HemaSphere

Leukemic stem cells (LSCs) sustain acute myeloid leukemia (AML) and are implicated in therapy resistance and relapse. Yet, it remains unknown how LSCs remodel the bone marrow niche. AML is known to alter stromal and vascular microenvironments, but these effects are difficult to separate from bulk leukemic burden and immune inflammation. Here, we use isogenic human AML xenografts with distinct LSC characteristics but comparable engraftment to define LSC-associated niche remodeling in vivo. Single-cell profiling revealed that LSC-high AML shifts the mesenchymal niche toward fibro-inflammatory states, expanding Fmod+ fibroblasts and Cd34+ perivascular fibroblast-like cells while suppressing osteolineage differentiation. The leukemic compartment remained heterogeneous, with a specific MEP-like LSC population expressing niche-remodeling ligands including TGFB1, IL1B, and ANGPT1. LSC-high AML activated a TGF{beta}-responsive, CREB3L1-controlled fibroblast trajectory, and perturbing TGF{beta} signaling or CREB3L1 activation reduced stromal support for AML cells. These findings identify a specific LSC subtype as a source of niche-remodeling cues that shape specialized leukemia-supportive niches.

Although B-cell acute lymphoblastic leukemia (B-ALL) is highly responsive to antigen-directed immunotherapies, treatment resistance remains a major barrier to achieving durable responses in patients. We recently developed a novel VpreB1 (CD179a)-directed antibody-drug conjugate with calicheamicin (VpreB1-ADC) that exploits the restricted expression of VpreB1 within the surrogate light chain in early B cells, including B-ALL. In the present work, we investigated mechanisms of resistance to the VpreB1-ADC. Mechanisms of resistance were evaluated using a TCF3::HLF B-ALL model, assessing target engagement parameters including VpreB1 surface expression and antibody internalization. The role of the multidrug resistance transporter ABCB1 (P-glycoprotein) was evaluated via pharmacologic inhibition, using tariquidar and zosuquidar, and enforced overexpression across multiple B-ALL cell lines. Sensitivity to alternative non-ABCB1 substrate payloads exatecan and PNU-159682 was also assessed. Resistant TCF3::HLF cells retained VpreB1 expression and efficient antibody internalization. Instead, resistance was driven by elevated ABCB1 expression and activity. ABCB1 inhibition with tariquidar or zosuquidar restored VpreB1-ADC sensitivity. Conversely, enforced ABCB1 overexpression conferred ADC resistance, which was reversed by ABCB1 inhibition. Cells with high ABCB1 activity remained fully sensitive to alternative payloads, including exatecan and PNU-159682, which are not ABCB1 substrates. ABCB1-mediated drug efflux drives intrinsic resistance to calicheamicin-conjugated ADCs in B-ALL. Combining ADCs with ABCB1 inhibitors or selecting payloads non-susceptible to ABCB1 efflux offer viable strategies to overcome resistance and optimize future ADC therapies.

Uncontrolled proliferation of myeloid progenitor cells in acute myeloid leukemia (AML) is counteracted in most patients by toxic and often ineffective systemic treatments. Poly (ADP-ribose) polymerase inhibitors (PARPi) show subtype-restricted activity - potent in RUNX1-RUNX1T1 and PML-RAR[a] fusions, limited in KMT2A-rearranged (KMT2A-r) disease - but the lack of molecular understanding has hampered their clinical implementation. We combined single-cell and single-molecule assays on DNA replication intermediates and DNA damage signalling with therapy response readouts to investigate the role of fork plasticity factors in response to PARPi and AML standard-of-care (cytarabine, araC). In PARPi-sensitive AML models, PARP inhibition deregulates RECQ1-mediated fork restart, initially triggering fork acceleration and later fork breakage within the same S phase. Conversely, PARPi resistant KMT2A-r AML lines are protected by PrimPol-dependent DNA synthesis and its inactivation promptly induces fork breakage and PARPi sensitivity. Strikingly, PrimPol overexpression in PARPi-sensitive AML models prevents fork collapse and PARPi/araC therapy response, both in vitro and in vivo, identifying PrimPol as novel predictive biomarker and therapeutic target in AML. Our data uncover novel tissue-specific mechanisms of action for PARPi and pinpoint replication fork plasticity as key molecular determinant of AML therapy response. HighlightsO_LIFork plasticity is a key molecular determinant of treatment response in leukemia. C_LIO_LIPARP inhibition triggers fork breakage via deregulated restart of reversed forks. C_LIO_LIBypassing fork reversal, PrimPol limits therapy-induced DNA damage and cytotoxicity in AML. C_LIO_LIPrimPol drives resistance to cytarabine and PARP inhibition in vitro and in vivo. C_LI

