Blood Advances

BackgroundSezary syndrome (SS) is a rare and aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) with limited therapeutic options and a median survival of fewer than five years. Despite advances in how single-cell RNA sequencing (scRNA-seq) has improved the understanding and treatment of other cancer types, such insights in SS remain limited. The drivers of disease progression and immunologic dysfunction are incompletely defined, underscoring the need to characterize both malignant T cells (MTCs) and their interactions with surrounding immune populations. ObjectivesTo systematically characterize malignant and non-malignant immune cells in CTCL, identify distinct malignant T-cell subtypes, and uncover transcriptional programs and immune evasion pathways with therapeutic relevance. MethodsWe analyzed scRNA-seq data on peripheral blood mononuclear cells (PBMCs) from 22 SS patients and 7 healthy controls. MTC subtypes were identified using a combination of transcriptional profiling, copy number variation (CNV) analysis, and T-cell receptor (TCR) clonotyping. CITE-seq was utilized on a subset of samples to correlate genetic findings with surface protein expression. ResultsWe identified three MTC subtypes: (1) MTC central memory (CM), a Th2-skewed CM phenotype that constituted the dominant malignant population; (2) MTC effector/effector-memory (E/EM), a subset enriched in Th1-associated genes; and (3) MTC regulatory (Reg), a regulatory-like, exhausted phenotype, along with shared and subtype-specific gene signatures. The predominance of MTC CM suggests a stable malignant state, while relative rarity of MTC E/EM and MTC Reg may reflect treatment effects or disease progression. In addition to KIR3DL2, we identified KIR2DL3 and KIR3DL1 as upregulated immune-evasive receptors on MTCs and surrounding cells. Tensor factorization of ligand-receptor interactions revealed pro-inflammatory yet immunosuppressive signaling in myeloid cells converging on STAT3 activation. ConclusionsThis study defines three transcriptionally and functionally distinct MTC subtypes in SS, highlighting subtype-specific vulnerabilities that may inform personalized treatment strategies. Our findings suggest that targeting not only MTCs but also KIR-family receptor signaling and JAK-STAT activation in the immune microenvironment may have therapeutic implications. The identification of novel immunosuppressive pathways and cell survival mechanisms opens avenues for tailored interventions - including widespread KIR inhibition, repurposing JAK inhibitors, and other novel therapies - to improve patient outcomes in SS. Contributor StatementB.A. Childs conducted the conceptualization (equal), data curation (equal), formal analysis (lead), investigation (lead), methodology (equal), validation (equal), visualization (equal), writing - original draft (lead), writing - review & editing (supporting). E. Elghonaimy provided conceptualization (equal), data curation (equal), formal analysis (equal), investigation (equal), methodology (equal), resources (equal), supervision (equal), validation (equal), visualization (equal), writing - original draft (supporting), and writing - review & editing (supporting). P. R. Geethakumari provided data curation (supporting), investigation (supporting), resources (supporting), writing - review & editing (supporting). K. Kumar contributed conceptualization (supporting), data curation (supporting), resources (supporting), and writing - review & editing (supporting). J. F. Merola provided conceptualization (supporting), investigation (supporting), resources (supporting), writing - review & editing (supporting). H.W. Goff contributed conceptualization (equal), data curation (equal), formal analysis (supporting), funding acquisition (lead), investigation (supporting), methodology (supporting), resources (supporting), supervision (equal), visualization (equal), writing - review & editing (equal). T. A. Aguilera provided conceptualization of the project (equal), data curation (equal), formal analysis (equal), funding acquisition (equal), investigation (equal), methodology (equal), project administration (equal), resources (lead), supervision (lead), visualization (supporting), writing - original draft (supporting), writing - review & editing (equal). T.A. Aguilera is the guarantor and accepts full responsibility for the overall content, had access to all the data, and controlled the decision to publish. Bulleted StatementsO_ST_ABSWhat is already known about this topic?C_ST_ABSO_LISezary syndrome is a rare, aggressive CTCL subtype associated with poor survival; a minority of patients achieve long-term remission with allogeneic stem cell transplant. C_LIO_LIKIR3DL2 is an investigational target with ongoing trials (e.g., lacutamab, anti-KIR3DL2 agent). C_LIO_LIJAK inhibitors have shown mixed effects, with anecdotal benefit and potential harm. C_LIO_LIThe heterogeneous biology of Sezary cells remains poorly characterized, limiting diagnostic and therapeutic advancement. C_LI What does this study add?O_LIWe define three distinct malignant T-cell subtypes in Sezary syndrome, each with clinical and therapeutic implications, and a unifying genetic signature. C_LIO_LIIn addition to KIR3DL2, a known CTCL marker, we identify KIR2DL3 and KIR3DL1, inhibitory receptors implicated in viral and autoimmune regulation, as additional immune evasion mechanisms. C_LIO_LIJAK/STAT activation in neighboring myeloid cells may be driven by Sezary cell signaling through S100A8, S100A9, CD74, IL-10, and TNF, a potentially targetable axis. C_LI What is the translational message?O_LIThe predominance of a central memory-like malignant phenotype, alongside other distinct subsets reveal an opportunity for subtype-guided treatment strategies. C_LIO_LIOptimal therapeutic targeting may include both malignant T cells and interacting immune cells. C_LIO_LIOur findings identify targets that may be actionable, including KIR-family receptors and STAT3-related signaling. C_LIO_LIInsights from other T-cell disorders may guide repurposing of existing therapies to improve outcomes. C_LI Lay Summary: How Sezay Syndrome, a Type of Skin Lymphoma, Impacts the Immune SystemSezary syndrome (SS) is a rare and aggressive form of skin cancer that starts in the blood. It is a type of cutaneous T-cell lymphoma (CTCL), where cancerous T cells (a kind of white blood cell) spread from the bloodstream to the skin. SS mainly affects adults and has limited treatment options, with most people living less than five years after diagnosis. This study, based in the United States, aimed to understand how cancerous T cells behave in SS and how they interact with other immune cells. We used a method called single-cell RNA sequencing to examine over 100,000 individual immune cells from the blood of 22 patients with SS and 7 healthy individuals. This allowed us to see which genes were active in each cell. We found that the cancerous T cells in SS are not all the same. Instead, they fall into three distinct groups: one resembled central memory T cells that typically live in the blood, one looked more aggressive and inflammatory, and one resembled exhausted regulatory immune cells. We also discovered that cancer cells and surrounding immune cells expressed molecules called KIRs, which may help them avoid being attacked. Other immune cells released signals that turned on a pathway called JAK-STAT, which may further protect the cancer. These findings reveal new ways that SS protects itself and avoids the immune system. Understanding these escape routes may help guide future treatments, including drug combinations that block these signals and improve patient outcomes. Abbreviated AbstractSingle-cell RNA and protein profiling of Sezary syndrome reveals three distinct malignant T-cell subtypes and uncovers convergent immunosuppressive signaling through KIR-family receptors and STAT3 activation. These findings expose targetable pathways driving immune evasion and offer a foundation for more personalized therapeutic strategies in this aggressive form of cutaneous T-cell lymphoma.

