that both fermentation and oxidation are dispensable for the survival and function of hematopoietic stem cells (HSC). Combined LDHA and LDHB deletion to ablate LDH did not impair HSC function, suggesting that HSCs and rapidly proliferating hematopoietic progenitors surprisingly do not require fermentation. Combined LDHA, LDHB, and PDH deletion abolished both glucose oxidation and fermentation, but did not impair HSC function. Glycolysis was preserved, suggesting the operation of an alternative endpoint. LDH/PDH-deficient HSCs terminated glycolysis through pyruvate export. Pyruvate export by HSCs and progenitors was a physiological response to changing nutrient levels. Quadruple deletion of LDHA/B, PDH, and the pyruvate transporter MCT1 impaired HSC function. This suggested that an essential role of glycolysis termination is not to produce acetyl-CoA or lactate but to remove pyruvate. Therefore, in contrast to classical theories and to in vitro metabolism, carbohydrate metabolism in vivo does not require oxidation or fermentation but can terminate directly in pyruvate export, and this alternative pathway is sufficient to support stem cell function.

BackgroundCutaneous melanoma (CM) is an aggressive skin cancer with rising incidence, representing a growing public health concern. Despite the remarkable success of immune-checkpoint inhibitors (ICIs) in the management of advanced disease, mortality remains high due to therapy resistance. Identifying reliable prognostic and predictive biomarkers is therefore essential to improve patient stratification, optimize treatment selection, and minimize unnecessary toxicity. MethodsWe comprehensively profiled the circulating immune landscape of 54 treatment-naive CM patients by integrating flow cytometry immunophenotyping with clinicopathological data, and performed tumor gene expression analysis in a subset of 26 patients. ResultsElevated HLA-DR and CD69 expression on circulating CD4+ T cells, together with reduced circulating CD8+ T cell frequency, emerged as candidate prognostic biomarkers associated with improved survival. Prognostic models combining these immune variables with clinical covariates accurately stratified patients by overall survival (89.5% sensitivity, 72.7% specificity; AUC = 0.872, p < 0.0001) and progression/recurrence risk (75% sensitivity and 71.4% specificity; AUC = 0.763, p = 0.001). In a subset of 43 patients subsequently treated with ICIs, elevated baseline HLA-DR and CD69 expression on circulating CD4+ T cells was also associated with therapeutic benefit. A predictive model integrating these markers with clinical covariates achieved good discriminatory performance (65.2% sensitivity, 88.9% specificity; AUC = 0.775, p = 0.0027). Tumor gene expression profiling supported the role of IFN-{gamma}-related signatures, previously linked to ICI response, as complementary prognostic and predictive tools. ConclusionThese findings highlight systemic CD4+ T cell activation status as a promising, easily measurable biomarker in CM, laying the foundation for future strategies to refine patient stratification and guiding immunotherapy decisions.

Antibody-drug conjugates (ADCs) require high and homogeneous target expression for optimal efficacy, yet the spatial, temporal, and cellular heterogeneity of clinically approved ADC targets in high-grade serous ovarian cancer (HGSC) remains incompletely defined. We analyzed bulk RNA-sequencing, single-cell RNA-sequencing, and whole-genome sequencing data from 867 samples across 304 patients enrolled in the real-world DECIDER cohort to systematically evaluate 11 approved ADC targets. FOLR1, TACSTD2, and ERBB2 emerged as highly expressed candidates. Inter-patient variability exceeded intra-patient heterogeneity, which further decreased following neoadjuvant chemotherapy. Target expression was highly concordant across anatomical sites and largely stable from diagnosis to relapse. Single-cell RNA-sequencing results revealed that TACSTD2 and FOLR1 showed the most frequent cancer cell-restricted expression. In rare cases of gene amplification, ERBB2 and F3 emerged as potential targets alongside TACSTD2 and FOLR1. Overall, 80% of patients displayed homogeneous expression of at least one actionable target, with frequent co-expression of TACSTD2 and FOLR1. These findings indicate that ADC target expression in HGSC is broadly stable across space and time and support the prioritization and strategic integration of TACSTD2- and FOLR1-directed ADCs in this disease.