The immunosuppressive bone marrow microenvironment is an important contributor to the limited success of immunotherapy in acute myeloid leukemia (AML), but the cellular interactions underlying AML immune evasion are incompletely understood. We therefore generated a single-cell spatial transcriptomic and proteomic atlas using 148 bone biopsies from 113 individuals comprising pediatric and adult AML at diagnosis and non-leukemic controls. We observed an expansion of regulatory T cells (Tregs) in AML, with stronger colocalization between Tregs and macrophages compared to non-leukemic bone marrow. Distinct cellular neighborhoods were enriched for myeloid progenitor-like cells together with macrophages and T cells, which correlated with higher macrophage and T cell immune checkpoint expression. Moreover, these neighborhoods were associated with specific AML subtypes, especially KMT2A-rearranged and RUNX1::RUNX1T1 AML. These spatial patterns were validated by identification of malignant cells via in situ fusion detection in RUNX1::RUNX1T1 cases. Functional experiments revealed that macrophages and AML cells not only actively recruit Tregs, but also promote naive T cell differentiation into Tregs. Spatially informed ligand-receptor analysis predicted the involvement of the Galectin-9 - CD44/TIM-3 axis in this immunosuppressive crosstalk, which was supported by in vitro inhibition of CD44 and/or TIM-3 preventing macrophage- and AML-induced Treg differentiation. Collectively, this comprehensive spatial map of the AML bone marrow identified tripartite crosstalk between AML, macrophages, and T cells mediated by the Galectin-9 - CD44/TIM-3 axis as a key component of the immunosuppressive microenvironment. Targeting Galectin-9 - CD44/TIM-3 interactions may be a promising strategy to overcome immune evasion and enhance immunotherapeutic success in AML. HighlightsO_LISpatial transcriptomic and proteomic atlas of pediatric and adult acute myeloid leukemia (AML) bone marrow C_LIO_LIIncreased colocalization of macrophages and regulatory T cells (Tregs) in AML C_LIO_LIMacrophages and AML cells induce differentiation of naive T cells to Tregs in vitro, which can be prevented by inhibition of CD44 or TIM-3 C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/743431v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@5887f4org.highwire.dtl.DTLVardef@45d17corg.highwire.dtl.DTLVardef@1bc3264org.highwire.dtl.DTLVardef@9059aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

Acute myeloid leukemia (AML) is a clinically heterogeneous disease. Although the genetic abnormalities associated with poor prognosis are well defined, how they drive unfavorable outcomes remains unclear. Using published gene-expression and dependency datasets, we searched for cell-surface protein-coding genes associated with poor survival and required for AML growth, prioritizing this class of proteins for its accessibility to biologics. This search identified CD59, a GPI-anchored protein with a canonical role in complement regulation, whose high mRNA expression correlates with adverse-risk genetics and stemness signatures. CD59 silencing impaired proliferation across genetically diverse AML cell lines, reduced leukemic burden, and extended survival in cell xenograft models. Moreover, CD59 expression was enriched on leukemic stem cells (LSCs), and its depletion impaired LSC self-renewal and primary AML engraftment in vivo while sparing normal hematopoiesis. Mechanistically, these effects reflected a non-canonical role for CD59 in sustaining Ras-MAPK signaling, whereby its loss depleted inner-leaflet phosphatidylserine and impaired Ras and c-Raf membrane recruitment and activation. rILYd4, a recombinant fragment of the bacterial toxin intermedilysin that binds and degrades CD59, recapitulated these effects and sensitized cells to venetoclax in vivo. These findings reveal CD59 as a critical regulator of Ras-MAPK signaling required for AML growth and nominate its rILYd4-mediated degradation as a therapeutic strategy.

