Leukemia

TP53 mutations represent one of the strongest adverse prognostic factors in myelodysplastic neoplasms (MDS). While multi-hit TP53 (TP53multiHit) alterations uniformly lead to very poor outcomes, the prognostic relevance of monoallelic TP53 (TP53mono) mutations remains controversial. TP53 variants can cause loss-of-function, dominant-negative, or gain-of-function effects. We hypothesized that functional heterogeneity among TP53 variants contributes to the variable clinical behavior observed in monoallelic TP53-mutated MDS. Therefore, we analyzed pretreatment samples from 4,505 patients with MDS from two independent cohorts (IWG, n=3,173; J-MDS, n=1,332), including 271 patients with TP53mono and 499 with TP53multiHit. Functional annotation of TP53 variants was performed using a previously published phenotype score (PS) derived from saturation mutagenesis screens, capturing dominant-negative and loss-of-function effects. Median overall survival (OS) differed significantly by TP53 allelic state (TP53 wild-type (TP53wt) 42.4 months; TP53mono 22.9 months; TP53multiHit 9.2 months; p < 0.001). Within the TP53mono subgroup, functional annotation identified marked heterogeneity. Patients with high PS ([&ge;]7) showed significantly inferior OS compared with those with low PS (median OS: 13.8 vs. 39.2 months; HR 1.68, 95% CI 1.16-2.42; p = 0.006), particularly for IPSS-R and IPSS-M low-risk cases. Combining PS and variant allele frequency (VAF) further improved risk stratification. TP53mono patients with PS [&ge;]7 and VAF [&ge;]22% had outcomes comparable to TP53multiHit (median OS: 8.8, p = 0.2), whereas those with PS <7 and VAF <22% exhibited survival similar to TP53wt (median OS: 49.7, p = 0.9). Overall, functional annotation of TP53 variants refines prognostication in TP53mono-mutated MDS and may enhance individualized risk assessment.

BackgroundChildhood T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with poor prognosis. Differently from B-cell precursor ALL, T-ALL lacks effective risk stratification strategies. A recent study has integrated whole genome and whole transcriptome data to define over 15 distinct molecular subtypes with prognostic significance. However, clinical translation of this knowledge remains challenging due to the complexity of interpreting high-dimensional multi-omics-based data. MethodsHere, we present clinTALL, a deep learning based multi-task pipeline for pediatric T-ALL subtype classification and treatment outcome estimation. The model integrates multimodal input data and uses a neural network architecture to generate a shared latent embedding for jointly learned multi-task prediction. The competing risk-based model was used to predict event-specific outcomes. The model was trained on a publicly available multimodal dataset comprising clinical, genomic and transcriptomic features of 1309 pediatric T-ALL samples. ResultsWe observed that the transcriptomic-only model achieved superior single modality results, with 92.2% accuracy for subtype prediction and a 65.9% concordance index (C-index) for event-free survival (EFS) in a cross-validation setup. Integrating all data modalities maintained high subtype classification accuracy (91.7%) and improved the overall concordance index for EFS estimation to 67.5%. The competing risk-based model enables accurate predictions of induction failure (C-index = 96.0%) and second malignant neoplasm (C-index = 62.1%). We validated molecular subtype predictions on an internal dataset of 120 pediatric T-ALL samples and obtained an accuracy of 81.8%. To facilitate the broad application of multi-omics based subtype prediction and treatment outcome inference, we provide clinTall as a Docker based application, allowing for user friendly access to the tool. The full source code of clinTALL is available on GitHub (https://github.com/UKWgenommedizin/clinTALL). ConclusionTogether, our machine learning-based framework allows for automated, accurate sub-type classification and treatment outcome inference using multimodal input data, advancing precision risk stratification for pediatric T-ALL.

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.