Currently, there are at least a dozen recognized hereditary hematopoietic malignancies (HHMs), some of which phenocopy others. Among these, three HHMs driven by germline mutations in ANKRD26, ETV6, or RUNX1 share a phenotype of thrombocytopenia, qualitative platelet defects, and an increased lifetime risk of hematopoietic malignancies (HMs). Prior work has demonstrated that RUNX1 germline mutation carriers experience an elevated lifetime risk (66%) for developing clonal hematopoiesis (CH) prior to age 50. Germline mutations in ANKRD26 or ETV6 phenocopy RUNX1 germline mutations, but no studies have focused on the risk of CH in individuals with germline mutations in ANKRD26 or ETV6. To determine the prevalence of CH in individuals with germline mutations in ANKRD26 or ETV6, we performed next generation sequencing on hematopoietic tissue from twelve individuals with either germline ANKRD26 or germline ETV6 mutations. Each patient had thrombocytopenia but had not developed HMs. Among the seven individuals with germline ANKRD26 mutations, one patient had a CH clone driven by a somatic SF3B1 mutation (p.Lys700Glu). This mutation increased from a variant allele frequency (VAF) of 9.4% at age 56 to 17.4% at age 60. None of the germline ETV6 mutation carriers had evidence of CH at the limits of detection of the NGS assay (5% VAF). Unlike individuals with germline mutations in RUNX1, no individuals under the age of 50 with germline mutations in ANKRD26 or ETV6 had detectable CH. This work demonstrates that ANKRD26 germline mutation carriers, but not ETV6 mutation carriers, experience elevated risk for CH.