Large genomically annotated acute myeloid leukaemia (AML) datasets exist, but population-based contemporary cohorts remain scarce. Here we report clinicopathological, genomic, and outcome data from Danish AML patients. 2,512 AML patients were identified between 2015-2022, of whom 33.8% had available NGS data (NGS+). In patients [≤]70 years, baseline characteristics and outcomes were comparable between NGS+ and NGS- groups. In patients >70 years, more NGS+ patients received intensive treatment, but survival was similar among intensively treated patients. The distribution of mutations varied significantly by age and sex, with older age and male sex exhibiting higher frequencies of adverse-risk gene mutations. In intensively treated NGS+ patients, ELN2017 stratified 5-year OS: 58.4% (favorable), 43.4% (intermediate), and 28.2% (adverse), with hazard ratios (HRs) of 0.63 (favorable) and 1.45 (adverse) relative to intermediate. ELN2022 yielded corresponding OS rates of 56.9%, 51.8%, and 29.7%, with HRs of 0.78 and 1.86. The two models had comparable predictive performance for OS in a time-dependent model. In conclusion, outcomes of intensively treated AML patients were comparable irrespective of NGS status, underscoring the representativeness of the REFORM-AML database for the Danish AML population. Age and male sex correlated with adverse-risk mutations, and both ELN2017 and ELN2022 robustly predicted survival.

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma (PTCL) are aggressive hematological malignancies requiring novel therapeutic strategies. The majority of T-ALL and PTCL tumors display metabolic activation and addiction to endogenous serine/glycine synthesis (SSP), providing opportunities for targeted therapy with the clinically used antidepressant sertraline, inhibiting SSP enzymes SHMT1/2. However, sertraline monotherapy only induces cell cycle arrest and has limited efficacy in suppressing disease progression in vivo. MethodsDrug synergy of sertraline combined with clinically used proteasome inhibitors carfilzomib and bortezomib was evaluated. Drug effects on cell cycle, proliferation and apoptosis were assessed in T-ALL, PTCL and healthy blood cells using flow cytometry assays. Proteomic, lipidomic and metabolic analyses on drug treated T-ALL cells were performed to elucidate the molecular mechanisms underlying drug synergy, followed by validation of changes of interest, metabolic rescues and shRNA-knockdown of SSP enzymes in T-ALL cells. In vivo therapeutic efficacy and immune remodelling were evaluated in an immunocompetent MYCN-overexpressing PTCL mouse model. ResultsSertraline acted synergistically with clinically used proteasome inhibitor carfilzomib to induce cell cycle arrest and apoptosis in T-ALL and PTCL cells with SSP activity, with minimal effects on SSP-inactive T-ALL cells or healthy blood cells. Adding carfilzomib also enhanced the therapeutic efficacy of sertraline in an aggressive MYCN PTCL model. Sertraline rewired cell metabolism towards increased cholesterol uptake and biosynthesis in SSP-active T-ALL cells, and this effect was not obtained by other means of SSP inhibition. In contrast to sertraline, carfilzomib promoted cholesterol efflux. Moreover, carfilzomib reduced total lipid levels, further restricting nutrients in sertraline - carfilzomib treated cells. Additionally, the drug combination impaired mitochondrial respiration and elevated reactive oxygen species (ROS) levels and DNA damage in SSP-active tumor cells, which was rescued by citrate supplementation. Interestingly, these metabolic changes were associated with microenvironmental changes in our mouse model, where the drug combination elevated natural killer T-cells, neutrophils and eosinophils. ConclusionsOur study identifies synergy of sertraline - carfilzomib combination treatment mediated through metabolic impairment and is associated with remodelling of the immune microenvironment. This invites for further clinical investigation of this drug combination as a therapeutic strategy for SSP-active T-cell malignancies.