Despite considerable advances, the emergence of treatment resistance to tyrosine kinase inhibitors (TKIs) therapy remains a significant challenge in chronic myeloid leukemia (CML). Here, we report the first clinical case of resistance to combined ponatinib and asciminib therapy in a CML patient who relapsed with B lymphoblastic blast crisis. While at presentation the patient harbored the canonical e13a2 BCR::ABL1 fusion, at relapse his disease harbored the T315I mutation together with a novel e6a3 BCR::ABL1 fusion, arisen by internal deletion in the original translocated allele. Structural modeling and biochemical analyses demonstrated that deletion of exon 2-encoded residues of ABL1 destabilizes the autoinhibited conformation, resulting in a hyperactive kinase with increased propensity for B-cell differentiation. Functional studies revealed that both BCR::ABL1e6a3 and BCR::ABL1e6a3/T315I conferred resistance to ponatinib and asciminib, alone or in combination. BCR::ABL1e6a3 demonstrated enhanced sensitivity to active-state selective inhibitors dasatinib and bosutinib, whereas BCR::ABL1e6a3/T315I remained resistant. Combined drug sensitivity assays showed that axitinib restored inhibitory activity when combined with ponatinib or asciminib. Strikingly, a combination of axitinib and asciminib with low dose ponatinib fully suppressed enzymatic activity of BCR::ABL1e6a3/T315I and cellular proliferation. These data show that treatment with asciminib and ponatinib can select for mutations with notably elevated enzymatic activity, effectively targeted by an axitinib-based triple combination. These data highlight the remarkable mutability of the BCR::ABL1 kinase, including through novel isoforms and provides a strong rationale for the clinical assessment of a triple inhibitor combination as a strategy to overcome resistance to dual ponatinib and asciminib therapy.

Prognostic stratification in multiple myeloma (MM) relies on staging systems that assign patients to fixed categories at diagnosis and discard the temporal information that accumulates during treatment. We developed a dynamic multimodal framework that predicts residual overall survival using observation windows ranging from 1 to 18 months post-diagnosis. The model integrates DeepInsight-transformed gene expression representation, longitudinal laboratory measurement trajectories across 10 analytes, and treatment history for three drug classes through an adaptive fusion mechanism that accounts for missing clinical observations. On the MMRF CoMMpass cohort (n = 752), five-fold cross-validation yielded a concordance index (C-index) of 0.773 {+/-} 0.024 and a time-dependent AUC at a 1-year prediction horizon (tdAUC1yr) of 0.789 {+/-} 0.021, outperforming all evaluated baseline methods including DeepSurv (0.633 {+/-} 0.095) and random survival forests (0.636 {+/-} 0.024) on matched cross-validation splits. Modality ablation identified longitudinal laboratory measurements as the strongest individual contributor (C-index 0.693); the DeepInsight spatial encoding of gene expression yielded higher discrimination than a multilayer perceptron (MLP) baseline operating on the same features (0.624 vs. 0.596). Kaplan-Meier analysis showed significant prognostic group separation at all primary landmarks (log-rank p < 0.001; hazard ratios 3.46-3.93). A distilled student model retaining only the DeepInsight representation and five baseline clinical features achieved C-index 0.672 and tdAUC1yr 0.740 on an independent microarray cohort (GSE24080, n = 507) without retraining. Interpretability analysis identified prognostic associations consistent with established myeloma biology, including ubiquitin-proteasome pathway genes, endoplasmic reticulum stress markers, and Interferon Alpha Response pathway enrichment.