Acquired aplastic anemia (AA) is a rare blood disorder that results from immune-mediated destruction of bone marrow (BM) progenitor cells. Improved understanding of the mechanisms that favor T cell attack in BM could help to improve early diagnosis and disease treatment. Diacylglycerol kinase {zeta} (DGK{zeta}) limits T cell responses through phosphorylation of diacylglycerol into phosphatidic acid. This reaction attenuates diacylglycerol-dependent activation of the Ras/ERK/CD69 and PKC{theta}/NF{kappa}B pathways in response to antigen. Here we show that, in contrast to the lack of basal activation observed in peripheral lymphoid organs, DGK{zeta}-/- mice showed increased numbers of activated T cells in BM, together with a significant increase in IFN{gamma} as well as perforin and granzyme B and C levels. The enhanced presence of T cells in DGK{zeta}-/- mouse BM correlates with reduced BM cellularity, impaired hematopoiesis, and lower frequency of circulating red cells, granulocytes, and platelets. Our studies coincide with the recent characterization of lower DGK{zeta} expression in T cells isolated from the BM of patients with acquired AA, and suggest that limited DGK{zeta} expression and/or functions predispose to T cell-mediated BM destruction. This study identifies the BM as a niche particularly sensitive to DGK{zeta} deficiency and indicates that this mouse model could be of interest for studying the mechanism that contributes to AA development. Key pointsO_LIDGK{zeta}-deficiency in mice results in larger numbers of CD69-positive T cells in bone marrow, with enhanced expression of IFN{gamma} and lytic enzymes. C_LIO_LIDGK{zeta} loss recapitulates many clinical aspects of human aplastic anemia, identifying a critical hub for immune system-dependent bone marrow failure. C_LI Visual abstract O_FIG O_LINKSMALLFIG WIDTH=140 HEIGHT=200 SRC="FIGDIR/small/136390v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@d485b2org.highwire.dtl.DTLVardef@9599c1org.highwire.dtl.DTLVardef@1a17842org.highwire.dtl.DTLVardef@1de640d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Immunoglobulin light-chain amyloidosis (AL) has been reported to be associated with multiple myeloma (MM) in approximately 10-15% of cases, with the two conditions often coexisting. Understanding the interaction between these diseases is vital for improving patient outcome and developing targeted treatments. Our study investigates cellular heterogeneity in immunoglobulin light-chain amyloidosis (AL) and coexisting multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS) (AL_MM) using single-cell RNA sequencing (scRNA-seq). A total of 21 bone marrow samples from 20 patients were analyzed, including 9 with AL_MM. We observed exclusive association of the 1q gain chromosomal aberration with the AL_MM group (p<0.01). The scRNA-seq data revealed 27 distinct cell clusters representing 14 unique cell types, with 6 clusters identified as plasma cell (PC) subpopulations. These subpopulations exhibited considerable inter- and intra-individual heterogeneity. Comparative analysis between the AL and AL_MM groups showed higher proportions of GMP, HSC, cDC2, and T cells in the AL group (p<0.05), suggesting a distinct tumor microenvironment. Differential gene expression analysis between the two groups identified 152 up-regulated and 134 down-regulated genes in AL_MM samples, with 28 of the up-regulated genes and 1 downregulated gene located on chromosome 1q. Furthermore, the application of inferCNV to the scRNA-seq data revealed amplifications in chromosome 4, 7, 11, 20, and 22 of AL samples. Our findings enhance the understanding of the molecular mechanisms underlying AL and its progression in patients with coexisting MM or MGUS. The study provides valuable insights into disease progression and cellular variability, supporting improved patient stratification and the development of targeted therapies for these complex hematological conditions. KEY POINTSO_LI1q gain is mainly restricted to amyloidosis patients with co-occurring sMM, MM, or MGUS. C_LIO_LIMultiple genes located on chromosome 1 are upregulated in plasma cells of patients with co-occurring AL and MM, potentially contributing to disease progression. C_LI

Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma, is characterized by infiltration of malignant T-cells into the skin. While early-stage disease (IA-IIA) follows an indolent course, approximately 25% of patients progress to late-stage disease (IIB-IVB) with significantly worse prognosis and a median survival of 1-5 years. Identifying which early-stage MF patients are at high risk for disease progression remains difficult. This may be affected by the complex interaction between malignant tumor cells and the tumor microenvironment. Increased numbers of cancer-associated fibroblasts (CAF) are found in early-stage MF and have been shown to support tumor growth, but the subtypes have not yet been classified in lesional MF tissue. We performed single-cell RNA sequencing on skin samples from healthy individuals, early-stage MF patients, and late-stage MF patients to investigate the fibroblast population. Analysis of the highly differentially expressed genes in the fibroblast populations revealed nine distinct subclusters, comprising five major types: ECM/structural, vascular/metabolic, immune-modulatory, antigen-presenting, and developmental fibroblasts. Stage-specific differences revealed that the vascular/metabolic subcluster was enriched in early-stage MF, while the ECM/structural subcluster was enriched in late-stage MF. An antigen-presenting subcluster, a novel and underrecognized subtype, was identified by its high expression of MHC class II genes and pathways essential for antigen processing and presentation of exogenous peptides. These inappropriate antigen-presenting fibroblasts may play a role in chronic T-cell exhaustion seen in late-stage diseases. Further studies with additional patient samples will validate these findings and clarify the role of these subtypes in diagnosing and predicting the outcome of mycosis fungoides. Translational RelevanceOur findings reveal heterogeneity among fibroblasts in mycosis fungoides (MF) skin lesions, highlighting potential prognostic biomarkers and therapeutic targets with direct implications for improving the risk assessment and treatment of advanced disease. Using single-cell RNA sequencing, nine transcriptionally distinct fibroblast subclusters were identified that are involved in ECM remodeling, immune modulation, and metabolic adaptation. Examining both early- and late-stage MF disease, in a racially diverse patient cohort, offers new insights into how fibroblast composition changes during disease progression and across demographics. Additionally, we characterize an antigen-presenting fibroblast population in MF that may play an underappreciated role in the chronically exhausted T cells of advanced-stage disease.