Background: Stomach adenocarcinoma is driven by heterogeneity, limiting therapeutic success. Although ROS acts as a continuous redox rheostat for tumor evolution, it is categorized based on binary models that are masked by tumor-microenvironment (TME) confounders. Here, we have defined a continuous, TME-independent ROS axis to help identify intrinsic vulnerabilities and improve patient stratification. Methods: Non-negative matrix factorization (NMF) defined a ROS-Axis in TCGA-STAD which was validated in ACRG Cohort. Multivariate regression model isolated intrinsic signatures via residual ROS scores by adjusting for TME confounders. Survival was assessed using Cox hazard models. Drug sensitivities were mapped using GDSC2/ElasticNet modeling with cross-cohort replication. Results: Our results define a reproducible ROS gradient, driven by effectors like NQO1 and SOD1, characterizing ROS-high tumors as proliferative, epithelial and immune -cold. High residual ROS score was associated with an improved prognosis, regardless of TNM stage and age. Pharmacogenomic mapping revealed an overlapping sensitivity to mTOR inhibitors in ROS-high gastric cancer tumors which persisted after TME confounder adjustment. Conclusion: The continuous ROS axis provides a functional readout of metabolic dependency that refines traditional anatomical staging. By identifying mTOR dependent cold tumors, our framework offers a precision strategy for immunotherapy-resistant patients like those affected by microsatellite-stable gastric cancer.

Myeloid derived suppressor cells (MDSCs) are key players in the immune-suppressed tumor microenvironment (TME) and significantly contribute to immune checkpoint inhibition (ICI) resistance, making them favorable targets for cancer immunotherapy. Epigenetic reprogramming of MDSCs using histone deacetylase (HDAC) inhibitors shows promise to sensitize the TME to ICIs. However, the molecular mechanism of HDAC inhibition in MDSCs has yet to be elucidated. Murine and human MDSC models treated with Entinostat revealed that the long non-coding RNA Malat1 downregulates pSTAT3 and decreases MDSC-mediated suppression of T cell proliferation. Through HDAC inhibitor screens, we identified HDAC1 as preferentially regulating Malat1 expression, STAT3 activation, and MDSC suppression. We also show that HDAC1 inhibition increases MDSC apoptosis by shifting pro-vs. anti-apoptotic signals and increases G0/G1 cell cycle arrest via decreasing G1-S transition cyclin-CDK complexes. Collectively, our findings provide a multi-pronged mechanism of HDAC inhibition in MDSCs that inform the development of future rational combination therapies. One Sentence SummaryHDAC1 inhibition in MDSCs increases Malat1, decreases pSTAT3, induces apoptosis/cell cycle arrest, and decreases suppression of T cells

PURPOSE: Newly-diagnosed glioblastoma (nGBM) is a devastating tumor with median survival of only 14-18 months despite aggressive standard of care (SOC). Dendritic cell (DC) homologous antigenic double-loading provides a powerful pattern-based signal that initiates cDC1-like skewing of monocytic precursors, inducing downstream development of CD8+ memory effectors. Here we report phase I results for DOC1021 (dubodencel), a novel DC vaccine regimen integrated with SOC. METHODS: In this dose-escalating study, DC prepared from mobilized peripheral blood were doubly loaded with autologous tumor lysate and amplified tumor mRNA and administered bilaterally near the deep cervical node chains in three biweekly courses given with weekly peg-IFN after conclusion of chemoradiation. Four dose levels from 3.5x106 to 3.6x107 total cells were tested. Patients with subtotal resection or tumor progression prior to vaccination were not excluded. RESULTS: Eighteen patients (median age 61 years (range 47-73), 94% MGMT unmethylated, 25% subtotal/partial resected) completed vaccination (16 nGBM, 2 recurrent) with no dose-limiting toxicities. Attributable AE were mostly mild and flu-like or injection-site reactions. Twelve-month OS among the newly-diagnosed cohort was 88% compared to an expected ~60% for SOC alone. Patients who received observation rather than reoperation in response to worsening MRI contrast-enhancement demonstrated gradual lesional resolution and improved OS. Immunophenotyping revealed post-vaccination elevations in CD4 and CD8 memory T-cells in peripheral blood, and spatial transcriptomic analysis revealed foci of activated inflammatory complexes at the primary tumor site. CONCLUSIONS: DOC1021 was safe, feasibly integrated within SOC, and associated with more favorable outcomes in this challenging patient population. Patients who received observation rather than reoperation for worsening MRI contrast-enhancement exhibited superior survival, suggesting an immune-reactive tumor microenvironment manifesting as pseudo-progression. These data supported initiation of a randomized Phase II trial (NCT06805305) for nGBM.