Somatic mutations in the RAS pathway are highly prevalent in B-Cell Acute Lymphoblastic Leukemia (B-ALL), yet their impact on the bone marrow immune microenvironment and response to immunotherapy remains poorly defined. In this study, we integrated bulk RNA-sequencing, single-cell RNA-sequencing (scRNA-seq), and spectral flow cytometry to characterize the immune landscape of RAS-mutant B-ALL. We identified pathogenic mutations in KRAS, NRAS, PTPN11, or BRAF in 42% of the cohort, predominantly as clonal events. Despite similar T-cell frequencies by flow cytometry, bulk transcriptomes from RAS-mutant samples showed suppression of immune-response and T-cell-activation pathways, and T cells from RAS-mutant patients exhibited impaired proliferation ex vivo. Single-cell analysis revealed higher CD8 dysfunction scores and enrichment of regulatory T cells (Tregs) in RAS-mutant bone marrow. These findings were validated by spectral flow cytometry and by CIBERSORTx deconvolution of bulk data. Trajectory analysis supported a higher CD4 to Treg differentiation in the RAS-mutant niche, and CellChat mapping identified contact-dependent and checkpoint interactions (including TIGIT-NECTIN2 and CTLA-4-CD86/ICOSL) enriched in RAS-mutant samples. Functionally, blinatumomab produced limited leukemic-cell killing ex vivo overall, but addition of CTLA-4 blockade (ipilimumab) selectively restored blinatumomab efficacy in RAS-mutant samples. Together, these results indicate that RAS-pathway activation associates with a Treg-enriched, immunosuppressive bone-marrow microenvironment and point to CTLA-4-targeted strategies to enhance T-cell-engager efficacy in this subgroup.

Children diagnosed with cancer typically receive standardized treatment regimens. Despite highly protocolized care, children living in poverty experience a greater risk of cancer relapse and higher mortality compared to their more affluent peers.1,2 Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer, and children with ALL exposed to poverty are more likely to experience early relapse.3 Using single-cell RNA sequencing to analyze leukemic blasts and their microenvironment at diagnosis we found that poverty-exposed patients with standard-risk B-ALL exhibit transcriptional signatures of steroid resistance at time of diagnosis. Additionally, we observe increased expression of inflammatory signatures in myeloid cells and reduced effector signatures in CD8+ T-cells in children with B-ALL living in poverty. Further investigation of the mechanisms underlying these associations may identify opportunities for risk-adapted therapeutic strategies to improve disease outcomes in pediatric ALL.

Extramedullary acute myeloid leukemia (eAML) represents a clinically challenging manifestation of acute myeloid leukemia (AML), but its molecular drivers remain poorly defined. We performed targeted sequencing in 85 eAML biopsies, representing one of the largest molecular analyses of eAML to date. We detected mutations in RAS or RAS-modifying genes (RASMUT; NRAS, KRAS, PTPN11, CBL, and NF1) in 41% of cases, representing a significant enrichment compared to bone marrow (BM) samples of more than 1300 AML patients not selected for eAML. Analysis of paired eAML and BM specimens revealed expansion and/or de-novo appearance of RASMUT clones at the extramedullary site. Functional studies using primary murine leukemia cells and CRISPR/Cas9-engineered isogenic human leukemia cell lines demonstrated that RASMUT increase the migration and invasion of leukemic cells compared to RAS-wildtype controls. Consistently, RASMUT cells showed increased infiltration into the chorioallantoic membrane of chicken embryos and demonstrated enhanced extramedullary growth after injection into immunocompromised mice. RNA sequencing revealed increased expression of junctional adhesion molecule-like (JAML) and activation of PI3K/AKT signaling in RASMUT cells. JAML silencing and pharmacologic AKT inhibition reversed the RASMUT-driven effects on leukemic cell migration, demonstrating a causal role of the JAML-PI3K/AKT axis in RASMUT-driven eAML formation. In conclusion, these findings delineate the molecular landscape of extramedullary AML and show that RASMUT are enriched within this AML subform. They further demonstrate that RASMUT actively contribute to leukemic tissue infiltration through activation of a RASMUT-JAML-PI3K/AKT axis, highlighting AKT signaling as a potential therapeutic vulnerability in RASMUT-associated eAML.