Hematopoietic stem cell transplantation is a common treatment for many blood disorders and can be a life-saving therapy for patients with leukemias, lymphomas and multiple myeloma. Umbilical cord blood (UCB) serves as a valuable source of hematopoietic stem and progenitor cells (HSPCs) for transplantation, particularly for patients lacking a matched donor. However, the limited number of repopulating cells in UCB units restricts its clinical utility. Our prior studies showed that genetic deletion of the polycomb repressive complex 2 (PRC2) co-factor Jarid2 in mouse multipotent progenitors (MPPs) conveyed ectopic self-renewal capacity. Here, we hypothesized that the function of human HSPCs could be enhanced through JARID2 inhibition. In this study, we demonstrate that both constitutive and transient knockdown of JARID2 increases the number and enhances the functionality of human HSPCs both in vitro and in vivo. This phenotype was distinct from inhibition of EZH2 in UCB cells, suggesting the mechanism was independent of PRC2 co-factor activity of JARID2. Mechanistically, JARID2 knockdown promotes a quiescent, long-term self-renewal gene expression program governed by upregulating STAT1 and characterized by an MHC class II immunophenotype. Analogous to mice, these mechanisms conferred HSC-like potential to human MPPs in vivo. Taken together, these findings highlight JARID2 inhibition as a novel and reversible approach to expand functional UCB-derived HSPCs ex vivo, potentially improving access to stem cell transplantation for a wider patient population. One Sentence SummaryGenetic inhibition of JARID2 enhances repopulating activity of human hematopoietic stem and progenitor cells in vivo via STAT1 upregulation.

Over the last decade, more data has revealed that increased surface expression of the "dont eat me" CD47 protein on cancer cells plays a role in immune evasion and tumor progression, with CD47 blockade emerging as a new therapy in immuno-oncology. CD47 is critical in regulating cell homeostasis and clearance, as binding of CD47 to the inhibitory receptor SIRP can prevent phagocytosis and macrophage-mediated cell clearance. The purpose of this study was to examine the role of the CD47-SIRP signal in platelet homeostasis and clearance. Therapeutic reagents targeting the CD47-SIRP axis are very promising for treatment of hematologic malignancies and solid tumors, but lead to transient anemia or thrombocytopenia in a subset of patients. We found that platelet homeostatic clearance is regulated through the CD47-SIRP axis and that therapeutic blockade to disrupt this interaction in mice and in humans has a significant impact on platelet levels. Furthermore, we identified genetic variations at the SIRPA locus that impact platelet levels in humans such that higher SIRPA gene expression is associated with higher platelet levels. SIRPA expression at either end of the normal range may affect clinical outcomes of treatment with anti-CD47 therapy. Key pointsO_LIPlatelet homeostasis is regulated through the CD47-SIRP axis and therapeutic blockade to disrupt this interaction impacts platelet levels C_LIO_LICommon genetic variants at SIRPA locus associate with platelet levels C_LI