Acute myeloid leukemia (AML) is an aggressive blood cancer characterized by high rates of relapse and poor outcomes, especially in elderly or unfit patients, who cannot tolerate intensive chemotherapy. While the BCL2 inhibitor venetoclax has improved initial responses in this high-risk population, relapses remain nearly universal, highlighting the need for novel therapeutic strategies. Here, we identify the RNA-binding protein PTBP1 as a critical dependency in AML. PTBP1 depletion impairs leukemic growth in vitro and in vivo, and is associated with widespread splicing alterations and global disruption of protein synthesis. Integrative transcriptomic and iCLIP analyses reveal that PTBP1 orchestrates a splicing program centered on Rho GTPase signaling, with CDC42 as a key downstream effector. Mechanistically, PTBP1 loss triggers a splicing switch from CDC42-v1 to CDC42-v2, leading to reduced GTPase activity and impaired protein synthesis. Pharmacological inhibition of CDC42 selectively induces cytotoxicity in AML cells, while sparing healthy hematopoietic cells. Importantly, CDC42 inhibition markedly enhances venetoclax anti-leukemic efficacy. These findings establish PTBP1 as a critical regulator of AML cell fitness and identify a clinically actionable therapeutic combination that exploits AML dependency on PTBP1-CDC42 signaling to enhance the efficacy of venetoclax-based regimens.

Pediatric acute myeloid leukemia (AML) is driven by aberrant transcriptional programs sustained by poorly defined cis-regulatory mechanisms. To systematically identify functional enhancer dependencies, we developed an integrative enhancer discovery strategy that combines H3K27ac CUT&Tag profiling, enhancer-associated transcription, and CRISPR interference (CRISPRi) screening. By leveraging enhancer-associated transcription to prioritize candidate regulatory elements, we identified 321 leukemia-associated enhancers for functional interrogation. This approach uncovered the hematopoietic MYB enhancer (H-ME) within the HBS1L-MYB-AHI1 locus as a critical regulator of leukemic growth. H-ME repression reduced chromatin accessibility and active histone marks at the MYB promoter, suppressed MYB expression, and induced differentiation-associated transcriptional programs. In contrast, selective depletion of the enhancer-associated transcript had no effect on MYB expression or leukemic proliferation, demonstrating that enhancer activity resides within the underlying regulatory DNA element rather than its mature RNA product. H-ME exhibited preferential activity in megakaryocytic leukemia, and its perturbation impaired leukemic growth in primary patient-derived models in vitro and in vivo. Together, our findings establish an integrative framework for the systematic discovery of functional enhancer dependencies and identified H-ME as an RNA-independent regulator of MYB in pediatric AML.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer, yet the mechanisms by which inherited risk variants predispose to leukemia development remain poorly understood. A major challenge to studying these mechanisms has been the lack of model systems that faithfully capture the transient developmental states in which predisposition alleles are thought to act. Here, we establish a human B-cell differentiation platform from hematopoietic stem/progenitor cells that enables CRISPR-based engineering, recapitulates early B-cell lymphopoiesis, and enriches for rare developmental intermediates. By applying systematic perturbations with multiplexed single-cell transcriptomic profiling to mimic the effects of mutations in nine familial B-ALL predisposition genes, we decipher mechanisms by which B-cell development can be altered by such inherited variation to predispose to B-ALL. Through these studies, we identify convergent delays in B-cell differentiation at progenitor stages characterized by high-level RAG1/2 recombination activity. We propose that these delays at progenitor stages increase the likelihood that cells can undergo illegitimate RAG-mediated recombination to promote transformation, a finding consistent with similar rates of illegitimate RAG-associated genomic alterations in those with B-ALL associated with familial predisposition variants compared to sporadic cases.