BackgroundChronic myelomonocytic leukemia (CMML) is a clinically heterogeneous myeloid malignancy with limited therapeutic options and suboptimal risk stratification. Although single-cell RNA sequencing has refined disease classification through gene expression profiling, post-transcriptional mechanisms--particularly adenosine-to-inosine (A-to-I) RNA editing--remain unexplored at single-cell resolution. We hypothesized that cell-specific RNA editing programs contribute to CMML heterogeneity and define distinct, clinically actionable cellular states in CMML. MethodsWe developed a single-cell-aware computational framework for high-confidence identification and quantification of RNA editing events. Candidate sites were detected at pseudo-bulk depth using stringent filters and subsequently quantified at single-cell resolution. The pipeline incorporated dual alignment, barcode correction, artifact removal, and exclusion of genomic variants to ensure specificity. We applied this framework to discovery and independent validation CMML cohorts. Editing-defined cellular states were identified by unsupervised clustering of single-cell editing profiles and evaluated for associations with clinical stage, TET2 status, survival, and response to hypomethylating agent (HMA) therapy. Regulatory mechanisms were assessed by analyzing ADAR1/ADAR2 expression and relationships between editing levels and target gene expression. ResultsWe identified 3,326 high-confidence A-to-I RNA editing sites and delineated reproducible editing-defined cellular states. A granulocyte-monocyte progenitor-like editing state (edClu1_sub0) aligned with an inflammatory, monocytic-biased transcriptional program and was significantly associated with adverse survival, advanced-stage disease and TET2-mutant CMML, supporting it as a high-risk biomarker-defined subpopulation. In contrast, states such as edClu3 and edClu6 were enriched in earlier-stage, TET2-wild-type CMML and correlated with improved outcomes. Editing-defined states demonstrated systematic remodeling following HMA therapy, indicating treatment-responsive post-transcriptional programs. The high-risk state exhibited elevated ADAR1 and reduced ADAR2 expression, suggesting enzyme-specific regulatory imbalance as a potential therapeutic vulnerability. Integrative analyses further nominated immune-related genes--including LAPTM5, CTSS, and CD83--as CMML-specific oncogenic RNA editing targets, with coordinated increases in editing and expression within the aggressive state. ConclusionsRNA editing represents a clinically informative and mechanistically relevant layer that refines CMML stratification at single-cell resolution, independent of gene expression. These findings provide a framework for integrating post-transcriptional regulation into precision oncology and highlight RNA editing signatures as biomarkers for risk assessment, treatment monitoring, and therapeutic targeting in hematologic malignancies.

ObjectivesThe therapeutic efficacy of rituximab has reduced the discriminatory power of the International Prognostic Index (IPI) in diffuse large B-cell lymphoma (DLBCL), particularly within intermediate-risk categories. To address this "risk dilution," we aimed to develop and internally validate the AB-IPI (Albumin-BCL2 Refined Prognostic Index) using a hypothesis-driven approach that integrates tumor burden, host fitness, and tumor biology. MethodsThis multi-center retrospective study analyzed 289 patients with de novo DLBCL treated uniformly with R-CHOP immunochemotherapy. We combined the standard IPI with serum albumin < 3.6 g/dL (representing host fitness/rituximab pharmacokinetics) and BCL2 protein expression > 50% (representing tumor biology). The model was validated internally using bootstrapping with 1,000 resamples in accordance with TRIPOD Type 1b guidelines. This study adhered to the TRIPOD (Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis) statement for model development and internal validation (Type 1b). ResultsDuring the observation period, 115 death events were recorded. Multivariate Cox regression identified albumin < 3.6 g/dL (Hazard Ratio 2.62), IPI score > 2 (HR 2.13), and BCL2 > 50% (HR 1.72) as independent prognostic factors. The model maintained a robust Events Per Variable (EPV) ratio of 38.3. The AB-IPI stratified patients into four distinct risk groups with 5-year overall survival rates of 88.0% (Low), 76.1% (Intermediate-1), 45.0% (Intermediate-2), and 29.0% (High). The calibration plot demonstrated excellent agreement between predicted and observed probabilities, with a calibration slope of 0.98, indicating minimal optimism and robust risk estimation. Decision Curve Analysis (DCA) demonstrated that the AB-IPI provided a superior Net Benefit across a wide range of clinically relevant threshold probabilities. ConclusionsThe AB-IPI demonstrates superior clinical utility and calibration compared to the standard IPI. By identifying patients with compounded biological risks who are unlikely to be cured by R-CHOP alone, this score offers a practical framework for optimizing therapeutic strategies, such as the allocation of polatuzumab vedotin.