Reliable, minimally invasive biomarkers for predicting immunotherapy response in head and neck squamous cell carcinoma (HNSCC) remain an unmet clinical need. Here, using patients from a prospective, multi-institutional phase II clinical trial (NCT02641093), we performed whole genome sequencing of 185 plasma cell-free DNA (cfDNA) samples collected longitudinally from 68 patients with locally advanced, surgically resectable HNSCC undergoing neoadjuvant and adjuvant pembrolizumab treatment. We developed the regional motif diversity score (rMDS), a novel fragmentomic metric quantifying the entropy of cfDNA 5' end motifs across genomic regions. Remarkably, unsupervised analysis revealed that rMDS robustly distinguished immunotherapy responders from non-responders, outperforming established cfDNA fragmentomic metrics and copy number alterations, while demonstrating independence from technical confounders. Longitudinal analysis revealed dynamic rMDS changes in genomic regions enriched for immune, lectin, and keratinization-related genes, hallmarks of squamous cell carcinoma, reflecting the interplay between tumor and peripheral immunity during the immunotherapy treatment. Interestingly, the regions with the most dynamic rMDS changes were highly enriched in telomere proximal loci, suggesting a novel link between telomere biology and cfDNA fragmentation. A machine learning classifier based on rMDS achieved robust predictive performance across multiple validation settings (AUC 0.89-0.99), with the highest accuracy at post-treatment timepoints and superior to PD-L1 expression and tumor fraction in the same sample. Predicted responders demonstrated significant trends toward improved disease-free survival (log rank test p=0.035, hazard ratio: 2.67, 95% confidence interval: 1.03-6.92), underscoring the clinical utility of rMDS-based stratification. These findings position rMDS as a biologically meaningful and clinically actionable biomarker for immunotherapy response in HNSCC, supporting its integration into future risk assessment frameworks and broader cancer care.

5-Azacytidine (5-AzaC) is a cytidine analog and is widely used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Although its therapeutic activity is primarily attributed to hypomethylation resulting from DNA incorporation, the majority of 5-AzaC is incorporated into RNA. However, the functional consequences of 5-AzaC incorporation into RNA have been unknown. Here, we show that 5-AzaC treatment of cells leads to inhibition of protein synthesis. Ribo-seq, Disome-seq, and RNA-seq in cells treated with 5-AzaC exhibit a time-dependent C-to-G transversion signature in mRNAs within 2 h of treatment. These transversion events are enriched within footprint positions corresponding to the A-site of monosomes or leading stalled ribosome in a disome complex. Consistently, ribosome and disome footprints are accumulated at sites with C-rich codons in the A-site, specifically with the codons containing a C in the second position. 5-AzaC activates the integrated stress response (ISR) and the ribotoxic stress response (RSR) in a GCN2- and ZAK-dependent manner, consistent with disome-mediated signaling. Furthermore, loss of the Ribosome Quality Control (RQC) factor, ZNF598, sensitizes cells to 5-AzaC. Collectively, our results support a model where 5-AzaC is rapidly incorporated into mRNAs, disrupts decoding, and triggers disome-mediated signaling pathways, which contribute to its cytotoxicity. These findings suggest that translation disruption represents an additional layer of 5-AzaCs mechanism of action, alongside its known DNA-mediated effects.

Most patients with acute myeloid leukemia (AML) initially respond to standard chemotherapy. However, relapse and refractory disease remain common. The responses to targeted therapies are often transient and the efficacy of immunotherapy is limited. Although single-cell RNA sequencing (scRNA-seq) studies have provided insights into the cellular diversity and immune landscape of AML, many have primarily focused on limited, or newly diagnosed patient cohorts, leaving cellular dynamics across advanced disease incompletely defined. Here, we profiled 72 samples from AML patients across different disease stages using scRNA-seq and compared these against healthy donor samples. We observed selective enrichment of immature progenitor populations, along with widespread upregulation of oxidative phosphorylation in AML. The immune microenvironment of AML was characterized by CD8+ effector memory T cell expansion with reduced IL2-STAT5 and increased mTORC1 pathways and exhaustion markers, suggesting a functional imbalance. Several AML-specific genes were identified providing potential therapeutic opportunities. Cell communication analysis revealed reduced HLA interactions in relapsed/refractory samples compared to diagnosis samples, suggesting impaired antigen presentation and defective T cell priming. Together, these results improve the understanding of cellular and immune changes in AML during disease progression and provide a basis for new therapeutic strategies.