Therapy resistance in acute myeloid leukemia (AML) is linked to metabolic plasticity and mitochondrial fitness of leukemic stem and progenitor cells. Clever-1 is a scavenger receptor with established immunoregulatory functions, but its leukemia cell-intrinsic roles remain unclear. Here we identify Clever-1 as a regulator of mitochondrial integrity and lipid-dependent oxidative metabolism in AML. Using the anti-Clever-1 antibody bexmarilimab, we show that Clever-1 inhibition induces early mitochondrial transcriptional reprogramming, followed by suppression of oxidative phosphorylation (OXPHOS) in AML cell lines. Immunoelectron microscopy demonstrates mitochondrial localization of Clever-1, while proteomic analyses reveal altered association with mitochondrial-linked proteins, including ATAD3. Functionally, Clever-1 inhibition reduces mitochondrial delivery of lipoprotein-derived lipids, resulting in selective changes in mitochondrial lipid composition. These changes are accompanied by impaired respiratory complex IV assembly, disrupted cristae architecture, accumulation of dysfunctional mitochondria, and reduced spare respiratory capacity. AML models with high baseline OXPHOS activity are particularly sensitive to Clever-1 inhibition, with mitochondrial dysfunction exacerbated under lipid-restricted or metabolically stressful conditions. Together, these findings define Clever-1 as a regulator of mitochondrial bioenergetic resilience and a targetable metabolic vulnerability in AML.

Acquired aplastic anaemia is caused by immune-mediated loss of haematopoietic stem and progenitor cells (HSPCs), but the regulatory states that sustain cytotoxic immunity and their relationship to inherited susceptibility remain incompletely understood. We integrated two single-cell RNA-sequencing cohorts spanning healthy, non-severe and severe aplastic anaemia with single-cell chromatin accessibility profiling, genome-wide association meta-analysis, Bayesian fine-mapping and stratified LD-score regression. Single-cell transcriptomics revealed a coordinated shift across the immune and haematopoietic compartments. Cytotoxic CD8 and {gamma}{delta} T cells converged on a shared NKG7/CCL5/PRF1 effector program, indicating that cytotoxic differentiation extends across T-cell lineages. Effector-memory T cells combined inflammatory signalling with SOCS, DUSP, TNFAIP3, RGS1 and TOX, consistent with sustained stimulation accompanied by extensive feedback regulation. With increasing disease severity, these inflammatory states were further coupled to hypoxic, oxidative and unfolded-protein-response programmes, suggesting qualitative remodeling of the immune compartment rather than uniform amplification of perforin-granzyme expression. Single-cell chromatin accessibility provided a regulatory counterpart to these transcriptional states. Naive and memory-associated cells retained TCF7/LEF1/BACH2 accessibility, whereas cytotoxic cells acquired coordinated accessibility across CCL5, NKG7, PRF1, granzymes and killer-receptor loci. Pseudotime, motif activity and integrated RNA-chromatin profiles positioned AP-1, NFAT and TBX21 along this transition, linking loss of memory-associated regulation to acquisition of cytotoxic effector competence. Genetic meta-analysis independently recovered association at the HLA-B region, reinforcing antigen presentation as the principal inherited susceptibility axis. Fine-mapping additionally prioritized a non-HLA locus without resolving its effector gene, while stratified LD-score regression found no detectable preferential enrichment of common-variant heritability within effector-memory or cytotoxic regulatory elements. Integrated with the cellular data, these findings support a mechanistic hierarchy in which HLA-linked antigen presentation establishes the selective context, persistent cytotoxic T-cell state remodeling maintains pathogenic immune pressure, and IFN{gamma}-responsive HSPC suppression translates this pressure into haematopoietic failure.