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.

BackgroundChimeric antigen receptor (CAR) T cell therapy has transformed the treatment of B cell malignancies, but translation to acute myeloid leukemia (AML) has been hindered by on-target, off-tumor (OTOT) toxicity. In particular, endothelial cell (EC)-specific toxicity has limited clinical translation of promising leukemia stem cell-enriched targets such as CD93. Innovative strategies to mitigate EC damage while preserving antileukemic efficacy are needed. MethodsWe hypothesized that a NOT-gated CAR T cell strategy could circumvent EC toxicity associated with CD93 targeting. Considering CAR target antigen density and the pro-inflammatory microenvironment of CAR T cells, we identified VE-cadherin (VC), a highly specific EC marker, as an optimal inhibitory CAR target. We engineered a novel VC-specific single chain variable fragment (scFv), confirmed EC specificity in the context of a VC-specific second-generation activating CAR, then evaluated VC/CD93 NOT-gated CAR T cells for EC protection and antileukemic activity in in vitro cytotoxicity assays and in a three-dimensional vascularized microphysiological system. ResultsVC/CD93 NOT-gated CAR T cells maintain potent cytotoxicity against AML across multiple effector-to-target ratios, but preserve EC integrity, including in a three-dimensional vascular model system. Importantly, prior AML exposure did not impair the EC-protective function of the VC-specific iCAR, indicating durable NOT-gate activity under inflammatory conditions. Conversely, EC-induced iCAR inhibitory functions did not limit downstream antileukemic cytotoxicity, confirming a reversibility of both activation and inhibitory signals. Conclusions: These findings establish NOT-gated CAR T cells as an effective strategy to overcome EC-specific OTOT toxicity. Our results underscore the importance of CAR target discovery and validation across a spectrum of inflammatory states that can influence antigen expression and available therapeutic windows. This approach expands the potential CAR target landscape for AML and may be more broadly applicable to other malignancies where OTOT toxicity limits clinical translation.

Background/ObjectivesIdentification of prognostic biomarkers that capture biologically aggressive disease remains a major need in chronic lymphocytic leukemia (CLL). Aberrant calcium signaling contributes to leukemic survival; however, the clinical relevance of Ca{superscript 2}/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a calcium-responsive kinase, has not been defined. This study evaluated CaMKK2 as a candidate prognostic biomarker and functional regulator in CLL. MethodsCaMKK2 expression was quantified in purified CD19 CLL cells from a clinically annotated cohort balanced by immunoglobulin heavy chain variable region (IGHV) mutation status. Associations with time-to-treatment and overall survival were analyzed. Functional relevance was assessed by pharmacologic inhibition of CaMKK2 in primary CLL cells using metabolic (MTS) and apoptosis (Annexin V/PI) assays. Correlations between CaMKK2 expression and inhibitor sensitivity were determined. The impact of CaMKK2 inhibition on nurse-like cell (NLC) differentiation and macrophage-mediated leukemic support was evaluated in ex vivo culture systems. ResultsElevated CaMKK2 expression was enriched in IGHV-unmutated CLL and associated with shorter time-to-treatment and inferior overall survival. CaMKK2 inhibition reduced primary CLL viability in a dose-dependent manner and induced apoptosis, with sensitivity correlating with CaMKK2 expression levels. Inhibition also attenuated CD163 macrophage polarization and impaired NLC-mediated support of leukemic cells. ConclusionsCaMKK2 expression identifies biologically aggressive CLL and functionally contributes to leukemic persistence. These findings position CaMKK2 as a prognostically relevant biomarker with therapeutic implications, supporting further evaluation of CaMKK2-targeted strategies in high-risk CLL. Sample SummaryChronic lymphocytic leukemia (CLL) shows marked variability in clinical outcome, highlighting the need for biomarkers that identify patients at higher risk of progression and guide therapeutic strategies. Calcium signaling supports leukemia cell survival, yet the clinical relevance of the calcium-responsive enzyme CaMKK2 has not been established. In this study, we demonstrate that elevated CaMKK2 expression in patient-derived leukemia cells is associated with more aggressive disease and earlier need for treatment. Laboratory experiments further show that inhibiting CaMKK2 reduces leukemia cell survival and disrupts supportive macrophage-like cells within the tumor microenvironment. These results position CaMKK2 as a candidate prognostic biomarker that reflects biologically high-risk disease and may inform therapeutic development. Future studies are warranted to determine whether CaMKK2-based risk stratification or targeted inhibition could improve management of patients with CLL.