Mastocytosis is a clonal disorder driven by KIT mutations, but resistance to tyrosine kinase inhibitors (TKIs) remains a major challenge. Following the discovery of an AXL L197M mutation in a patient with congenital aggressive mastocytosis, we demonstrated unexpected wild-type AXL expression in neoplastic mast cells (MCs) across mastocytosis subtypes, challenging current views concerning mastocytosis pathophysiology. AXL was undetectable in steady-state MCs but several factors, including IFN- and IFN-{beta}, induced its expression, consistent with the inflammatory nature of mastocytosis and the high interferon levels in patient plasma. Ectopic expression of WT or L197M AXL in the ROSA KIT D816V cell line enhanced proliferation and survival by upregulating pSTAT5, pSTAT3, pFAK, p-p38, survivin and BCL2. Both AXL forms conferred resistance to the KIT inhibitor PKC412/midostaurin by sustaining BCL2, MCL1, and BCL-XL expression while reducing caspase-3 activation. L197M AXL induced slightly stronger resistance to apoptosis than WT, but this difference was not significant. Combined KIT and AXL targeting (PKC412+R428) restored TKI sensitivity by downregulating BCL-XL, Livin and cIAP1, and activating caspase-3, highlighting the therapeutic potential of dual KIT/AXL pathway inhibition. Importantly, neoplastic MCs from a mast cell leukemia patient harboring the KIT F522C mutation and unresponsive to PKC412 strongly expressed AXL and displayed marked in vitro sensitivity to R428 alone, highlighting AXL as a potential therapeutic target in aggressive mastocytosis not driven by KIT D816V. These findings identify AXL as a previously unrecognized driver of malignant MC survival and TKI resistance, and support AXL inhibition as a promising therapeutic strategy in aggressive mastocytosis. Key Points- AXL is aberrantly expressed in neoplastic mast cells, driving survival and resistance to KIT inhibition in mastocytosis. - Dual KIT and AXL inhibition restores TKI sensitivity in KIT-mutant mastocytosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/686205v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1569c9eorg.highwire.dtl.DTLVardef@1445b32org.highwire.dtl.DTLVardef@bf3565org.highwire.dtl.DTLVardef@14a94f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Clonal hematopoiesis (CH) is an early indicator of hematologic malignancies, driven by mutations in hematopoietic stem cells (HSCs) such as TET2 or TP53. Mutations in ZBTB33 have been implicated in MDS and suggested as a potential driver of CH. However, the role of ZBTB33 in hematopoiesis and its involvement in CH remains unclear. We generated a Zbtb33-knockout mouse strain to elucidate its role in hematopoiesis and the immune system. Our findings indicate that hematopoiesis in Zbtb33-defecient mice appeared grossly normal, and competitive bone marrow transplantation assays demonstrated that loss of Zbtb33 in HSCs did not confer expansional advantage. Introducing the Zbtb33 mutation into Tet2- or Tp53-mutation background yielded no synergistical effects. Tumor challenging assays suggested that Zbtb33 influences cancer immunity response, rather than directly driving CH or myeloid malignancies. In summary, ZBTB33 deficiency was insufficient to induce clonal hematopoiesis but may have a regulatory role in tumor microenvironment. Statement of significanceClonal hematopoiesis (CH) is linked to mutations in hematopoietic stem cells, but the role of Zbtb33 in CH remains unclear. To investigate this, we examined the function of Zbtb33 under physiological conditions and in response to external stimuli. Additionally, we explored whether Zbtb33 mutations cooperate with other genetic mutations to drive clonal hematopoiesis. Key pointsO_LILoss of Zbtb33 fails to induce clonal hematopoiesis and does not synergize with Tet or Tp53 mutations. C_LIO_LIHowever, it plays a significant role in regulating cancer immunity and the tumor microenvironment. C_LI

The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPR-mediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome. Key PointsO_LIGLUT1 knockout does not affect erythroid differentiation and minimally impacts reticulocyte membrane composition C_LIO_LIMetabolic adaptation facilitates reticulocyte tolerance of GLUT1 absence C_LI

Bone marrow mesenchymal stromal cells (MSCs) are a major source of secreted factors that control hematopoietic stem and progenitor cell (HSPC) function. We previously reported the generation of revitalized MSCs (rMSCs), which more effectively support HSPCs in culture. In a secretome screen using rMSCs, we identified semaphorin 3A (SEM3A) as a secreted factor upregulated as part of a pro-inflammatory signature that may contribute to HSPC expansion by rMSCs. We show that recombinant SEM3A acts directly on HSPCs to inhibit their cycling ex vivo. Analysis of a SEM3A loss of function mutation in vivo revealed hematopoietic progenitor expansion and accelerated recovery after myeloablation, consistent with a role for SEM3A in regulating HSPCs at steady state and during hematopoietic stress. This work highlights proteomic screening using rMSCs as a method to identify novel secreted niche factors and uncovers a novel role for SEM3A in controlling HSPC proliferation in stress hematopoiesis. SummaryBorger et al. characterize the secretome of revitalized bone marrow stromal cells and identify a novel role of the protein semaphorin 3A in regulating hematopoietic stem and progenitor cell proliferation in steady state and stress conditions.