Although the human genome encodes a vast repertoire of noncoding RNAs that regulate gene expression, the noncoding genome remains underexplored due to technical challenges. Specifically, during transcriptomic sequencing data alignment, the overlap between noncoding and coding loci can create ambiguous read alignments that are subsequently discarded from downstream analysis. For this reason, most of the noncoding genome is excluded from standard genomic annotations used for sequencing alignment. To address this challenge and enable concurrent profiling of the coding and noncoding transcriptome, we systematically integrated standard coding (GENCODE) and noncoding (LncBook) genome annotations, preserving coding gene annotations and removing overlapping noncoding regions. The resulting integrated genome annotation expanded the number of annotated noncoding genes from 40,785 to 138,296 while preserving all coding genes and reducing ambiguous read assignment. To evaluate the utility of our integrated genome annotation for uncovering novel, biologically relevant noncoding RNAs (ncRNAs), we realigned CD138-positive bulk RNA-seq (N = 942) and CD138-negative single-cell RNA-seq (N = 478) data from the MMRF CoMMpass study, generating a comprehensive coding-noncoding atlas of the myeloma bone marrow microenvironment with noncoding genes representing 51% of highly variable genes and displaying significant cell type specificity. Tumor expression profiling based on this integrated profiling identified 15 clusters, including two enriched for amp(1q21) or t(4;14) and associated with shorter progression-free survival (PFS). Differential expression and systematic filtering yielded 19 candidate high-risk ncRNAs, including previously uncharacterized ENSG00000310209, which was associated with poor PFS (HR = 1.141, P = 0.0025), increased IRF4 activity, Wnt pathway activation, CCL5 signaling, and the accumulation of anergic-like CD8+ T cells. These findings establish integrated coding-noncoding analysis as a strategic approach for discovering functional ncRNAs from transcriptomic sequencing data.

The genetic risk factors of immune cytopenias are unclear. Immune cytopenias have been reported in various genetic contexts: 1) inherited error of immunity genes, mainly due to rare germline variants, 2) systemic lupus erythematosus, associated with common germline variants, 3) hematological malignancies, and 4) clonal hematopoiesis, the latter two due to somatic variants. However, the respective contribution and interaction of these variants remain to be investigated. Here, we used two large biobanks with whole genome sequencing data to systematically investigate the genetic contribution to immune cytopenia. We found that the four types of genetic variants independently contribute to immune cytopenia risk. We notably found that carriers of variants in some autosomal recessive genes of inherited error of immunity had an increased risk of immune cytopenia. Additionally, common variant-mediated risk of systemic lupus erythematosus also increased the risk of immune cytopenia. Overall, a third to a half of patients with immune cytopenia carried at least one of the four genetic risk variants investigated. Combining the four variants allowed stratifying the risk of immune cytopenia in both general and high-risk population. In general population, the 10-year incidence of immune cytopenia in the lowest and highest risk groups was 0.08% and 1.5%, respectively. In sum, this work identified that different genetic risk factors can lead to immune cytopenia. A large proportion of individuals with immune cytopenia carried an underlying genetic risk factor. Finally, combining these genetic risk factors enabled risk stratification.

Abstract Background: The stem cell factor receptor or c-Kit is a type III receptor tyrosine kinase, activated by its ligand Stem cell factor (SCF). Up on activation, c-kit induces signaling pathways that regulates blood cell proliferation, survival, differentiation, and migration. Several studies reported that c-Kit/SCF signaling, contributes to the development and progression of acute myeloid leukemia (AML) in patients. However, the downstream proteins regulated by c-kit activation and their clinical significance in AML remain poorly explored. Methods: Human Acute megakaryoblastic leukemia (Mo7e) cells, were-stimulated with SCF and global protein expression were profiled using two-dimensional gel electrophoresis coupled with MALDI-TOF and LC-MS/MS. Differentially expressed proteins were functionally characterized and validated using patient data from the TCGA-LAML and matched normal data from GTEx, GEO datasets, and quantitative RT-PCR. Their diagnostic and prognostic significance was assessed using ROC, Cox regression, LASSO, Kaplan Meier survival analyses, and a prognostic nomogram model. Results: Proteomic profiling identified 14 differentially expressed proteins in SCF-stimulated Mo7e cells, which are predicted to involved in cytoskeletal organization, protein folding, metabolism, vesicular trafficking, and translational regulation. Transcriptomic analysis of the TCGA-LAML cohort revealed significant dysregulation of CFL1, CCT8, HSP90B1, MDH2, EIF5A, GSN, and TPI1. Integrated ROC, Cox regression, and LASSO analyses identified CFL1, CCT8, and GSN as the most robust prognostic biomarkers associated with poor overall survival in LAML patients. Their expression patterns were validated in independent GEO datasets and by qRT-PCR in SCF stimulated Mo7e cells. Finally, a three-gene nomogram model was developed and validated to predict the overall survival probability of AML patients at 1-, 3-, and 5-year time points. Conclusions: This study identifies CFL1, CCT8, and GSN as key downstream effectors of c-Kit signaling as prognostic biomarkers for AML. These findings provide mechanistic insights into c-Kit-driven leukemogenesis and establish a clinically relevant three-gene signature for AML risk stratification and potential therapeutic targeting.