Diffuse Large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma in the Western world. First-line immunochemotherapy fails in approximately 30-40% of patients, with refractory and relapse patients presenting a dismal prognosis. Currently, these high-risk patients cannot be accurately identified at diagnosis. Using statistical modeling and machine learning approaches applied to large public DLBCL datasets, we identified a novel predictive signature based on the reactivation of eight normally silent tissue-dependent genes associated with survival. We then developed a multiplex RT-MLPseq based assay, compatible with formalin-fixed paraffin-embedded (FFPE) samples and transferable into routine clinical practice, enabling analysis of expression of these eight genes and validated their prognosis impact in an independent real-life cohort. This signature could be integrated with current prognostic indices and molecular classifications to improve patient stratification and guide treatment selection toward a personalized theragnostic approach, thereby enhancing management of non-responder patients.

BackgroundChimeric antigen receptor (CAR)-T cells are therapeutic breakthroughs against advanced non-Hodgkin lymphomas and myelomas. On the other hand, no CAR-T cell product has been so far clinically approved for therapy of Hodgkin Lymphoma (HL), T cell lymphoma (TCL), or Epstein-Barr-Virus (EBV)-associated lymphoproliferative diseases (EBV-LPDs). CD30 (TNFRSF8) is commonly expressed on HL and on subsets of TCL and EBV-LPDs. CD30CAR-T cells generated via transduction with viral vectors have been tested in clinical trials, showing overall good responses against HL. CAR-T cells produced entirely with locus-specific gene editing methods are emerging as attractive next-generation engineered cell products for ease of multiple seamless cell modifications. MethodsUsing CRISPR/Cas9-mediated techniques, we optimized homology-directed repair templates (HDRTs) and performed all-in-one multiplex editing to knock-in (KI) CD30CAR within the TCR constant (TRAC) locus and to simultaneously knock-out (KO) PD-1 or/and {beta}2M. CD30CAR-T cells were tested in CD30+ cell models of HL, TCL, and EBV-LPDs. ResultsWe compared mouse versus human anti-CD30 scFv designs in HDRTs incorporating TRAC homology arms, FcIg spacer/detection domain, and CD28 / CD3{zeta} signaling domains. We obtained an average of 30% TRACKICD30CAR-T cells and efficient in vitro cytotoxicity with CD30+ cell targets. CARs incorporating the high-affinity humanized 5F11 scFv showed the highest CAR expression, and the editing templates were further modified to incorporate a truncated CD34 (tCD34) spacer/detection domain. 5F11-CD30CAR-tCD34-T cells showed high CAR-KI rates (approx. 50-80% 12-14 days after editing) and potency in vitro and in vivo. Subsequently, we tested all-in-one CAR KI with additional KOs by co-electroporation of guide RNAs (gRNAs) targeting the genes encoding PD-1 or /and {beta}2M to improve function and allow for improved cell persistence in allogeneic recipients, respectively. Compared with CD30CAR-T cells, CD30CAR-{beta}2MKO-T cells were similarly viable and functional and showed low risk of translocations. PD1KO enabled CD30CAR-T cells to produce higher levels of cytotoxic features upon exposure to targets. However, simultaneous {beta}2MKO and PD-1KO compromised the expansion capacity of CD30CAR-T cells and resulted in detectable translocations. ConclusionsNon-virally engineered 5F11-CD30CAR-T cells represent a novel cell therapy modality against CD30+ lymphomas. Multiplex editing remains to be optimized to avoid unwanted genomic alterations and chromosomal translocations.