Mycosis fungoides (MF) is a rare cutaneous T cell lymphoma that, in its early stages, can closely mimic eczema and psoriasis in both clinical appearance and histopathologic features, leading to frequent misdiagnosis, inappropriate treatment, and delayed care. Reliable adjunctive biomarkers are lacking, underscoring the need for improved diagnostic strategies. We developed a biomarker discovery framework based on bulk RNA sequencing of skin biopsies (19 MF, 112 psoriasis, 105 eczema), which identified seven candidate diagnostic genes. RT-PCR analysis in FFPE tissue specimens from 65 MF and 101 eczema/psoriasis samples verified LCK and HOMER1 as robust discriminator genes. A logistic regression model based on LCK and HOMER1 gene expression differentiated MF from psoriasis and eczema with 91% sensitivity and 94% specificity. Independent validation on 7 additional international cohorts (MF n=58, eczema/psoriasis n=55) confirmed robust performance. Spatial and single-cell transcriptomic analyses revealed biological underpinnings of classifier accuracy: LCK was enriched in malignant and specific T cell subsets in MF, whereas HOMER1 was confined to keratinocytes in eczema and psoriasis but nearly absent in MF. Case studies demonstrated that the classifier identified MF in routine biopsies earlier than histopathology. This molecular diagnostic approach enables earlier and more reliable distinction of MF from common inflammatory dermatoses, offering a clinically applicable tool to reduce diagnostic uncertainty, accelerate appropriate treatment, and might improve patient outcomes. One Sentence SummaryA two-gene classifier reliably distinguishes mycosis fungoides from eczema and psoriasis, enabling early and accurate diagnosis.

The transplantation of gene-modified autologous hematopoietic stem and progenitor cells (HSPCs) offers a promising therapeutic approach for hematological and immunological disorders. However, this strategy is often limited by the toxicities associated with traditional conditioning regimens. Antibody-based conditioning strategies targeting cKIT and CD45 antigens have shown potential in mitigating these toxicities, but their long-term safety and efficacy in clinical settings require further validation. In this study, we investigate the thrombopoietin (TPO) receptor, cMPL, as a novel target for conditioning protocols. We demonstrate that high surface expression of cMPL is a hallmark feature of long-term repopulating hematopoietic stem cells (LT-HSCs) within the adult human CD34+ HSPC subset. Targeting the cMPL receptor facilitates the separation of human LT-HSCs from mature progenitors, a delineation not achievable with cKIT. Leveraging this finding, we developed a cMPL-targeting immunotoxin, demonstrating its ability to selectively deplete host cMPLhigh LT-HSCs with a favorable safety profile and rapid clearance within 24 hours post-infusion in rhesus macaques. These findings present significant potential to advance our understanding of human hematopoiesis and enhance the therapeutic outcomes of ex vivo autologous HSPC gene therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/581887v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@10b265aorg.highwire.dtl.DTLVardef@102ec94org.highwire.dtl.DTLVardef@fa269dorg.highwire.dtl.DTLVardef@fd2583_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

We employ and extensively characterise an ex vivo culture system to study terminal erythroid maturation of CD34+ progenitors from the peripheral blood of normal individuals and patients with Congenital Dyserythropoietic Anaemia type 1 (CDA-I). Using morphological analysis, FACS analysis and the proteomic approach CyTOF, we analysed patient-derived erythroblasts stage-matched with those from healthy donors during the expansion phase and into early differentiation. In patient cells, aspects of disordered erythropoiesis manifest midway through differentiation, including increased proliferation and changes in the DNA accessibility profile. We also show that cultured erythroblasts from CDA-I patients recapitulate the pathognomic feature of this erythroid disorder with up to 40% of the cells having abnormal spongy chromatin morphology by electron microscopy, as well as upregulation of GDF15, a marker of ineffective erythropoiesis. In the tertiary phase of culture, patient cells show significantly less enucleation and there is persistence of earlier erythroid precursors. Furthermore, the enucleation defect appears to be more severe in patients with mutations in C15orf41, as compared to the other known causative gene CDAN1, indicating a genotype/phenotype correlation in CDA-I. Such erythroblasts are a valuable resource for investigating the pathogenesis of this disease and provide the opportunity for streamlining diagnosis for CDA-I patients and ultimately other forms of unexplained anaemia.