Purpose: Adults with high- or very high-risk acute leukemia (AL) or myelodysplastic syndrome (MDS) face substantial relapse risk after allogeneic hematopoietic stem-cell transplantation. We evaluated single-unit cord blood (CB) transplantation after ex vivo expansion with UM171 in this population. Patients and Methods: Two prospective, single-arm phase II trials at four centers enrolled 64 adults with high- or very high-risk AL or MDS; 60 received a UM171-expanded CB transplant and comprised the analysis population. CB units were preferentially selected at a 5/8 HLA match to maximize the graft versus leukemia effect. Patients received intermediate- or high-intensity conditioning with tacrolimus/mycophenolate mofetil graft-versus-host-disease (GVHD) prophylaxis. Endpoints included safety, feasibility, non-relapse mortality (NRM), relapse-free survival (RFS), overall survival (OS), GVHD, GVHD-free relapse-free survival (GRFS), chronic GVHD-free relapse free survival (CRFS). Results: Thirty-two percent of patients had undergone previous transplantation, 17% of patients with AL were not in remission and 24% of those with AML/MDS had TP53 mutations. Of 62 patients who remained eligible for transplantation, 60 had a graft successfully manufactured and infused. Median times to neutrophil and platelet engraftment were 17 and 38 days, respectively. NRM was 5.1% at day 100 and 15.2% at 1 year. Two-year cumulative incidence of relapse was 22.3%. Two-year OS and RFS were 63.9% and 60.4%, respectively. Grade III-IV acute GVHD incidence was 20.3% at 1 year and moderate-to-severe chronic GVHD incidence was 6.8% at 2 years. Conclusion: UM171-expanded CB transplantation was feasible and provided prompt engraftment, durable disease control, and infrequent clinically significant chronic GVHD in adults with high- and very high-risk AL/MDS. Comparative studies are warranted to define its role relative to contemporary donor platforms.

Background: Promoters have been traditionally seen as contiguous gene-adjacent cis-regulatory elements. Yet, substantial studies corroborate that Epromoters (promoters with enhancer activity) engage in distal forms of gene regulation. Although in the three-dimensional (3D) space enhancer-promoter networks have been well studied, the contribution of the circuits of promoter-promoter (P-P) interactions is poorly understood. Furthermore, whether the regulatory aspects of P-P interactions in cancer may be controlled by physical 3D-mediated Epromoter interactions remains elusive. Results: We show that Epromoter-mediated 3D interactions regulate target genes and participate in cluster co-regulation, playing a critical role in T-cell acute Lymphoblastic Leukemia (T-ALL). To achieve this, we first leveraged survival CRISPR screenings in T-ALL model cells (Jurkat) to identify potential Epromoters. By integrating these findings with an H3K27ac HiChIP dataset from T-ALL cells, we characterized a set of Epromoters that establish 3D genome interactions with other promoters. We observed that promoters organize into dense, promoter-rich genomic clusters, and that among them, the clusters enriched with Epromoters actively regulate complex gene expression networks. To investigate gene coregulation, we integrated transcriptomic data from T-ALL patients and found that promoter-promoter (P-P) pairs exhibit positive correlation at multiple levels, and that several Jurkat Epromoter candidate clusters are significantly co-regulated in the patient cohort. To experimentally validate these candidates, we utilized CRISPRi to inhibit Epromoters, which revealed direct transcriptional regulation of multiple target genes within each hub. Finally, we performed cell competition assays to confirm that these Epromoters are vital for T-ALL cell survival. Conclusions: Our analysis provides support for the role of Epromoters in the regulation of 3D P-P interactions and co-regulation of promoter hubs, and how these interactions play a critical part in T-ALL cell survival.