BackgroundAcute myeloid leukemia (AML) is characterized by marked cellular heterogeneity and immune dysregulation. Adenosine-to-inosine (A-to-I) RNA editing, primarily catalyzed by ADAR and ADARB1, represents an important post-transcriptional regulatory mechanism, yet its condition- and cell type-specific landscape in AML remains poorly defined, particularly at single-cell resolution. MethodsWe analyzed publicly available single-cell RNA sequencing data from healthy donors (HL), newly diagnosed AML (ND), remission (RM), and persistent disease (PO), integrating single-cell and pseudo-bulk analyses in a multiscale framework. RNA editing sites were identified using a stringent discovery pipeline and quantified at both pseudo-bulk and cell type-resolved levels. Differential RNA editing was assessed using regression-based read-count models, primarily beta-binomial regression with subject-specific random effects when applicable. Pairwise contrasts between clinical conditions were evaluated using delta-method inference, with statistical significance defined by false discovery rate and a minimum effect-size threshold. Selected editing sites were examined in independent human AML cohorts for validation and clinical association. ResultsWe identified 2,875 recurrent A-to-I RNA editing sites enriched in intronic and 3' untranslated regions and linked to immune and inflammatory pathways. At the pseudo-bulk level, 150 sites were differentially edited across clinical states, and global RNA editing varied by condition, showing an overall negative association with ADAR and ADARB1 expression with context-dependent exceptions. Cell type-resolved analyses identified 148 differentially edited sites with strong lineage specificity. In ND, leukemia-associated cell states consistently exhibited lower editing than lineage-matched healthy counterparts. T cells consistently harbored differential editing signals across all condition contrasts, while progenitor-like cells showed the strongest RM-versus-ND differences despite minimal changes in global editing. Notable editing events were observed in GBP4, SPN, TNFSF10, EMB, and FKBP5. Several candidate sites were validated in independent AML cohorts and were associated with clinical features. ConclusionsThis multiscale analysis reveals that RNA editing in AML is condition- and cell type-specific and is not fully captured by bulk transcriptomic measures. Site-specific, lineage-restricted RNA editing represents a distinct regulatory layer that reflects disease state and cellular context, highlighting its potential relevance for understanding AML biology and informing future biomarker development.