The high heterogeneity of pediatric cancers presents significant diagnostic challenges, underscoring the need for accurate classification. Although molecular profiling supports first-line diagnostics and guides treatment, it can delay final diagnosis. While Nanopore-based methylation analysis has enabled rapid CNS tumor diagnosis, its application to pediatric solid tumors and lymphomas has remained largely unexplored. We developed Tucan, a deep-learning classifier trained on 3,818 methylation array profiles representing 84 subtypes, designed to classify tumors from sparse Nanopore methylation data. In retrospective validation (n=514), Tucan generated confident predictions (CFT[&ge;] 0.7) within 30 minutes of sequencing in 385 cases, achieving 372 correct diagnoses (F1-score: 0.98). In prospective testing (n=74; 63 classifiable), 52 samples reached the confidence threshold with 96% accuracy, confirming the original diagnosis in 47 cases and correctly refining or revising it in three. Together, Tucan enables rapid, high-confidence molecular classification of pediatric solid tumors and lymphomas.

Genetically engineered human induced pluripotent stem cells (hiPSCs) represent a promising platform for regenerative medicine and next-generation immunotherapies. While recent advances enable stroma-free differentiation of hiPSCs into mature CD3TCR{beta} cytotoxic T lymphocytes (CTLs), overall efficiency remains limited. Here, we identify small-molecule modulators that enhance T cell output, particularly at the ProT cell stage. Targeted and stage-specific inhibition of AHR, DOT1L, or GSK3 drives robust maturation from ProT to CD4 immature single-positive (ISP) cells, markedly increasing CD4CD8 populations and augmenting CTL production of up to 2000 fold. hiPSC-derived T (iT) cells matured under these conditions display superior activity in cytotoxicity assays using AMG-701 (BCMAxCD3) or Blinatumomab (CD19xCD3). These effects were reproducible across independent hiPSC lines, diverse hematopoietic progenitor generation methods, and multiple stroma-free differentiation platforms, and were further validated in cord blood CD34 cells. Notably, AHR inhibition enhanced T cell development and promoted B lymphopoiesis, revealing shared regulatory pathways in lymphoid lineage specification. We also demonstrate that the Oct4-activating compound OAC1 functions as a weak AHR inhibitor, partially recapitulating the effects of canonical AHR blockers in both cellular and zebrafish AHR reporter systems. Collectively, our findings define key molecular circuits governing human lymphoid differentiation and establish practical strategies to optimize the yield and function of hiPSC-derived cytotoxic T cells. This work advances the development of both universal and autologous hiPSC-derived T cell therapies, offering a path forward even for patient-specific hiPSC lines with suboptimal T cell differentiation potential.

KRAS mutations drive multiple cancers and represent an important therapeutic target, together with other oncogenic regulators such as MYC, KIT, and BCL2 that are critically involved in pancreatic cancer. Here we describe a novel therapeutic strategy based on stable nucleolipid-modified G-quadruplexes (NLG4). Cell viability assays demonstrate that NLG4 strongly inhibit pancreatic cancer cell proliferation, whereas non-lipidic G-quadruplex sequences display minimal activity under comparable conditions. Owing to their distinctive physicochemical properties, including stabilization of parallel G-quadruplex structures and self-assembly into micellar aggregates, NLG4 efficiently internalize into cells and interact with key G-quadruplex unfolding factors such as UP1. This interaction leads to a marked downregulation of KRAS, c-MYC, c-KIT, and BCL2 expression. Suppression of these oncogenes profoundly affects pancreatic cancer cell fate, as evidenced by reduced expression of proliferation (Ki67) and anti-apoptotic (BCL2) markers. In addition, NLG4 treatment decreases inflammatory signaling mediated by NF-{kappa}B and inhibits major pro-proliferative kinase pathways, including ERK, AKT, and phosphorylated AKT. The therapeutic relevance of this decoy strategy is further supported by the observed potentiation of gemcitabine antitumor activity. Overall, these findings highlight NLG4 as a promising anticancer approach that simultaneously targets multiple oncogenic pathways through G-quadruplex-based decoy mechanisms, with translational potential for future pancreatic cancer treatment.