Multiple myeloma (MM) exhibits profound molecular heterogeneity, yet current risk stratification relies on cytogenetics or single-omics signatures that often fail to capture cross-layer regulatory complexity. We re-analyzed a multi-omics dataset integrating copy-number, transcriptomic, proteomic, and phosphoproteomic data to dissect how common genomic driver alterations propagate through the molecular cascade. Supervised classification demonstrated that downstream layers, particularly the proteome and phosphoproteome, classify genomic events more accurately than primary genomic or transcriptomic data. Intriguingly, trans-acting features alone were sufficient for classification, indicating that while direct dosage effects manifest at the RNA level, downstream network responses dominate the proteomic state. Multi-omics factor analysis (MOFA2) identified a continuous latent axis predicting progression-free and overall survival independent of R-ISS. This factor captured a gain(1q)/del(13q) axis modulated by immune infiltration and NSD2 expression, integrating variance across all four modalities. To enable clinical translation, we derived sparse, single-modality proxies using elastic net regression. An RNA proxy faithfully recapitulated the multi-omic factor and validated independently in published microarray and RNAseq cohorts, demonstrating robust prognostic utility across treatment eras. These findings reveal that multi-omics integration uncovers hidden prognostic axes obscured by single-omics analyses, and that sparse proxies can bridge the gap between complex discovery and clinical implementation.

Inherited genetic variation substantially increases the risk for developing childhood B-cell acute lymphoblastic leukemia (B-ALL), the most common cancer in children, yet the underlying mechanisms remain poorly understood. To address this limitation, we employ a single-cell multiomic framework to functionally dissect common regulatory variants associated with B-ALL risk. Coupling this multiomic analysis with assessment of allelic skews in chromatin accessibility, we reveal the impact of risk alleles and disruptions in transcription factor networks specific to B-cell progenitors, thereby providing mechanistic insights into altered regulatory programs underlying B-ALL predisposition. By constructing long-range variant-to-target gene maps, we identify 34 high-confidence B-ALL susceptibility genes. Among these, we uncover and functionally validate a risk allele that selectively upregulates expression of ELK3, a previously unrecognized regulator of B-cell development and leukemogenesis. Together, these findings establish a comprehensive variant-to-function map of cell state-specific regulatory disruptions underlying inherited predisposition to B-ALL and define new risk mechanisms, which could pave the way for future targeted prevention approaches.

Relapse in cancer is frequently driven by therapy-resistant quiescent cancer stem cells. Conventional chemotherapy has been designed to target proliferating tumor cells and is generally presumed to be ineffective against non-cycling cancer stem cells. Using acute myeloid leukemia (AML) as a model, we challenge this prevailing view by showing that inhibition of the mitotic master regulator Polo-like kinase 1 (PLK1), a kinase extensively pursued for antiproliferative cancer therapy, unexpectedly eradicates quiescent leukemia stem cells (LSC) through a mechanism distinct from its canonical mitotic function. In proliferating AML cells, PLK1 inhibition (PLK1i) induced G2/M arrest and mitotic catastrophe. In contrast, quiescent LSC underwent apoptosis independent of mitotic arrest, revealing a cell-state-dependent mode of drug action. Mechanistically, PLK1i initiated a multi-step process through disruption of a previously unrecognized, stem cell-specific interaction between PLK1 and MAP1A, resulting in perturbed vesicle trafficking and endolysosomal homeostasis characterized by altered receptor internalization, vesicle accumulation and lysosomal dysfunction, ultimately culminating in apoptotic cell death. Combinatorial pharmacologic perturbation studies established microtubule regulation as a critical determinant of quiescent LSC survival, while ex vivo and in vivo assays demonstrated depletion of functionally-defined LSC following PLK1i. These findings identify a previously unrecognized role for PLK1 in intracellular trafficking and establish MAP1A-dependent control of vesicle homeostasis as a mechanistic determinant of cancer stem cell survival. More broadly, this study demonstrates that classical antimitotic compounds, including microtubule-targeting agents and PLK1 inhibitors, can eradicate both cycling leukemic blasts and quiescent LSC through distinct, cell state-dependent mechanisms, challenging proliferation-centric models of chemotherapy action.