Mature erythrocytes are under tight homeostatic control with the need for constant replacement from progenitors to replace damaged or obsolete red blood cells (RBCs). This process is regulated largely by erythropoietin (Epo) which promotes the survival of erythroid progenitors and facilitates their differentiation and proliferation. Ablation of Bcl2l1 (which encodes BCL-xL) results in embryonic lethality with a lack of mature erythrocytes but does not perturb erythroid progenitors. Similarly, conditional Bcl2l1-deletion results in severe anemia with the death of late erythroid progenitors and induction of extramedullary erythropoiesis. While BCL-xL is critical to the survival of mature erythrocytes, it is still unclear whether other anti-apoptotic molecules mediate survival during earlier stages of erythropoiesis. Here, we demonstrate that erythroid-specific Mcl1-deletion results in embryonic lethality due to severe anemia caused by a lack of mature RBCs. Mcl1-deleted embryos exhibit stunted growth, ischemic necrosis, and decreased RBCs in the blood. Furthermore, we demonstrate that the dependence on MCL-1 is only during early erythropoiesis, whereas during later stages the cells become MCL-1-independent and upregulate the expression of BCL-xL. Functionally, MCL-1 relies upon its ability to prevent apoptosis to promote erythroid development since co-deletion of the pro-apoptotic effectors Bax and Bak can overcome the requirement for MCL-1 expression. Furthermore, ectopic expression of human BCL2 in erythroid progenitors can compensate for Mcl1 deletion, indicating redundancy between these two anti-apoptotic family members. These data clearly demonstrate a requirement for MCL-1 in promoting survival of early erythroid progenitors.

Cyclin-dependent kinase 9 (CDK9) drives transcriptional elongation and supports the expression of short-lived oncogenic and anti-apoptotic proteins such as MYC and MCL1. PRT2527 is a potent, selective CDK9 inhibitor currently in early clinical development. We evaluated its preclinical activity in marginal zone lymphoma (MZL) models, including cell lines with acquired resistance to BTK, PI3K, and BCL2 inhibitors. Short exposure (4 hours) to PRT2527 produced nanomolar cytotoxicity across all tested MZL cell lines, with efficacy maintained in resistant derivatives. Transcriptomic profiling of VL51 cells showed broad gene repression, including MYC, IRF4, NF-{kappa}B-related genes, and MCL1, alongside increased expression of HLA class II genes. Moreover, comparison with additional CDK inhibitors revealed a similar transcriptional repression signature, underscoring a conserved CDK-dependent regulatory network. Protein analyses confirmed rapid depletion of MCL1, MYC, RNA polymerase II, and IRF4. Flow cytometry validated increased HLA class II and decreased HLA class I surface expression. Combination studies demonstrated additive to synergistic effects with BTK inhibition (ibrutinib) or dual PI3K/BCL2 inhibition (copanlisib plus venetoclax), independent of baseline drug sensitivity. Mechanistically, these combinations may enhance apoptosis by concurrently suppressing survival signaling and transcriptional addiction. Analysis of patient samples revealed high CDK9 expression, further supporting the biological relevance and therapeutic rationale for targeting CDK9 in this disease. Our findings support the development of CDK9-based combination strategies for relapsed/refractory MZL and other B-cell malignancies, with an additional potential for integration with immunotherapies. Key PointsO_LICDK9 inhibitor PRT2527 kills marginal zone lymphoma cells, regardless of whether they are resistant to other targeted drugs. C_LIO_LIPRT2527 boosts the effects of BTK, PI3K, and BCL2 inhibitors and alters immune-related gene expression. C_LI Draft of graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/705084v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@37b9e6org.highwire.dtl.DTLVardef@8de6a5org.highwire.dtl.DTLVardef@219771org.highwire.dtl.DTLVardef@15dabfe_HPS_FORMAT_FIGEXP M_FIG C_FIG

Platelet -granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germline RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMM), is associated with thrombocytopenia, platelet dysfunction and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen and IgG levels were decreased in a FPDMM patient. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, siRNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared to control cells, with increases in caveolin-1 and flotillin-1 (two independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes) and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 knockdown increased colocalization of albumin with flotillin and of fibrinogen with RAB11 suggesting altered trafficking of both. The increased albumin and fibrinogen uptake and levels of caveolin-1, flotillin-1, LAMP2 and IFITM3 were recapitulated by shRNA RUNX1 knockdown in CD34+-derived MK. These studies provide the first evidence that in RUNX1-haplodeficiency platelet endocytosis of albumin and fibrinogen is impaired and that megakaryocytes have enhanced endocytosis with defective trafficking leading to loss of these proteins by distinct mechanisms. They provide new insights into mechanisms governing endocytosis and -granule deficiencies in RUNX1-haplodeficiency. Key pointsO_LIPlatelet content and endocytosis of -granule proteins, albumin, fibrinogen and IgG, are decreased in germline RUNX1 haplodeficiency. C_LIO_LIIn RUNX1-deficient HEL cells and primary MK endocytosis is enhanced with defective trafficking leading to decreased protein levels. C_LI