Immunosuppressive tumor microenvironment (TME) inactivates CD8+ cytotoxic lymphocytes (CTLs). Here, we identify SPTBN2 spectrin as a key immunosuppressive regulator induced in CTLs in response to nutritional deficit. In human pancreatic and colorectal cancers, SPTBN2 expression negatively correlated with CTL infiltration and patients survival. In TME of mouse pancreatic and colorectal adenocarcinomas, SPTBN2 inactivated intratumoral CTLs, stimulated tumor growth and conferred cross-resistance to anti-cancer therapies. SPTBN2 knockout protected CAR T-cells from trogocytosis and increased their memory state. SPTBN2 maintained levels of cell surface proteins such as BTLA that undermine CAR T-cell cytotoxicity and promote exhaustion. Re-expression of BTLA largely reversed phenotypes in SPTBN2-deficient CAR T-cells. In manufactured CAR T cells, SPTBN2 was associated with their clinical failure in pediatric patients with leukemia. Accordingly, ablation of SPTBN2 in CAR T-cells increased their cytotoxicity, in vivo persistence and therapeutic effects indicating that SPTBN2 can be targeted to increase the efficacy of anti-cancer therapies.

Inflammation is a key driver of hematopoietic dysfunction in myeloid malignancies, but its role in the context of hypomethylating therapy remains incompletely understood. Although 5-Azacytidine is used posttransplant in high-risk myelodysplastic syndrome (MDS), only 50% of patients show a clinical response. We provide evidence that inherent inflammatory properties of healthy donor CD34+ stem cells exist that are likely to contribute to the "response" seen in MDS patients. These are linked to epigenetic priming of the myeloid niche, resulting in S100A8/A9-driven inflammatory program that promotes functionality of immature NK cells. Using in vitro differentiation systems, multi-omic profiling, and a S100A9-/- mouse model, we find that 5-AzaC modulates inflammatory transcriptional programs through epigenetic rewiring of upstream regulatory elements. Loss of S100A9 disrupts myeloid differentiation, impairs NK cell maturation, and alters key developmental regulators including CEBPB, JUN, and NFIL3. In vivo, 5-AzaC restores these defects and primes NK cells in a time- and context-dependent manner. Re-analysis of the published Australian MDS/CMML cohort shows that "responders" display increased S100A8/A9 expression together with enhanced IFN-{gamma}, IL6-JAK-STAT3, and TNF signaling. These findings suggest that inflammatory myeloid programs may serve as predictive biomarkers and therapeutic targets to enhance NK cell-mediated graft-versus-leukemia activity posttransplant. SummaryO_LIWe provide compelling evidence that inherent properties of healthy donor CD34+ hematopoietic stem cells (SCs) exist that are likely to contribute to the "response" seen upon pre-emptive posttransplant 5-AzaC therapy of patients with high-risk myelodysplastic syndrome (MDS). C_LIO_LIThese properties are linked to a distinct form of epigenetic plasticity at upstream-located transcription factor (TF) binding sites. This may indirectly contribute to acute S100A8/A9-driven inflammation, which is demonstrable in distinct monocyte subsets and, importantly, also in NK cells thereby determining the characteristics of inflammatory monocyte-NK cell crosstalk. C_LIO_LIMice with a targeted deletion of S100A9 fail to upregulate CEBPB / JUN and NFIL3 which results in impaired myeloid priming and dysfunctional NK cell maturation, respectively. C_LIO_LIRe-analysis of the Australian MDS/CMML cohort confirms that MDS patients that "respond" to 5-AzaC exhibit activated IFN-{gamma}, IL6-JAK-STAT3, and TNF-signaling pathways in the context of upregulated S100A8/A9 after six months of treatment. C_LIO_LIOur study indicates that screening of healthy donors SCs for specific inflammatory markers in early developing monocytes could be used as a marker to predict which donor will have the potential of generating a S100A8/A9-driven inflammatory response. This may help identify patients with MDS as well as AML who are likely to benefit from low-dose, short-term 5-AzaC therapy as early as day 7 after transplantation, potentially resulting in increased graft-versus-leukemia (GvL) activity. C_LI