Targeted immunotherapies have revolutionized outcomes for lymphoid malignancies, but success in myeloid neoplasms is limited by the lack of amenable targets and immunologically hostile tumor microenvironment (TME). Myeloproliferative neoplasms are chronic myeloid blood cancers, a third of which are driven by mutations in calreticulin (mutCALR). This yields a common neoepitope that binds to, and activates, the thrombopoietin receptor and results in display of the oncoprotein on the extracellular membrane of disease-driving cells, exposing a therapeutic vulnerability. Here, we present a first-in-class chimeric antigen receptor (CAR) T-cell therapy that specifically targets mutCALR+ cells, both in vitro and in vivo. The CAR T-cell therapy selectively depleted mutCALR+ stem cells from patients with myelofibrosis while sparing healthy stem cells, and improved survival in mutCALR leukemia xenografts. To mimic myelofibrotic marrow, we developed a bespoke human chimeroid model and showed no decrease in the potency of CAR T cell-mediated target cell killing even in a fibrotic tumor microenvironment. We also devised a method to boost cell surface expression of mutCALR in CD34+ cells isolated from patients with accelerated/blast phase MPN (defined as >10 % blasts in peripheral blood or bone marrow), enhancing CAR T targeting. This study presents a therapeutic with potential to eradicate mutCALR-driven malignancies and highlights an innovative strategy to evaluate blood cancer-targeting immunotherapies in a relevant TME. One Sentence SummaryA first-in-class CAR T-cell therapy targeting mutant calreticulin selectively depletes malignant stem cells in vivo and in fibrotic human organoids.

The BCL-2 inhibitor venetoclax (VEN) in combination with hypomethylating agents (HMAs) has improved treatment responses in acute myeloid leukaemia (AML), but the mechanisms underlying their synergy remain unclear. We investigated the role of DNA demethylation in the enhanced cytotoxicity of VEN-HMA combinations. Using AML cell lines, we compared the effects of azacitidine (AZA), decitabine (DAC), cytarabine (ARA-C) and the DNMT1-selective inhibitor GSK-3685032 (GSK5032) with VEN. As expected, VEN showed strong synergy with AZA, DAC, and the DNA-damaging agent ARA-C, but not with GSK5032, despite the latter inducing extensive DNA demethylation. Genome-wide methylation profiling confirmed that loss of DNA methylation did not correlate with increased cytotoxicity or synergy with VEN. Moreover, combining GSK5032 with ARA-C did not enhance cytotoxicity, indicating that DNA demethylation and DNA damage do not act additively. Instead, synergy was consistently associated with the DNA damage-inducing properties of AZA, DAC, and ARA-C. Extensive DNA demethylation tended to antagonize VEN activity, suggesting that the epigenetic effects of HMAs may limit their synergistic potential. Overall, our findings demonstrate that DNA damage-related cytotoxicity, rather than DNA demethylation, is the dominant mechanism driving VEN-HMA synergy and provide evidence that VEN-mediated cytotoxicity arises primarily from genotoxic stress, supporting refinement of treatment strategies.

Bi-allelic CEBPA mutations occur in 5-15% of acute myeloid leukemia (AML) patients. The precise molecular consequences of CEBPA mutations, especially in combination with frequently co-occurring mutations in TET2, WT1, and GATA2, remain incompletely understood. Here, we present a robust human model of CEBPA-mutant AML through gene editing of healthy bone marrow-derived hematopoietic stem cells. Loss of the CEBPA-p42 isoform expressed in healthy cells with concomitant upregulation of the leukemic CEBPA-p30 isoform resulted in a myeloproliferative phenotype. Concurrent loss-of-function mutations in TET2 or WT1 drove full leukemic transformation, while GATA2 haploinsufficiency promoted erythroid precursor accumulation without overt AML. Single-cell transcriptomics and low-input proteomics revealed enhanced myeloid output, increased interferon signaling and elevated cholesterol biosynthesis in leukemic cells. Targeting cholesterol synthesis enhanced chemosensitivity, highlighting a potential therapeutic vulnerability, particularly relevant for CEBPA-mutant patients harboring co-mutations in TET2 or WT1, which have poor outcomes. Statement of significanceInduction of CEBPA-p30 by CRISPR/Cas gene editing in healthy human BM HSCs drives overt AML in vivo. TET2 and WT1 loss accelerate leukemogenesis, while GATA2 haploinsufficiency redirects differentiation toward erythroid precursors potentially driving acute erythroid leukemia. CEBPA-p30 AML exhibits cholesterol biosynthesis dependency, revealing a therapeutic vulnerability to statins.