To explore the role of clonal hematopoiesis (CH) on chimeric antigen receptor (CAR) T-cell therapy outcomes, we performed targeted deep-sequencing on 114 large B-cell lymphoma patients treated with anti-CD19 CAR T-cells. We detected CH in 42 (36.8%) pre-treatment patient samples, most frequently in PPM1D (19/114) and TP53 (13/114) genes. The incidence of grade [&ge;]3 immune-effector cell-associated neurotoxicity syndrome (ICANS) was higher in CH-positive patients compared to CH-negative patients (45.2% vs. 25.0%, p=0.038). Higher toxicities with CH were primarily driven by three CH genes, DNMT3A, TET2 and ASXL1 (DTA mutations). The incidence of grade [&ge;]3 ICANS [58.9% vs. 25%, p=0.02] and grade [&ge;]3 cytokine release syndrome [17.7% vs. 4.2%, p=0.08] were higher in patients with DTA mutations than those without CH. The estimated 24-month cumulative incidence of therapy-related myeloid neoplasms after CAR-T therapy was higher in patients with CH than those without CH (19% [95%CI: 5.5-38.7] vs. 4.2% [95%CI: 0.3-18.4], p=0.028). Statement of SignificanceOur study reveals that clonal hematopoiesis mutations, especially those associated with inflammation (DNMT3A, TET2, ASXL1), are associated with severe grade toxicities in lymphoma patients receiving anti-CD19 chimeric antigen receptor therapy. Further studies to investigate the mechanisms and interventions to improve toxicities in the context of CH are warranted.

Diffuse large B cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma and the most frequently diagnosed hematologic malignancy in the United States. DLBCL exhibits significant molecular and clinical heterogeneity, and at least a third of patients are left uncured with standard frontline chemoimmunotherapy. As such, there is a critical need to identify novel targeted therapies to improve outcomes. We conducted a phenotypic screen of kinase inhibitors against DLBCL cell lines and non-malignant controls. We identified the cyclin G-associated kinase (GAK) as a tumor-selective, readily druggable target whose inhibition killed DLBCL cell lines, while sparing non-malignant blood cells. Upon investigation of GAKs cellular function, we discovered that inhibition results in G2/M-phase cell cycle arrest. Immunofluorescent confocal microscopy revealed significant chromosome misalignment and spindle distortion in DLBCL cells following GAK-inhibition, disrupting progression through mitosis. Analysis of RNA-seq data from clinical samples showed increased GAK expression associates strongly with RB1 deficiency in DLBCL cases, suggesting dependency on GAK for proper mitotic progression linked to retinoblastoma associated protein (RB) loss of function, a common DLBCL driver. In cell-cycle analyses and under microscopy, RB-deficient DLBCL cells treated with an exquisitely selective GAK inhibitor showed complete arrest at G2/M, pronounced distortion of mitotic spindles, and widespread chromosomal damage. Finally, in vivo studies of DLBCL xenograft-bearing NSG mice achieved a dramatic tumor-burden reduction in response to targeted GAK inhibition. These results reveal a novel cell cycle kinase suitable for therapeutic exploitation in DLBCL patients and linked to the common, undruggable biomarker of RB loss of function. KEY POINTSO_LIPhenotypic screening and target deconvolution revealed GAK as a novel therapeutic target in diffuse large B-cell lymphoma C_LIO_LIGAK kinase activity is a key requirement for successful mitosis in DLBCL linked to deficiency of the retinoblastoma (RB) tumor supressor C_LI

The development of antibody responses to SARS-CoV-2 is an indicator of seroprevalence and may afford protection from infection. It has been presumed that antibody responses to SARS-CoV-2 will be impaired in patients with aggressive haematological malignancy (PHM) due to underlying immunological dysfunction caused by malignancy or systemic anti-cancer treatment (SACT), placing them at increased risk. Here we analysed longitudinal serum samples from ten hospitalised PHM with aggressive disease and on SACT, collected up to 103 days post-onset of COVID-19 symptoms. We found that the majority (8/9) of PHM with confirmed SARS-CoV-2 infection seroconverted and developed antibodies to the major SARS-CoV-2 antigens (S1 and N) with most (6/8) produced neutralising antibody responses. Furthermore, the dynamics of antibody responses were broadly similar to that reported for the general population, except for a possible delay to seroconversion. Our finding that PHM on SACT can make functional antibody responses to SARS-CoV-2 has important implications for patient management and serological monitoring of SARS-CoV-2 in high-risk groups.